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[南尧说生]

"This theory is currently the most promising theory for ending aging. I have integrated the mainstream aging theories through metabolic flux and temporal sequencing. In subsection 5.7, I have factually falsified the replicative senescence theory, which was previously over-extrapolated to the in vivo context, reducing its absoluteness and status as a primary cause in the field of aging. Here, I welcome individuals to discuss and research the theory with me, and to jointly explore and unravel the mysteries of aging."

 

 Email:   DTKK4863@outlook.com

 

Comprehensive Decoding of Aging Pathways: The Metabolic Homeostasis Cascade Collapse Convergence and Re-Drive Model Hypothesis  

 

  Abstract  

 

Static wear-and-tear theories cannot explain the diversity, non-linear acceleration, and cliff-like phenotypes of aging. This paper proposes that the essence of aging is the cascade collapse and self-locking of metabolic flux homeostasis. The core is a central axis model composed of AMPK, mTOR, PGC-1α, and PPARα: the axis slips from rhythmic oscillation to a non-steady state, converging on a decline in mitochondrial functionality (the first internal drive node), subsequently triggering stepwise disorders in glycolipid metabolism, GH/IGF-1 decline, antioxidant network collapse, inflammatory-glucocorticoid hijacking, and senescent cell accumulation, forming a six-factor self-locking cascade. This theory is strictly limited to the human physiological context, provides testable predictions (e.g., adolescent erythrocyte deformability as an early marker of central axis imbalance), and shifts aging intervention from "single-target repair" to "restoring systemic rhythm."

 

  Keywords:   Aging; Mitochondria; Metabolic Homeostasis; AMPK; mTOR; PGC-1α; PPARα; Inflammation; Growth Hormone; Paradigm Shift

 

  Author:   Yingjie Jin

  Email:   DTKK4863@outlook.com

 

  Introduction  

 

Current static aging theories define the essence of aging as static wear-and-tear, yet they struggle to explain three core paradoxes: inter-individual heterogeneity, non-linear acceleration, and cliff-like senescence. These three phenomena themselves constitute a falsification of single-causation models.

 

This paper constructs a dynamic aging theory based on metabolic flux homeostasis, whose architecture possesses a threefold explanatory power. First, through the multi-parameter phase space of metabolic flux homeostasis, it can computationally explain aging diversity—the set-point bias of different individuals on the central axis (AMPK-mTOR-PGC-1α-PPARα) determines the differential emergence of pathological phenotypes. Second, through multi-factorial convergence onto the critical node of declining mitochondrial functionality, it explains the phenotypic convergence of aging. Ultimately, by revealing the mechanism through which the dominant factors of aging switch with the phase transitions of metabolic homeostasis, it elucidates the non-linear dynamic nature of aging acceleration and cliff-like senescence.

 

However, the core parameters of this dynamic framework (VC antioxidant buffering capacity, GH/IGF-1-NRF1 mitochondrial regulatory axis, local skin IGF signaling network, etc.) have unique metabolic configurations in humans. Traditional research has long extrapolated observational data from rodent models and plant systems (such as bamboo) to humans, ignoring fundamental differences in metabolic architecture between species—humans lack the capacity for endogenous vitamin C synthesis, the regulation mode of the GH/IGF-1 axis is qualitatively different from that in rodents, and the skin exhibits a local IGF signaling dilemma. Such extrapolations carry a significant risk of metabolic context misalignment.

 

This study establishes a directionally specialized human dynamic aging theory framework, covering multi-level mechanisms including cellular energy metabolism, epigenetic clocks, tissue microenvironment interactions, and systemic homeostasis regulation, forming a comprehensive explanation for the asynchrony, heterogeneity, and intervenability of human aging. This theory is strictly limited to the human physiological context; if applied to rodents, bamboo, or other species, targeted corrections must be made for their metabolic characteristics (such as endogenous VC synthesis, IGF-1 axis patterns, NRF1 tissue expression, etc.) to achieve cross-species compatibility.

 

  Key Argument: Mitochondrial Functional Decline is the Endogenous Driver of Aging  

 

The core argument of this paper is that the decline in mitochondrial functionality, driven by metabolic homeostasis imbalance, is the fundamental, endogenous factor driving the entire aging process.

 

This argument does not negate the importance of other aging mechanisms but repositions them, describing who is the background driver at this stage, who is the primary driver at this stage, and who is a downstream phenomenon in the aging process, thus forming a temporal sequence. Mitochondrial functionality is the critical node that converges and integrates all upstream factors to re-drive aging. This hypothesis posits that mitochondria are not merely passive "victims" damaged during aging, but active "instigators." The decline in mitochondrial functionality, as the starting point of the aging program, initiates and amplifies a subsequent series of aging-related pathways through its core position as the cell's energy factory and signaling hub. This viewpoint is strongly supported by recent research. For example, a study published in  Scientific Reports  found that by reprogramming aged individual fibroblasts into induced pluripotent stem cells (iPSCs), age-related mitochondrial respiratory defects could be completely reversed, restoring them to levels comparable to fetal fibroblasts. This result indicates that the aging state of mitochondria is not determined by irreversible mtDNA mutations but is subject to dynamic, reversible epigenetic regulation. Combined with empirical research on epigenetic drift aging, which shows that aging is not caused by gene mutation but by the distortion of information during transmission, it can be jointly deduced that aging is reversible.

 

Furthermore, based on the empirical study of increased inflammatory factor concentrations in adolescents aged 9-17 [216], the two modes of inflammatory factor increase mentioned in that literature, and the literature on the effects of inflammatory factors on mitochondrial functionality [159][158], and considering that multiple aging pathways commonly point towards pro-inflammatory states, I identify the decline in mitochondrial functionality as the first initiating node of convergence and re-driving of multiple aging factors, while inflammatory factors serve as the linking node running through all aging drivers [50].

 

  1.3 Definition and Scope of Mitochondrial Functional Decline  

 

To precisely articulate our theory, "mitochondrial functional decline" must be strictly defined. In the framework of this paper, mitochondrial functional decline specifically refers to a situation where, under conditions of normal mitochondrial quantity and quality, the efficiency of its physiological function, ATP output, declines. This situation may be caused by multiple factors, such as interference from inflammatory factors, weakened antioxidant function, decreased erythrocyte oxygen release efficiency, shortened duration of AMPK signaling, or excessively weak AMPK signaling—multiple multi-factorial causes triggering mitochondrial functional decline. This functional decline does not lead to cell apoptosis but is sufficient to disrupt the cell's normal physiological rhythm, initiating a series of adaptive or pathological signaling pathways.

 

For example, literature [173] points out that when erythrocyte deformability decreases, their oxygen affinity increases and oxygen release capacity decreases. Therefore, if human erythrocytes experience decreased deformability due to factors like hyperlipidemia, oxidative stress, and insufficient nitric oxide production, the oxygen released by erythrocytes to normal body cells within the same time window will decrease. Consequently, the cellular oxidative phosphorylation efficiency will decline. This point does not depend on a decrease in mitochondrial quantity and quality.

 

  [Conceptual Distinction]  

  Epigenetic Inertia:   In this hypothesis, this specifically refers to a state where key genes such as PPARα, AMPK, mTOR, and PGC-1α are locked into a stable, low-expression level that is difficult to self-recover after being suppressed. This is a specific dysfunctional pattern in early aging.

 

  Epigenetic Disorder:   Refers to the systemic, global dysregulation of genome-wide epigenetic modifications (such as DNA methylation) in the late stages of aging, caused by reasons such as the deficiency of metabolic substrates like α-ketoglutarate (AKG).

 

  2. Core Regulatory Network of Mitochondrial Functional Decline - The Central Axis Model  

 

In literature [216], within a latent class growth analysis model, a total of 6,556 participants were assigned to a group/trajectory class. The optimal model identified three CRP trajectories: 1. Reference group (n=6109; 93%) – persistently low CRP levels. 2. Early-peak group (n=197; 3%) – CRP levels peaked at age 9. 3. Late-peak group (n=250; 4%) – CRP levels peaked at age 17. The mechanism of this inflammation cannot be explained by traditional inflammatory models. Attributing it to developmental, gene-specific drives of different inflammations cannot explain why so many people have the same genetic mutations predisposing them to inflammation. However, the model of erroneous metabolic homeostasis driving inflammatory factor production can explain the source of inflammation in children aged 9-17 and the different patterns of its formation.

 

  2.1 Based on theoretical deduction of existing pathway interaction patterns, I constructed the central axis model, which can integrate past aging theories.  

 

The central axis model is a metabolic network based on the combination of four key metabolic pathways: Peroxisome Proliferator-Activated Receptor Alpha (PPARα), AMP-Activated Protein Kinase (AMPK), Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha (PGC-1α), and Mammalian Target of Rapamycin (mTOR). This integrates the multiple aging mechanisms from past traditional theories, forming a key central robust mechanism that determines which aging mechanism an organism is more biased towards at the early stages of aging, explaining the diversity of aging and, more profoundly, the underlying principles of this diversity.

 

Its core can be defined by seven rules.

 

1.  AMPK can inhibit mTOR. Literature [14] points out that mTOR can be inhibited by enhancing AMPK.

2.  mTOR can inhibit AMPK. Literature [15] points out that AMPK can be inhibited by enhancing mTOR.

3.  Synergy and antagonism between PPARα and PGC-1α. Literature [59] indicates that PGC-1α serves as the basis for PPARα function. Literature [62] points out that if PPARα expression is insufficient, PGC-1α is more inclined to initiate NRF1/NRF2-related antioxidant genes. Therefore, it can be inferred here that PGC-1α is functionally inhibited by PPARα. Conversely, if PGC-1α is highly expressed and PPARα is insufficient, a tendency towards NRF1/NRF2 signaling dominance will occur.

4.  AMPK can increase PGC-1α expression, and mTOR can increase PPARα expression. Literature [98] indicates the promotion mechanism of mTOR on PPARα, while literature [14] indicates the promotion mechanism of AMPK on PGC-1α.

5.  Oxidized PTEN activates mTOR. Literature [78] indicates that when PTEN is oxidized, the mTOR signaling pathway is activated.

6.  The flux of the Pentose Phosphate Pathway (PPP) is a key factor for which AMPK, mTOR, PGC-1α, and PPARα must compete within the same time window. Literature [162] indicates that ribose-5-phosphate (R5P) from the PPP is the sole carbon skeleton source for synthesizing purine/pyrimidine nucleotides, meaning a decrease in its flux will lead to a decrease in the level of the shared resource pool for gene expression. Simultaneously, literature [56] indicates that NADPH, a product of the PPP pathway, provides reducing equivalents for oxidative protein folding; its deficiency increases protein misfolding, increases the proportion of ineffective proteins and inhibits signaling, which can also be viewed as a resource that AMPK, mTOR, PGC-1α, and PPARα must jointly compete for.

7.  The interaction mechanism of PPARα/PGC-1α: The transcriptional self-maintenance of PPARα depends on the de novo synthesis of very long-chain fatty acids (VLCFA), a process that consumes NADPH. Conversely, the NRF1/NRF2 antioxidant pathway mediated by PGC-1α also requires NADPH for glutathione regeneration. When PGC-1α is highly expressed, NADPH flux is siphoned off by antioxidant demands, inhibiting VLCFA synthesis, thereby blocking the self-maintaining positive feedback loop of PPARα. This competition for Pentose Phosphate Pathway (PPP) flux constitutes the molecular basis for the 'fatty acid oxidation-antioxidant defense' zero-sum game in the central axis model, determining whether the cell tends towards PPARα-dominated anabolic homeostasis or PGC-1α-dominated catabolic/antioxidant homeostasis. [268]

 

  2.2 The specific operational mechanism of the central axis model.  

 

In the central axis model, the four key gene pathways—PPARα, AMPK, mTOR, PGC-1α—jointly control four mechanisms crucial to organisms: the tricarboxylic acid (TCA) cycle, oxidative phosphorylation, biosynthetic metabolism, and antioxidant networks. These four mechanisms are interconnected. By simplifying the expression and activity state of each core factor (AMPK, mTOR, PGC-1α, PPARα) into three basic levels: too weak, steady state, and too strong, the total number of combinations formed by these four variables is theoretically 3 to the power of 4, i.e., 81 possible "functional state combinations."

 

Among these 81 combinations, there is one and only one combination representing the perfect internal homeostasis enjoyed in early life: all four cores are in their precise "steady-state" interval. This state is the sole ideal state where mitochondrial function is optimized and energy metabolism is clean and efficient. It is the only stable state where the organism could halt aging before it begins. The remaining 80 combinations all constitute the starting point that triggers the decline in mitochondrial functionality, the primary driver of aging.

 

  2.2.1 Normally functioning central axis model.  

 

Starting with the change in AMP/ATP ratio caused by decreased biosynthetic metabolism controlled by the mTOR pathway, which promotes AMPK, the AMPK pathway is activated due to the AMP/ATP signal. During this process, the high-intensity AMPK signal drives PGC-1α, leading to antioxidant defense and mitochondrial maintenance. However, this path only initiates PGC-1α rather than enhancing its self-maintenance axis strength. When the cell obtains sufficient ATP and completes energy production, mTOR is activated by the energy signal, promoting the TCA cycle by activating PPARα, manufacturing energy, maintaining methylation and demethylation homeostasis, and completing biosynthesis and protein assembly, the key goals for organismal survival. This continues until the mTOR pathway, due to the synthesis process, causes ATP levels to drop, activating the AMPK pathway again, thus starting the next metabolic cycle of the central axis model.

 

  2.2.2 Epigenetic inertia formed by erroneous operation of the central axis's self-maintenance mechanisms.  

 

AMPK, mTOR, PPARα, and PGC-1α all possess self-metabolic maintenance mechanisms, which determine the expression strength of these four pathways.

 

  2.2.2.1 The imbalance mechanism of the first cause: Epigenetic inertia formed by competitive collapse of homeostatic circuits.  

 

This hypothesis posits that the essence of mitochondrial functional decline is the loss of dynamic balance in the core regulatory circuits maintaining its functional homeostasis. These circuits depend on the self-positive feedback maintenance of key molecules and antagonize other circuits. Once external stress persists too long, the positive feedback loops are disrupted, and their expression levels become locked in a pathological range, thus driving mitochondrial function decline through various pathways, forming a convergent model that drives aging.

 

  Definition of Co-activator Factor:   Co-activator factor refers to the dynamic resource pool of ribose-5-phosphate and NADPH determined by the flux of the Pentose Phosphate Pathway (PPP).

The Pentose Phosphate Pathway has two main functions: producing NADPH and producing ribose-5-phosphate.

Ribose-5-phosphate sustains cellular gene transcription. If it is deficient, the cell will lack a source of ribonucleotides. NADPH is responsible for the synthesis of cellular deoxyribonucleotides, maintaining intracellular gene repair, and the redox folding of proteins during transcription. The normal expression of the four core genes AMPK, mTOR, PPARα, and PGC-1α requires the joint collaboration of ribose-5-phosphate and NADPH. Therefore, the dynamic resource pool of ribose-5-phosphate and NADPH determined by the upstream Pentose Phosphate Pathway can be identified as the co-activator factor. Although the PPP pathway itself cannot be competed for, the metabolic efficiency and flux of the pathway determine the output quantity of the co-activator factors, maintaining the definition of the co-activator factor.

 

  2.2.2.2 Competition in the energy metabolism hub: Self-maintenance mechanisms and rhythmic inhibition of PPARα and PGC-1α.  

 

PGC-1α achieves its self-maintenance mechanism through the NRF1-lipoic acid-PGC-1α pathway. PPARα achieves its self-maintenance mechanism through co-regulation formed by multiple downstream metabolites of acetyl-CoA during the TCA cycle metabolism. Changes in AMPK and mTOR pathways determine whether PGC-1α dominates or PPARα dominates. Excessive self-maintenance mechanisms will lead to mutual signal antagonism.

 

  Checks and Balances of the Energy Sensing Center: Mutual Inhibition and Self-Maintenance of AMPK and mTOR  

A similar "mutual inhibition-self-maintenance" mechanism also exists between the energy sensing centers AMPK and mTOR.

  AMPK Self-Maintenance:   After AMPK is activated, by inhibiting mTORC1 activity and promoting self-maintenance, this signal would normally reverse through changes in the intracellular AMP/ATP ratio. However, if the pathway where AMPK pathway dominance upregulates NAMPT expression to promote NAD+ regeneration and the NAD+-SIRT1-AMPK pathway malfunctions, then the AMPK pathway will exhibit high or low expression, and the expression intensity will be disordered, failing to maintain rhythmic regulation of PPARα and PGC-1α. This creates an energy environment conducive to its own sustained activation, forming positive feedback.

  mTOR Self-Maintenance:   After mTORC1 is activated, it promotes the synthesis of macromolecules like proteins and lipids. During this process, a large number of free radicals are generated inside the cell. In the normal process, these free radicals would moderately oxidize and modify PTEN to relieve the inhibition of the mTOR pathway, thereby further enhancing the mTOR pathway. However, NRF2 is induced by oxidative stress to express and reduce oxidized PTEN. When the mTOR pathway is erroneously overexpressed, the oxidative stress it triggers will persistently cause PTEN to be oxidatively modified, while the overexpressed NRF2 also triggers oxidative stress, preventing PTEN redox reduction, forming a self-sustaining high-maintenance state of the mTOR cycle.

The two constitute a precise bistable switch. Under physiological conditions, they dynamically oscillate based on energy status, maintaining overall balance. However, under persistent energy stress or nutrient excess, one side's self-maintenance mechanism may be excessively strengthened, completely suppressing the other. For example, long-term nutrient excess consolidates mTOR's "high expression-high activity" steady state and suppresses AMPK to "low expression-low activity," leading to impaired energy sensing, inhibited autophagy, and directly contributing to a decline in mitochondrial quality.

 

  2.3 Connection points where excessive or deficient expression of the four gene pathways AMPK, mTOR, PPARα, and PGC-1α jointly lead to mitochondrial functional decline.  

 

  2.3.1 Mitochondrial functional decline caused by AMPK pathway imbalance  

  Excessive AMPK Signaling:   The AMPK pathway regenerates NAD⁺ through NAMPT. NAD⁺ maintains its own gene expression self-maintenance through the SIRT1-AMPK pathway. When this metabolic level is too high, the AMPK pathway signal will be frequently activated, inhibiting the initiation of mTOR signaling, leading to decreased biosynthetic metabolism. For example, this causes a decrease in nitric oxide (NO) levels, leading to loss of microvascular dilation, and a simultaneous decrease in erythrocyte deformability due to NO deficiency. This triggers microcirculatory disturbance, causing ischemia and hypoxia. Ischemia and hypoxia will promote glycolysis and transcription of inflammatory factors released into the blood.

  Deficient AMPK Signaling:   When the AMPK self-maintenance mechanism is in a weakened state, although the AMPK pathway is not completely inhibited, it leads to decreased energy metabolism. Both oxidative phosphorylation and glycolysis will have reduced ATP production capacity, leading to a decline in mitochondrial functionality. Simultaneously, due to decreased ATP levels, ATP generation in erythrocytes will also decrease, resulting in erythrocyte deformability disorders, causing hypoxia-induced glycolysis and release of inflammatory factors into the blood. At the same time, as DNA repair-related genes are downstream of the AMPK pathway, their decreased expression will lead to reduced gene repair efficiency, increased genomic instability, and weakened inhibition of NF-κB, which also triggers the secretion of inflammatory factors into the blood.

 

  2.3.2 Mitochondrial functional decline caused by mTOR pathway imbalance  

  Excessive mTOR Signaling:   The mTOR pathway promotes itself through ROS free radicals generated during anabolic metabolism, which oxidize and modify the PTEN pathway. Persistent stimulation by ROS free radicals induces high NRF2 expression, leading to the downstream HO-1 pathway decomposing heme to form free iron. Free iron further promotes oxidative stress by generating hydroxyl radicals. This pathway causes erythrocyte deformability disorders through oxidative stress, leading to microcirculatory disturbance, and achieves mitochondrial functional decline through hypoxia. Simultaneously, excessive ROS radicals inhibit mitochondrial metabolic efficiency, and the mTOR pathway itself inhibits the AMPK pathway. ROS itself is also a pro-inflammatory signal. Ischemia and hypoxia caused by decreased erythrocyte deformability can further produce inflammatory factors via microcirculatory disturbance, forming a combined force that jointly inhibits mitochondrial function.

  Deficient mTOR Signaling:   This pathway leads to decreased anabolic metabolism, causing abnormalities in the arginine metabolic pathway, resulting in decreased nitric oxide levels and reduced erythropoiesis. This leads to microcirculatory disturbance, causing ischemia and hypoxia, promoting inflammation, and leading to mitochondrial functional decline due to inflammatory factors. However, unlike excessive AMPK expression, this pathway originates from a malfunction of mTOR's own self-maintenance mechanism and does not imply that AMPK pathway expression is too high.

 

  2.3.3 Mitochondrial functional decline caused by PGC-1α pathway imbalance  

  Excessive PGC-1α Signaling:   This pathway, through its antagonism with PPARα, inhibits the efficiency of the TCA cycle, causing cells to fall into a state of reduced ATP output. This pathway can also inhibit erythrocyte energy metabolism. Simultaneously, by limiting the synthesis of AKG and acetyl-CoA, it induces epigenetic abnormalities, leading to decreased nitric oxide synthesis, reduced erythrocyte deformability, and a reduction in erythrocyte count. The hypoxia-inflammatory factor pathway, combined with its own weakening of the TCA cycle, will jointly cause mitochondrial functional decline.

  Deficient PGC-1α Signaling:   This pathway leads to decreased NRF1 expression, a decline in mitochondrial quality and quantity, and a decrease in the entire antioxidant network's basal antioxidant performance against oxidative stress. However, this pathway can also cause mitochondrial functional decline due to oxidative stress and electron leakage. Simultaneously, it interferes with erythrocyte energy metabolism, increases the risk of erythrocyte oxidative stress, leading to affected erythrocyte deformability. Mitochondria will experience functional decline through the hypoxia-inflammation pathway, and also through electron leakage oxidative stress. If this bias is too high, it may lead to a decrease in mitochondrial quantity and quality even in adolescence.

 

  2.3.4 Mitochondrial functional decline caused by PPARα pathway imbalance  

  Excessive PPARα Signaling:   Overexpression of this pathway triggers oxidative stress and simultaneously inhibits NRF1/NRF2 expression by excessively binding the PGC-1α pathway, achieving oxidative stress effects. This pathway can affect erythrocyte deformability through oxidative stress, forming microcirculatory disturbance. Mitochondrial functional decline occurs due to inflammatory factors induced by hypoxia and electron leakage.

  Deficient PPARα Signaling:   This pathway mainly affects the TCA cycle, leading to a decrease in cellular ATP generation, and simultaneously affects the generation of AKG and ketone bodies. A decrease in AKG will impair the overall cellular demethylation function, leading to epigenetic disorder. Genes related to nitric oxide production become inhibited in demethylation and cannot be expressed, thus reducing nitric oxide levels, causing decreased erythrocyte deformability, forming microcirculatory disturbance. Mitochondrial functional decline occurs due to inflammatory factors induced by hypoxia, combined with its own weak expression.

  Summary:   Theoretically, a perfect central axis should produce more energy during the TCA cycle, and promote mitochondrial regeneration and antioxidant function regeneration during the PGC-1α phase. However, this paper deduces that in reality, such a perfect central axis is very difficult to maintain normally and stably. For example, just one instance of staying up late can disrupt the operation of this central axis, causing its imbalance through hormones, hypoxic microcirculatory disturbance, and oxidative stress.

Simultaneously, the daily diet, such as vegetables containing ferulic acid and antioxidants, can correct this process. The entire process exhibits a dynamically changing state, like a constantly swinging pendulum. Seeking the right path can improve the state of the central axis. The central axis itself provides a reasonable explanation for the appearance of inflammatory factors in 9-17 year olds and their different emergence models, where metabolic errors in biological functions lead to inflammation.

 

  2.4 Correspondence of central axis proportional imbalance with current mainstream aging theories and the rationale.  

 

  I. AMPK Imbalance: Dysregulation of the Energy Sensing Center  

When AMPK is in a "too weak" state, it directly corresponds to the theory of "  autophagy dysfunction and proteostasis imbalance  ." The rationale is that AMPK is the primary kinase for sensing energy crisis (increased AMP/ATP ratio) and initiating autophagy to remove damaged components and recycle resources. Its insufficient activity means the cell cannot activate this crucial "quality control and recycling system" under energy stress. Simultaneously, AMPK's regulation of the balance between protein synthesis and degradation also fails, jointly leading to the accumulation of misfolded proteins and senescent organelles within the cell, dismantling cellular homeostasis from the inside. This is the source of garbage accumulation in aging cells—directly corresponding to the   "Autophagy Dysfunction"   and   "Proteostasis Imbalance"   theories.

When AMPK is in a "too strong" state, it points to the pathological logic of "  anabolic failure and nutrient utilization disorder  ." The physiological role of AMPK is "energy conservation" and "catabolism." Persistently overactive AMPK signaling excessively inhibits its key target mTORC1, which is the main "anabolic" switch driving the biosynthesis of proteins, lipids, etc. Therefore, the abnormal hyperactivity of AMPK is not beneficial but creates a false and persistent state of "cellular famine," inappropriately shutting down the anabolic pathways necessary for cell growth, repair, and regeneration, leading to impaired protein-driven cell function maintenance, repair, and renewal despite sufficient genes and energy—directly corresponding to the   "Anabolic Failure"   and   "Nutrient Sensing Dysregulation"   theories.

 

  II. mTOR Imbalance: Uncontrolled Master Switch of Growth Signals  

When mTOR is in a "too strong" state, this is the direct molecular interpretation of the "  nutrient sensing dysregulation and cellular senescence  " theory. mTORC1 is the signaling hub most sensitive to nutrients and growth factors. Its sustained hyperactivity means the cell erroneously perceives itself in a nutrient-rich environment, uncontrollably driving anabolism, consuming large amounts of resources, and producing metabolic waste. More importantly, it strongly inhibits autophagy and impedes the normal apoptosis program of damaged cells, ultimately pushing cells towards a state of metabolic activity but functional abnormality, secreting large amounts of inflammatory factors, becoming an engine of tissue inflammation and dysfunction—directly corresponding to the   "Nutrient Sensing Dysregulation"   and   "Cellular Senescence"   theories.

When mTOR is in a "too weak" state, it corresponds to "  anabolic failure and impaired tissue regeneration  ." mTOR signaling is the fundamental command for all constructive cellular activities like growth, proliferation, and protein synthesis. Its insufficient signal means the cell loses the fundamental driving force to execute repair and renewal. Whether it's muscle protein synthesis, healing of damaged tissues, or immune cell proliferation, all will stagnate, directly manifesting as age-related tissue atrophy, delayed wound healing, and decreased immune function—directly corresponding to the   "Anabolic Failure"   theory.

 

  III. PGC-1α Imbalance: Command Disorder of the Mitochondrial Factory  

When PGC-1α is in a "too weak" state, it directly corresponds to the core of the "  mitochondrial dysfunction  " theory. PGC-1α is the master transcriptional co-activator for mitochondrial biogenesis, regulating a series of mitochondrial generation and antioxidant genes, including NRF1/2. Its low expression prevents the construction of new mitochondrial "factories" and the renewal of old "equipment," manifesting as reduced mitochondrial quantity, decreased quality, and lowered respiratory efficiency. This is not only an energy crisis at the cellular level but also the starting point of the systemic antioxidant defense network collapse—directly corresponding to the   "Mitochondrial Dysfunction"   theory.

When PGC-1α is in a "too strong" state, it may lead to "  metabolic rhythm disorder and reductive stress/signaling interference  ." Although less studied, theoretically, the persistent overactivation of PGC-1α disrupts the dynamic balance between mitochondrial biogenesis and clearance, oxidation and antioxidation. It may force cells to consume large amounts of resources to build mitochondria when unnecessary, and may excessively suppress normal reactive oxygen species signals, which are essential for many physiological signal transductions. This imbalance interferes with the cell's metabolic flexibility and normal signal transduction rhythm—linking to the   "Metabolic Disorder"   and   "Signal Integration Dysregulation"   theories.

 

  IV. PPARα Imbalance: Dysfunctional Speed Regulation of the Metabolic Engine  

When PPARα is in a "too weak" state, it is the direct driver of the "  loss of metabolic flexibility  " (the molecular basis of systemic glycolipid metabolic disorder) theory. PPARα is the core transcriptional regulator of fatty acid oxidation and the TCA cycle in tissues like the liver and muscle. Its weakened function means that fatty acids, an important fuel source, cannot be effectively "burned," forcing cells to rely more on glucose metabolism. This rigidity and switching failure of metabolic pathways is the upstream culprit for insulin resistance, ectopic lipid accumulation, and systemic metabolic inefficiency during aging—directly corresponding to the   "Loss of Metabolic Flexibility / Glycolipid Metabolic Disorder"   theory, and initiating the   "Epigenetic Alteration"   pathway.

When PPARα is in a "too strong" state, it is closely associated with "  oxidative stress and lipotoxicity  ." Excessively activated PPARα indiscriminately drives the influx of fatty acids into mitochondria for β-oxidation. If this process exceeds the processing capacity of mitochondria or the carrying limit of the electron transport chain, it leads to massive production of reactive oxygen species, exacerbating oxidative damage. Simultaneously, intermediate products of incomplete lipid oxidation may accumulate, producing lipotoxicity and further impairing cell function—directly corresponding to the   "Free Radical/Oxidative Stress"   theory.

 

  2.5 Chronic hypoxic microenvironment drives stem cell aging, the primary driver of stem cell aging.  

 

The chronic hypoxic microenvironment continuously acts on stem cells through microcirculatory disturbance, triggering metabolic reprogramming and a cascade decompensation of the telomere regulatory network. This process has significant temporal dependency: Telomerase reverse transcriptase (TERT), whose expression level directly determines telomerase activity. Literature [177] indicates that moderate intermittent hypoxia can induce TERT activity and telomere stability, which explains the improvement of telomeres by a single hypoxic event. Literature [133] provides reverse evidence, showing FBP1 specifically removes TERT phosphorylation modification, blocking its nuclear translocation or telomere recruitment, proving that TERT is crucial for telomere elongation.

 

However, hypoxia is not always beneficial. Literature [173] points out that when erythrocyte deformability declines, oxygen affinity increases, and oxygen release capacity decreases. Therefore, it can be inferred that within the same time window, due to increased oxygen affinity and decreased oxygen release of erythrocytes, the oxygen available to local tissues under the metabolic background of that same time window will decrease, forming persistent hypoxia. When cells transition to persistent hypoxia, the proportion of glycolytic metabolism increases. Literature [179] points out that under hypoxic conditions, through the fermentation branch of the glycolytic pathway, NADH is used to reduce pyruvate to lactate by cytoplasmic lactate dehydrogenase (LDH), and also points out that lactate regulates pro-inflammatory cytokine expression through the NF-κB pathway.

 

Literature [180] points out that the expression level of nuclear factor kappa B (NF-κB) increases, and NF-κB can bind to the TINF2 promoter, thereby increasing the expression of TRF1-interacting nuclear factor 2 (TIN2).

When TIN2 is overexpressed, it inhibits telomere elongation. Literature [178] points out that TIN2 overexpression can prevent telomere elongation in various human cell lines. Literature [132] jointly verifies this point: high TIN2 expression leads to telomere shortening.

Therefore, it can be inferred that the sustained increase in the proportion of glycolytic metabolism induced by continuously insufficient oxygen supply within the same time window will have the property of inhibiting stem cell self-renewal, leading to replicative senescence.

 

  2.6 Microcirculatory disturbance intensity and central axis imbalance ratio, and alternative aging entry pathways.  

 

Although theoretically, any non-steady-state pathway will eventually lead to microcirculatory disturbance, the disturbance itself may have significant temporal differences depending on the pathway causing it. Based on the intensity of the central axis imbalance itself, the degree of microcirculatory disturbance varies significantly among individuals. Only some will exhibit microcirculatory disturbance in adolescence, entering the stage of mitochondrial functional decline through this path.

 

In certain aging subtypes, for example, states characterized by high mTOR expression and low PGC-1α expression, the accumulation of senescent cells and their Senescence-Associated Secretory Phenotype (SASP) can itself act as an upstream driver, persistently inducing mitochondrial functional decline. Research has shown that various inflammatory factors can directly impair mitochondrial energy metabolism and homeostasis maintenance by inhibiting mitochondrial biogenesis, interfering with electron transport chain activity, and disrupting mitochondrial dynamic homeostasis [157]. Further molecular mechanism studies show that in inflammation-driven senescent cells, impaired mitochondrial integrity leads to the abnormal leakage of mitochondrial RNA into the cytoplasm. This process activates innate immune sensor pathways like RIG-I/MDA5-MAVS, thereby amplifying SASP production and forming a self-sustaining cycle of inflammation and mitochondrial functional damage [156]. In this type, mitochondrial functional decline does not originate from initial energy supply deficiency or microcirculatory disturbance but is a direct result of long-term inflammatory signal input.

 

It can be said that while microcirculatory disturbance may not be obvious in everyone during early aging, mitochondrial functional decline will first be realized through various pathways, thus initiating the entire cascade collapse of aging from the first cause to the sixth cause—a self-reinforcing, self-maintaining, self-locking aging metabolic homeostasis. However, microcirculatory disturbance is the easiest feature to reveal; its eighty non-steady-state correlations are stable. When the central axis imbalance is strong enough, microcirculatory disturbance will inevitably be revealed.

 

Therefore, the first cause is the decline in mitochondrial functionality itself. Its core lies not in the appearance of previous traditional aging theories, but in the fact that the first cause itself forms a convergence of the damage pathways caused by multiple aging factors on the body. The decline in mitochondrial functionality, the first cause, is the node where multiple aging factors converge and drive the cascade collapse of the aging process. Once this first cause pushes the aging process to the second cause, it itself becomes a supporting role to the second cause, resulting in a switch of the primary aging factors.

 

  2.7 Model of the Sodium-Potassium Pump's Functional Impairment Due to Decreased ATP Levels Affecting Ion Channels.  

 

Literature [137] points out that the sodium-potassium pump requires ATP for energy. Based on this, it can be inferred that when the ATP produced by mitochondria drops to a level insufficient to meet the sodium-potassium pump's demand, the pump's energy supply decreases, leading to a reduced efficiency of potassium ion entry into the cell against its gradient. The intracellular potassium ion concentration decreases. When intracellular potassium ion concentration drops, the cell's capacity to maintain potassium ion efflux decreases. Literature [143] points out that in immune cells, reduced potassium efflux and cell membrane depolarization trigger calcium influx. Literature [136] similarly points out that in vascular smooth muscle, inhibition of potassium efflux and cell membrane depolarization trigger calcium influx. Through this common mechanism in two unrelated cell systems, it can be inferred that this is a shared mechanism of human cells. Literature [138] points out that the plasma membrane calcium pump, driven by ATP, pumps calcium ions out of the cell against their gradient. It can be inferred that if intracellular ATP levels decrease, the function of pumping calcium ions out will also decline. Literature [141] points out that the sodium-calcium exchanger NCX does not depend on ATP; it expels one calcium ion and imports three sodium ions by utilizing the sodium ion gradient. Literature [251] points out that aging leads to a significant increase in intracellular sodium concentration, accompanied by decreased cellular water content and increased cytoplasmic colloidal density.

 

Therefore, it can be inferred that when cellular ATP levels decrease, the function of the sodium-potassium pump declines due to a lack of ATP. This decline leads to prolonged depolarization and repolarization times, while hyperpolarization time remains unchanged. This results in more calcium ions entering the cell during depolarization. For survival needs, the cell will expel a calcium ion at the cost of importing three sodium ions through the NCX pathway. During this process, cellular sodium ion levels increase. The process of repolarization, involving pumping potassium in and sodium out via the sodium-potassium pump, cannot completely eliminate the negative effects of this pathway, leading to an age-related increase in intracellular sodium concentration.

 

  2.7.1 Convergence of Multi-factorial Mitochondrial Functional Decline to Microcirculatory Disturbance, Metabolic Switch Elevating Lactate Levels, Inhibiting Nitric Oxide and Forming a Self-Perpetuating Cycle.  

 

When the proportion of ATP produced by mitochondrial oxidative phosphorylation decreases, cells, needing to maintain metabolism, will drive glycolysis to compensate for the insufficiency of oxidative phosphorylation.

The metabolic switch increases the proportion of glycolytic metabolism, and the byproduct lactate will similarly increase. Lactate is not just metabolic waste but also an efficient signal regulatory factor. Lactate can promote vascular endothelial growth factor (VEGF) expression through the HIF-1α-VEGF pathway [168]. Previous studies examining individuals aged twenty to eighty-five did detect increased levels of vascular growth factors in the blood [167]. However, when persistent angiogenic factor levels increase while blood vessels need to maintain homeostasis, this involves the negative feedback regulation mechanism of angiogenic factors. According to this study [169], sustained stimulation of endothelial cells with VEGF leads to the downregulation of Flk-1 protein. This results in impaired eNOS activation and NO release upon subsequent VEGF challenge. Consistent with VEGF-stimulated Flk-1 degradation, it was demonstrated that VEGF stimulates Flk-1 ubiquitination, and Cbl mediates this effect through its ubiquitin ligase activity. This study proves that with increased angiogenic factor levels, the signaling quality of angiogenic factors declines.

 

Nitric oxide (NO) is a key substance promoting vasodilation and improving erythrocyte deformability, acting downstream of VEGF [169]. The increase in angiogenic factor levels will, through a negative feedback mechanism [169], lead to an age-related decrease in NO levels [170][171]. Subcutaneous blood supply depends on capillaries. The decrease in NO will lead to structural hypoxia in subcutaneous tissues. Although this hypoxia can induce increased levels of angiogenic factors, the blood angiogenic factors are already above normal levels, inducing desensitization [167][169]. Meanwhile, the body will also increase secretion of the inhibitory factor endostatin in response to excessive angiogenic factors [172]. Therefore, in a state of desensitized pro-angiogenic signaling and increased anti-angiogenic signaling, subcutaneous capillary tissues prone to structural hypoxia will exhibit a tendency towards reduced vascular density.

 

This pathway leads to weakened nitric oxide, and the consequent decrease in erythrocyte deformability and reduced oxygen release efficiency [173] will inevitably, because oxygen is a necessary substrate for the NAD+/NADH cycle and the FAD/FADH2 cycle, result in decreased efficiency of the FAD/FADH2 cycle, slowed lipid metabolism, and obstructed AKG and ketone body generation. The decreased efficiency of the NAD+/NADH cycle will force cells to increase the proportion of glycolytic metabolism as compensation. At the overall metabolic cycle level, this forms a harmful negative feedback loop.

 

  Clarification:   This mechanism was placed in the third cause, metabolic switch subsection, in the old theory. However, its mechanism may be a key convergence node linking the first and second causes. Therefore, in this version, it has been elevated to the stage of the first cause. Nevertheless, in the third cause section, this content is retained to maintain the flow of the preceding and following text.

 

  2.7.2 Decreased Mitochondrial ATP Production Efficiency and Erroneous Ratio of GR Nuclear Translocation Signals.  

 

Literature [288] points out that dysfunction of mitochondrial DNA polymerase γ (POLG) leads to alterations in mitochondrial DNA, resulting in the loss of mitochondrial oxidative phosphorylation (OXPHOS) and mitochondrial ATP generation. Total cellular ATP is generated by two energy pathways: glycolysis and mitochondrial OXPHOS. Mitochondrial dysfunction leads to reduced mitochondrial ATP generation, inducing compensatory upregulation of cytoplasmic glycolysis, thereby increasing the contribution of glycolysis to total cellular ATP generation.

 

Literature [289] indicates that through the glycolysis process, one glucose molecule is broken down into two pyruvate molecules. Depending on the microcellular environment (especially oxygen supply, energy demand, and the presence of mitochondria), pyruvate has several different fates:

In cells containing mitochondria, pyruvate can enter the citric acid cycle in the mitochondrial matrix and undergo oxidative phosphorylation. Because it relies on oxygen as the final electron acceptor, oxidative phosphorylation cannot occur under hypoxic conditions. Furthermore, as the enzymes for the citric acid cycle and electron transport chain are located inside mitochondria, cells lacking mitochondria (like erythrocytes) cannot rely on oxidative phosphorylation for energy production.

In erythrocytes and hypoxic tissues, pyruvate remains in the cytoplasm and is converted to lactate, a process called anaerobic glycolysis. This final reaction allows the regeneration of NAD+, a cofactor that must be present at sufficiently high intracellular concentrations for earlier glycolytic reactions to remain favorable. However, compared to oxidative phosphorylation, anaerobic glycolysis is significantly less efficient, netting 2 ATP molecules per glucose molecule (compared to 32 ATP in oxidative phosphorylation).

 

Literature [290] points out that AMPK is involved in maintaining bioenergetic homeostasis and may therefore regulate energy recovery following excitotoxicity or OGD-induced energy crisis. We and others have previously demonstrated that ATP depletion during excitotoxic injury increases protein levels of active threonine-172 phosphorylated AMPK, indicating higher AMPK activity. To directly monitor AMPK activity in a single-cell system, we used a FRET-based AMPK activity reporter. The probe contains a synthetic peptide that is phosphorylated by AMPK, altering probe conformation and increasing FRET, thereby enhancing the ratiometric fluorescent signal. As a positive control for this probe, we confirmed that administration of the AMP "mimetic" AICAR or exposure of CGNs and cortical cells to 2-DG increased the AMPKAR ratiometric fluorescent signal, consistent with other measurements observing elevated AMPK following these drug exposures. Using this probe, we found that AMPK activity increased rapidly and transiently during glutamate treatment in CGNs and NMDA treatment in cortical cells.

Literature [291] points out that AMPK induces acetylation of importin α1 at the Lys22 site in a p300-dependent manner and directly phosphorylates its Ser105 site. These modifications work together to enhance the nuclear import function mediated by importin α1.

Literature [292] points out that KPNA2 interacts with KPNB1 and upregulates its expression to promote M2 polarization of macrophages in the tumor microenvironment, thereby accelerating the proliferation and metastasis of gastric cancer cells. The interaction between KPNA2 and KPNB1 was confirmed through Co-IP and IF detection. Knocking down KPNB1 reversed a series of cellular phenotypes caused by KPNA2 overexpression. This functionally strongly proves that the role of KPNA2 largely depends on its regulation of KPNB1.

Literature [293] points out that ovariectomized mice exhibited elevated nuclear GR, decreased mitochondrial GR, and increased PBR expression in BMSCs. High-dose dexamethasone impaired osteogenic differentiation, manifested as nuclear GR accumulation, reduced mitochondrial translocation, and increased PBR expression. Co-immunoprecipitation confirmed a direct interaction between mitochondrial GR and PBR. Importazole reduced nuclear GR levels, promoted bone formation, and alleviated osteoporosis, while Emapunil enhanced GR mitochondrial translocation, improved mitochondrial function, and strengthened bone health.

 

Based on the above literature, it can be inferred that within the same time window, the amount of ATP produced by glycolysis is less than that produced by oxidative phosphorylation. This pathway leads to a prolonged state where ATP is at a low level, increasing the time AMPK is activated by low ATP levels. This step increases KPNA2 gene expression, raising importin α1 levels. Elevated importin α1 drives KPNB1 gene expression to produce importin β. The result of this pathway is that even if glucocorticoid levels do not change, the ratio of its signaling acting on mitochondria versus the cell nucleus will change, still leading to osteoporosis, with bones in areas of faster metabolism being affected first. The content of this subsection can explain the differential skeletal aging observed in literature [294].

 

  2.8 ATP Decline in Cells Reduces Sodium Pumping Capacity, Interstitial Fluid and Extracellular Matrix Cannot Return to Blood via Atypical Pathways.  

 

Literature [146] points out that a high-salt diet increases sodium content in interstitial fluid and the extracellular matrix, discovering that under high-salt conditions, extracellular sodium transfers intracellularly. As mentioned in Section 2.7 above, a decrease in ATP leads to functional decline of the sodium-potassium pump. Literature [252] points out that sodium content in skin and muscle tissues increases with age, inconsistently with blood levels, and simultaneously indicates that osmotically inactive skin Na+ can be mobilized by salt deprivation, thereby reducing the content of negatively charged skin glycosaminoglycans. Literature [253] points out that cartilage also contains glycosaminoglycans.

 

Literature [150] and [149] indicate that the assembly of elastin requires precise hydrophobic interactions and hinges. Literature [151] points out that heat and sodium can affect hydrophobic structures. Literature [254] indicates that liver aging drives aging in other organs by releasing transforming growth factor (TGF). Literature [255] points out that TGF drives glycosaminoglycans. Literature [256] points out that hypoxia drives glycosaminoglycan synthesis. Literature [152] points out that high sodium itself is a pro-inflammatory signal. Literature [257] points out that high sodium itself inhibits the lymphatic pathway's sodium reclamation function. Literature [258] points out that M1-polarized macrophages secrete matrix metalloproteinases (MMPs), degrading collagen. Literature [259] points out that inflammatory factors can drive macrophage M1 polarization.

 

Therefore, it can be inferred that during aging, TGF drives glycosaminoglycans, which have the effect of immobilizing sodium through their negative charge. This leads to increased sodium content in the skin and cartilage, forming a differential change in sodium content between the blood and the extracellular matrix. This differential change causes high-sodium interstitial fluid to inhibit the clearance function of lymphatic interstitium. Sodium, based on the ion gradient, will move towards the vascular cells, which act as an ionic low-concentration sink, and be pumped into the blood by vascular cells, reducing sodium load. However, during aging, vascular cells also experience reduced pumping efficiency due to a lack of ATP, leading to a state dominated by the glycosaminoglycan negative charge aggregation effect, where blood sodium ions preferentially transfer to the extracellular matrix.

 

In human articular cartilage, spinal cartilage, blood vessels, and skin, collagen and elastin are present. When the sodium content bound to glycosaminoglycans increases, the pro-inflammatory signals formed by sodium and its effect on hydrophobic structures jointly lead to the decline of elastin and collagen. In bones, this forms decreased structural toughness of articular and spinal cartilage, and inflammation-induced protein degradation, leading to osteoarthritic degenerative changes. In blood vessels and skin, it promotes the decline of skin and vascular elasticity. Hypoxia promoting glycosaminoglycans can explain, based on differences in oxygen release, whether non-age-related, pathological functional decline occurs in joints, spine, skin, and blood vessels.

 

  2.9 Model of the Effect of Reduced Sodium-Potassium Pump Potassium Flux on Cell Membrane Receptors.  

 

Literature [160] points out that the Growth Hormone Releasing Hormone Receptor (GHRHR) maintains depolarization by inhibiting delayed rectifier potassium channels to reduce potassium efflux, while the Somatostatin Receptor (SSTR) activates GIRK channels promoting potassium efflux and inducing hyperpolarization [161]. Therefore, it can be inferred that when ATP decline leads to slower refilling of the potassium pool by the sodium-potassium pump, causing prolonged depolarization time while hyperpolarization remains unchanged, a phenomenon of receptor blunting occurs. Prolonged depolarization means a longer GHRHR signaling time, leading to more vigorous growth hormone secretion. However, since growth hormone secretion is pulsatile and requires a large amount of ATP for secretion within a single time window, when sodium-potassium pump function decreases due to ATP decline, this results in decreased pulsatile growth hormone levels, increased basal, constant growth hormone levels, and the blunting/desensitization of relevant growth hormone receptors.

 

Simultaneously, as the downstream pathway of nuclear estrogen receptors can improve sodium-potassium pump function [145], this becomes a connection point for gender differences in aging. Because estrogen receptors improve the sodium-potassium pump, maintaining cell membrane receptor density, including that of the cytokine receptor family's growth hormone receptor GHR, the pro-aging effects related to sodium-potassium pump issues are lower in female individuals compared to age-matched male individuals.

 

Literature [143] points out that when Kv1.3 or KCa3.1 are inhibited, membrane potential depolarizes, directly inhibiting CRAC (Orai1)-mediated Ca²⁺ influx, leading to reduced IL-2 production and impeded proliferation. Therefore, it can be inferred that prolonged depolarization time will weaken immune function to a certain extent.

Literature [144] points out that long-term depolarization upregulates inhibitory G-protein signaling. From this, it can be inferred that functional decline of the sodium-potassium pump can inhibit the normal function of most receptors other than nuclear receptors. However, this effect is more confined to the middle and late stages of aging and cannot constitute a primary pro-aging factor in early aging. Literature [260] points out the blunted growth hormone secretion in aged individuals. Females <50 years: basal secretion (mU/L·24h): 15.3; pulsatile secretion (mU/L·24h): 128. Females ≥50 years: basal secretion (mU/L·24h): 19.5 (↑27%); pulsatile secretion (mU/L·24h): 77 (↓40%). Males <50 years: basal secretion (mU/L·24h): 7.0; pulsatile secretion (mU/L·24h): 78. Males ≥50 years: basal secretion (mU/L·24h): 7.5; pulsatile secretion (mU/L·24h): 79.

Thus, it can be deduced that the increase in basal growth hormone secretion caused by depolarization exists but has age limits. Beyond the current age stage, as aging deepens and the pituitary gland ages, a dual decline in both basal and pulsatile growth hormone will occur.

 

  2.10 Self-locking Mechanism of the Sodium-Potassium Pump on ATP.  

 

The functional decline of the sodium-potassium pump pushes up the sodium ion concentration in the extracellular matrix. This process itself is a pro-inflammatory signal [152], with literature pointing out that elevated sodium levels in the ECM induce the transcription of inflammatory factors. "TNF-α, via TNFR1, recruits TRADD/RIP1, prompting CypD-dependent sustained opening of the mPTP, leading to a 45% decrease in mitochondrial membrane potential and a 38% reduction in ATP synthesis; CypD deletion or cyclosporin A treatment can block this effect, directly proving that TNF-α activates mPTP is a key mechanism for membrane potential collapse and energy inhibition" [158][159]. From this point, the first cause forms a logically closed loop, capable of self-reinforcement, self-maintenance, and self-perpetuation, becoming a pro-aging factor.

 

  3.1 The Second Cause of Aging: Glycolipid Metabolic Disorder  

 

The decline in mitochondrial function will lead to a decrease in the efficiency of normal cellular fat metabolism, primarily targeting organs capable of direct fatty acid metabolism, such as the heart, liver, and muscles.

 

The core mechanism is that the final step of fatty acid β-oxidation, involving FADH, cannot be oxidized. The problem might be a decrease in blood oxygen release within the same time window, as indicated in literature [173] regarding altered erythrocyte oxygen affinity, reduced oxygen release, and increased oxygen affinity. Alternatively, as indicated in literature [159], TNF-α-induced oxidative stress damages the MPTP proteins adenine nucleotide translocator and voltage-dependent anion channel, altering redox homeostasis and leading to pore opening. Literature [158] points out that sustained mPTP opening leads to mitochondrial energy metabolism disorder.

 

Therefore, combining the various driving pathways of the first cause mentioned above, it can be inferred that the emergence of the second cause is a cascade collapse triggered by the decline in mitochondrial functionality (the first cause). Simultaneously, it can be inferred that in a metabolic context where hepatic fatty acid metabolism and the resulting ketone body supply decline, the body's cells will increasingly rely on blood glucose to maintain overall ATP levels.

 

  3.1.1 Decline in Lipid Metabolism: Reduction of Free Carnitine and Accumulation of Multiple Carnitines  

 

Mitochondria are the primary site for fatty acid β-oxidation, responsible for converting long-chain fatty acids into energy. Literature [86] points out that free L-carnitine is crucial for transporting fatty acids into the mitochondrial matrix and is essential for fatty acid metabolism. Literature [87] demonstrates that various acylcarnitines accumulate due to reduced fatty acid β-oxidation, causing damage to mitochondrial function. This forms a self-locking mechanism for the second cause and simultaneously demonstrates the negative impact on the human body of the altered metabolic ratio between glucose and fatty acids, specifically the decrease in lipid metabolism proportion.

 

  3.2 Increased Proportion of Glucose Metabolism: Accumulation of Advanced Glycation End Products (AGEs) and Initiation of Inflammation  

 

In the shared metabolic state of most human cells, it can be observed that even with sufficient blood glucose, cells still opt to use some ketone bodies for energy supply; this is the basis of cellular metabolic flexibility. Literature [92] points out that in non-obese, non-diabetic healthy adults, a portion of the body's energy in the fasting state comes from the oxidation of free fatty acids (FFA). Therefore, it will become more inclined to obtain energy through glucose oxidative phosphorylation. Literature [88] points out that accelerating carbohydrate metabolism directly inhibits fatty acid oxidation, with glucose oxidation increasing from a basal 6.2 +/- 0.8 to 22.3 +/- 1.4 mumol.kg-1.min-1 (P < 0.01). Total (indirect calorimetry) and plasma fatty acid oxidation (isotope determination) decreased from 2.6 +/- 0.2 to 0.4 +/- 0.3 (P < 0.01) and 2.2 +/- 0.2 to 1.4 +/- 0.1 μmol.kg-1.min-1 (P <0.05), respectively.

Therefore, by combining the phenomena from these two papers, it can be concluded that the decline in lipid metabolism caused by mitochondrial functional decline will reversely reinforce the proportion of glucose metabolism in the human body.

 

Literature [181] points out that the aerobic oxidation of glucose and other biomolecules within cells to produce carbon dioxide and water is not entirely clean. Molecular oxygen, even in its most stable triplet ground state, contains two unpaired electrons, which determines the easy emergence of reactive oxygen species (ROS) as alternative partial reduction products instead of water. Literature [182] points out that ketone-based metabolism produces less destructive reactive oxygen species at the mitochondrial and cellular level compared to glucose metabolism.

 

Therefore, it can be deduced here that in a metabolic background where ketone body levels decline, the glucose metabolic pathway will produce more ROS radicals in a state of mixed glycolipid metabolism.

Based on literature [181], it is indicated that the level of free radicals is positively correlated with the accumulation of advanced glycation end products (AGEs). It also points out that this pathway activates NOX-2, mitochondrial ROS, and NF-κB through AGE–RAGE binding, amplifying TNF-α/IL-6/ICAM-1 signals; ROS, in turn, promotes new AGE formation, forming a 'glycation-oxidation-inflammation' self-perpetuating cycle. Inflammation, based on the two core mechanisms mentioned in [159] and [158]—where TNF-α-induced oxidative stress damages MPTP proteins, altering redox homeostasis and causing pore opening, and sustained mPTP opening leads to mitochondrial energy metabolism disorder—will strengthen the effect of mitochondrial functional decline in reverse.

 

Therefore, glycolipid metabolic disorder not only reflects a change in the energy supply mode but is also the first core stage in the amplification of erroneous signals from the steady-state collapse that the human body inevitably experiences, starting from the convergence node of mitochondrial functional decline in aging.

 

  3.3 Fat Accumulation Dilutes Vitamin D3 within Fat Reserves, Shifting Adipocyte Extracellular Vesicles Towards Pro-Inflammatory State.  

 

Literature [120] points out that Vitamin D is a fat-soluble vitamin. After ingestion, most of it is taken up by adipose tissue and stored long-term. However, this form of Vitamin D3 has low bioactivity, yet this storage of Vitamin D3 in the body is crucial for inflammation regulation.

When body fat percentage is healthy, the concentration of Vitamin D3 (cholecalciferol) within adipocytes is relatively full, forming a non-genomic weak regulation of fat cells through its quantity. Literature [122] points out that 1α,25-(OH)₂D₃, upon binding to membrane-localized VDR (mVDR), activates the PLC-IP₃ pathway, prompting the endoplasmic reticulum to release Ca²⁺, rapidly increasing cytosolic free Ca²⁺; the Ca²⁺/CaM complex binds and activates CaMKKβ, which directly phosphorylates the Thr172 site of the AMPKα subunit, leading to a rapid increase in AMPK activity within minutes; activated AMPK then phosphorylates and inhibits NF-κB p65 nuclear translocation, while reducing the expression of pro-inflammatory genes like IL-6 and IL-1β, thus constituting Vitamin D's non-genomic anti-inflammatory mechanism.

 

Therefore, it can be inferred that when glycolipid metabolic disorder progresses continuously, leading to fat accumulation such as visceral fat, Vitamin D3 cholecalciferol is diluted in the expanded adipose tissue, causing a decrease in its intracellular concentration. Exogenous supplementation would require extremely high doses to restore its biological concentration to youthful levels, leading to the failure of its original anti-inflammatory non-genomic regulatory mechanism. This triggers adipocytes to shift towards a pro-inflammatory state, and the extracellular vesicles they secrete also undergo a phenotypic switch, from anti-inflammatory to pro-inflammatory.

 

These pro-inflammatory vesicles further promote lipolysis, breaking down stored fat into the blood. This mechanism can be deduced as the body's metabolic regulatory mechanism to cope with local excess body fat. However, in the context of glycolipid metabolic disorder and decreased fat metabolic efficiency, this mechanism forms an erroneous regulatory function, failing to achieve the lipolytic fat-reducing effect, and instead acts as a persistent negative factor through sustained low-grade inflammatory signals, raising the level of inflammatory factors in the body. Literature [183] supports this view, based on the fact that obese patients have higher levels of inflammation compared to normal-weight individuals.

 

  3.4 The Metabolic Core of the Second Cause of Aging: Functional Decline of the α-Ketoglutarate (AKG) Axis  

 

α-Ketoglutarate (AKG) is an intermediate in the tricarboxylic acid (TCA) cycle, a key nexus linking energy metabolism, epigenetic regulation, and the synthesis of cysteine, the precursor for the cellular antioxidant glutathione. Its levels primarily originate from intracellular self-synthesis through the TCA cycle and liver synthesis and release into the blood. Literature [184] provides strong evidence for the release of AKG from the liver into the blood, while confirming that increasing the level of fat metabolism through fasting can raise blood AKG levels.

 

  3.4.1 Decreased AKG Levels Impair Epigenetic Demethylation Efficiency, Forming Epigenetic Drift.  

 

Literature [185] points out that α-ketoglutarate (α-KG), as an essential co-substrate for the Fe(II)/α-KG-dependent dioxygenase family (including TET1-3 and ALKBH1-8), catalyzes the sequential oxidation of 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC), and 5-carboxylcytosine (5-caC) by providing oxidative equivalents. Subsequently, it achieves active DNA demethylation through the base excision repair pathway mediated by thymine DNA glycosylase (TDG), thereby reversing abnormal hypermethylation states and reactivating downstream gene expression.

Therefore, it can be inferred that a decrease in AKG levels will impair the efficiency of epigenetic demethylation, forming epigenetic drift. Literature [186] provides the core mechanism, replicating the aforementioned hypermethylation drift within 7 days, accompanied by typical aging phenotypes such as AMPK↓, mTOR↑, and enhanced secretion of SASP factors. Supplementation with exogenous AKG or restoration of TET expression reverses the drift and reduces the release of inflammatory factors, confirming the correlation between AKG and epigenetic drift.

 

  3.4.2 Decreased Synthesis of the Key Antioxidant Precursor Cysteine Due to Decreased AKG Levels.  

 

Cysteine is a key raw material for glutathione synthesis [186]. The synthesis of cysteine requires serine to provide the carbon skeleton, which is generated from homocysteine through a two-step transsulfuration reaction catalyzed by CBS/CTH [186]. Literature [187] points out that serine biosynthesis is located in the cytoplasmic segment downstream of glycolysis. 3-PG is first oxidized by phosphoglycerate dehydrogenase (PHGDH) to 3-phosphohydroxypyruvate, then catalyzed by phosphoserine aminotransferase (PSAT1) to 3-phosphoserine, and finally dephosphorylated by phosphoserine phosphatase (PSPH) to produce serine.

AKG mediates the transamination process of PSAT1. If AKG levels decline, the flux through PSAT1 will decrease. Literature [188] precisely points this out: the "cell-autonomous" function of Psat1 is to transfer the amino group from glutamate to α-KG, which then proceeds to synthesize glutamine. Once α-KG or glutamine is scarce, the transamination reaction stalls due to a lack of amino donors/acceptors, causing a synchronous drop in Psat1 flux.

Therefore, it can be inferred that if AKG levels decline, the metabolic pathway of serine will be obstructed, subsequently leading to an increased ratio of homocysteine in the blood and decreased cysteine levels. Ultimately, this results in the declining metabolic steady-state of the human body's functions during the aging process.

 

  3.4.3 Decreased α-Ketobutyrate Levels, Weakening NAD+ Regeneration and Related Signaling Pathways.  

 

Literature [189] points out that homocysteine condenses with serine to form cystathionine, a reaction catalyzed by cystathionine-β-synthase (CBS), the rate-limiting step of the pathway. Cystathionine is then hydrolyzed by cystathionine-γ-lyase (CGL) to produce cysteine and α-ketobutyrate. Literature [190] points out that α-ketobutyrate increases NAD+ production via lactate dehydrogenase LDH-1.

According to literature [191], Sirtuins (SIRTs) are class III histone deacetylases dependent on nicotinamide adenine dinucleotide (NAD⁺). Members of this gene family, SIRT1 through SIRT7, each have unique subcellular localizations and functional characteristics: SIRT1, primarily located in the nucleus and cytoplasm, regulates DNA damage repair, apoptosis inhibition, and metabolic homeostasis by deacetylating key proteins like p53, FOXO transcription factors, and PGC-1α; SIRT2 shuttles between the cytoplasm and nucleus, primarily deacetylating α-tubulin and histones H3 and H4, regulating cell cycle progression and microtubule dynamics; SIRT3, SIRT4, and SIRT5 are localized in mitochondria, where SIRT3 enhances antioxidant defense and regulates oxidative phosphorylation by deacetylating SOD2, SIRT4 mainly exhibits ADP-ribosyltransferase activity regulating insulin secretion and amino acid metabolism, and SIRT5 controls the urea cycle and cellular respiration through its potent desuccinylase activity; SIRT6 and SIRT7 are localized in the nucleus, with SIRT6 maintaining genomic stability and DNA repair by deacetylating H3K9, H3K18, and H3K56 while also possessing ADP-ribosyltransferase activity, and SIRT7, mainly located in the nucleolus, regulates ribosomal DNA transcription and chromatin structure by deacetylating H3K18ac and H3K36ac, protecting homologous recombination from damage. These sirtuin members, through their specific enzymatic activities and substrate selectivities, play coordinated and unique roles in metabolic regulation, stress response, aging processes, and disease pathogenesis.

Therefore, it can be inferred here that when serine decreases due to declining AKG levels, the downstream conversion efficiency of homocysteine to cysteine decreases. The metabolic intermediate α-ketobutyrate synchronously decreases, subsequently leading to a decrease in NAD+ levels, forming a negative signal regulation spreading from a single point to multiple targets.

 

  3.5 The Arachidonic Acid-Nuclear Membrane Mechanics Axis, the Second Factor in Stem Cell Replicative Senescence.  

 

Cysteine is a precursor substance for glutathione [186]. As stated in paragraph 3.4.2, its levels at this stage have already been affected by impaired metabolic synthesis due to glycolipid metabolic disorder. Paragraph 3.4.3 indicates that α-ketobutyrate's precursor homocysteine metabolism is hindered, causing a decline in NAD+ levels. Paragraph 3.2 indicates that the increased proportion of glucose metabolism drives up oxidative stress. These three metabolic pathways jointly increase the pressure on intracellular antioxidant resources.

 

Therefore, through the mechanism in literature [224] stating that glutathione can regenerate VC, and literature [192] stating that VC can regenerate VE through electron transfer, it can be inferred that the aging model described in the current hypothesis shows an impact on the ratio of oxidized to reduced Vitamin E on the cell membrane, with the proportion of oxidized VE increasing.

 

Literature [193] points out that oxidation of the cell membrane can lead to a decrease in membrane fluidity, and the reduced form of VE can prevent oxidation. Therefore, an increase in the proportion of oxidized VE indicates membrane oxidation, which will lead to decreased cell membrane fluidity.

 

Literature [166] points out that the lateral migration and aggregation of insulin receptors on the cell membrane depend on lipid fluidity; membrane fluidity is dynamically regulated by lipid composition and acts as a 'lipid switch' determining the initiation strength and duration of insulin signaling. Therefore, it can be inferred that when the proportion of oxidized VE increases and the cell membrane is oxidized, the decrease in membrane fluidity will inhibit the activation and transduction of insulin receptor signals, forming insulin resistance.

 

Literature [125] indicates that the FADS2 gene encodes Δ6-desaturase, the rate-limiting enzyme in the metabolic pathway from linoleic acid (LA) to arachidonic acid (AA), and insulin upregulates the FADS2 gene encoding Δ6-desaturase.

 

Literature [124] points out that the arachidonic acid pathway endogenously activates RhoA, leading to H3K9me2-mediated heterochromatin loss and dysfunction, thereby driving hematopoietic stem cell aging.

 

Therefore, combining the above literature, it is inferred that during aging, the increased oxidative load and declining antioxidant function driven by glycolipid metabolic disorder elevate the level of oxidized VE on the cell membrane, causing membrane oxidation and desensitization of the insulin receptor. The insulin resistance formed by this desensitization weakly drives up insulin levels.

 

Subsequently, the increase in insulin levels drives the expression of Δ6-desaturase (FADS2), increasing the conversion flux from linoleic acid (LA) to arachidonic acid (AA). This endogenously activates RhoA through the arachidonic acid pathway, leading to H3K9me2-mediated heterochromatin loss and dysfunction, thereby inhibiting hematopoietic stem cell function [124][125]. Through the membrane-nucleus mechanics, cell membrane oxidation, and the commonality of the hematopoietic stem cell microenvironment with other stem cells, it can be inferred that this mechanism has the potential to extend outward, encompassing the replicative senescence of other non-hematopoietic stem cells.

 

  3.6 Vitamin E (VE) Decline Due to Decreased Glutathione Synthesis Leads to Decreased Mitochondrial Burst Energy Metabolism.  

 

When the level of oxidized VE on the cell membrane is too high, the cell membrane becomes oxidized, which drives the activity of SREBP genes. As pointed out jointly in literature [73] and [75], elevated levels of oxysterols can relieve INSIG-mediated inhibition, promoting the nuclear translocation of SREBP-2 and upregulating the expression of cholesterol synthesis genes.

 

Simultaneously, literature [75] points out that the synthesis of coenzyme Q10 and cholesterol share the mevalonate (EVA) pathway. Therefore, it can be inferred here that if the expression of the cholesterol synthesis gene SREBP is upregulated due to oxidative pressure, the flux of the EVA pathway will be more biased towards synthesizing cholesterol rather than CoQ10.

 

Literature [76] indicates that for athletes or those needing sudden bursts of high-intensity exercise, insufficient CoQ10 limits the capacity for rapid mitochondrial ATP generation; supplementing CoQ10 can restore the maximum oxidative phosphorylation rate and reduce dependence on anaerobic glycolysis.

In the field of biological metabolic timing, this can be understood as: within the same time window, the upper limit of energy production by mitochondrial energy metabolism through oxidative phosphorylation is dependent on the sufficiency of CoQ10.

 

Literature [46] points out that growth hormone secretion exhibits a high-amplitude, intermittent, pulsatile release pattern. In the field of biological metabolic timing, this can be understood as: within the same time window, the strength of a growth hormone secretion pulse is not limited to the strength of promoting signals or whether receptors have become blunted/desensitized, but is also directly related to instantaneous energy, i.e., the burst power of ATP production. Literature [194] also corroborates this point: the transport of growth hormone from the Golgi apparatus to the release site and its subsequent secretion is completed within 5–7 minutes and primarily depends on oxidative phosphorylation; inhibiting mitochondrial ATP synthesis (oligomycin, dinitrophenol) can reduce the release rate by 60–70% without affecting protein synthesis.

 

Therefore, it can be inferred that when the disordered glycolipid metabolic network increases oxidative stress, elevating oxidized VE and creating a vulnerability in the antioxidant defense, the process of cholesterol oxidation promoting cholesterol synthesis will weaken growth hormone synthesis by reducing CoQ10 synthesis. This results in a CoQ10-dependent decline in growth hormone due to insufficient burst energy during the extremely short secretion window, without involving decreased promoting signals or receptor desensitization.

 

  4 The Third Cause of Aging: Decline in Growth Hormone (GH) Levels and Cliff-like Senescence.  

 

The functional decline of the Growth Hormone (GH)-Insulin-like Growth Factor-1 (IGF-1) axis is a landmark event in the aging process. Its starting point primarily relies on the decline in CoQ10 levels, which triggers a decrease in the cell's instantaneous ATP production capacity, leading to a decline in GH pulsatile levels. However, the true node where it replaces the second cause and becomes the current primary aging factor is when the GH pulse can no longer induce effective conformational changes in cell membrane receptors. This situation results in a drastic decline in GH's regulatory signals to the body.

 

  4.1 The Effect of Decreased Growth Hormone (GH) Levels on NRF1 and Mitochondrial Quantity.  

 

The pulsatile concentration of GH drives conformational changes in cell membrane growth hormone receptors [195]. The GH-IGF-1 axis maintains NRF1 expression through the PGC-1α pathway [79], and NRF1 sustains antioxidant function and mitochondrial biogenesis [69,81,157]. Literature [65,63,62] points out NRF1's role in maintaining antioxidant function.

 

The signaling pathways activated by conformational and non-conformational changes of the GH receptor are different [80]. The conformational change pathway involves the JAK2-STAT5 pathway [80], and JAK2-STAT5 signaling is an upstream signal for PGC-1α-NRF1 [100]. Literature [80] indicates that if GH only binds through the high-affinity site-1 without causing significant rotation, the JAK2-STAT signal is weak. Therefore, it can be inferred that when GH levels drop to a point insufficient to induce conformational changes, humans will experience a drastic decline in NRF1-maintained mitochondrial biogenesis and antioxidant function, forming a self-sustaining, self-stabilizing, self-amplifying erroneous metabolic mechanism.

 

  4.2 The Effect of GH-Deiodinase-T4/T3 Pathway Obstruction on Carnitine Synthesis.  

 

Literature [81] points out that intervention through the GH-IGF-1 pathway, restoring its signaling, can increase T3 levels and decrease T4 levels. Literature [82] reviews how the GH-IGF-1 pathway promotes the conversion of T4 to T3 by enhancing the activity of type II deiodinase (D2). Literature [196] indicates that T3 promotes carnitine synthesis by transcriptionally upregulating hepatic γ-butyrobetaine hydroxylase (BBH).

 

Therefore, at this step, it can be inferred that the weakening of the GH-IGF-1 pathway synchronously affects carnitine synthesis, impairing fat metabolism efficiency, and further locking and strengthening the lipid metabolism disorder inherent in the second cause (glycolipid metabolic disorder), forming a self-locking erroneous metabolic homeostasis.

 

Simultaneously, carnitine is an important transporter for fatty acids entering mitochondrial metabolism [86]. Its reduced level means that the quantity of the raw material (fatty acids) for mitochondria to produce AKG and ketone bodies decreases, forming inhibitory effects on AKG and ketone body generation.

 

  4.3 Decreased T3, Increased T4 Ratio Co-regulates Abnormal Expression of NF-κB Downstream Gene Spectrum, Shifting Towards Pro-inflammatory Factor Expression.  

 

During aging, the balance between thyroid hormones thyroxine (T4) and triiodothyronine (T3) and the switching of their action pathways is a core link leading to the dysregulation of the NF-κB signaling pathway.

1.   Interference with p65-DNA binding efficacy:   TR can competitively hinder the binding of p65 to κB sites on its target promoter.

2.   Alteration of co-regulator recruitment landscape  

This mechanism primarily operates when T3, at physiological concentrations, enters the cell and mainly binds to its nuclear receptors (TRα/β), forming a T3-TR complex. This complex does not directly inactivate NF-κB but, through protein-protein interactions with the p65 subunit (RelA), interferes with p65-DNA binding efficacy [105]. Furthermore, it alters the co-regulator recruitment landscape, excluding histone acetyltransferases (HATs) like CBP/p300, weakening chromatin opening, and recruiting NCoR/SMRT-HDAC3 complexes to promote local chromatin compaction. Strongly pro-inflammatory genes like IL-1β and TNF-α, which are highly dependent on p300, are thus preferentially silenced. Ultimately, even if NF-κB is partially activated for immune surveillance needs, the intensity of its driven transcriptional output for pro-inflammatory factors is biased towards low levels, coexisting with the output of anti-inflammatory factors.

 

This mechanism demonstrates the unique nature of T3's inflammatory control. Compared to other anti-inflammatory mechanisms that primarily focus on inhibiting NF-κB, this pathway primarily works by altering the transcriptional spectrum downstream of NF-κB.

 

However, in the context of aging mentioned earlier, growth hormone declines, cortisol levels rise, and during the conversion of T4 to T3, more T4 is converted to the bio-inactive reverse T3 (rT3). In this metabolic disorder, the body experiences a T3 shortage. This point will cause the TR pathway to lose its regulatory control over the downstream spectrum of the NF-κB pathway, leading NF-κB to exhibit a greater tendency to transcribe inflammatory factors.

 

Simultaneously, the relatively elevated T4 instead binds to integrin αvβ3 on the cell membrane, activating the PI3K-AKT signaling cascade, thereby triggering potent ROS production, leading to the release of NLRP3 inflammasomes and pro-inflammatory cytokines.

 

Ultimately, this pathological shift from the "T3-TR anti-inflammatory axis" to the "T4-integrin αvβ3 pro-inflammatory axis" causes a fundamental reprogramming of the downstream gene expression spectrum regulated by NF-κB. The transcriptional output shifts from anti-inflammatory factor dominance to a pattern more prone to massively expressing pro-inflammatory factors like IL-1β and TNF-α, thus becoming one of the significant sources of inflammatory factors in the body.

Literature [105] comprehensively elaborates on the functional differences between T3 and T4 in anti-inflammatory and pro-inflammatory roles.

 

Therefore, this section allows the inference that the decline of the GH-IGF-1 signaling pathway will, following a decrease in T3 levels, create a pro-inflammatory effect. This can explain the observed increase in inflammatory levels accompanying the aging process.

 

  4.4.1 The Regulatory Network of Triiodothyronine (T3) and Protein Quality Control  

 

T3's regulation of protein quality control is mainly achieved by activating the transcription factor FOXO3a. As indicated in literature [36], T3 directly activates FOXO3a via the Thyroid Hormone Receptor-PI3K axis. Literature [197] points out that FOXO3 can directly regulate the transcription of autophagy pathway genes ATG12, BNIP3, PINK1, etc., by binding to their promoter regions, simultaneously activating macroautophagy and mitophagy. The result is the timely clearance of protein aggregates.

 

Therefore, it can be inferred here that a decrease in T3 hormone levels not only leads to uncontrolled inflammation but also results in impaired protein quality control mechanisms and the accumulation of damaged mitochondria. This is the first factor contributing to the impairment of protein quality control mechanisms.

 

  4.4.3 Disruption of the TH-AMPK Positive Feedback Loop and Central Axis Imbalance.  

 

Literature [35] points out that thyroid hormone (T3) transiently triggers the AMPK-ULK1 axis by increasing mitochondrial reactive oxygen species (ROS), selectively inducing mitophagy. Simultaneously, literature [198] points out that the AMPK pathway can directly phosphorylate TFEB, promoting its transcriptional activity.

Therefore, based on these two studies and inferred through the central axis theory, during the decline of T3 levels, the body's autophagy function is comprehensively reduced. At this stage, the body's central axis undergoes a constrictive collapse. The signaling rhythm of AMPK will experience a metabolic decline in its self-maintenance homeostasis due to the drop in T3 levels. The central axis exhibits a convergent and rigidifying change during aging: the AMPK pathway is inhibited, and the mTOR pathway's signal is enhanced.

 

If the central axis in the first cause stage was a neutral pendulum, then during the constrictive collapse of the central axis caused by the T3 decline in the third cause stage, the central axis is already a tilted pendulum that continues to swing.

 

  4.4.4 Lysosomal Dysfunctional Hyperactivity: The Synergistic Collapse of the Autophagy-Lysosome Network Triggered by T3 Signal Attenuation, Resulting in Decreased Stem Cell Self-Renewal and Replicative Senescence.  

 

Literature [126] points out that aged hematopoietic stem cells (HSCs) exhibit high acidity. By inhibiting excessive lysosomal acidification and increasing TFEB expression, autophagy can be restored, simultaneously improving HSC self-renewal. Literature [36] indicates that FOXO3 directly drives the expression of autophagy genes, and TFEB is one of its downstream genes. Literature [37] points out that T3 hormone can drive FOXO3 expression. Concurrently, literature [33] describes the phenomenon of accumulating backlog due to reduced flux in the autophagy-to-lysosome process.

 

Combining these four papers and the mechanisms from 4.4.3 regarding T3 promoting AMPK [35] and AMPK promoting TFEB [198], the following deduction can be made: the decline in GH-IGF-1 signaling leads to decreased T3. The decrease in T3 causes reduced FOXO3 expression, leading to a comprehensive decline in the expression of autophagy pathway genes. However, due to cellular stress caused by the accumulation of misfolded proteins, genes related to lysosomal biogenesis are excessively promoted, resulting in an over-acidified state of stem cell lysosomes. This means the cell's capacity to package, load, and destroy misfolded proteins is comprehensively reduced. Yet, the stress signal from the accumulation of misfolded proteins increases the synthesis of lysosomes, the destruction pathway. However, because the packaging and loading pathways are not restored, the flux of misfolded proteins to lysosomes decreases, while the misfolded protein stress signal is persistently maintained. This ultimately triggers cellular over-acidification, leading to decreased HSC self-renewal. Given that different stem cells all possess lysosomes and share characteristics of high acidity, this mechanism has the basis to be extrapolated to other stem cells. Literature [36] corroborates this point: in adult mouse neural stem cells, FOXO3 directly binds to the promoter regions of a set of core autophagy genes (e.g., Map1lc3b, Becn1, Atg12), transcriptionally upregulating them to maintain autophagic flux and protein homeostasis. Once FOXO3 is lost, the autophagy network collapses, protein aggregation increases, and stem cell function rapidly ages.

 

  4.5 Metabolic Pathway Switch: From Oxidative Phosphorylation to Glycolysis Energy Crisis.  

 

The ATP output of mitochondria is key to maintaining cellular homeostasis. Studies [199][200] indicate that in cells with mitochondrial damage, the AMPK pathway promotes ATP production through glycolysis via HIF-1, while in cells with normal mitochondrial function, it promotes oxidative phosphorylation metabolism. Literature [3] points out that when a cell faces an energy crisis, the ATP/AMP ratio decreases, activating AMPK.

 

Therefore, combining the content of these two papers, it can be inferred that when cellular energy supply from oxidative phosphorylation is insufficient, the cell will increase the proportion of glycolytic metabolism within the same time window to cope with this imminent energy crisis. However, glycolysis occurs in the cytoplasm. While it can rapidly break down glucose to pyruvate, netting 2 molecules of ATP, it also produces a large amount of lactate catalyzed by lactate dehydrogenase (LDH). Literature [22] demonstrates, using the glycolytic mode of tumor cells, that glycolytic metabolism produces lactate. Literature [201] validates two points in mice: lactate levels increase with age. In mice with mitochondrial DNA mutations, the rate of lactate increase in the cerebral cortex between 6–9 and 35–38 weeks of age was 2.1% per week. In the striatum, lactate levels in mtDNA mutation mice aged 6 to 9 weeks were also twice as high, and these levels remained elevated as the mtDNA mutation mice aged. During normal aging, we found that lactate levels determined by 1H-MRS increased in later life; between 20 and 42 weeks of age, the average increase was 1.6% per week in the cerebral cortex and 1.7% in the striatum. Simultaneously, this paper also validates the inference that mitochondrial functional decline triggers glycolytic metabolism.

 

  4.5.1.1 The Impact of Metabolic Switch on Epigenetics: Acetyl-CoA Decline as the Second Factor in Epigenetic Drift.  

 

Accompanied by the decline in oxidative phosphorylation metabolism and the increased proportion of glycolytic metabolism, pyruvate, a key precursor for acetyl-CoA, must be catalyzed by the mitochondrial pyruvate dehydrogenase complex (PDC). However, mitochondrial functional impairment limits the efficiency of this process. Consequently, within the same time window, the pyruvate flux generated by glycolysis does not actually enter the mitochondria but is instead converted to lactate and expelled from the cell through the pathway of pyruvate regenerating NAD+.

 

This point can be used to explain the phenomenon mentioned in literature [201] of lactate increasing with age. Therefore, it can be inferred that the decline in acetyl-CoA levels is caused by the restriction of pyruvate metabolism due to decreased oxidative phosphorylation. Literature [106] points out that acetyl-CoA is an essential substrate for histone acetylation; a decrease in its level directly leads to insufficient histone acetylation modification, subsequently affecting chromatin openness and gene transcriptional activity, forming transcriptional inhibition at the epigenetic level.

 

  4.5.2.1 The metabolic pathway switch affects NADH to NAD+ metabolism, causing the CD38-NAD+-AMPK inflammatory signal reversal pathway to fail.  

 

Literature [41] indicates that CD38, as an inflammation-sensitive molecule, its expression can be directly induced by classic inflammatory factors like TNF-α and IL-6, thereby rapidly mediating the regulation of NAD+ levels under inflammatory conditions. CD38 catalyzes the hydrolysis of NAD+ to generate ADP-ribose (ADPR) and cyclic ADP-ribose (cADPR). cADPR can be hydrolyzed by the ectonucleotide pyrophosphatase/phosphodiesterase CD203a (NPP1) to form AMP [202]. AMP can drive the AMPK signaling pathway [203], and the AMPK signaling pathway has the effect of inhibiting NF-κB. Literature [56] supports this: AMPK blocks NF-κB nuclear translocation by inhibiting the NLRP3 inflammasome.

 

Therefore, it can be inferred that this mechanism serves as a reversal of inflammatory signals—a stress emergency stop mechanism. Activation of this mechanism will effectively improve the body's inflammatory state.

 

However, if this anti-inflammatory mechanism is continuously activated, the CD38-NAD+-AMPK pathway will consume a large amount of NAD+, leading to a persistent decline in the flux of the AMPK self-maintenance axis (NAD+-SIRT1-AMPK pathway). This shortens the window for cells to enter gene repair, causing a tendency towards genomic instability. Concurrently, the central axis shifts again, and the positive anti-aging pathways triggered by the SIRT gene family are once again comprehensively inhibited, creating an amplified aging effect. It is important to note here that this mechanism is not a negative one but a self-protective mechanism. Its core problem stems from the increased concentration of inflammation, and the negative consequences result from the compensatory effects of altered flux.

 

Literature [250] points out that iNKT immune cells depend on CD1d for antigen presentation. Under α-GalCer stimulation, iNKT cells exhibit impaired proliferation and IFN-γ production capacity, and PD-1 levels increase. Blocking experiments show that most of these functional defects are unrelated to PD-1. Simultaneously, literature [249] points out that iNKT cells exhibit unique immunometabolic characteristics, and AMP-activated protein kinase (AMPK) is essential for their function. AMPK deficiency impairs the physiological function of AT-iNKT cells. Concurrently, literature [248] points out that activating iNKT cells can efficiently clear senescent cells.

 

Therefore, it can be inferred that iNKT immune cells possess the ability to bypass immune checkpoints. If NAD+ in the blood is diverted by conventional cells for anti-inflammatory purposes within the same time window, the NAD+ available to iNKT cells decreases, weakening AMPK promotion. This step weakens the function of iNKT immune cells in clearing senescent cells. As blood NAD+ levels decline during aging, the iNKT pathway for clearing senescent cells in the immune system will gradually be inhibited, leading to an increased rate of senescent cell accumulation.

 

  4.5.2.3 The increased proportion of glycolytic metabolism due to metabolic switch further promotes stem cell replicative senescence through TIN2.  

 

Section 2.5 previously demonstrated that hypoxia-induced sustained glycolytic metabolism can trigger stem cell replicative senescence. Therefore, against the backdrop of an unbalanced proportion of glycolytic metabolism due to metabolic switch, it can be inferred that the increased proportion of aerobic glycolysis may have the same effect as anaerobic glycolysis, reinforcing this core factor driving stem cell aging.

 

  4.5.2.4 The increased proportion of glycolytic metabolism and changes in substrate flow involved in the Pentose Phosphate Pathway within the glucose metabolic network.  

 

The substrate of the Pentose Phosphate Pathway (PPP), glucose-6-phosphate (G6P), faces competitive diversion among three destinations during aging: 1. Glycolysis for energy (coping with ATP crisis); 2. Glycogen synthesis (energy storage); 3. PPP generation of the co-activator factor resource pool (NADPH and ribose-5-phosphate). When metabolic pathways are forced to tilt towards glycolysis due to declining mitochondrial function, G6P is massively diverted to pathway ①, exerting a continuous siphoning effect on the substrate supply for the PPP. Literature [162] confirms the three metabolic pathways of the PPP.

 

Therefore, it can be inferred that although this diversion changes slowly in instantaneous concentration, it leads to a decrease in the regeneration rate of NADPH and ribose-5-phosphate, limiting the upper driving demand of the four-gene program and creating a "metabolic flux supply-demand imbalance." The expression of AMPK, mTOR, PPARα, and PGC-1α does not depend on static stocks of substrates but on the continuous delivery rate of co-activator factors. When the delivery rate lags, the self-maintenance mechanisms of the four factors fall into a state of "relative energy supply deficiency."

 

The contraction of resource pool availability forces the originally rhythmically alternating four genes from "synergistic homeostasis" into "competitive involution": the strengthening of each party's self-maintenance mechanism (e.g., excessive mTOR activation) comes at the expense of compressing the repair and regeneration rates of the other three. This process compresses the central axis from a resilient oscillating system into "tendential rigidity," escalating epigenetic inertia from "biased tendency" to "self-sustaining pathway solidification," systematically reinforcing the aging tendency driven by the first cause.

 

Simultaneously, as NADPH serves as the electron donor for glutathione reductase (GR) and thioredoxin reductase (TrxR), it can directly regenerate GSH and reduced thioredoxin. Its declining level will further decrease the cellular antioxidant function already compromised by the disordered glycolipid metabolic network (the second cause). The protein disulfide isomerase (PDI)-ERO1α oxidative folding system in the endoplasmic reticulum relies on the reducing power provided by TrxR to continuously reactivate oxidized PDI into active isomerase. The decline in NADPH levels reduces the rate at which oxidized PDI is reduced, causing a sharp increase in the rate of disulfide bond mismatches in nascent peptide chains, intensifying the imbalance of intracellular proteostasis at the folding stage. This constitutes the second mechanism of proteostasis imbalance.

 

  4.5.2.5 The decline in NADPH levels and T3 levels triggers compensatory acceleration of histone synthesis, but further worsens proteostasis imbalance.  

 

The insufficient reducing power supply at the folding end (governed by NADPH) and the declining clearance function (maintained by T3) simultaneously appear. This dual decline in folding and clearance leads to the "net available amount" of functional proteins persistently falling short of cellular metabolic demands. Consequently, the cell misidentifies this roughness as a genuine lack of effective functional proteins, rather than recognizing the issue of protein quality itself. This activates stress signals like IRE1α-XBP1s, non-rhythmically upregulating the transcription of histones H3/H4, attempting to meet the demand for effective functional proteins through a higher transcription rate. While this logic can restore the synthesis of effective functional proteins in early aging, as the rate of histone transcription increases, this process itself similarly affects protein quality control.

 

Thus, a vicious cycle forms: insufficient cellular effective functional proteins → increased histone transcription rate to compensate → but the transcribed proteins are ineffective misfolded proteins → effective functional proteins remain insufficient → histones continue to increase transcription speed to meet the most basic survival needs of the cell for effective functional proteins. This is the third mechanism of proteostasis imbalance.

 

  4.5.2.6 The decline in PPP flux triggers changes in cellular hyaluronic acid molecular weight levels, forming a pro-inflammatory mechanism.  

 

In the human body, hyaluronic acid (HA) can be categorized into two types based on molecular weight: low molecular weight HA (LMW-HA, 10–300 kDa) and high molecular weight HA (HMW-HA, ≳1000 kDa). When PPP flux is squeezed by glycolysis, although HA itself will not decrease because glycolysis intermediates (glucose-1-phosphate leading to UDP-glucuronic acid via the glucuronic acid pathway, and fructose-6-phosphate leading to UDP-N-acetylglucosamine via the amino sugar synthesis pathway) sustain its production, the squeezing of PPP flux leads to a decline in NADPH levels, triggering a shift in HA molecular weight from high to low molecular weight forms.

 

HMW-HA binds to CD44 with high affinity, can block TLR4 dimerization, and inhibits the NF-κB pathway, manifesting anti-inflammatory effects, stabilizing the extracellular matrix, and maintaining tissue barrier function. Literature [163][164] demonstrated the anti-inflammatory properties of HMW-HA.

 

LMW-HA binds to TLR2/4 and RHAMM, activating the MyD88-NF-κB and ERK1/2 signaling pathways, inducing the expression of IL-1β, TNF-α, and MMP-9, manifesting pro-inflammatory, pro-cell migration, and pro-angiogenic effects. Literature [165][164] demonstrated the pro-inflammatory properties of LMW-HA.

 

Therefore, the two molecular weight forms of HA constitute functionally "antagonistic-agonistic" opposing signals and can be viewed as functionally opposite bioactive molecules, rather than a single substance. This mechanism can explain the core factor behind the gradual increase in inflammatory factor concentrations with aging and serves as a key explanatory mechanism for the scope of inflammation.

 

  4.5.3 Metabolic Switch Elevates Lactate Levels, Promotes Sustained Increase in Blood Vascular Growth Factor Levels, Inducing VEGF Receptor Desensitization.  

 

The metabolic switch increases the proportion of glycolytic metabolism, and the byproduct lactate is not just metabolic waste but also an efficient signal regulatory factor. Lactate can promote vascular endothelial growth factor (VEGF) expression through the HIF-1α-VEGF pathway [168]. Previous studies examining individuals aged twenty to eighty-five did detect increased levels of vascular growth factors in the blood [167]. However, when persistent angiogenic factor levels increase while blood vessels need to maintain homeostasis, this involves the negative feedback regulation mechanism of angiogenic factors. According to this study [169], sustained stimulation of endothelial cells with VEGF leads to the downregulation of Flk-1 protein. This results in impaired eNOS activation and NO release upon subsequent VEGF challenge. Consistent with VEGF-stimulated Flk-1 degradation, it was demonstrated that VEGF stimulates Flk-1 ubiquitination, and Cbl mediates this effect through its ubiquitin ligase activity. This study proves that with increased angiogenic factor levels, the signaling quality of angiogenic factors declines.

 

Nitric oxide (NO) is a key substance promoting vasodilation and improving erythrocyte deformability, acting downstream of VEGF [169]. The increase in angiogenic factor levels will, through a negative feedback mechanism [169], lead to an age-related decrease in NO levels [170][171]. Subcutaneous blood supply depends on capillaries. The decrease in NO will lead to structural hypoxia in subcutaneous tissues. Although this hypoxia can induce increased levels of angiogenic factors, the blood angiogenic factors are already above normal levels, inducing desensitization [167][169]. Meanwhile, the body will also increase secretion of the inhibitory factor endostatin in response to excessive angiogenic factors [172]. Therefore, in a state of desensitized pro-angiogenic signaling and increased anti-angiogenic signaling, subcutaneous capillary tissues prone to structural hypoxia will exhibit a tendency towards reduced vascular density.

 

  4.5.5 Decreased Erythrocyte Deformability Mediates Metabolic Pathway Switch in Osteoarticular Tissues, Affecting Acetyl-CoA Levels, Epigenetic Disorder Promotes 15-PGDH Expression, Inhibiting Articular Cartilage Regeneration.  

 

Articular chondrocytes maintain stable HIF-1α expression through physiological glycolysis and resulting lactate, thereby promoting COX-2 transcription. However, due to the structurally hypoxic environment within joints, chondrocytes must maintain a stable glycolytic metabolism. Bone joint cells must push the TCA cycle with limited oxygen, and this oxygen concentration is strictly regulated, exhibiting a U-shaped curve. It needs to be maintained in a low-oxygen range, but not too low; both excessively high and low oxygen levels lead to decreased acetyl-CoA generation. Decreased erythrocyte deformability leads to erythrocytes having higher oxygen affinity but reduced oxygen release capacity [173]. This results in a decrease in the oxygen released by erythrocytes entering the joint within the same time window.

 

Consequently, the intra-articular TCA cycle experiences flow attenuation due to hypoxia, leading to a reduced supply of acetyl-CoA to articular chondrocytes, directly weakening the H3K27ac modification of the COX-2 promoter and causing the synthesis capacity of Prostaglandin E2 (PGE2) to fall below the negative feedback regulation threshold. This step is akin to the epigenetics having given the initiation signal, but at the execution port of acetylation, it is affected by the decreased metabolic flow of acetyl-CoA, possessing high expression potential but unable to convert it into effective gene expression. [128][129]

 

At this point, the transcriptional inhibition of 15-hydroxyprostaglandin dehydrogenase (15-PGDH) by PGE2 significantly relaxes. The promoter of this enzyme, which should be silenced due to insufficient histone acetylation, is instead aberrantly occupied by low-grade inflammatory signals (NF-κB/AP-1), forming "de-inhibited transcription." More critically, the ubiquitin-proteasome clearance capacity of aged chondrocytes decreases, causing the degradation rate of 15-PGDH to lag behind its synthesis, ultimately resulting in its net accumulation in the extracellular matrix. The increased level of 15-PGDH in articular cartilage degrades PGE2. Under normal biological levels, a small increase in PGE2 can promote articular cartilage regeneration [127]. However, the increase in local 15-PGDH concentration disrupts the normal ratio between 15-PGDH and PGE2, forming a reverse inhibition of PGE2 by 15-PGDH. Even if PGE2 levels rise in the blood with aging, due to the blood supply differences between the red and white zones of joints, the white zone can only rely on passive diffusion. With poor blood flow signals, it cannot effectively be influenced by blood-borne PGE2 [130]. Even if a small amount of PGE2 can enter the white zone through its small molecular structure, it will still be degraded under the metabolic context of high 15-PGDH concentration, leading to a decline in the self-repair function of articular cartilage. [127]. This study [174] demonstrated that persistent non-intermittent hypoxia can exacerbate osteoarthritis and increase the probability of OA onset, providing strong evidence for decreased erythrocyte deformability mediating the inhibition of cartilage regeneration.

 

Simultaneously, this mechanism operates at a fundamental level and has the basis to extend to other avascular areas such as the spine.

 

  4.6 Lactate/Ketone Bodies/Immune System, The Dilemma of Immune Clearance in Aging.  

 

Literature [246] points out that both ketone bodies and lactate can enter cells via monocarboxylate transporters (MCTs). Literature [57] indicates that ketone bodies directly inhibit the transcriptional activity of p53 through β-hydroxybutyrylation (Kbhb) modification. Literature [237] points out that increased P53 expression upregulates immune checkpoints. Literature [247] points out that it can similarly upregulate the immune checkpoint PD-L1, inhibiting immune clearance. Literature [58] indicates that ketone body metabolism requires the consumption of NAD+. Literature [54] points out that PARP1 activation consumes NAD+ and activates AMPK. Literature [53] points out that cells excessively express PARP1 when initiating the apoptosis program.

 

Therefore, it can be inferred that in the dilemma of senescent cell apoptosis, one aspect of decreased immune clearance is caused by a decline in ketone body levels. When the cell apoptosis program is initiated, the cell drives the AMPK pathway via PARP1 using NAD+, enhancing the P53-BAX pathway and inhibiting mTOR to achieve senescent cell apoptosis. When this pathway is blocked due to mTOR activation and poor cell membrane fluidity, the cell would use ketone bodies to enter the senescent cell. The P53 inhibitory effect formed by β-hydroxybutyrylation (Kbhb) modification achieves the suppression of P53-mediated immune checkpoint PD-L1 upregulation and P53-BAX downregulation, thereby inhibiting inflammation and relieving the immune checkpoint PD-L1's suppression on immune cells (NK and T cells), thus completing the immune clearance of senescent cells. However, the occurrence of metabolic switch and the ongoing imbalance caused by increased lactate proportion and decreased ketone body proportion will alter the ratio of lactate to ketone bodies entering the senescent cell. Lactate will increase the immune checkpoint PD-L1 of the senescent cell, assisting the senescent cell in immune evasion, leading to a decrease in the efficiency of senescent cell clearance.

 

  4.7.1 Skeletal Muscle Decline: GH Directly Induces Decorin Expression and Its Anti-Atrophy Mechanism  

 

Literature [83] points out that GH can directly induce the expression of Decorin in skeletal muscle through a non-IGF-1 pathway. Decorin, a small leucine-rich proteoglycan, has the key function of inhibiting Myostatin, a crucial negative regulator of muscle growth.

 

Therefore, it can be inferred that the decline in GH leads to weakened inhibition of myostatin, resulting in a decrease in skeletal muscle mass, manifesting as age-related muscle loss and symptoms of skeletal muscle decline.

 

  4.7.2 Loss of Skeletal Muscle Function as an Anti-Inflammatory Endocrine Organ  

 

Literature [84] points out that IL-6 induced by exercise in muscles possesses anti-inflammatory properties. Literature [245] indicates that muscles can secrete the anti-inflammatory factor IL-10. Literature [246] points out that TNF-α increases myostatin expression via the NF-κB-dependent pathway.

 

Therefore, it can be inferred that this constitutes a multiple vicious cycle. When GH declines, the inhibitory pathway on myostatin weakens, and the control over inflammatory factor secretion further fails. TNF-α will further enhance the myostatin pathway, inhibiting muscle regeneration. Simultaneously, inflammation inhibits mitochondrial functionality, further reducing the instantaneous ATP generation capacity, leading to a further decline in GH pulsatile levels.

 

Concurrently, accompanying the loss of muscle mass and the lipid metabolism predicament jointly driven by the second and third causes, this itself symbolizes an increase in pro-inflammatory fat levels and a decrease in inflammation-suppressing muscle levels. Hence, it can be inferred that the proportional change in muscle and fat levels in the body, coupled with the decreased efficiency of muscle in consuming fat, makes it easier for fat to accumulate, creating an inflammatory environment.

 

From this, it can be concluded that the decline of skeletal muscle is not only a loss of muscle function but also a crucial link in the collapse of the systemic anti-inflammatory defense. Acting as a direct effector and amplifier downstream of the GH-IGF-1 axis, it transforms the attenuation of hormonal signals into a tangible, vicious cycle of uncontrolled inflammation and erroneous metabolic evolution of the body's functions.

 

  4.7 Failure of the Conventional Anti-Inflammatory Pathway of Growth Hormone.  

 

Literature [100] points out that GH signaling can be rapidly initiated through three pathways: JAK2-STAT5, PI3K-Akt, and MAPK-ERK, enhancing cell survival, promoting proliferation, and inhibiting apoptosis. Literature [204] indicates that C-Jun N-terminal kinases (JNKs) belong to the mitogen-activated protein kinase (MAPK) family. Therefore, when growth hormone drives the MAPK pathway, the MAPK-JNK pathway will also be driven. Literature [205] indicates that a decrease in JNK signaling triggers inflammation. Literature [206] points out that activating JNK upregulates interleukin-10. However, there are counterexamples; literature [207] points out that inhibiting JNK can decrease inflammation.

 

Therefore, it can be inferred here that the activity of JNK exhibits a U-shaped biphasic pro-inflammatory nature. During the decline of GH signaling, the JNK signal will trigger inflammation through over-suppression.

 

Literature [208] points out that the PI3K-Akt pathway of GH signaling can initiate the expression of the ST6GAL1 gene. Concurrently, literature [101,104] points out that ST6GAL1, as a sialyltransferase, plays a key role in cell membrane glycosylation modification. Its upregulation can reduce the affinity of membrane receptors for inflammatory ligands like TNF-α and IL-6, thus achieving "feedforward inhibition" of inflammatory signals. This axis constitutes the anti-inflammatory homeostatic pathway of GH.

 

Therefore, it can be inferred here that as GH secretion declines during aging, the weakening of the PI3K-Akt-ST6GAL1 pathway and the MAPK-JNK pathway of GH forms a dual pro-inflammatory effect, triggering a sustained low-grade inflammatory state [102,103,104].

 

  4.8 Co-Regulation of Immunosenescence by GH Signaling and Glucocorticoids  

 

  4.8.1 GH Decline Directly Leads to Thymic Atrophy and its Synergy with Cortisol for Fatty Infiltration of the Thymus.  

 

Literature [107] points out that IGF-1 increases the number of thymocytes in PSGL-1KO mice. Literature [209] points out how GH affects T-cell development and immune reconstitution post-transplantation.

 

Therefore, combining the content of these two papers, it can be inferred that thymic atrophy during aging is related to the weakening of the GH-IGF-1 axis signal. Simultaneously, literature [108] points out that low growth hormone and high glucocorticoid levels lead to increased visceral fat. Literature [109] finds that BMI is positively correlated with thymic fatty infiltration, and the thymus of obese patients shows accelerated degeneration. Literature [110] points out that damage to the thymic microenvironment leads to the exhaustion of naive T-cell output, with obesity being an important factor.

 

Therefore, combining these three papers, it can be inferred that the weakening of the GH-IGF-1 signal leads to fat accumulation. High glucocorticoids promote the transfer of fat to the viscera, forming a fatty infiltration of the immune organ, the thymus, destroying the thymic regeneration microenvironment. Obese patients will exhibit stronger immunosuppression at this stage.

 

  Summary:   The weakening of the GH-IGF-1 signal not only inhibits the regeneration of the thymus itself but also, through glucocorticoids, directs erroneous fat accumulation in the thymus, destroying the thymic regeneration microenvironment.

 

  4.8.2 GH Deficiency Synergizes with Cortisol to Damage the Bone Marrow Immune System.  

 

A similar process also occurs in the bone marrow, the root of the immune system. Literature [111] points out that mesenchymal stem cells (MSCs) are precursor cells for both osteoblasts and adipocytes, with a bidirectional competitive relationship in their differentiation. It also points out that osteoporosis involves adipocyte occupation and active secretion of RANKL to promote osteoclast activation, damaging bone homeostasis. Literature [210] points out that cortisol drives the transformation of MSCs into adipocytes, and bone marrow adipose tissue (BMAT) is a dynamic fat organ within the bone marrow microenvironment, integrating energy storage, endocrine function, and immune regulation. Literature [112] indicates that BMAT induces oxidative stress in hematopoietic stem cells (HSCs) through excessive secretion of free saturated fatty acids, causing mitochondrial dysfunction and inhibiting HSC self-renewal capacity, leading to immunosuppression.

 

The preceding text has detailed the failure of multiple inflammatory factor control mechanisms, and literature [211] points out that elevated levels of inflammatory factors drive up glucocorticoid levels.

 

Therefore, it can ultimately be inferred that: the metabolic homeostasis imbalance (first cause) drives up inflammatory factors. The glycolipid metabolic disorder (second cause) and the declining GH levels (third cause) jointly drive the failure of multiple inflammation control points, forming a chronic inflammatory metabolic homeostasis, which collectively drives up glucocorticoid levels. Combined with the failure of GH pulse-induced conformational changes (third cause), this further causes atrophy of immune organs and the invasion of fat tissue into immune organs, initiating the fatty infiltration program and "hollowing out" the thymus and bone marrow from the inside. This constitutes the phenomenon of immunosenescence during the aging process.

 

  4.9 Imbalance of the GH/IGF-1 Signal Ratio: From Physiological Synergy to Pathological Divergence  

 

The incomplete intersection of GH signaling and IGF-1 axis signaling was previously described in Section 4.7, where GH signaling can inhibit myostatin independently of the IGF-1 pathway, thereby promoting muscle growth and maintaining skeletal muscle health. Multiple studies demonstrate that while GH and IGF-1 signals are synergistic, they also exhibit antagonism and independence. Literature [113] points out that inhibiting IGF-1 signaling can improve aging. However, a problem with this paper is its failure to strictly differentiate between GH and IGF-1. IGF-1 is not entirely driven downstream by GH but is synthesized through multiple pathways. For instance, increased insulin levels can drive IGF-1 synthesis, independent of GH signaling [213]. Literature [212] provides contrary empirical evidence showing that GH signaling improves aging. Literature [285] indicates that deficiency of human growth hormone induces skin premature aging.

 

Combining this with the formation mechanism of insulin resistance mentioned earlier in Section 3.5, and the tendency for insulin levels to slowly increase with age, alongside the previous findings that IGF-1 inhibition improves aging and GH signal enhancement also improves aging, a deduction can be made. During aging, the GH/IGF-1 signal ratio becomes imbalanced in its decline, exhibiting a decoupled and uncoordinated state at the signal level. The regulatory effects attributed to IGF-1 exceed those of GH signaling; the IGF-1 signal becomes background noise in the aging process, while GH experiences a drastic signal weakness due to its inability to effectively achieve conformational changes. Based on the fact that IGF-1 signaling can activate the mTOR signal, it can be speculated that this pathway further enhances mTOR in the aging process, strengthening its self-sustaining homeostasis, forming a continuous mTOR activation signal, increasing the inhibition of the BAX apoptosis pathway, reducing the self-apoptosis probability of senescent cells, and becoming a significant factor in accelerating senescent cell accumulation.

 

  4.10 Decline in GH Pulse Leads to Decreased IGF-1 Signaling in Skin Fibroblasts, Causing Collagen Decline. Inflammatory Factors Synergistically Damage Skin and Drive TGF-β to Protect Blood Vessels.  

 

Literature [214] points out that a decrease in circulating IGF-1 levels in the blood does not affect wound repair maintained by local IGF-1 signaling. Literature [215] indicates that fibroblasts are responsible for collagen production and maintenance. Literature [216] points out that IGF-1 signaling significantly promotes the secretion of COL1 and COL3 by fibroblasts via the PI3K/AKT pathway in a time- and dose-dependent manner.

 

Therefore, through the above three papers and the content of Section 4.1, it can be inferred that dermal fibroblasts depend on GH pulse-induced conformational changes to form local IGF-1 signals that maintain and promote collagen secretion by dermal fibroblasts. This process is unrelated to the background level of circulating IGF-1 in the blood and is solely governed by the local IGF-1 signal dominated by GH pulsatile secretion. When the third cause becomes the primary pro-aging factor, it directly leads to a drastic decline in the visual effects of skin plumpness and moisture maintained by collagen produced by fibroblasts, forming a visually perceptible cliff-like aging effect. Concurrently, combined with the content of Section 4.6, it can be speculated that this process will be accompanied by a decrease in muscle mass. Literature [219] points out that as IGF-1 signaling decreases, fibroblasts lose their proliferative capacity and their ability to maintain ECM integrity, leading to dermal thinning and reduced skin elasticity.

 

  4.10.1 Synchronous Aging of Blood Vessels and Skin.  

 

The metabolic homeostasis imbalance (first cause) triggers an increase in inflammatory factor levels. Glycolipid metabolic disorder (second cause) promotes a secondary increase in inflammatory factor levels. By the time of the third cause, the core T3 anti-inflammatory mechanism and GH anti-inflammatory mechanism collectively decline. Concurrently, two major senescent cell clearance mechanisms gradually fail. The decrease in GH's guidance on fibroblasts jointly promotes the damaging effects of inflammatory factors on collagen.

 

Both blood vessels and skin contain fibroblasts. Among the multiple functions of fibroblasts is the production of elastin and collagen [217]. Literature [218] points out that inflammatory factors can destroy collagen. Literature [52] indicates that inflammatory factors can drive the secretion of Transforming Growth Factor-β (TGF-β). Literature [51] indicates that abnormal sustained activation of the TGF-β pathway leads to organ tissue fibrosis. Literature [219] further points out that the TGF-β signaling pathway is another key mediator of fibroblast aging, particularly through its role in fibrosis and ECM remodeling. TGF-β is a potent regulator of fibroblast function, and its signaling through SMAD proteins promotes the expression of ECM components like collagen. In aged fibroblasts, TGF-β signaling is upregulated, leading to excessive ECM deposition and fibrosis, ultimately forming wrinkles and skin hardening.

 

Combining these papers, it can be inferred that during aging, the increase in inflammatory factors, while destroying collagen, simultaneously promotes the secretion of TGF-β as a negative regulator. This negative regulation can maintain the homeostasis of blood vessels. However, because TGF-β secretion simultaneously exacerbates fibrosis, this triggers increased fibrosis in blood vessels and skin, leading to the phenomena of skin aging and vascular aging. Literature [220] points out that blood vessels and skin exhibit a synchronous aging trend.

 

  5 The Fourth Cause of Aging: Antioxidant Network Collapse  

 

Both NRF1 and NRF2 can bind to the Antioxidant Response Element (ARE). This mechanism provides the basis for their competition at the signaling level [62]. Literature [63] points out that NRF2 is responsible for emergency antioxidant responses, while NRF1 is responsible for maintaining GSH recycling regeneration and proteasome homeostasis; dual gene knockout renders cells extremely sensitive to the pro-oxidant tBHQ. Literature [62] also points out that upon knocking out NRF1, although NRF2 can still be activated, it cannot compensatorily maintain the normal expression of mitochondria-related antioxidant genes, leading to impaired mitochondrial function.

 

As mentioned earlier in Section 4.1, the third cause, the decline in GH-IGF levels, leads to decreased PGC-1α expression, and NRF1 levels decline accordingly. Therefore, it can be inferred that with aging, the body's antioxidant function is increasingly handed over to NRF2. The typical activation mode of NRF2 involves its release through the oxidation of Keap1 by ROS. This means that when the antioxidant function maintained by NRF1 is lost and only that maintained by NRF2 remains, the activation frequency of NRF2 will increase.

 

Literature [42] indicates that sustained activation of NRF2 drives the transcription of HO-1, which decomposes heme, increasing intracellular free iron levels and triggering the ferroptosis pathway via oxidative stress.

 

Based on this, combined with the inference of frequent NRF2 activation, it can be further deduced that although frequent NRF2 expression may not necessarily lead the cell to ferroptosis, it will increase cellular oxidative stress and the cellular antioxidant load. This creates chronic oxidative stress. Chronic oxidative stress promotes the frequent expression of NRF2, accelerating it. Through frequent binding to the ARE element, it can be deduced that NRF2 will inhibit NRF1 expression by competing for the ARE, forming a vicious cycle of self-maintenance, self-reinforcement, and self-perpetuation in a locked state. This is the starting point of the fourth cause.

 

  5.1 The Antioxidant Network, Electron Donor Transfer and Reserves.  

 

Literature [221] indicates that the total amount of Vitamin C in the human body is 3000mg. Literature [222][223] points out that a healthy adult has 1500mg of Vitamin C in its reduced form, ascorbic acid (AA). Literature [224] points out that the synthesis of glutathione can regenerate VC and also indicates that when dehydroascorbic acid (DHA) concentration exceeds 400 μM, it consumes intracellular reduced glutathione (GSH). Literature [225] points out that the DHA/AA ratio changes in the elderly, with the proportion of DHA increasing, and that elderly individuals with low AA are more susceptible to age-related diseases. Literature [226] points out, through pharmacokinetics, the absorption dilemma of reduced VC (AA) in the human body. At low to moderate oral doses (30–180 mg/day), absorption efficiency is as high as 90%, with bioavailability up to 90%. However, exceeding 1000 mg/day causes the fractional absorption rate to drop sharply to less than 50%, primarily due to high saturation of intestinal SVCT1. For oral doses exceeding 400 mg/day, due to limited absorption and increased renal clearance, plasma ascorbic acid only increases modestly. The kidneys reabsorb ascorbic acid through SVCT1 in the proximal tubules; however, once plasma levels exceed the renal reabsorption threshold, the kidneys actively excrete excess ascorbic acid through urine, reducing its systemic retention. Additionally, tissue saturation adds another barrier. Therefore, higher plasma ascorbic acid concentrations do not necessarily lead to elevated intracellular levels, especially in already supplemented tissues, where SVCT2-mediated uptake is strictly controlled. Literature [227] points out that depletion of inducible Thioredoxin 1 (TRX1) slows the reactivation of PTEN in intact living cells. Literature [228] points out that Thioredoxin can regenerate VC. Literature [229] indicates that taking 500mg of VC can promote an increase in the ratio of reduced glutathione in erythrocytes, facilitating glutathione regeneration. Literature [230] points out that the MRP2 (multidrug resistance-associated protein 2) pump mediates the excretion of GSH and GSSG, as well as endogenous and exogenous substances bound to GSH, glucuronic acid, or sulfate. NRF2 overexpression can activate MRP2. Literature [231] points out that exogenous intake of VC can lower the level of GSSG in the blood. Literature [78] indicates that PTEN can inhibit mTOR.

 

Therefore, we can infer that Vitamin C is the largest electron reserve pool in the human body. Its primary use is to regenerate VE, maintain membrane antioxidant function, and counteract the damage from ROS free radicals produced by endogenous and exogenous oxidants in the blood. Human cells prefer to export reduced VC to form the blood's antioxidant defense line and then reclaim oxidized VC, regenerating it through pathways like glutathione and thioredoxin to maintain the stability of the blood's antioxidant defense.

 

Once the proportion of oxidized VC in the antioxidant network increases, cells will draw upon reduced glutathione to regenerate VC. This step may lead to increased intracellular GSSG levels and decreased intracellular GSH. However, cells can oxidatively modify Keap-1 to release NRF2, excrete intracellular GSSG via MRP2, and then synthesize GSH to remedy the situation, restoring intracellular reduced glutathione levels. In this pathway, the proportion of oxidized VC in the blood determines whether cells need to initiate emergency rescue via the NRF2 pathway.

 

Low-frequency rescue via the NRF2 pathway will directly lower blood GSSG levels, while high-frequency rescue will increase blood GSSG levels. Simultaneously, when the proportion of oxidized VC within the cell increases, it will divert NADPH expenditure away from other antioxidant demands, such as the Thioredoxin (Trx) pathway, within the same time window.

 

The Thioredoxin (Trx) pathway is also a key route for VC regeneration. When the proportion of oxidized VC increases, the main flux of Trx shifts towards VC. The maintenance of PTEN's reduced state will then be disrupted. One core function of PTEN is to degrade phosphatidylinositol trisphosphate (PIP3), thus negatively regulating the PI3K-AKT-mTOR signaling axis. Once PTEN is inactivated by oxidation, its degradation of PIP3 weakens, leading to sustained activation of AKT, which in turn triggers the overactivation of the mTORC1 signaling pathway. This pathway promotes glutathione synthesis by enhancing biosynthetic metabolism.

 

  5.3 The weakening of the antioxidant network, Vitamin A, and the reverse regulatory mechanism of the PTEN-mTOR pathway.  

 

Literature [23] points out that retinol binds to RBP. Literature [25] indicates that reduced Vitamin A (retinol) bound to RBP (holo-RBP) activates the STRA6 receptor. Literature [24] confirms that the holo-RBP/STRA6 complex has a pro-survival (anti-apoptotic) function. Literature [26] confirms that STRA6 activates the ILK/AKT/mTOR signaling axis. Literature [232] points out that oxidized Vitamin A (retinoic acid) can also bind to RBP, but STRA6 cannot take up retinoic acid from this complex as it does with retinol. The uptake of retinoic acid from the retinoic acid/RBP complex requires the participation of CRABP-I (cellular retinoic acid binding protein-I), and cannot be accomplished by STRA6 alone. Literature [27] points out that retinoic acid promotes apoptosis. Literature [78] indicates that PTEN oxidation can activate mTOR. Literature [233] points out that increased mTOR activation enhances the expression of anti-apoptotic proteins. Literature [234] points out that senescent cells maintain high levels of mTOR activity. Literature [235] indicates that VE regulates Bax translocation to the mitochondria, inhibiting Bax-initiated apoptosis. Literature [236] points out that VE deficiency leads to apoptosis sensitivity.

 

Therefore, it can be inferred that when cellular redox resources are ample and electron transfer is smooth, Vitamin A is generally maintained in its reduced state. Holo-RBP (retinol bound to plasma retinol-binding protein) binds to the membrane receptor STRA6, triggering STRA6-dependent cell membrane signal transduction. This complex can induce the recruitment and activation of JAK2 in the intracellular domain of the receptor after binding, subsequently inducing tyrosine phosphorylation of the STAT family transcription factor STAT5. Tyrosine-phosphorylated STAT5 can directly bind to Raptor, a core component of mTORC1, activating the mTOR signal. However, PTEN, due to ample antioxidant resources, inhibits mTOR, forming an opposing regulatory signal. Because VA's reduced state activates mTOR through a milder pathway, it is more easily regulated by the body, and the signal is more stable compared to the oxidized PTEN pathway.

 

Similarly, VE in its reduced state inhibits BAX. However, all of this does not inhibit normal cell apoptosis. When a cell undergoes apoptosis, its intrinsic apoptotic program initiates the vigorous activation of the PARP1 gene. PARP1, as a molecular sensor of DNA damage, rapidly consumes the intracellular NAD+ pool through its extraordinary enzymatic activity, aiming to mark and initiate repair by synthesizing poly(ADP-ribose) chains. This step drastically pushes up p53 expression via ADP through the AMPK pathway. The whole system is akin to a slope: when cellular reducing resources are ample, cells do not easily enter the apoptotic program and instead try their best to repair. Moreover, because the cell membrane's degree of oxidation is low and fluidity is sufficient, cells entering the apoptotic program are more easily killed by their own intrinsic apoptotic program.

 

Conversely, if cellular antioxidant resources are insufficient and electron transfer is abnormal, PTEN becomes oxidized, mTOR is continuously activated, and although VA and VE no longer exert anti-apoptotic effects but instead initiate pro-apoptotic effects, the cell must face two major inhibitory pathways for apoptosis: cell membrane oxidation and sustained mTOR activation. These jointly lead to a decrease in the rate of cell apoptosis, increasing the proportion of senescent cells in the human body.

 

  5.4 Persistent mTOR Signal Inhibits AMPK, Shortening the Expression Window of DNA Repair Enzymes Downstream of the AMPK Pathway and Increasing Cellular Genomic Instability.  

 

Literature [175] points out that there are multiple DNA repair enzymes downstream of the AMPK pathway. Literature [237] points out that increased P53 expression will elevate the expression of immune checkpoints.

 

Therefore, it can be inferred that when mTOR is uncontrollably activated due to PTEN oxidation, AMPK is inhibited. DNA repair pathways downstream of AMPK—such as homologous recombination repair, nucleotide excision repair, base excision repair, and mismatch repair—will experience a decrease in maintenance frequency and quality. The genome will consequently experience increased instability. This genomic instability triggers an elevation in P53 levels, and P53 upregulates immune checkpoints, leading to the inhibition of immune clearance.

 

  5.5 Persistent mTOR Signaling Mediates Changes in Downstream Gene Spectrum Expression.  

 

Literature [176] points out that under energy stress conditions, eIF4E mediates systematic translational spectrum rearrangement by differentially binding to cap-proximal sequences.

 

Therefore, it can be inferred that against the backdrop of persistent mTOR activation signals inhibiting AMPK, cellular ATP will remain continuously insufficient. However, with PTEN being oxidized, mTOR will be prematurely re-activated. That is, before intracellular ATP energy has recovered, mTOR will already promote cellular biosynthetic metabolism again. Due to ATP insufficiency, the duration of this biosynthetic metabolism will not increase significantly. However, each mTOR expression cycle will de novo promote the expression of related genes. In each cycle, eIF4E must dissociate from 4E-BP1 again, but the phosphorylation-dephosphorylation cycle shortens its half-life. Simultaneously, due to different binding sites and affinities, the expression of genes like the transcription factor PAX8 will decrease due to its own structure, while ribosomal proteins like RPS6 may increase in expression due to simpler structures. This represents a change in the downstream gene spectrum expression.

 

In the context of persistent mTOR mismatched activation caused by PTEN oxidation, this translational rearrangement shifts from a short-term adaptive mechanism to a long-term aging driver.

 

In this rearrangement, SASP factors, chemokines, acute inflammatory factors, pro-apoptotic factors, DNA damage monitoring genes, hypoxia genes, and amino acid starvation signal molecules will prevail. Meanwhile, tissue-specific transcription factors, cell junctions, hormone receptors, anti-apoptotic factors, growth factors, and oncogenes will decline. Among these, the decrease in anti-apoptotic-related genes and the increase in pro-apoptotic genes do not mean an improvement in the harms of the fourth cause.

 

When mTOR is persistently expressed in normal cells, the result is the decreased expression of anti-apoptotic genes, increased expression of the DNA damage monitoring gene P53, and increased expression of pro-apoptotic genes. This makes normal cells more prone to entering senescence, while senescent cells are conversely less likely to undergo apoptosis.

 

  5.6 Damage to Telomeres from Elevated P53 Expression Levels and the Vicious Cycle.  

 

Literature [267] points out two points in its theory: First, SIRTs bind to telomeres and rDNA, maintaining the stability of telomeric and rDNA regions. P53 inhibits the expression of the seven SIRT proteins, thereby causing instability in telomeres and nucleolar rDNA. Second, as the telomeric DNA array and/or rDNA array (the clock) shorten, P53 (the primary command) produces a 'concentration gradient' along the time axis... driving programmed expression of gene clusters on chromosomes.

 

Combining this paper with the preceding text, it can be inferred that when the fourth cause is triggered, human cells, due to frequent and persistent mTOR signals, will have inhibited AMPK activation. DNA repair efficiency decreases, forming genomic instability, and P53 levels increase. Elevated P53 inhibits the expression of the seven SIRT proteins, leading to instability in telomeres and nucleolar rDNA. As the telomeric DNA array and/or rDNA array (clock) shortens, P53 levels further increase. From this point, a vicious cycle forms, possessing self-maintaining and self-reinforcing effects.

 

  5.7 The Field Damage Theory of rDNA Shortening and Recovery Mechanisms.  

 

Literature [269] points out that rRNA constitutes the structural and functional core of ribosomes. ROS can affect RNA in multiple ways [34,35,36,37], including chemical modification of bases and sugar groups, generation of non-basic sites, and strand breaks. Guanine is the most easily oxidized nucleobase and is most fully studied in this regard. One oxidized form of guanine, 8-hydroxyguanine (8-oxoguanine; 8-oxoG), is a ubiquitous oxidative lesion easily detected in cellular nucleic acids. When 8-oxoG is present in mRNA, it interferes with decoding, possibly by forming Hoogsteen pairs with adenine upon rotation around the N-glycosidic bond. Changes in the base-pairing capacity of 8-oxoG can also perturb RNA folding. A recent study revealed diverse outcomes when 8-oxoG was incorporated into model RNA substrates, ranging from stabilization of existing structural motifs to destabilization and rearrangement into new structures. Because ribosomal translation activity depends on many precisely tuned conformational changes and movements within its rRNA framework, oxidation of key bases that maintain correct rRNA structure could impair ribosomal function. Therefore, the need to tolerate some oxidation was likely a factor shaping ribosomal evolution. Many guanines are significantly depleted from mitochondrial rRNA, accompanied by a decrease in overall RNA content.

 

Literature [270] points out that the muscle regeneration process encompasses four interconnected phases: necrosis, inflammation, satellite cell activation and differentiation, maturation of newly formed myofibrils, and muscle remodeling. During the inflammatory phase, i.e., during muscle injury repair, reactive oxygen species (ROS) are produced in large quantities, mainly present in neutrophils and M2 macrophages. Furthermore, ROS-activated MAPK (mitogen-activated protein kinase), NFκB (nuclear factor kappa B), and AP-1 (activator protein 1) induce protective responses in injured muscle through ROS activation. Additionally, antioxidant enzymes such as superoxide dismutase 2, glutathione peroxidase, and catalase increase in the initial days following injury. Therefore, ROS activate important signaling pathways for muscle repair. However, impaired oxidative stress resolution may lead to secondary damage to uninjured fibers.

 

Literature [271] indicates that resistance exercise training (RET) can be effectively applied to increase muscle mass and function in older adults aged 65-75 years. However, there is speculation that adults over 85 years may respond less well to the benefits of RET. This study compared the effects of RET on muscle mass and function in healthy older adults aged 65-75 years versus those over 85 years. We treated 17 healthy older adults aged 65-75 years (65-75yr, n=13/4 [female/male]; 68±2 years; 26.9±2.3 kg/m²) and 12 healthy older adults over 85 years (85+ yr, n=7/5 [female/male]; 87±3 years; 26.0±3.6 kg/m²) with 12 weeks of whole-body RET (three times per week). Quadriceps and L3 lumbar muscle cross-sectional area (computed tomography), whole-body lean mass (dual-energy X-ray absorptiometry), strength (one-repetition maximum test), and physical performance (timed up-and-go and short physical performance battery) were assessed before and after 6 and 12 weeks of training. After 12 weeks of RET, quadriceps cross-sectional area increased by 10%±4% and 11%±5% (from 46.5±10.7 to 51.1±12.1 cm², and from 38.9±6.1 to 43.1±8.0 cm²; p<.001; η2 = 0.67); whole-body lean mass increased by 2%±3% and 2%±3% (p=.001; η2=.22); one-repetition maximum leg extension strength increased by 38%±20% and 46%±14% (p<0.001; η2 = 0.77) in the Old 65-75 and Old 85+ groups, respectively. RET responses did not differ between groups (time × group, all p>0.60; all η2 ≤ 0.012). Physical performance on the Short Physical Performance Battery and timed up-and-go both improved (all p < 0.01; η2 ≥ 0.22), with no differences between groups (time × group, p > 0.015; η2 ≤ 0.07). Prolonged RET increases muscle mass, strength, and physical performance in aging populations, with no significant difference between older adults aged 65-75 years and those aged 85+ years.

 

Literature [272] points out that this study demonstrates resistance training significantly increases muscle stem cell content... In older adults, satellite cell numbers significantly increased after 12 weeks of resistance training. Resistance training significantly increased satellite cell numbers in type II muscle fibers of older adults... This systematic review and meta-analysis showed that resistance training in older adults can significantly increase muscle stem cells.

 

Based on the four articles above, it can be inferred that the current scope of application for the replicative senescence theory has boundary issues. Replicative senescence is a classic wear-and-tear theory of aging. Within this theoretical framework, there is no concept of recovery, and unidirectional irreversible wear is its core definition of aging. But this is an erroneous inference that overgeneralizes the in vitro passage model to the in vivo context.

 

Oxidative stress can damage rDNA, leading to loss of rDNA array copies. If one follows the logic of replicative senescence, human exercise would generate free radicals, causing oxidative stress. This oxidative signal drives muscle growth, while these free radicals would simultaneously damage the rDNA array. However, this logical deduction is falsified by resistance training. The empirical evidence showing that individuals engaging in regular, long-term resistance training maintain more muscle mass cannot be explained by this logic. Because if one maintains long-term resistance training, ROS would damage the rDNA array, inhibiting the self-renewal of muscle satellite stem cells. During aging, due to severe loss of rDNA array copies from excessive exercise, muscle satellite stem cells would be unable to maintain their proper numbers, leading to muscle atrophy. But reality is the opposite, indicating that either oxidative stress cannot drive the loss of rDNA array copies, or human stem cells possess a recovery mechanism for the rDNA array.

 

Given that oxidative stress driving the loss of rDNA array copies has been confirmed, human stem cells must necessarily possess a recovery mechanism for the rDNA array. Otherwise, the beneficial effects of many forms of exercise, such as resistance training, on aging would not exist.

 

Literature [273] points out that unequal sister chromatid exchange (USCE) allows cells to restore rDNA copy number on one chromatid at the expense of its sister chromatid. This phenomenon occurs specifically in Drosophila male germline stem cells (GSCs)—cells that undergo asymmetric division to produce one self-renewing stem cell and one differentiating daughter cell. In this process, the chromatid that gains rDNA copy number restoration is preferentially retained in the stem cell lineage, while the copy-damaged one segregates into the differentiating daughter cell.

 

Literature [274] points out that in Saccharomyces cerevisiae, the maintenance of rDNA copy number (CN) depends on competitive occupancy between the Pol I-UAF complex and the Sir2-UAF complex. When CN is high, Pol I-UAF binds to the rDNA promoter, blocking further amplification of copy number by inhibiting transcription-replication coupling negative feedback. When CN decreases due to oxidative stress or recombination, the Sir2-UAF complex relieves the inhibition of the bidirectional E-pro promoter, necessarily initiating intra-array homologous recombination (intra-array HR), driving rDNA gene conversion and amplification to achieve copy number recovery. This "suppress when high, promote when low" bidirectional homeostatic switch enables the rDNA array to possess active counting and self-recovery capabilities, unlike the unidirectional depletion of telomeres.

 

Literature [275] points out that when rDNA undergoes a double-strand break (DSB), a specialized nucleolar DNA damage response (n-DDR) is activated. Upon activation, rDNA relocates from the nucleolar interior to the nucleolar periphery, forming "nucleolar caps." Within this subnuclear structure, homologous recombination (HR) repair is exclusively performed. During this process, the BLM (Bloom syndrome) helicase plays a crucial role by unwinding the DNA duplex to facilitate recombinational repair—BLM-deficient cells exhibit recombination rates in rDNA exceeding 10%, demonstrating that this enzyme is a core regulatory factor for maintaining genomic stability of the human rDNA array by ensuring its integrity through the HR pathway.

 

Based on the above literature, it can be inferred that rDNA recovery mechanisms exist in both humans and animals. Combined with the empirical effectiveness of resistance training and the phenomenon of oxidation damaging rDNA, a field theory of dynamic recovery inhibition for rDNA copy number can be proposed. "Field" refers to different interfering factors producing an effect on a single target. The preceding text has already mentioned that genomic instability can inhibit rDNA through P53, and oxidative stress directly damages rDNA, confirming that rDNA copy number loss is multi-source inhibition. Meanwhile, rDNA itself possesses repair mechanisms. The intersection of the two forms a dynamic phenomenon of recovery and loss.

 

However, this loss is not irreversible. Merely removing inhibitory factors or strengthening recovery factors holds the promise of restoring the normal level of stem cell rDNA arrays.

 

  5.8 Aging Diversity and Central Axis Correlation.  

 

As demonstrated earlier, changes in the antioxidant pattern occur during the fourth cause stage, and this reinforces the pathogenic nodes mentioned earlier, forming the phenomenon of diverse aging-related diseases.

 

This subsection will explain the central axis changes corresponding to many aging-related diseases under the framework of aging-related central axis changes. However, the scope of this subsection is limited to aging-related frameworks; non-aging-related frameworks may present incompatibility.

 

  5.8.1 Type 2 Diabetes: Decline in Antioxidant Function and Local AMPK/mTOR Signal Imbalance.  

 

In subsection 3.5, the principle behind the origin of insulin resistance was demonstrated: its core is cell membrane oxidation causing decreased membrane fluidity, restricting insulin receptors due to membrane fluidity and failing to complete normal activation and signal transduction. The fourth cause reinforces this process. Among those particularly affected in this stage are individuals with intrinsically weak PGC-1α signaling, weak AMPK signaling, strong mTOR signaling, and strong PPARα signaling. They will be the first to experience enhanced insulin resistance due to increased oxidative stress and decreased cell membrane fluidity. Blood glucose signals stimulate β-cells to increase mTOR to secrete insulin. During this process, if mTOR-driven oxidative stress further pushes the local β-cell NRF2-HO-1-free iron-hydroxyl radical-PTEN oxidation-mTOR promotion-AMPK inhibition pathway, it will reinforce the inhibition of AMPK and NRF1, forming a self-locking dilemma of local cellular metabolic vicious cycle.

 

Such a metabolic self-locking dilemma will damage β-cells through the ROS pathway, ultimately triggering β-cell dysfunction. Research on the correlation of glycine and cysteine with type 2 diabetes demonstrates that glycine and cysteine indeed have the effect of reversing type 2 diabetes, but do not possess the ability to reverse the local metabolic homeostasis imbalance. Their intervention targets the more fundamental downstream oxidative damage, rather than intervening at the more upstream erroneous metabolic homeostasis.

 

In literature [276], the improvement effect of glycine and cysteine on type 2 diabetes was empirically validated. When HFD-induced diabetic mice were administered NAC at an effective dose and duration, it significantly improved glucose tolerance and insulin sensitivity and successfully rescued β-cell overcompensation. NAC-treated HFD mice showed normalization of β-cell mass and size, significant improvement in β-cell identity, significant improvement in Pdx-1 nuclear localization, and reduced β-cell oxidative stress marked by 8-OHdG. Importantly, NAC reduced the activation of PaSCs within the islets and inhibited PaSC-induced collagen deposition, thereby preventing HFD-induced fibrosis. This study demonstrates that antioxidant treatment with NAC helps maintain healthy β-cells and a quiescent PaSC population within the islets of HFD-induced diabetic mice, and contributes to a better understanding of the effective dose and timing of antioxidant therapy for human obesity-related diabetes.

 

  5.8.2 Nitric Oxide, Cardiovascular Disease, and Commonalities with Heart Disease.  

 

Subsection 3.4.2 demonstrated the predicament of homocysteine metabolism into cysteine. Subsection 3.1 demonstrated the decline in AKG levels caused by reduced lipid metabolism.

 

  5.8.2.1 Homocysteine-Induced Vascular Endothelial Damage – Atherosclerosis and Heart Disease  

 

Homocysteine has the effect of damaging vascular endothelium [278]. However, if it could be normally metabolized into cysteine, it would not have this damaging effect. The core metabolic homeostasis error is that during the fourth cause stage, nitric oxide becomes oxidized and disabled, further decreasing erythrocyte deformability. The oxygen release function of erythrocytes in liver tissue further declines. Lipid metabolism is further affected by hypoxia, the proportion of lipid metabolism decreases, and AKG levels drop, triggering metabolic accumulation of homocysteine, which damages the cardiovascular system. Simultaneously, due to decreased oxygen release rate, the myocardium will experience occult hypoxia. This forms a situation where non-identical tissues exhibit interrelated pathologies. This metabolic imbalance type corresponds to individuals with weak AMPK signaling, strong mTOR signaling, and weak PGC-1α.

 

  5.8.2.2 Non-Endothelial Damage Myocardial Hypoxic Heart Disease  

 

Individuals with excessive PPARα have lower homocysteine accumulation at the cellular metabolic level and can avoid the metabolic accumulation of homocysteine. However, PPARα itself possesses metabolic pro-oxidative properties, which will exacerbate the oxidative damage of nitric oxide, leading to a decline in oxygen release rate and triggering an occult hypoxic metabolic state in the heart itself.

 

Literature [279] and [280] confirm the correlation between high-altitude hypoxia and the occurrence of heart disease. On the Qinghai-Tibet Plateau, the prevalence of high-altitude heart disease in immigrant Han children reaches 2.5%, and increases with altitude. Adult high-altitude heart disease is directly related to chronic high-altitude hypoxia, manifesting as significant pulmonary hypertension, right ventricular hypertrophy, and right heart failure. In areas above 3000 meters altitude, the prevalence of hypertension increases by 2% for every 100-meter increase in elevation.

 

Long-term exposure to high-altitude hypoxic environments induces pulmonary vascular remodeling, leading to pulmonary hypertension (PH), which in extreme cases progresses to right heart failure (RHF). High-altitude exposure exacerbates the risk of decompensation in heart failure patients, resulting in more cardiac events and re-hospitalizations.

 

  5.8.3 Association between AMPK/mTOR Ratio Imbalance and Hypertension.  

 

During sleep, blood flow distribution changes from peripheral to central circulation—this is classic textbook content. Based on this principle, nerve cells have high energy metabolism dependence. In a metabolic background where nitric oxide is oxidatively damaged, the imbalance state jointly formed by weak AMPK signaling and strong mTOR signaling manifests in the pineal gland, which secretes melatonin. Pineal gland melatonin synthesis requires tryptophan hydroxylase (TPH), a high-energy-consuming rate-limiting enzyme (requires NADPH and O₂). This will lead to decreased melatonin secretion, causing difficulty falling asleep, shortened sleep time window, and abnormal blood flow redistribution during sleep, among other negative factors. Due to the oxidation of nitric oxide, the oxygen release capacity of erythrocytes decreases within the same time window. Against the backdrop of a shortened time window, the recovery resources provided will sharply decrease. The internal organs will not receive effective recovery, thus pushing up blood pressure to compensate for occult visceral ischemia.

 

Literature [277] confirms the association between sleep deprivation and hypertension. During acute sleep deprivation, a significant enhancement of sympathetic nervous system activity can be observed, accompanied by activation of the hypothalamic-pituitary-adrenal (HPA) axis, leading to abnormally elevated cortisol levels. Furthermore, markers of oxidative stress (including NRF2, HO-1, and NQO1 pathways) are upregulated under sleep insufficiency, collectively contributing to increased peripheral vascular resistance and significant increases in both systolic and diastolic blood pressure.

 

  5.8.4 Nitric Oxide and Degenerative Joint and Spine Disease  

 

Subsection 4.5.5 demonstrated the dependence of articular cartilage regeneration on oxygen release rate. Subsection 5.8.2 demonstrated the metabolic homeostasis error of nitric oxide being oxidized and disabled during the fourth cause stage.

 

In the metabolic context of the fourth cause stage, where nitric oxide's oxidative damage increases and it becomes disabled, individuals with intrinsically weak PGC-1α signaling, weak AMPK signaling, strong mTOR signaling, and strong PPARα signaling will be the first to experience increased oxidative stress, further decreasing erythrocyte deformability. This leads to a further decline in oxygen release rate in joints and spine, forming occult hypoxia in these areas. This hypoxia will exacerbate the flow attenuation of the TCA cycle, reducing the supply of acetyl-CoA, weakening the H3K27ac modification of the COX-2 promoter, and causing PGE2 synthesis capacity to fall below the negative feedback regulation threshold. Consequently, the transcriptional inhibition of 15-PGDH by PGE2 significantly relaxes, leading to a decline in joint and spine repair efficiency. When repair efficiency falls below the daily wear-and-tear rate, it results in the occurrence of degenerative joint and spine diseases.

 

  5.8.5 AMPK/mTOR Ratio Imbalance, Inflammatory Factors – Osteoporosis.  

 

Subsection 2.3.1 demonstrated the mechanism by which weak AMPK signaling leads to reduced inhibition of NF-κB. Subsection 4.8.2 demonstrated the damage of GH deficiency synergizing with cortisol to the bone marrow immune system.

In the fourth cause stage, against the metabolic background of central axis constrictive collapse (weak AMPK signaling, strong mTOR signaling), AMPK's inhibition of NF-κB further weakens. Glucocorticoid levels rise due to increased chronic inflammation levels, jointly inducing adipogenic differentiation of bone marrow mesenchymal stem cells (MSCs). Individuals with weak AMPK signaling will be the first to experience weakened NF-κB inhibition, leading inflammatory factors to drive the transformation of MSCs into adipocytes, forming fatty infiltration of the bone marrow microenvironment by Bone Marrow Adipose Tissue (BMAT).

Inflammatory factors can also drive M1 polarization of macrophages via the NF-κB pathway, secreting matrix metalloproteinases (MMPs) that degrade collagen [259]. Simultaneously, due to weakened AMPK inhibition of NF-κB, RANKL (Receptor Activator of NF-κB Ligand) secretion abnormally increases, while Osteoprotegerin (OPG) expression is suppressed. The imbalance of the RANKL/OPG ratio triggers excessive osteoclast activation, ultimately leading to osteoporosis.

This metabolic imbalance type corresponds to individuals with weak AMPK signaling, strong mTOR signaling, and weak PGC-1α signaling.

 

  5.8.6 Correlation of Fatty Liver and Hyperlipidemia with Metabolic Homeostasis.  

 

Subsection 3.1 demonstrated the mechanism of lipid metabolism decline. PPARα is the master transcriptional regulator of hepatic fatty acid β-oxidation, maintaining the flux of fat metabolism. A decrease in its level vacates spare NADPH resources for PGC-1α to maintain antioxidant function, weakening the situation of nitric oxide being oxidatively disabled due to the fourth cause metabolic background. Therefore, an isolated decline in PPARα signaling is decoupled from the fourth cause. Its metabolic decline can independently complete the phenomena of fatty liver and hyperlipidemia. However, against the metabolic background of the fourth cause, if PGC-1α and PPARα decline simultaneously, the pathway of nitric oxide being oxidatively disabled and the subsequent decrease in oxygen release rate can inhibit the liver's efficiency in fat metabolism. Inhibited lipid metabolism triggers the collapse of PPARα's self-maintenance mechanism, forming a coupled decline in PPARα signaling, leading to fatty liver and hyperlipidemia.

 

  5.8.7 Nitric Oxide – Microcirculatory Disturbance – Increased Cancer Incidence.  

 

Literature [281] points out that through a retrospective cohort study (median follow-up 9 years) of 1042 patients with non-obstructive coronary artery disease (NOCAD), it was confirmed that coronary microvascular dysfunction (CMD, defined as coronary flow reserve ≤2.5) is significantly associated with cancer incidence: CMD patients had a cancer incidence rate of 15.5%, and their cancer-free survival rate was significantly lower than those without CMD (log-rank P = 0.005). Multivariate Cox regression analysis showed that after adjusting for age, sex, hypertension, diabetes, smoking, and renal function, CMD was an independent predictor of cancer (hazard ratio 1.40, 95% confidence interval 1.09–2.04, P = 0.04). This study only established a clinical correlation without elucidating the molecular mechanism. Literature [282] points out the mechanism: when local microcirculatory disturbance (nitric oxide disability leading to decreased erythrocyte deformability) triggers structural hypoxia, cells are forced to initiate glycolytic metabolism. The continuous siphoning effect of glycolysis on the Pentose Phosphate Pathway (PPP) substrate glucose-6-phosphate (G6P) leads to decreased NADPH levels. The decline in NADPH triggers a shift in hyaluronic acid (HA) molecular weight from high molecular weight (anti-inflammatory) to low molecular weight (pro-inflammatory) state, activating the TLR2/4-NF-κB signal and inducing the expression of inflammatory factors like IL-1β and TNF-α. Under continuous hypoxia and inflammatory pressure, cells acquire advantageous mutations in the unfolded protein response (UPR) through natural selection, ultimately heading towards hypoxic carcinogenesis.

Therefore, in the fourth cause stage, nitric oxide oxidative disability → decreased erythrocyte deformability → collapse of microcirculatory oxygen release rate → tissue occult hypoxia is the metabolic bridge connecting CMD and cancer. Hypoxia induces HIF-1α stabilization, driving the glycolysis-lactate-inflammation cascade, ultimately forming the pathological loop of "hypoxic carcinogenesis."

 

  5.8.8 Sodium-Related Alzheimer's Disease Due to Decreased Energy Metabolism.  

 

Subsection 2.7 demonstrated that sodium-potassium pump dysfunction reversely inhibits lymphatic drainage. The efflux of cerebrospinal fluid (CSF) is achieved through the glymphatic pathway. When intracranial neurons or glial cells fall into energy metabolism disorder due to central axis imbalance—such as weak AMPK signaling causing energy metabolism decline, or weak PGC-1 signaling causing oxidative stress-induced mitochondrial damage and mitochondrial regeneration dilemma—the sodium-potassium pump suffers from insufficient energy supply. This leads to a situation where sodium ions reversely inhibit the glymphatic pathway for CSF efflux.

The glymphatic pathway for CSF efflux is the core mechanism for clearing misfolded proteins from the brain. Its inhibition can induce the onset of Alzheimer's disease.

Literature [283] points out that in AD patients, [Na] and Na/K ratio are elevated in CSF and brain tissue.

Na/K ratio is often used as an indicator of cardiovascular disease risk, including hypertension. Therefore, we assessed Na and K levels in the prefrontal cortex, thalamus, and CSF of Alzheimer's disease patients (n=67) and controls (n=30). [Na] in the prefrontal cortex was similar in AD patients and controls, while [K] was lower in AD patients compared to those without hypertension (HTN) (Fig 1A, B). Despite no significant difference in [Na], the Na/K ratio in the prefrontal cortex was significantly higher in AD patients regardless of HTN status. In thalamic tissue, [Na] was significantly higher in the hypertensive AD group compared to hypertensive controls, and generally higher in normotensive AD group, though this difference was not statistically significant. [K] was similar across all groups, resulting in a significantly higher Na/K ratio in the hypertensive AD group compared to hypertensive controls. In CSF, compared to controls, [Na] was significantly higher and [K] significantly lower in AD patients, irrespective of HTN status; thus, the Na/K ratio was significantly higher in AD patients compared to controls.

In the frontal cortex and thalamus, as well as within the CSF, [Na] and Na/K ratio were consistently and positively correlated with Braak stage. Neither frontal cortex nor thalamic [K] correlated with Braak stage, whereas [K] in CSF was negatively correlated with Braak stage. [Na], [K], and Na/K ratio at different Braak stages (0–VI) are displayed via bar graphs, showing a gradual increase in Na and Na/K ratio as Braak stage progresses from 0 to VI. Taken together, [Na] and Na/K ratio in CSF and the examined brain tissues are positively correlated with the severity of AD as determined by Braak stage.

This research finding proves that indeed, a phenomenon of insufficient sodium-potassium pump energy supply occurs in the brains of Alzheimer's disease patients. Simultaneously, this paper further demonstrates the positive correlation between Alzheimer's disease patients and sodium, providing empirical support for subsection 5.8.8.

 

Against the background of the fourth cause comprehensively elevating the body's oxidative stress baseline, PTEN is oxidatively activated, leading to sustained and frequent initiation of mTOR signaling, and the activation window for AMPK is significantly compressed. In this metabolic pattern, individuals with the central axis subtype characterized by decreased AMPK signaling and decreased PGC-1α signaling in the brain will develop Alzheimer's disease.

 

  Conclusion:   Although subsection 5.8 has not exhaustively covered the full spectrum of aging-related diseases, using the above representative pathological models of cardiovascular, metabolic, skeletal, and neoplastic diseases as examples, it has fully demonstrated the dialectical explanatory power of the metabolic flux homeostasis theory for both aging diversity (differential manifestation across multiple organs) and convergence (collapse of the oxidative stress baseline in the fourth cause as the common convergence node). Based on different individuals, different organs, and the innate differences in genetic gene expression superimposed with acquired homeostatic shaping, different central axis homeostatic preferences (specific combinations of AMPK/mTOR/PGC-1α/PPARα imbalances) are formed. Under the same metabolic background of the fourth cause, they manifest pathogenic pathways that are phenotypically diverse yet mechanistically isomorphic, confirming that aging is a continuous phase transition of metabolic homeostasis hierarchical collapse, rather than a simple superposition of discrete diseases.

 

  6 The Fifth Cause of Aging: Inflammatory Factors Hijack the Glucocorticoid Axis, Signal Desensitization Reduces Regulatory Effects, Amplifying Immunosuppressive Negative Effects.  

 

The increase in senescent cells driven by the fourth cause promotes the elevation of chronic inflammation levels. The fifth cause will be a stage dominated by inflammatory factors. This stage will expand the severity of damage through the SLAM family-IP3 pathway, synchronously affecting stem cell self-renewal, antioxidant functions, hormone secretion, and other pathways to promote the aging process.

 

  6.1 Collapse of Senescent Cell Clearance: Failure of the Glucocorticoid-Senescent Cell Apoptosis Axis  

 

Literature [114] points out that ketone bodies can inhibit HDAC. Literature [115] indicates that inhibiting HDAC enhances the transcription of the glucocorticoid receptor (GR). Literature [116] points out that in thymocytes and lymphocytes, glucocorticoids strongly induce apoptosis via the mitochondrial pathway and the death receptor pathway. Literature [117] indicates that cortisol can adjust the competitive balance between pro-survival and pro-apoptotic factors.

 

Therefore, it can be inferred that cortisol can initiate a reverse regulatory effect on mTOR when mTOR inhibits apoptosis. However, literature [118] points out that the p65 (RelA) component of NF-κB and the glucocorticoid receptor (GR) mutually inhibit each other's ability to activate transcription.

 

This constitutes a decreased sensitivity of senescent cells to the negative regulatory hormone for mTOR, thereby causing both cortisol's inhibition of inflammatory factor secretion by senescent cells and its inhibition of mTOR activation to decrease. Simultaneously, literature [119] points out that a high inflammatory factor environment also upregulates key anti-apoptotic proteins like c-FLIP and Bcl-2 through the TNFR2-TRAF2-NF-κB axis and the parallel PI3K-Akt axis. Therefore, it can be inferred that the increase in senescent cells caused by the fourth cause simultaneously inhibits the glucocorticoid-mediated mitochondrial apoptotic pathway and the TNFR1-mediated death receptor apoptotic pathway through inflammatory factor mediation, preventing senescent cell apoptosis from multiple pathways and exacerbating the rate of increase in senescent cell accumulation.

 

  6.2 The Inhibitory Effect of Elevated Glucocorticoid Levels on Immunity and Metabolism  

 

Literature [238] points out that glucocorticoids have the effect of inducing apoptosis in immature thymocytes and eosinophils. Literature [239] points out the suppressive effect of glucocorticoids on various immune cells.

 

Combined with the hijacking and inhibition of GR caused by the drastic increase in inflammatory factor concentration due to senescent cell accumulation mentioned earlier in Section 6.1, it can be inferred that the body will further increase glucocorticoid concentration due to chronic inflammation, forming inhibition on immune cells and further promoting immune cell exhaustion.

 

  6.3 Glucocorticoid Axis Hijacked by Inflammatory Factors, Sustained Activation of the SLAMF Family-IP3 Pathway by Inflammatory Factors, Triggering Calcium Oscillation Rhythm Collapse.  

 

The SLAM family can be induced and upregulated in various cells by inflammatory factors via the NF-κB pathway. Literature [28] points out that inflammatory factors can increase SLAMF7 expression via the NF-κB pathway. Literature [29] indicates a mechanism of ectopic expression for SLAMF1. Literature [240] points out that the SLAMF family is expressed in multiple immune cells and responds to induction by inflammatory factors TNF-α/IL-1β through the NF-κB pathway. Literature [241] indicates that SLAMF3 is expressed in hepatocytes. Literature [13] points out that SLAMF1 is expressed in hematopoietic stem cells (HSCs), and that removing HSCs with high SLAMF1 can reverse the aging phenotype. Literature [30] points out that SLAMF1 recruits PLCγ via ITSM, and PLCγ requires its PH domain to bind PIP3 for membrane targeting to achieve calcium release. Literature [31] points out that calcium oscillations in mesenchymal stem cells (MSCs) control cell cycle progression, determining whether cells proceed to proliferation or differentiation. Literature [242] points out the relationship between calcium release and mitochondrial flashes. Literature [243], a review, provides a more systematic description of the phenomenon from IP3 to mitochondrial flashes. Literature [244] points out that mitochondrial flashes can reshape the epigenome. Literature [245] points out the relationship between epigenetic methylation and the release mechanism of endogenous retroviruses.

 

Therefore, it can be inferred that under the sustained backdrop of chronic inflammation, chronic inflammation induces increased expression of a single SLAMF family gene in multiple cells, achieving sustained calcium release signals through the IP3 pathway. This forms an erroneous calcium metabolic homeostasis, causing stem cells to be more inclined towards differentiation rather than self-renewal, leading to stem cell aging. Simultaneously, due to excessive calcium release, the efficiency of refilling the depleted calcium stores decreases, leading to a decrease in the frequency of mitochondrial flashes. Since mitochondrial flashes can reshape the epigenome, this pathway also leads to epigenetic aging of stem cells and conventional somatic cells.

 

The decline in epigenetic homeostasis triggers the release of endogenous retroviruses (ERVs) previously suppressed by the epigenome. This will lead to ERVs triggering inflammation, and inflammation will again upregulate the expression of the SLAMF family. This forms an erroneous cycle, possessing self-reinforcing and self-maintaining metabolic homeostasis.

 

Concurrently, the depolarization pathway and the SLAMF family pathway are convergent. Depolarization inputs calcium into the cytoplasm, and the SLAMF family-IP3 pathway similarly inputs calcium into the cytoplasm. The core of both is inputting calcium into the cytoplasm. To avoid calcium-induced cell death, cells pump calcium out via the sodium-calcium exchanger. Therefore, the depolarization aging pathway cannot form a negative regulatory effect on the erroneous calcium release.

 

  7 The Sixth Cause of Aging: Senescent Cell Accumulation and Reverse Reinforcement of Damage Pathways.  

 

Senescent cell accumulation is driven jointly by the preceding five causes. The iNKT immune cell clearance pathway fails due to declining NAD+ levels. NK and T cell clearance fails due to decreased ketone body proportion, increased lactate proportion, and upregulation of immune checkpoints. The sustained elevation of mTOR due to PTEN oxidation causes the self-apoptosis pathway of senescent cells to fail. Senescent cells themselves secrete inflammatory factors, which can, in turn, reinforce the previously collapsed homeostatic environment by inhibiting mitochondrial functionality and NAMPT-mediated NAD+ regeneration, and through the SLAMF family pathway. This forms a vicious cycle, making it the ultimate driver of aging.

 

Simultaneously, through the sustained release of inflammatory factors, senescent cells can be viewed as the most fundamental anchor of the aging metabolic homeostasis. As long as the number of senescent cells or their secreted SASP cannot be effectively reduced, the human body should not possess the possibility of reverse homeostatic restoration.

 

  8.1 Gender Differences and Hormonal Associations.  

 

Literature [261] points out that both estrogen receptors ERα and ERβ can promote growth hormone secretion. Literature [43] indicates the correlation between obese patients and decreased growth hormone. Literature [42] points out gender differences in fat distribution: male fat tends to accumulate in the abdomen, while female fat tends to accumulate in the legs and subcutaneously. Literature [44] indicates the correlation between fat and inflammatory factor secretion. Literature [45] points out the effect of adiponectin on skin function. Literature [47] points out the phenomenon of cortisol promoting lipolysis. Literature [48] points out that cytokines promote lipolysis in 3T3-L1 adipocytes by inducing NADPH oxidase 3 expression and superoxide production. Literature [46] points out that breast development requires ERα signaling. Literature [262] points out the phenomenon of fat converting into estrogen, and how estrogen exerts its biological functions by interacting with its receptors (ER). These receptors can be either nuclear or membrane-bound. The main nuclear forms are two: α and β, which differ in tissue expression and function. Although ERα has a stronger physiological role in females, ERβ activity is similar in males and females. Upon ligand binding, ER undergoes a conformational change, allowing heterodimer formation and interaction with the estrogen response element (ERE) in target gene promoters. Literature [263] points out the phenomenon of male pubertal gynecomastia, affecting up to 50% of adolescents of the appropriate age. Literature [264] indicates that ER receptors have been shown to directly interact with Peroxisome Proliferator-Activated Receptor gamma (PPARγ), the main driver of lipid storage and adipogenesis, thereby blocking its transcriptional activity. Literature [265] points out the effects of pulsatile versus continuous estradiol treatment on two key aspects of estradiol action: gene expression and cell proliferation. Cells were stimulated with estradiol in 1-hour pulses or continuously for 24 hours, with the concentration × time exposure being identical for both conditions. In MCF7 cells, the transcriptional activity of a transiently transfected responsive estrogen response element luciferase reporter construct by the estrogen receptor (ER) was significant (approximately 10-fold). Literature [266] points out that E2 induces short-term translation of ERβ mRNA (2 and 4 hours post-stimulation) followed by a late (24 hours post-stimulation) enhancement of transcription. Both processes require E2-induced sustained and palmitoylation-dependent p38/MAPK activation.

 

Based on the above literature, the following inference can be made: during human puberty, adolescents, due to abundant subcutaneous fat, have a higher local conversion efficiency of estrogen. This conversion efficiency can maintain a local high signal without globally stimulating the negative feedback of ERβ signaling. Estrogen maintains the adolescent's skin condition through ERα signaling. Pubertal gynecomastia can self-resolve and disappear, indicating that ERα signaling declines in the later stages of development. This pathway may be due to inflammatory factors driving lipolysis, leading to the loss of subcutaneous fat, resulting in a decline in the local conversion rate of androgens to estrogen.

 

Androgens induce fat to accumulate in the abdomen, again affecting the efficiency of subcutaneous fat in converting androgens to estrogen and secreting adiponectin. Abdominal fat accumulation further increases inflammatory factor levels, which in turn promotes glucocorticoid secretion. Glucocorticoids also have the function of promoting lipolysis and abdominal accumulation, collectively forming the erroneous metabolic state of stable abdominal obesity. Simultaneously, the estrogen secreted by abdominal obesity itself is non-pulsatile. This pathway, due to its duration, reinforces ERβ signaling, forming an inhibition of ERα signaling by ERβ signaling.

 

The decline in local estrogen conversion rate may be the first node where metabolic homeostasis collapse occurs earlier in males. This homeostatic collapse is the decline of beneficial subcutaneous estrogen conversion. Due to physiological structural differences between males and females, ERα signaling is easily disrupted by abnormalities in one's own fat homeostasis, forming an erroneous hormonal secretion state of decreased ERα signaling and increased ERβ signaling.

 

As abdominal fat continues to accumulate, it will suppress growth hormone. Compared to estrogen's effect in females of promoting growth hormone secretion, this pathway exhibits a clear difference in the timing of homeostatic collapse. The second cause in females, because estrogen promotes growth hormone secretion, is weaker in its malignancy compared to males of the same age. During the progression from the third cause to the fourth cause, estrogen maintains higher growth hormone levels than in males. Even in the early fourth cause, estrogen can still maintain the promotion of growth hormone. These factors collectively become key reasons for the difference in average lifespan between males and females.

 

  9 Self-Locking Mechanism of Aging Pathways: Interlocking Cascade Amplification  

 

The core of aging is the collapse of metabolic homeostasis, which will form an erroneous homeostatic maintenance. This is the core self-locking mechanism of this paper. Erroneous homeostatic maintenance—the self-locking mechanism—is the main factor behind the current irreversibility of aging. Single-pathway drug regulation cannot break through the homeostatic self-locking.

 

The first self-locking mechanism originates from the central axis imbalance. The central axis itself possesses self-maintenance mechanisms that can persistently maintain an erroneous homeostasis, biasing it towards the aging pathway it maintains. Through non-homeostatic damage convergent pathways like inflammatory factors, decreased erythrocyte oxygen release-induced hypoxia, and multi-causal oxidative stress, it dominates the maintenance of the erroneous metabolic state of mitochondrial functional decline. The decline in CoQ10 levels (second cause) further impairs mitochondrial functionality, leading to a situation where the central axis can be restored, but mitochondrial energy metabolism remains decreased. This means that restoring the metabolic homeostasis of the central axis cannot effectively reverse aging.

 

The second self-locking mechanism comes from the decline in T3-carnitine synthesis triggered by decreased growth hormone levels. This pathway fundamentally causes the metabolic error of glycolipid metabolic disorder. Even by restoring the central axis and supplementing CoQ10, normal energy metabolism cannot be restored. Glycolipid metabolic disorder is persistently maintained by the decline in carnitine levels.

 

The third self-locking mechanism is the inhibition of NRF1 expression formed by the increased frequency of NRF2 expression due to oxidative stress, excessively binding the ARE element. This pathway dictates that even if the central axis is restored, CoQ10 is supplemented, and drugs promote NRF1 expression, as long as the state of frequent NRF2 expression is maintained due to oxidative stress, NRF1 expression will decline, mitochondrial quantity will synchronously decrease, and the pituitary gland will still be limited by the problem of insufficient ATP within the same time window.

 

The fourth self-locking mechanism is the accumulation of senescent cells secreting SASP. The SLAMF family-driven decline in mitochondrial flashes affects epigenetic homeostasis and stem cell self-renewal. The disruption of epigenetic homeostasis triggers the expression of endogenous retroviruses, causing inflammation, which in turn promotes SLAMF family expression. Simultaneously, inflammation can also promote mitochondrial functional decline. This means that even if the central axis is restored, CoQ10 is supplemented, NRF1 expression is enhanced by drugs, and ketone body levels are increased through fasting to improve glycolipid metabolic disorder, as long as senescent cells are not cleared and the inflammation triggered by the first three causes does not cease, this pathway cannot be reversed or corrected.

 

The emergence of self-locking mechanisms describes the key nodes and obstacles in the process of aging reversal through changes in metabolic homeostasis, thereby enabling a clearer understanding of aging, decoding aging, and reversing aging.

 

  Evolution Model of Dominant Aging Factors, i.e., the Aging Chronological Model.  

 

  Latent Phase (Adolescence):   The "first cause" of aging has already been initiated. Its early manifestation may be the dysregulation of the "four axes," causing mitochondrial functional decline through one of the eighty non-homeostatic pathways, for example, triggering occult tissue hypoxia, thereby decreasing mitochondrial ATP output.

 

However, this stage is strongly compensated by extremely vigorous growth hormone secretion. The exceptionally high capacity for mitochondrial biogenesis and quality maintenance masks the decline in mitochondrial functionality, temporarily preventing the system from collapsing downstream. This represents the earliest form of aging: sub-health.

 

  Young Adulthood (Approx. 18-25 years):   The plateau period of growth hormone ends and it begins to decline. Glycolipid metabolic disorder triggered by sub-health starts to emerge. This stage is primarily driven by aging caused by central axis imbalance sub-health, i.e., the first cause, mitochondrial functional decline. This stage continues until around the age of 25, after which it transitions into aging driven by glycolipid metabolic disorder. The previous driver of sub-health aging, mitochondrial functional decline, becomes a background aging driver and is no longer the primary aging driver.

 

  Prime Adulthood (Approx. 25-34 years):   This stage is the primary phase where glycolipid metabolic network disorder drives aging. The key aging characteristics of this stage include the emergence of antioxidant function decline caused by decreased lipid metabolism network, an increase in the duration of oxidized VE state, and the first acceleration of senescent cell accumulation. Simultaneously, growth hormone undergoes a slow decline, but it has not yet become a primary aging driver. NRF1 expression levels decrease, and CoQ10 levels decline. This aging driver reaches its limit around age 34, subsequently falling into the next imbalance: aging driven by declining growth hormone levels.

 

  Middle Age (Approx. 34-45 years):   In this stage, the decline in growth hormone becomes the primary aging factor. The key transition node occurs when the growth hormone pulse fails to reach the functional threshold required for growth hormone receptor conformational change. The resulting decline in NRF1 expression becomes the main factor driving the self-maintenance of the growth hormone decline. NRF2 expression levels will begin to increase, but the antioxidant network has not yet collapsed. The main features are a natural decline in muscle mass. Inflammation levels have not significantly increased due to glucocorticoid inhibition, but the efficiency of immune cells in clearing senescent cells decreases due to glucocorticoid-mediated immunosuppression. Senescent cell accumulation accelerates again, forming a vicious cycle where senescent cells secrete inflammatory factors, and inflammatory factors drive glucocorticoid secretion.

 

  Middle Age (Approx. 45-60 years):   Sustained metabolic pressure causes NRF2's compensatory antioxidant mechanism to reach its limit. The collapse of the antioxidant network becomes apparent, with a typical marker being an increase in the proportion of oxidized Vitamin E, which reversely inhibits CoQ10 synthesis, further decreasing growth hormone levels. Simultaneously, the antioxidant network, like a snapped spring, forms a state where chronic oxidative stress inhibits the recovery of antioxidant function. By oxidizing PTEN and enhancing mTOR activity, the rate of senescent cell accumulation suddenly increases, differing from previous rates.

 

Against the backdrop of GH-IGF-1 axis decline, the rapidly increasing senescent cells and the soaring inflammatory factors due to their concentration jointly drive down mitochondrial ATP output. This decline will trigger a cliff-like drop in gonadal hormone secretion levels. This drop will cause a second decrease in energy metabolism, i.e., another decline in mitochondrial ATP output. These two steps of decline in baseline mitochondrial ATP output will cause the calcium pump's energy demands to go unmet. Mitochondrial flashes depend on the calcium pump to transport calcium ions into the endoplasmic reticulum. This dual decline in baseline mitochondrial ATP output will lead to decreased efficiency in refilling calcium stores. The SLAMF pathway will become a secondary factor in aging at this point. The mechanism by which stem cells compensate for diversity through quantity will, under the influence of the SLAMF-IP3 pathway, gradually move towards a collapse in numbers, causing a drastic decline in tissue repair capacity.

 

  Old Age (Approx. 60-70 years):   The inflammatory-glucocorticoid axis (fifth cause) is continuously hijacked by inflammatory factors. The stem cell pool continues to be exhausted as the SLAMF family pathway affects calcium homeostasis, preventing self-renewal. The system falls into a vicious cycle of "inflammatory factors-inflammatory factors." The rate of senescent cell accumulation continues to increase until the stage where senescent cells become the primary driver of aging, driving inflammation, which in turn drives more inflammation.

 

  Senescence Phase (Approx. 70-105 years):   Aging in this stage is primarily driven by senescent cells. Senescent cells are the main driver at this stage, capable of influencing the other five causes in reverse by secreting inflammatory factors, thus driving aging.

 

  Terminal Phase (Approx. 105+ years):   The aging process halts. At this age, the increase in mortality risk due to aging ceases. The core mechanism behind the cessation of this increasing mortality risk is that all positive maintenance pathways in the human body—including antioxidant, anti-inflammatory, and energy generation functions—have collapsed to an extreme point. The body can no longer collapse towards a further imbalanced homeostatic state, analogous to the true vacuum state in vacuum decay.

 

  11 Discussion  

 

This hypothesis, through my process of tracing back from initial observations of high mTOR causing senescent cell accumulation, step-by-step back to growth hormone, the necessity of peak ATP levels within the same time window for the extremely short secretion window of growth hormone, and then reaching the construction of the higher-level central axis theory, ultimately resulted in the current version of the theory.

 

  11.1 The current mainstream understanding is that AMPK is good, but the theory states that excessive AMPK expression is bad. Why?  

 

In ketogenic diet research, one study result showed that the ketogenic diet promotes senescent cell generation. If AMPK is good, why would ketone bodies, whose primary function is to promote AMPK, produce a contrary result? The answer mainly lies in the difference between the oxidized and reduced states of VE. If a cell maintains a high proportion of reduced VE, then BAX will be inhibited by reduced VE. The AMPK-P53 pathway driven by ketone bodies cannot effectively drive BAX expression, preventing the cell from effectively entering the apoptosis program and keeping it more focused within the repair window. However, if the proportion of oxidized VE increases, cell membrane fluidity decreases. Promoting the AMPK pathway then easily enables the P53-BAX pathway downstream to take effect, initiating the cell into the apoptosis program. But because oxidized VE leads to decreased cell membrane fluidity and a stiffer membrane, the pore formation driven by this apoptosis program cannot effectively kill the cell. This creates a state where promoting AMPK is paradoxically harmful and pro-aging.

 

  11.2 Why should we first verify the hypoxic non-steady state?  

 

The hypoxic non-steady state is easier to verify in reality. Metabolic homeostasis imbalance driving mitochondrial functional decline is the key convergence node of the theory. If it cannot be verified, the whole theory's driving factors would be left hanging. The difficulty in verifying the central axis lies in the multitude of non-steady states, but changes in their strength combinations ultimately converge downwards onto eight main factors following roughly convergent paths. Therefore, microcirculatory disturbance caused by structural ischemia and hypoxia has a universal tendency in reality. However, this verification cannot be done too late; it must be conducted on young people. This is because middle-aged and elderly individuals may, due to increased levels of angiogenic factors, have microcirculatory disturbance become a permanent steady state unrelated to central axis metabolic imbalance.

 

  11.3 If intervention were to be undertaken, how should it be carried out according to the theoretical pathway?  

 

Ferulic acid: This drug can directly bypass the microcirculatory disturbance caused by central axis imbalance and has a strong improving effect on the entire branch pathway of the metabolic switch, i.e., improving the stem cell aging pathway caused by sustained hypoxia. Oxygen inhalation promotes nitric oxide synthesis, promotes the NAD+/NADH cycle, promotes the FAD/FADH2 cycle, clears lactate to break the self-locking pathway formed by lactate.

 

Simultaneously, using NRF1 activators like PQQ to restore mitochondrial biogenesis, combined with spermidine to restore mitophagy, and Coenzyme Q10 to strengthen mitochondrial energy metabolism, enhances mitochondrial energy output, reduces the proportion of glycolytic metabolism in normal cells, suppresses blood lactate levels, and, together with ferulic acid, shuts down the impact of the metabolic switch's downstream pathways on aging. This restores the maximum efficiency of erythrocyte oxygen transport within the same time window.

 

Regarding anti-inflammatory pathways, ginseng extract can be used to take the glucocorticoid anti-inflammatory pathway. However, because this anti-inflammatory pathway is activated by ginseng extract, it does not affect the immune system. When inflammation levels decrease, glucocorticoid levels subside, the conversion ratio of T4 to T3 increases, and the ratio of conversion to rT3 decreases, facilitating the recovery of the T3 anti-inflammatory axis. Omega-3 fatty acids can functionally mimic the T3 anti-inflammatory mechanism. At this step, VE must be supplemented; otherwise, it can trigger an increase in cell membrane lipid peroxidation, as mentioned in related literature [284].

 

VD supplementation can mimic the anti-inflammatory mechanism of T3.

 

Growth hormone relies on signaling and energy metabolism within the same time window. Based on the intervention strategy of the preceding drugs, by this point, energy metabolism has been restored, inflammation levels have decreased, and increased NRF1 expression improves the body's antioxidant function. The metabolic background now supports the recovery of growth hormone pulsatility. At this time, high-intensity intermittent exercise should be used to increase daytime growth hormone secretion. At night, a combination of lysine and arginine promotes growth hormone secretion.

 

This pathway cannot use direct injection of growth hormone as a replacement means; it must center on restoring the body's normal biological rhythm. Exogenous growth hormone injection can disrupt the sensitivity of human growth hormone receptors.

 

  Recovery of the antioxidant network and clearance of senescent cells:  

 

Supplement glycine and cysteine, combined with VC shock to replace oxidized VC in the body, to complete the rebuilding of the antioxidant network. Simultaneously, the combination of cysteine and glycine can clear senescent cells. For NAD+ supplementation, niacin can be used. In the metabolic background where inflammation levels have decreased, NAMPT is no longer inhibited by inflammatory factors, negating the need for more expensive NR and NMN. Concurrently, the increase in NAD+ levels will promote the activation of iNKT immune cells, increasing their probability of clearing senescent cells.

 

Fasting: This is a mandatory component of the theory. The ketone bodies it generates can, together with ginseng extract, strengthen the sodium-potassium pump. AKG can promote epigenetic demethylation. The enhancement of fat metabolism promotes the TCA cycle, increasing acetyl-CoA output, achieving the reversal of epigenetic drift. Simultaneously, it can enhance autophagy, compensating for the missing T3 autophagy signal, relieving the stress response of lysosomes, and improving the aging pathway of stem cell lysosomes. By lowering insulin levels, it inhibits the stem cell aging pathway of arachidonic acid. Ketone bodies, by inhibiting P53 in senescent cells, can inhibit the expression of immune checkpoints on senescent cells, increasing the probability of their clearance by NK and T cells.

 

Adjusting central axis metabolic homeostasis imbalance is difficult. Restoring it through cold stimulation is one path, but the elderly cannot easily bear its risks. Fasting can reverse the problem of decreased fat metabolism proportion. Combined with ferulic acid, this effectively bypasses the difficulty of restoring the first cause.

 

  11.4 Why are there relatively many drugs that functionally mimic the T3 anti-inflammatory pathway?  

 

The abundance of T3-mimicking drugs is mainly due to VD being limited by its own metabolic flux. Although its anti-inflammatory pathway most closely matches T3's mechanism, its hormonal nature creates toxicity when its flux is increased. Therefore, to replicate T3's anti-inflammatory mechanism, one must approach it from the perspective of multi-pathway, weak modulation, avoiding single-pathway toxicity.

 

When the body's T3 levels recover and achieve a positive cycle homeostasis with growth hormone itself, most drugs that mimic the T3 anti-inflammatory mechanism and functionality can be withdrawn. This drug modulation pathway is not intended to have drugs completely simulate the body's rhythm—nor can drugs perfectly simulate the body's rhythm—but to provide support for the recovery of the body's rhythm, forming a reverse cascade restoration of metabolic homeostasis.

 

  11.5 Senescent Cell Clearance Plan.  

 

Through pretreatment to enhance the body's antioxidant performance, use glycine + cysteine to reverse the phenomenon of accelerated senescent cell accumulation realized through the PTEN-mTOR pathway. Simultaneously supplement VC + VE + algal oil to restore cell membrane fluidity, creating the necessary conditions suitable for apoptosis. This process requires 4-8 weeks. Finally, promote the AMPK pathway through fasting, combined with AMPK activators, to further elevate the level of the AMPK pathway. Then use protocatechuic acid to lower anti-apoptotic proteins, supplement NAD+ boosters to increase NAD+ levels, allowing senescent cells to absorb NAD+ and form a high NAD+ state. This induces senescent cells to metabolize ketone bodies for energy, and through the promotion of the AMPK-P53 pathway, re-initiates the apoptotic program in senescent cells.

 

This pathway holds the promise of completing senescent cell apoptosis by restarting their intrinsic apoptosis program.

 

  11.6 Senescent Cell Clearance Plan via the Immune Pathway.  

 

Immune clearance never stops during aging; it merely becomes less efficient. Therefore, enhancing immunity through drugs holds the potential to clear senescent cells. This pathway can be achieved by supplementing NAD+ boosters, B6, AMPK activators, VC, combined with fasting to obtain ketone bodies. Ketone bodies, through modification pathways that inhibit P53 and lower immune checkpoints, can achieve enhanced immune clearance of senescent cells.

 

  11.7 Are the self-locking mechanisms in the theory all of them?  

 

They are not all the self-locking mechanisms. They are only a few key self-locking mechanisms deduced rationally based on the current level of knowledge of the author. Potential self-locking mechanisms have not been written into it. For example, genomic instability - increased P53 - increased immune checkpoints, forming an effect where senescent cells have higher immune checkpoints during apoptosis resistance, creating an effect of resisting immune clearance. However, this pathway can be breached by iNKT cells, so it was not mentioned among the four major self-locking mechanisms. This implies that the human body may have more potential self-locking mechanisms, restricting the body from breaking through the aging homeostasis and restoring youthful homeostasis through multiple pathways.

 

  11.8 Academic Statement on the Compatibility of this Theory with Genomics  

 

This hypothesis is fully compatible with genomics: it posits that aging rate depends on the dynamic interaction between genetic set points and metabolic flux homeostasis. Specifically, the coding genes of the four central axis pathways (PPARα, AMPK, PGC-1α, mTOR) and their regulatory region polymorphisms innately determine the activation threshold and self-maintenance gain of each pathway in an individual, forming a unique "homeostatic preference." Genetic backgrounds such as G6PD activity and mitochondrial DNA haplogroups modulate NADPH regeneration efficiency and oxidative stress tolerance thresholds. Extreme monogenic genetic diseases (such as Werner syndrome) are not counter-examples beyond the theory's scope but represent "fast-track passage"—where dysfunction of DNA repair genes causes the six-factor cascade to complete in a few years the phase transitions that normally take decades in aging. This is merely a phenomenon of mechanism isomorphism with compressed timing. Therefore, from physiological aging to pathological premature aging constitutes a continuous phase transition spectrum, to which this theory applies for the full spectrum of explanations.

 

  11.9 Clarification on the Theory's Applicability Limited to Humans, and the Need for Targeted Metabolic Flux Modifications for Rodent Studies  

 

When conducting rodent studies, targeted modifications for rodent-specific metabolic flux are needed. For example, humans cannot self-synthesize VC, but rodents can. Inhibiting growth hormone in rodents can extend lifespan. However, inhibiting growth hormone in humans leads to premature aging phenomena, such as the skin premature aging observed in Laron syndrome patients. This is because IGF-1 signaling is responsible for promoting collagen production by fibroblasts. Related literature [285] empirically demonstrates the skin premature aging phenomenon in Laron syndrome patients. Due to insufficient muscle development, milestones for walking and other gross motor skills are delayed. Hands and feet are small (acromicria). Hip dysplasia, especially avascular necrosis of the femoral head, is reported in up to 25% of patients. The skin is thin and finely textured, with premature aging wrinkles.

 

It is speculated that this is because rodents need to meet the demands of excessively rapid development, leading to excessively high growth hormone levels. When development reaches a threshold, it will directly drive phenomena akin to the fourth cause in humans. Growth hormone drives up mTOR levels, inhibiting AMPK, initiating an aging phenomenon starting from the fourth cause. Simply put, if we reorganize its aging metabolic flux, the process should be: a phenomenon akin to the fourth cause is initiated by growth hormone, the rate of senescent cell accumulation increases, inflammatory factors form self-perpetuating cycles affecting cellular calcium homeostasis, calcium homeostasis triggers epigenetic disorder, epigenetic disorder inhibits NRF1 and VC synthesis, leading to antioxidant network collapse. The entire initiation process can be said to be largely opposite to that in humans. However, this initiation process can explain why different species have different aging efficiencies.

 

If humans have multi-level networks that complement each other and exhibit homeostatic self-locking, rodents have a single aging pathway without an effective braking mechanism, plunging straight towards death. This also explains why many anti-aging therapies show good results in mice but exhibit reduced efficacy when translated to humans—because the metabolic backgrounds of aging they face are completely different.

 

  11.10 Clarification on the Non-Applicability of Synchronous Flowering in Moso Bamboo Clones to Humans  

 

Literature [286] points out that cloned moso bamboo asexual lines flower and die synchronously across geographies (60-120 year cycles). However, the scope of this theory cannot be fully replicated in humans. Literature [287] empirically demonstrates that the degree of difference in DNA methylation within monozygotic twin pairs is equivalent to the degree of difference between randomly paired unrelated individuals, indicating that even with identical genetic backgrounds, individual epigenetic clocks will move towards asynchrony.

 

Therefore, it can be demonstrated that the physiological mechanism of synchronous flowering and death across geographies as seen in moso bamboo asexual lines does not exist in humans.

 

  Research Limitations and Future Outlook  

 

The limitation of the current theory lies in the lack of decisive evidence to prove central axis imbalance. The hypoxic-ischemic model is a key phase for verifying aging homeostatic imbalance. However, because microcirculatory disturbance, according to the theory, becomes fixed with age, the subjects for inflammation measurement must be under 18 or just turned 18 years old to ensure measurement accuracy.

 

  Measurement pathway:   The benchmark is to measure whether erythrocyte deformability in the blood has decreased. If decreased erythrocyte deformability is observed, then central axis imbalance is an early factor in aging, and the central axis theory is confirmed. Otherwise, if no decrease in erythrocyte deformability is observed, central axis imbalance is not an early factor, i.e., the central axis theory is falsified.

 

Secondly, measure whether adolescents with microcirculatory disturbance exhibit increased senescent cells in the areas of microcirculatory disturbance and whether the level of inflammatory factors released due to their own hypoxia can, in the 18-25 age range, at the end of the growth hormone compensation stage, trigger mitochondrial functional decline through inflammatory factor levels. If mitochondrial functional decline occurs, it is confirmed; otherwise, the theory that mitochondrial functional decline is the key convergence node for multiple aging factors is falsified.

 

Simultaneously, this theory inevitably incorporates numerous mouse-related gene studies, which will lead to instability in the foundations of the current theory. This is an unavoidable predicament due to the limitations of current research.

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