Complete deciphering of the aging pathway: hypothesis of mitochondrial dysfunction as the primary cause of continuous decline in bodily functions leading to aging
1. Introduction: Defining the primary cause of aging: mitochondrial dysfunction
1.1 Limitations of existing aging theories
In the field of life sciences, the mechanisms of aging have always been the core and challenging aspect of research.
Although the mainstream scientific community has proposed various theories of aging, including telomere shortening, genomic instability, epigenetic alterations, protein homeostasis imbalance, nutrient sensing dysfunction, cellular senescence, stem cell exhaustion, and chronic inflammation, most of these theories focus on specific phenomena or downstream pathways in the aging process, failing to fundamentally define the "primary cause" that triggers this series of cascading reactions. [1]
These theories are akin to describing the different branches of a large tree, yet they fail to point out its common root system. For instance, the classical mitochondrial aging theory posits that the accumulation of mutations in mitochondrial DNA (mtDNA) is the primary cause of mitochondrial dysfunction and cellular energy crisis, thereby driving aging.
However, this theory is facing increasing challenges. Some studies have pointed out that the decline in mitochondrial respiratory function observed during aging may not be primarily due to the accumulation of mutations in mtDNA, but rather to epigenetic changes in the expression of mitochondrial-related genes encoded by nuclear genes. This discovery shakes the foundation of the traditional mitochondrial aging theory, suggesting that deeper regulatory mechanisms are at play.
Therefore, a core challenge in the current field of aging research lies in how to transcend these isolated and descriptive theoretical frameworks and construct a comprehensive model that integrates multiple markers of aging and elucidates their intrinsic causal relationships. This article aims to propose a novel perspective, defining mitochondrial dysfunction as the "primary cause" of aging, and on this basis, systematically analyzes how it ultimately leads to comprehensive systemic decline through a series of cascading reactions.
1.2 Core argument: Mitochondrial dysfunction is the intrinsic starting point of aging
The core argument of this article is that mitochondrial dysfunction is a fundamental and intrinsic factor driving the entire aging process. This argument does not negate the importance of other aging mechanisms, but rather repositions them. We believe that mitochondria are not merely a passive "victim" damaged during the aging process, but rather an active "culprit." The decline in mitochondrial function, as the starting point of the aging program, initiates and amplifies a series of subsequent aging-related pathways through its central role as the cell's energy factory and signaling hub. This viewpoint has been strongly supported by recent research. For example, a study published in Scientific Reports found that age-related mitochondrial respiratory defects could be completely reversed by reprogramming fibroblasts from elderly individuals into induced pluripotent stem cells (iPSCs), restoring them to levels comparable to those of fetal fibroblasts.
This astonishing result indicates that the aging state of mitochondria is not determined by irreversible mtDNA mutations, but rather is subject to dynamic and reversible epigenetic regulation. This provides crucial evidence for our hypothesis that "mitochondrial dysfunction is the primary cause": if the aging state of mitochondria can be "reset" by epigenetic reprogramming, then its functional decline must be an upstream and intervenable key node in the entire aging chain. Therefore, I regard mitochondrial dysfunction as a "primary endogenous factor" that creates the necessary pathophysiological conditions for the emergence and development of all subsequent aging characteristics by affecting cellular energy metabolism, redox balance, and signal transduction.
This model defines mitochondrial dysfunction as the 'primary cause', rather than considering mtDNA mutations as a parallel or alternative driver of aging. Instead, it explicitly states that mtDNA mutations themselves are a downstream consequence of impaired mtDNA maintenance function mediated by NRF1. Therefore, functional decline not only precedes structural damage but also serves as a causal prerequisite for structural damage.
1.3 Definition and scope of mitochondrial dysfunction
To precisely elaborate on our theory, a rigorous definition of "mitochondrial dysfunction" is necessary. Within the framework of this article, mitochondrial dysfunction specifically refers to the decline in the ability of mitochondria to perform core physiological functions, despite maintaining normal numbers and macroscopic structure (quality).
This definition is crucial as it shifts our focus from the physical existence of mitochondria (quantity and morphology) to the dynamic efficiency of their biochemical functions. This decline in functionality manifests in multiple aspects, including but not limited to: reduced efficiency of oxidative phosphorylation (OXPHOS), leading to decreased ATP production; imbalance in the production and clearance of reactive oxygen species (ROS), triggering oxidative stress; impaired ability to participate in key metabolic pathways such as fatty acid β-oxidation, amino acid metabolism, and the tricarboxylic acid cycle (TCA cycle); and decreased regulatory ability of calcium ion homeostasis. [2]
These subtle declines in function, although not immediately leading to cell death, can disrupt the normal physiological rhythms of cells and initiate a series of adaptive or pathological signaling pathways.
For instance, a slight decrease in energy metabolism activates AMP-activated protein kinase (AMPK), while persistent oxidative stress activates transcription factors such as nuclear factor E2-related factor 2 (NRF2). If these initial compensatory responses persist for a long period or are dysregulated, they will shift from protective mechanisms to driving forces that promote aging, thereby closely linking the decline in mitochondrial function, the "primary cause," with subsequent aging phenotypes.
[Conceptual Analysis]
Epigenetic inertia: In this hypothesis, it specifically refers to key genes such as PPARα, AMPK, mTOR, and PGC-1α, whose expression levels are locked in a stable, low-level state that is difficult to recover spontaneously after being inhibited. This represents a specific functional dysregulation pattern in early aging.
Epigenetic disorder: refers to the systemic and global loss of control of epigenetic modifications (such as DNA methylation) across the entire genome in later stages of aging, due to reasons such as the scarcity of metabolic substrates (such as α-ketoglutarate, AKG).
2. The core regulatory network-axis model of mitochondrial dysfunction.
Based on theoretical deduction of existing pathway interaction rules, I constructed a central axis model.
The central axis model is a metabolic network composed 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 model integrates multiple aging mechanisms from traditional theories, forming a key central robust mechanism that determines whether an organism tends to adopt a specific aging mechanism at the early stages of aging. It explains the diversity of aging while providing a clearer understanding of the underlying principles behind this diversity.
The central axis model, PPARα, AMPK, mTOR, and PGC-1α, four key gene pathways, collectively control four crucial mechanisms for organisms: the tricarboxylic acid cycle, oxidative phosphorylation, biosynthetic metabolism, and antioxidant network. These four mechanisms are interconnected, simplifying the expression and activity states of each core factor (AMPK, mTOR, PGC-1α, PPARα) into three basic levels: too weak, steady state, and too strong. Thus, the total number of combinations formed by these four variables is theoretically 3 to the power of 4, with a total of 81 possible "functional state combinations".
Among these 81 combinations, there is only one that represents the perfect homeostasis enjoyed in early life, where all four core elements are within their precise "homeostatic" ranges. This state is the only ideal state where mitochondrial function is optimized and energy metabolism is clean and efficient, serving as the golden benchmark for delaying the aging process of the body.
The remaining 80 combinations all constitute potential pathological origins that trigger the primary cause of "mitochondrial dysfunction".
2.1 A normally functioning axial model.
Starting from the decline in biosynthesis metabolism controlled by the ATP-dependent Mtor pathway, which leads to changes in the AMP/ATP ratio and promotes the activation of AMPK [134], the expression of AMPK increases due to the AMP/ATP signal [134]. In this process, the PGC-1α pathway, which is promoted by high expression of the AMPK pathway, is activated [3][4]. The AMPK pathway only determines whether to activate the PGC-1α pathway, not the intensity of PGC-1α pathway expression. The expression of the PGC-1α pathway is determined by its self-sustaining mechanism formed by the metabolic pathway of the pathway [3][61]. High expression of AMPK changes the AMP/ATP ratio through increased ATP levels, triggering the expression of the MTOR pathway [135]. At this point, the PPARα pathway increases its expression based on the weak expression of AMPK, promoting the tricarboxylic acid cycle [5][59]. The tricarboxylic acid cycle not only provides energy support for the biosynthesis metabolism maintained by the Mtor pathway, but also supplies substrate support for biosynthesis metabolism [5][87], until the biosynthesis metabolism of the Mtor pathway leads to a decrease in ATP levels [14][15][134]. This initiates the next round of metabolic cycling in the central model.
2.2 Epigenetic inertia resulting from the malfunctioning of the self-maintenance mechanism in the central axis model.
AMPK, mTOR, PPARα, and PGC-1α all possess self-sustaining metabolic mechanisms, which determine the expression intensity of these four pathways.
2.2.1 Imbalance mechanism of the first cause: epigenetic gene inertia formed by competitive collapse of the homeostatic loop.This hypothesis posits that the essence of mitochondrial dysfunction lies in the loss of dynamic balance in the core regulatory circuits that maintain its functional homeostasis. These circuits rely on self-positive feedback from key molecules and interact with other circuits in a mutually antagonistic manner. Once external stress persists for too long, the positive feedback loop will be disrupted, and its expression level will be locked in a pathological range, thereby driving mitochondrial dysfunction along various pathways and forming a convergent model that drives aging.
2.2.2 Competition in energy metabolism hubs: Self-maintenance mechanism and rhythmic inhibition of PPARα and PGC-1α.
PGC-1α achieves a self-sustaining mechanism through the -NRF1-lipoic acid-PGC-1α pathway, while PPARα achieves a self-sustaining mechanism of the PPARα pathway through the co-regulation of various downstream metabolites of acetyl-CoA in the tricarboxylic acid cycle metabolic process.
PPARα and PGC-1α respectively dominate the tricarboxylic acid cycle and the antioxidant network, and they both cooperate and compete in the regulation of mitochondrial function [5][59]. The main mechanism involves rhythmic changes achieved through the expression level of AMPK. When AMPK is highly expressed, PGC-1α is expressed, whereas when AMPK is weakly expressed, it promotes PPARα to achieve rhythmic regulation. This rhythmic mechanism is primarily achieved through differential regulation by the competition between PPARα and PGC-1α for coactivators [1], [2][50][96][97]
The balance of energy sensing hub: mutual inhibition and self-maintenance of AMPK and mTOR
A similar "mutual inhibition-self-maintenance" mechanism also exists between the energy-sensing hub AMPK and mTOR [14][15].
Self-maintenance of AMPK: After activation, AMPK reverses the change in the intracellular AMP/ATP ratio by inhibiting mTORC1 activity and promoting self-maintenance. However, if the downstream pathway dominated by the AMPK pathway increases NAMPT expression to promote NAD+ regeneration, leading to a problem in the NAD+-SIRT1-AMPK pathway, the AMPK pathway will exhibit high or low expression, resulting in a disorder in expression intensity and inability to maintain rhythmic regulation of PPARα and PGC-1α. This creates an energy environment conducive to its own sustained activation, forming a positive feedback loop [14][56].
Self-maintenance of mTOR: After activation of mTORC1, a large amount of free radicals are generated within the cell through the promotion of the synthesis of macromolecules such as proteins and lipids. In the normal process, these free radicals will moderately oxidatively modify PTEN to eliminate its inhibition on the mTOR pathway, thereby further enhancing mTOR expression. However, NRF2 is induced to express by oxidative stress, which oxidizes and reduces PTEN. When there is excessive expression in the incorrect mTOR pathway, the oxidative stress induced by itself will continuously trigger the oxidative modification of PTEN. Meanwhile, the overexpression of NRF2 also induces oxidative stress, preventing the oxidation and reduction of PTEN, forming a self-sustaining high-maintenance state of mTOR.
The two components constitute a precise bistable switch. Under physiological conditions, they dynamically oscillate based on energy status, maintaining overall balance [14][15]. However, in the presence of sustained energy stress or nutrient excess, the self-maintenance mechanism of one component may be overly strengthened, completely suppressing the other. For instance, long-term nutrient excess consolidates the "high expression-high activity" steady state of mTOR and inhibits AMPK to a "low expression-low activity" state, leading to energy sensing dysfunction, autophagy inhibition, and directly contributing to mitochondrial quality decline [15][35][38].
2.3 The four gene pathways of AMPK, MTOR, PPARα, and PGC-1α exhibit either excessive or insufficient expression, collectively leading to a decline in mitochondrial functionality.
2.3.1 Mitochondrial dysfunction triggered by imbalance in the AMPK pathway
Overexpression of the AMPK pathway
regenerates NAD⁺ through NAMPT, and NAD⁺ maintains self-expression of its own genes through the SIRT1-AMPK pathway [8][56]. When this metabolic level becomes excessively high, even with normal expression of PGC-1α and VA maintaining a reduced state, the expression level of the AMPK pathway cannot decrease, ultimately leading to inhibition of the MTOR pathway by the AMPK pathway [14][15]. This results in a decline in biosynthetic metabolism, such as blockade of the arginine metabolic pathway [22], insufficient nitric oxide synthesis, decreased erythrocyte deformability [16][17], microvascular circulation disorders [18][19], and hypoxia in most cells in most organisms. Mitochondrial function declines [18][20]. Simultaneously, the decline in biosynthetic metabolism also leads to a decrease in erythrocyte biosynthesis, resulting in an ischemic state [17][21]. Inflammatory factors will emerge due to microcirculation disorders,
When the AMPK autoregulation mechanism is in an overly weakened state, although the AMPK pathway will not be completely inhibited, it will result in a decline in energy metabolism, with simultaneous reductions in the levels of ATP produced by oxidative phosphorylation and glycolysis [2][10], leading to a decrease in mitochondrial functionality [18][20]. Simultaneously, due to the decline in ATP levels, ATP production in red blood cells will also decrease, resulting in red blood cell deformation disorders [16][17] and microvascular circulation disorders [18][19][21].
2.3.2 Mitochondrial dysfunction triggered by imbalances in the mTOR pathway
Overexpression of mTOR
initiates the ROS free radical oxidative modification of the PTEN pathway through anabolic processes [38][39], promoting mTOR expression. Due to the continuous stimulation of ROS free radicals, NRF2 forms a high expression state [62][63][65], leading to the downstream pathway HO-1 decomposing heme to form free iron [69][70]. The free iron further promotes oxidative stress by generating hydroxyl radicals [65][66][67], forming a vicious cycle. This pathway will induce erythrocyte deformation disorders through oxidative stress [16][17], leading to microcirculatory disorders [18][19][21], and mitochondrial dysfunction through hypoxia [18][20]. At the same time, excessive ROS free radicals will inhibit the metabolic efficiency of mitochondria [68][69], and the mTOR pathway inhibits the AMPK pathway itself [14][15], jointly inhibiting the normal function of mitochondria.
The pathway with excessively weak expression of mTOR
will experience a decline in anabolism, leading to abnormalities in the arginine metabolism pathway and a decrease in nitric oxide levels [22], as well as a reduction in erythrocyte synthesis [17]. This results in functional decline of mitochondria due to hypoxia [18][20]. However, unlike the over-expression of AMPK, this pathway is a malfunction arising from the self-maintenance function of mTOR itself, and does not imply over-expression of the AMPK pathway [14][15].
2.3.3 Mitochondrial dysfunction induced by imbalances in the PGC-1α pathway
Overexpression of PGC-1α in this pathway
inhibits PPARα through competition for coactivators [59][60][96][97]. This inhibition leads to a decrease in the efficiency of the tricarboxylic acid cycle [5][87], causing cells to enter a state of reduced ATP production. This pathway can also inhibit erythrocyte energy metabolism, affecting erythrocyte deformability [16][17], leading to microcirculatory disorders [18][19][21]. This results in mitochondrial dysfunction due to hypoxia [18][20], and simultaneously affects AKG anabolism [6][7], leading to decreased cellular demethylation function and epigenetic gene disruption [6][7][106]. This step inhibits the demethylation of nitric oxide genes, forming epigenetic repression [22], further inhibiting erythrocyte deformability, exacerbating microcirculatory disorders and mitochondrial dysfunction [16][17][18][19].
The pathway of PGC-1α under-expression
will lead to a decrease in the expression of NRF1 [3][4][61], a reduction in mitochondrial mass and number [3][4][81], and a decline in the basic antioxidant capacity of the entire antioxidant network against oxidative stress [62][63][64][65]. However, this pathway can also cause mitochondrial dysfunction due to oxidative stress and electron leakage [68][69]. Simultaneously, it will interfere with the energy metabolism of red blood cells [16][17], increase the risk of oxidative stress in red blood cells [68][69], affect the deformability of red blood cells [16][17], and lead to mitochondrial dysfunction due to hypoxia [18][20][21].
2.3.4 Mitochondrial dysfunction induced by imbalance of PPARα pathway
Overexpression of PPARα
can trigger oxidative stress due to the pathway's excessive expression [5][65][87], while inhibiting the PGC-1α pathway through competitive coactivators can amplify the effects of oxidative stress [59][60][96][97]. This pathway can affect the deformability of red blood cells through oxidative stress [16][17], leading to microcirculatory disorders [18][19][21], and mitochondrial dysfunction due to hypoxia [18][20].
The pathway primarily affected by the weak expression of PPARα
is the tricarboxylic acid cycle, leading to a decrease in cellular ATP production [5][87], as well as affecting the production of AKG and ketone bodies [6][7][91]. The decrease in AKG will affect the overall demethylation function of cells [6][7][106], leading to epigenetic gene disorders. The nitric oxide gene undergoes demethylation and is suppressed [22], unable to be expressed, thereby reducing nitric oxide levels, which in turn leads to a decrease in erythrocyte deformability [16][17], resulting in microcirculatory disorders [18][19][21]. Mitochondrial dysfunction due to hypoxia leads to a decline in mitochondrial function [18][20].
Summary: Theoretically, a perfect central axis should generate more energy during the tricarboxylic acid cycle, promote mitochondrial regeneration at the PGC-1α stage, enhance cellular mitochondrial regeneration, and regenerate antioxidant functions. However, this paper infers that in reality, maintaining such a central axis in a normal and stable state is challenging. For instance, just one night of staying up late can disrupt the operation of this central axis, leading to imbalances in hormones, blood-oxygen microcirculation disorders, oxidative stress, and ultimately, central axis imbalance.
However, at the same time, the diet that people consume daily, such as vegetables containing ferulic acid and antioxidants, can correct this process, presenting a dynamic state of change throughout. It is like a pendulum swinging at all times, and if the right path can be found, the state of the central axis can be improved.
2.4 Correspondence between axial disproportion and current mainstream theories of aging and its rationale.
1. Imbalance of AMPK: Dysregulation of the energy-sensing hub
When AMPK is in a "too weak" state, it directly corresponds to the theory of "autophagy dysfunction and protein homeostasis imbalance". The reason is that AMPK is the primary kinase that enables cells to sense energy crisis (increased AMP/ATP ratio) and initiate autophagy to eliminate damaged components and recycle resources [14][15]. Insufficient activity of AMPK means that cells cannot activate this key "quality control and recycling system" under energy stress [35][37]. At the same time, AMPK's regulation of the balance between protein synthesis and degradation also fails [33][36], leading to the accumulation of misfolded proteins and senescent organelles within the cell, disrupting cellular homeostasis from within. This is the source of waste accumulation in senescent cells, directly corresponding to the theories of "autophagy dysfunction" and "protein homeostasis imbalance" [1].
When AMPK is in an "overactive" state, it points to the pathological logic of "anabolic failure and nutrient utilization disorder". The physiological role of AMPK is "throttling" and "decomposing". Persistently overactive AMPK signaling excessively inhibits its key target mTORC1 [14][15], which is the "source-opening" master switch driving the biosynthesis of proteins, lipids, etc. [38][39]. Therefore, the abnormal hyperactivity of AMPK is not beneficial, but rather creates a false and persistent "cellular famine" state, inappropriately shutting down the anabolic pathways necessary for cell growth, repair, and regeneration, resulting in a fundamental impairment of tissue renewal and repair capabilities - directly corresponding to the theories of "anabolic failure" and "nutrient sensing disorder" [1][92].
II. Imbalance of mTOR: Loss of Control over the Master Switch of Growth Signals
When mTOR is in an "overactive" state, it serves as a direct molecular interpretation of the theory of "nutrient sensing dysregulation and cellular senescence". mTORC1 is the signal hub most sensitive to nutrients and growth factors [14][15]. Its persistently high activity implies that cells mistakenly perceive themselves to be in a nutrient-rich environment, thereby uncontrollably driving anabolic processes [38][39], consuming a large amount of resources, and generating metabolic waste. More importantly, it strongly inhibits autophagy [14][15] and hinders the normal apoptosis program of damaged cells [40], ultimately pushing cells into a "senescent cell" state characterized by active metabolism but abnormal function and the secretion of a large amount of inflammatory factors [10][11][50], becoming the engine of tissue inflammation and dysfunction - directly corresponding to the theories of "nutrient sensing dysregulation" and "cellular senescence".
When mTOR is in a "too weak" state, it corresponds to "anabolic failure and tissue regeneration impairment". mTOR signaling serves as the fundamental command for all constructive activities in cells, including growth, proliferation, and protein synthesis [14][15]. The deficiency of its signaling means that cells lose the fundamental driving force for repair and renewal [4][81]. Consequently, whether it is the synthesis of muscle proteins, the healing of damaged tissues, or the proliferation of immune cells, all will stagnate, directly manifesting as age-related tissue atrophy, delayed wound healing, and decreased immune function - directly corresponding to the theory of "anabolic failure" [38][39].
III. Imbalance of PGC-1α: Disorder of instructions in 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 coactivator of mitochondrial biogenesis [3][4][61], regulating a series of mitochondrial production and antioxidant genes, including NRF1/2 [3][4]. Its low expression leads to the inability to build new "mitochondrial factories" and the inability to update old "equipment" [79][81], manifested as a decrease in mitochondrial number, quality, and respiratory efficiency. This is not only an energy crisis at the cellular level, but also the starting point of the collapse of the systemic antioxidant defense network [62][63][64][65] - directly corresponding to the "mitochondrial dysfunction" theory.
When PGC-1α is in an "overactive" state, it may trigger "metabolic rhythm disorder and redox stress/signal interference". Although research is limited, theoretically, the excessive and sustained activation of PGC-1α can disrupt the dynamic balance between mitochondrial biogenesis and clearance, oxidation and antioxidation [61]. It may force cells to consume a large amount of resources to build mitochondria when not necessary, and may excessively suppress normal reactive oxygen species (ROS) signaling, which is essential for many physiological signal transducers [38][39]. This imbalance disrupts cellular metabolic flexibility and normal signaling rhythms, correlating with theories of "metabolic disorder" and "signal integration disorder" [5][87][92].
IV. Imbalance of PPARα: Failure of speed regulation in the metabolic engine
When PPARα is in a "too weak" state, it directly drives the theory of "metabolic flexibility loss" (i.e., the molecular basis of systemic glucose and lipid metabolism disorders). PPARα is a core transcriptional regulator of fatty acid oxidation and the tricarboxylic acid cycle in tissues such as the liver and muscles [5][87][92]. Its weakened function leads to the inability to effectively "burn" fatty acids, an important fuel, forcing cells to rely more on glucose metabolism [87][92]. This metabolic pathway rigidity and switching failure are the upstream culprits of insulin resistance, ectopic lipid accumulation, and systemic metabolic inefficiency during aging—directly corresponding to the theory of "metabolic flexibility loss/glucose and lipid metabolism disorders" and initiating the "epigenetic alteration" pathway [6][7][106].
When PPARα is in an "overactive" state, it is closely associated with "oxidative stress and lipotoxicity". Overactivated PPARα unrestrainedly drives the influx of fatty acids into mitochondria for β-oxidation [5][87]. If this process exceeds the mitochondrial processing capacity or the upper limit of the electron transport chain, it will lead to the excessive production of reactive oxygen species [65][68][69], exacerbating oxidative damage [65][66][67]. Simultaneously, incomplete oxidation of lipid intermediates may accumulate, resulting in lipotoxicity [87][92], further impairing cellular function - directly corresponding to the "free radical/oxidative stress" theory.
2.5 Molecular mechanism of stem cell replicative senescence driven by glycolysis-TERT-TIN2 axis under chronic hypoxic microenvironment
The chronic hypoxic microenvironment continuously exerts effects on stem cells through microcirculatory disorders, triggering metabolic reprogramming and a cascade of decompensation in the telomere regulatory network. This process exhibits significant temporal dependence: under single hypoxic stimulation, cells activate TERT through the enhanced glycolysis-FBP1 inhibition axis, achieving physiological repair of telomere length [133]; however, when the stimulation transitions to persistent hypoxia, this regulatory network is hijacked by a TIN2-mediated negative feedback mechanism [131][132], leading to the decoupling of telomerase activity and telomere length, and triggering replicative senescence.
2.6 The ratio of microcirculatory disturbance intensity to axial imbalance, and its alternative aging entry pathway.
In theory, although it can be argued that any non-steady-state pathway will ultimately lead to microcirculatory disorders, these disorders themselves may exhibit significant temporal differences depending on the triggering pathway. Furthermore, due to varying degrees of imbalance in the central axis, there are notable differences in the severity of microcirculatory disorders among individuals. Some individuals may enter a stage of mitochondrial dysfunction without experiencing significant microcirculatory disorders, such as those with a predominant weak expression of PPARα and AMPK. In this type, the decline in energy metabolism primarily affects mitochondrial function first, thus microcirculatory disorders may not manifest as distinct characteristics in such individuals. However, regardless of whether microcirculatory disorders occur, even in the absence of microcirculatory disorders, the imbalance in the central axis will lead to mitochondrial dysfunction through the preferred aging pathway, which is the primary cause of aging. For example, high expression of MTOR and weak expression of PGC-1 can lead to the accumulation of senescent cells, affecting mitochondrial function through the SASP pathway. Alternatively, local microcirculatory hypoxia can induce the transcription of inflammatory factors, which in turn lead to a decline in mitochondrial function, initiating a cascade collapse from the first to the sixth cause of aging. This self-reinforcing, self-sustaining, and self-locking metabolic homeostasis of aging is maintained, but microcirculatory disorders are the most easily manifested characteristic. When the imbalance in the central axis is too strong, microcirculatory disorders will inevitably manifest.
Therefore, the primary cause is the decline in mitochondrial function itself, and its core does not lie in the emergence of traditional aging theories previously, but rather in the convergence of the pathways through which the primary cause itself damages the body due to multiple aging factors. The decline in mitochondrial function itself is the node where multiple aging factors collectively cascade towards the aging process. Once the primary cause of mitochondrial dysfunction pushes the aging process towards the secondary cause, it will become a supporting role for the secondary cause, forming a switch in the main factors of aging.
3.1 The second cause of aging: disturbance of glucose and lipid metabolism
The decline in mitochondrial function directly leads to changes in the whole-body energy metabolism pattern, resulting in an imbalance in the utilization ratio of sugar and fat, which can trigger glucose and lipid metabolism disorders [86, 87]. The essence of glucose and lipid metabolism disorders lies in the altered proportion of differential gene expression in the gene expression network formed by lipid metabolism and glucose metabolism, such as the key impact on antioxidant function.
3.1.1 Decreased lipid metabolism: Reduction of free carnitine and accumulation of acylcarnitineMitochondria are the primary site for β-oxidation of fatty acids, responsible for converting long-chain fatty acids into energy. In this process, free carnitine (L-carnitine) acts as a "transporter" for fatty acids into the mitochondrial matrix, playing a crucial role in maintaining fat metabolism [86].
With the decline in mitochondrial function, acylcarnitine cannot be effectively oxidized and accumulates significantly in the cytoplasm [86, 87], leading to a decrease in free carnitine levels in the blood and an inability to effectively oxidize fatty acids. Simultaneously, these metabolic intermediates are not only markers of lipid metabolism disorders but may also interfere with cell membrane function and promote inflammatory responses [87]. Furthermore, the storage of triglycerides in adipose tissue increases, while the proportion of beneficial fatty acid metabolites such as unsaturated fatty acids decreases, further exacerbating metabolic inflammation.
The slowed repair and renewal of phospholipids in the inner mitochondrial membrane also affect membrane fluidity and membrane potential, further deteriorating mitochondrial function [87, 88].
3.2 Increased proportion of glucose metabolism: accumulation of advanced glycation end products (AGEs) and initiation of inflammationIn normal cells, even when nutrients are abundant, energy is still obtained through the metabolism of ketone bodies. When the efficiency of fat combustion decreases, due to energy requirements, cells, based on metabolic flexibility, will become more inclined to obtain energy through glucose oxidation and phosphorylation, which leads to metabolic rigidity and a significant increase in the proportion of glucose metabolism [88][92].
This metabolic shift brings about severe side effects, namely the accumulation of advanced glycation end products (AGEs) [88].
AGEs are stable covalent adducts formed through non-enzymatic reactions between reducing sugars (such as glucose) and free amino groups of proteins, lipids, or nucleic acids [88].
The accumulation of AGEs can cause various damages to the body:
Impaired protein function: Collagen and elastin undergo cross-linking after being glycosylated, leading to stiffness and fragility of the skin and vascular walls [88].
Inflammation response initiation: AGEs can bind to the RAGE receptor, activating pro-inflammatory signaling pathways such as NF-κB, inducing the release of inflammatory factors, and maintaining a chronic inflammatory state [88, 91].
Oxidative stress intensifies: Reactive oxygen species (ROS) are generated during the formation of AGEs, which can induce cells to further produce ROS, forming a vicious cycle [89, 90].
Microcirculatory disorders: AGEs accumulate on microvascular endothelial cells, increasing blood viscosity, blocking capillaries, leading to tissue ischemia and hypoxia, and further impairing mitochondrial function [88, 89].
Therefore, dysregulation of glycolipid metabolism not only reflects changes in energy supply patterns, but also serves as a crucial bridge connecting mitochondrial dysfunction with chronic inflammation and tissue damage [86-92].
3.3 Fat accumulation: Dilution within vitamin D3-fat reserves, with a pro-inflammatory shift in extracellular vesicles of adipocytes.
Vitamin D3, also known as cholecalciferol, is primarily stored in adipose tissue [120]. Although it is a low-biological activity form of vitamin D, the body's reserves of this vitamin D3 are crucial for regulating inflammation.
When body fat percentage is healthy, the concentration of vitamin D3 cholecalciferol within adipocytes is relatively high, forming a state of weak regulation of adipocytes by quantity [122]. In this state, both adipocytes and the extracellular vesicles they secrete exhibit an anti-inflammatory phenotype, which helps maintain metabolic homeostasis. [121]
However, when glycolipid metabolism disorders persist and progress to form fat accumulation, such as visceral fat, vitamin D3 cholecalciferol is diluted in the expanded adipose tissue, leading to a decrease in its intracellular concentration. Exogenous supplementation requires extremely high doses to restore its biological concentration to a youthful state, rendering the original weak anti-inflammatory regulatory mechanism ineffective. This triggers adipocytes to shift to a pro-inflammatory state, and the extracellular vesicles they secrete also undergo phenotypic changes, transitioning from anti-inflammatory to pro-inflammatory [121]. These pro-inflammatory vesicles further promote lipolysis, breaking down stored fat into the bloodstream, which is originally a metabolic regulatory mechanism for the body to respond to local excess body fat. However, in the metabolic context of glycolipid metabolism disorders and fat accumulation, this mechanism forms an erroneous regulatory function, transforming this beneficial regulation to the human body into a persistent negative factor. It promotes persistent low-grade inflammation and abnormal lipolysis, ultimately exacerbating age-related chronic inflammation and metabolic disorders.
3.4 Arachidonic acid-nuclear envelope mechanical axis: a structural compensatory trap for mitochondrial dysfunction
The disruption of glycolipid metabolism leads to decreased fat metabolism and increased blood glucose metabolism. This subtle metabolic change is itself a compensatory metabolic micro-regulation carried out by cells through metabolic flexibility to maintain ATP levels. This results in human cells requiring more blood glucose to maintain energy supply, which indirectly increases the body's demand for insulin. The slight increase in insulin levels will enhance the expression of Δ6-desaturase (FADS2), driving an increased conversion flux from linoleic acid (LA) to arachidonic acid (AA). This, in turn, activates RhoA endogenously through the arachidonic acid pathway, leading to H3K9me2-mediated heterochromatin loss and dysfunction, which subsequently inhibits the function of hematopoietic stem cells [124][125]. This pathway mechanism is primitive and has the basis for expansion to most stem cells.
3.4 Impaired fat metabolism leads to decreased pulse energy.
3.4 Metabolic core of the second cause of aging: Functional decline of the α-ketoglutarate (AKG) axis
In the metabolic disorders associated with aging, a core metabolic change is the progressive decline in α-ketoglutarate (AKG) levels. AKG is not only an intermediate in the tricarboxylic acid cycle, but also a key hub connecting energy metabolism, epigenetic regulation, and cellular defense systems. The decrease in its levels triggers a series of cascading but not absolute functional declines, significantly weakening the cell's ability to maintain homeostasis.
3.4.1 Decreased AKG levels impair epigenetic plasticity
AKG is an essential cofactor for various α-ketoglutarate-dependent dioxygenases, including the TET family enzymes responsible for active DNA demethylation and some histone demethylases [6]. During aging, mitochondrial dysfunction leads to a decrease in the flux of the tricarboxylic acid cycle, and the reduction in AKG production impairs the activity of these demethylases, making it more difficult to effectively remove methylation modifications in the promoter regions of numerous genes, including the key transcription factor TFEB involved in autophagy and lysosome biogenesis. As a result, the epigenetic plasticity of the genome decreases, some programs that maintain cellular homeostasis tend to be silenced, and cellular function declines are accelerated [22].
3.4.2 Limited supply of metabolic precursors for antioxidant defense
AKG participates in the synthesis of glutamate and glycine through transamination, with the latter being a key precursor for the synthesis of glutathione (GSH), the most important antioxidant in cells. Therefore, the AKG-glycine-serine-cysteine-GSH pathway is an important endogenous antioxidant synthesis pathway. When AKG levels decline, it restricts the supply of raw materials for this synthesis pathway. Although GSH synthesis does not completely cease, its synthesis rate and intracellular reserve levels will be slightly reduced due to insufficient precursors, leading to a weakened ability of cells to recycle reduced GSH and oxidized GSSG in the face of reactive oxygen species (ROS), resulting in a decrease in overall antioxidant defense efficacy.
3.4.3 NAD⁺ regeneration and reduced activity of related signaling pathways
The end product of cysteine metabolism, α-ketobutyrate (AKB), can promote the regeneration of nicotinamide adenine dinucleotide (NAD⁺) under the action of lactate dehydrogenase-1 (LDH-1). NAD⁺ is the core substrate of the deacetylase SIRT1. The decline in AKG axis function leads to a reduction in AKB production, thereby indirectly weakening the regenerative capacity of NAD⁺. The intracellular NAD⁺ level decreases for the first time, limiting the activity of SIRT1, which depends on it [3, 4]. This, in turn, affects multiple downstream processes jointly regulated by SIRT1 and AMPK:
Mitochondrial biosynthesis weakens: The activation of the transcriptional coactivator PGC-1α by SIRT1/AMPK decreases, leading to a reduction in the expression of its downstream target NRF1, which gradually diminishes the mitochondrial biogenesis and maintenance capabilities.
Rhythmic dysregulation of endogenous antioxidant programs: The timely activation ability of SIRT1 on peroxisomal fatty acid oxidase ACOX1 decreases, leading to a reduction or disruption in the rhythmic activation of ACOX1 to generate H₂O₂ signals. Since this rhythmic H₂O₂ signal serves as a critical physiological trigger for activating the antioxidant transcription factor NRF2, its deficiency significantly impairs the responsiveness of the cellular antioxidant gene expression program, and the rhythmic regulatory characteristics it should possess are also lost, thereby weakening the dynamic antioxidant defense capacity of cells at the transcriptional level [3, 4].
3.4. Vitamin E (VE) leads to a decline in mitochondrial pulse energy metabolism due to reduced glutathione synthesis.
Vitamin E (VE) is a major lipid-soluble antioxidant that protects cell membranes from lipid peroxidation [68,69]. The ratio of oxidized and reduced forms of VE serves as an important signaling source. When the proportion of oxidized VE increases, it indirectly affects SREBP activity by altering the level of membrane lipid peroxidation and oxidative stress status, thereby altering the distribution of precursors between cholesterol and coenzyme Q10 in the mevalonate pathway, indirectly influencing Q10 synthesis, and further affecting mitochondrial function [70,72,73,76,77]. Since coenzyme Q10 supports mitochondrial pulse-based energy metabolism, a decrease in Q10 synthesis ultimately leads to a decline in growth hormone levels, which rely on pulse-based energy metabolism and have a very short window period, due to energy deficiency during ATP synthesis.
Conclusion: The decline in pulse energy directly affects the pulsatile secretion rhythm of GH, rather than the baseline level of GH. This represents a direct causal link between metabolic disorders and hormone decline.
4. The third cause of aging: The decline in GH-IGF-1 axis function, mitochondrial dysfunction, and metabolic disorders can further affect the hypothalamic-pituitary-target gland hormone axis.
The functional decline of the growth hormone (GH)-insulin-like growth factor-1 (IGF-1) axis is a hallmark event in the aging process.
4.1 The impact of decreased growth hormone (GH) levels on NRF1 and mitochondrial quantityGH is not only a crucial hormone for promoting child growth but also continues to play a significant role in metabolic regulation in adulthood [80,81]. The decline in GH levels is closely associated with mitochondrial dysfunction [79,81]. Firstly, GH is one of the key hormones that promote the expression of NRF1 [81]. As the primary regulator of mitochondrial biogenesis, the expression level of NRF1 directly determines the number of mitochondria [3,4]. When GH levels decrease, the expression of NRF1 also decreases, leading to cells unable to maintain a sufficient number of mitochondria to meet energy demands. Even if the quality of individual mitochondria is acceptable, the overall energy output still drops significantly [79,81]. This also explains why, during the aging process, even with the supplementation of mitochondrial nutrients such as coenzyme Q10, it is difficult to completely reverse the decline in energy metabolism, as the fundamental issue lies in the reduction of the "number of factories" [79,81]. From this process onwards, growth hormone levels form a vicious cycle, self-locking and self-reinforcing.
4.2 The impact of the blockade of the GH-deiodinase-T4/T3 pathway on the production of catecholamines
GH not only affects mitochondrial quantity and function, but also influences metabolism by regulating thyroid hormone activity [80,81]. Specifically, GH can promote the activity of type II deiodinase (D2) [82], which is responsible for converting thyroxine (T4) into the active triiodothyronine (T3). D2 plays a crucial role, especially in peripheral tissues such as the liver and skeletal muscle [82]. T3 is a core hormone that maintains basal metabolic rate and promotes carnitine synthesis, thereby facilitating the entry of fatty acids into mitochondria for oxidation [82]. When GH levels decline, D2 activity decreases, leading to a block in the T4→T3 conversion, reduced levels of active T3, and subsequently inhibiting carnitine production [80,82].
Simultaneously, elevated glucocorticoid levels can also convert T4 into reverse triiodothyronine (rT3) by regulating the activities of type I deiodinase (D1) and type III deiodinase (D3), further reducing the effective T3 levels [85,82]. In conditions of glucose-lipid metabolism disorders or chronic inflammation, inflammatory factors can promote glucocorticoid secretion, exacerbating the decrease in T3 and forming a vicious metabolic cycle that overlaps with the decline in GH [80,82].
The decrease in total carnitine levels hinders the entry of fatty acids into mitochondria for oxidation, exacerbating lipid metabolism disorders and further affecting energy metabolism and mitochondrial function [79,81,82].
4.3 The decrease in T3 and increase in T4 ratio co-regulate the abnormal expression of downstream gene lineage of nuclear factor kappa B (NF-κB), shifting towards the expression of pro-inflammatory factors.
During the aging process, the balance between thyroid hormone thyroxine (T4) and triiodothyronine (T3) and the switching of their action pathways are the core links leading to the dysregulation of the Nuclear Factor-kappa B (NF-κB) signaling pathway. This mechanism exhibits a typical bidirectional regulatory pattern: Physiological levels of T3 primarily exert a genomic anti-inflammatory effect by binding to the Thyroid Hormone Receptor (TR). The key mechanism lies in the direct downregulation of the expression of key mediators such as Toll-like receptor 4 (TLR4), NF-κB, and its downstream NOD-like receptor protein 3 (NLRP3) inflammasome, as well as pro-IL-1β, thereby guiding the expression profile of downstream target genes towards an anti-inflammatory direction at the transcriptional level. However, in the state of aging-related metabolic disorders, the conversion efficiency of peripheral T4 to T3 decreases, leading to a relative scarcity of active T3, which loses its ability to balance the NF-κB pathway through the TR pathway. At the same time, the relatively elevated T4 binds to integrin αvβ3 on the cell membrane, activating the phosphatidylinositol 3-kinase-protein kinase B (PI3K-AKT) signaling cascade, inducing the production of a large amount of Reactive Oxygen Species (ROS), and subsequently strongly triggering the activation of the NLRP3 inflammasome. Ultimately, this pathological shift from the "T3-TR anti-inflammatory axis" to the "T4-integrin αvβ3 pro-inflammatory axis" leads to a fundamental reprogramming of the downstream gene expression profile regulated by NF-κB, shifting the transcriptional output from being dominated by anti-inflammatory factors to a pattern favoring the expression of pro-inflammatory factors such as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), thereby becoming one of the important sources of inflammatory factors in the body. [105]
4.4.1 Triiodothyronine (T3) and the regulatory network of protein quality controlTriiodothyronine (T3), as the active form of thyroid hormone, plays a crucial role in regulating the body's basal metabolic rate ([34, 37]). Recent studies have shown that T3 plays a central role in cellular protein quality control and forms a key metabolic-quality control cross-network with the energy-sensing molecule AMPK [35]. This network not only maintains protein homeostasis but also participates in the regulation of mitochondrial function and autophagy processes.
4.4.2 T3 regulates protein homeostasis through the FOXO3a-ULK1-autophagy axis
The regulation of T3 on protein quality control is primarily achieved through the activation of the transcription factor FOXO3a [36]. The specific mechanism is as follows:
After binding to the nuclear thyroid hormone receptor (TR) via upregulation of the ubiquitin-proteasome system (UPS)
T3, it can directly act on the FOXO3a gene promoter, significantly upregulating its transcription level. FOXO3a subsequently promotes the expression of a series of E3 ubiquitin ligases (such as MuRF1, Atrogin-1), marking misfolded or damaged proteins with "ubiquitin tags" and guiding them into proteasomal degradation [33, 36].
FOXO3a, which activates the autophagy-lysosome pathway,
is also a key inducer of autophagy. It can upregulate core autophagy genes such as LC3 and ATG12, and relieve the inhibition of the autophagy initiation complex ULK1, thereby initiating cellular autophagy and clearing aggregated proteins and damaged mitochondria [35, 36].
Therefore, at physiological T3 levels, FOXO3a forms a core pillar for maintaining protein homeostasis. With aging (such as the decline of the GH-IGF-1 axis), T3 levels decrease, FOXO3a expression and activity weaken, and UPS function and autophagic flux are impaired, leading to increased protein aggregation and cytotoxicity [33, 36].
4.4.3 Disruption of the T3-AMPK positive feedback loop and collapse of quality control
There exists a precise positive feedback loop between T3 and AMPK [35, 37]:
Activation of AMPK by T3
T3 increases the cellular AMP/ATP ratio by enhancing basal metabolic rate, thereby activating AMPK through allosteric changes; in addition, T3 can directly promote the phosphorylation of AMPKα subunit, enhancing its kinase activity [35].
Feedback regulation of AMPK on T3
Promoting T3 production: AMPK can phosphorylate and activate type II deiodinase (D2), converting T4 into active T3 and maintaining local T3 levels.
Collaborative downstream signaling: Both AMPK and T3 can phosphorylate ULK1, jointly initiating autophagy and optimizing mitochondrial function [34, 35, 37].
During the aging process, the decline in upstream mitochondrial function and GH levels leads to a decrease in T3 levels, thereby weakening AMPK activation. The reduced activity of AMPK, in turn, inhibits D2 enzyme function, resulting in the disruption of the T3-AMPK positive feedback loop. At this point, the ubiquitin-proteasome system (UPS) and autophagy system lose sufficient energy and transcriptional support, leading to a sharp decline in protein quality control ability, accelerating intracellular environmental disorder and the emergence of senescent phenotypes [33-37].
4.4.4 Lysosomal dysfunctional hyperactivity: Autophagy-lysosomal network collaborative collapse triggered by T3 signal attenuationThe aforementioned hypothesis points out that the decline of the GH-IGF-1 axis (the third factor) leads to a decrease in the level of active T3, which in turn impairs the initiation of autophagy by weakening signals such as AMPK and FOXO3a, that is, the source driving force of the cell's "waste packaging" ability is diminished [33, 36, 37]. However, the underlying pathology of aging lies not only in the reduction of "packaging", but also in the systemic disorder and collapse of the entire waste clearance pipeline. In the context of autophagy flow blockage, lysosomes exhibit a state of "disfunctional hyperactivity" characterized by "overactivity but loss of function", which is rooted in the decreased expression of the core coordinator TFEB [126].
Core pathological cascade:
Upstream command attenuation (T3↓ → autophagic flux blockage): The weakening of T3 signaling directly leads to the downregulation of clearance commands in the "master mode" of cells. The consequence is not the complete shutdown of autophagy, but a severe reduction in the efficiency of autophagic flux - insufficient autophagosome generation, impaired movement, or blocked fusion with lysosomes, resulting in the accumulation of degradation products within the cell, forming a "waste jam".
Central control hub dysfunction (decreased TFEB expression): The attenuation of T3/AMPK and other signals simultaneously leads to a decrease in the expression and activity of transcription factor EB. TFEB is the central control hub that coordinates the synchronous expression of autophagy genes and lysosomal genes, ensuring that the "packaging" and "incineration" capabilities are matched. Its functional weakening means that cells lose the ability to efficiently and synergistically expand the entire clearance system.
Downstream actuator disorder (stress signal ↑ → lysosomal disfunction): The continuous accumulation of cellular waste (such as damaged mitochondria and protein aggregates) itself constitutes a strong and uneliminable stress signal. In the absence of precise coordination by TFEB, these chaotic stress signals blindly drive lysosomal biogenesis through inefficient or abnormal bypasses. The result is the generation of a large number of lysosomes: an excessive and overly acidified problem. These lysosomes are in a state of "idling" - ready for degradation but with no cargo to clear, affecting the self-renewal of hematopoietic stem cells.
This state of "disinhibited hyperactivity" signifies the ultimate disorder of cellular quality control: not only is the clearance function paralyzed, but the system itself also becomes an energy consumer and a new source of damage.
[126] This study points out that the forced activation of TFEB through genetic means can reverse this state. The reactivation of TFEB is equivalent to bypassing the upstream attenuated T3 instructions and chaotic stress noise, directly issuing a top-level instruction to the cell to "reconstruct a complete set of efficient and coordinated clearance systems". This not only repairs lysosomal quality, but also simultaneously enhances autophagy ability, thereby dredging the autophagic flux, restoring intracellular environmental stability, and ultimately rejuvenating the function of senescent HSCs.
Conclusion: Therefore, the "disfunctional hyperactivity" of lysosomes is not an independent event, but rather the inevitable endpoint of a causal chain: "Third-party factor (T3 signal attenuation) → autophagic flux blockage and decreased TFEB expression → lysosomal compensatory imbalance driven by stress signals." It empirically demonstrates at the organelle level that the decline of the hormone axis leads not only to the weakening of a single function, but also to the coordinated collapse of the entire intracellular homeostasis regulatory network. This pathway mechanism is primitive and has the basis for expansion to most stem cells.
4.5 Vicious cycle of decreased ketone body metabolism and decreased GH levels
The decline in GH levels, coupled with the decrease in ketone body metabolism, constitutes the third factor of aging [79,80,81]. GH aids in fat decomposition and ketone body production [80,81], and ketone bodies are not only an important alternative energy source but also maintain mitochondrial function and the expression of basic antioxidant genes by activating the AMPK-PGC-1α-NRF1 pathway [3,4,14]. When GH levels decline and ketone body production decreases, this positive regulatory effect weakens accordingly [79,81].
Simultaneously, decreased ketone body levels can affect epigenetic homeostasis. The product of fat decomposition, α-ketoglutarate (AKG), is a crucial substrate for histone demethylation, and its decline may lead to epigenetic silencing of the GH gene itself, further suppressing GH levels [6]. Furthermore, the reduction in ketone bodies can also weaken their inhibitory effect on histone deacetylase (HDAC), affecting the synthesis of signaling molecules such as ceramide, thereby disrupting cellular metabolism and signal transduction [9,81].
4.6 Switching of metabolic pathways: energy crisis from oxidative phosphorylation to glycolysis
The decline in mitochondrial function triggers disturbances in glucose and lipid metabolism, one of the core manifestations of which is a fundamental shift in cellular energy metabolism patterns [79,81]. In a healthy, young state, cells preferentially utilize efficient mitochondrial oxidative phosphorylation (OXPHOS) to thoroughly decompose glucose, generating ample ATP. However, as aging progresses, the expression of NRF1 significantly decreases due to the decline in GH signaling, impeding the transcription and synthesis of mitochondrial respiratory chain complex subunits encoded by nuclear genes. This directly leads to a reduction in the number of mitochondria and functional abnormalities [81,3]. The scaling down and quality decline of this "energy factory" significantly reduce the efficiency of ATP production per unit of glucose through the tricarboxylic acid cycle (TCA cycle) and electron transport chain [79,3].
To address the looming energy crisis, cells are compelled to activate an evolutionarily conserved yet inefficient backup strategy—shifting towards glycolysis metabolic pathway [22,3]. Glycolysis occurs in the cytoplasm without the involvement of mitochondria, enabling rapid decomposition of glucose into pyruvate and net production of two molecules of ATP. However, in the context of impaired OXPHOS function, most pyruvate cannot enter mitochondria for further oxidation, but is instead largely reduced to lactic acid under the catalysis of lactate dehydrogenase (LDH) [22].
The switching of this metabolic pathway has two direct catastrophic consequences: firstly, a severe deficiency in energy production [3]. For the same unit of glucose, the ATP produced through glycolysis is less than 1/18 of that produced by OXPHOS, which traps cells in a state of "hunger" despite normal or even elevated glucose levels in the blood circulation [3,22]. Secondly, a systemic increase in lactate levels [22]. Lactate is no longer merely a local metabolite during exercise, but becomes a systemic metabolic marker as aging progresses [22]. The continuously elevated lactate levels in the blood not only exacerbate the acidic load on tissues and interfere with normal physiological functions, but more importantly, they compete with ketone bodies for entry into cells through monocarboxylate transporters (MCT) and play a role as an immune escape promoter in senescent cells (as described in the sixth factor [8]), directly hindering the immune system's clearance of senescent cells [8].
Therefore, the elevation of blood lactate levels is not an isolated metabolic phenomenon, but a crucial link connecting the upstream mitochondrial dysfunction and the downstream formation of an inflammatory microenvironment, as well as the suppression of stem cell function [79,3]. It signifies that the energy metabolism of cells has shifted from the efficient and clean "aerobic combustion" mode to the inefficient and polluting "anaerobic fermentation" mode, representing the metabolic aspect of systemic aging in the entire organism [3,8].
4.6.1.1 Impact of metabolic switch on epigenetics: dual deficiency of acetyl-CoA and α-ketoglutaric acid
As cellular energy metabolism shifts from efficient oxidative phosphorylation to inefficient glycolysis, it not only triggers an energy crisis and lactic acid accumulation, but also profoundly impacts the epigenetic maintenance system at the substrate level:
Acetyl-CoA deficiency: Pyruvate, as a key precursor of acetyl-CoA, cannot be effectively converted into acetyl-CoA under the background of enhanced glycolysis but blocked mitochondrial uptake. Acetyl-CoA is an essential substrate for histone acetylation, and its decreased level directly leads to insufficient histone acetylation modification, thereby affecting chromatin accessibility and gene transcriptional activity, forming "transcriptional repression" at the epigenetic level. [106]
The dual blow of AKG deficiency: Simultaneously, the decline in tricarboxylic acid cycle function leads to a decrease in α-ketoglutarate (AKG) levels, weakening the activity of demethylases such as TET, and rigidifying the DNA methylation landscape.
The simultaneous deficiency of these two key metabolites constitutes a "substrate crisis" for epigenetic maintenance, leading to the solidification and aging of the epigenetic landscape from both acetylation and demethylation directions, further transforming metabolic disorders into global dysregulation of gene expression.
4.6.1.2 Metabolic pathway switching affects acetyl-CoA levels, and epigenetic gene disruption promotes the expression of 15-PGDH, thereby inhibiting the health of articular cartilage.
Articular chondrocytes form glycolytic metabolic lactate through physiological glycolysis, stabilizing HIF-1α expression and thereby promoting the transcription of COX-2. However, due to the attenuation of the citric acid cycle flux, the supply of acetyl-CoA in the nucleus decreases, directly weakening the H3K27ac modification of the COX-2 promoter, and reducing the prostaglandin E2 (PGE2) synthesis capacity below the negative feedback regulation threshold. This step is akin to the epigenome having given the initiation signal, but during the execution of port acetylation, it is affected by the decreased metabolic flux of acetyl-CoA, resulting in high expression but unable to convert it into effective gene expression. [128][129]
At this point, the transcriptional inhibition of PGE2 on 15-hydroxyprostaglandin dehydrogenase is significantly relaxed, and the promoter of this enzyme, which should be silenced due to insufficient histone acetylation, is paradoxically occupied by low-grade inflammatory signals (NF-κB/AP-1), leading to "de-repression transcription". More critically, the ubiquitin-proteasome clearance ability of senescent chondrocytes decreases, causing the degradation rate of 15-PGDH to lag behind its synthesis, ultimately resulting in net accumulation in the extracellular matrix. An increase in 15-PGDH levels in articular cartilage degrades prostaglandin E2, whereas a slight increase in prostaglandin E2 at normal biological levels can promote articular cartilage regeneration [127]. However, the elevated local concentration of 15-PGDH disrupts the normal ratio between prostaglandin E2 and 15-PGDH, forming a reverse inhibition of PGE2 by 15-PGDH. The increased PGE2 in the blood due to aging cannot effectively affect the white zone through blood-borne prostaglandin E2 due to the blood supply differences between the red and white zones of the joint, and the poor blood flow signal [130]. Even if a small amount of prostaglandin E2 can enter the white zone through small molecular structures, it will still be metabolized against the background of high 15-PGDH concentration, leading to a decline in the self-repair function of articular cartilage. [127] Simultaneously, this mechanism lies at the bottom and has the basis to expand to avascular zones such as the spine.
4.6.2 The switching of metabolic pathways affects the metabolism of NADH to NAD+, leading to the failure of the CD38-NAD+AMPK inflammatory signaling reversal pathway.When the proportion of cellular oxidative phosphorylation decreases and the proportion of glycolysis increases, such a slight change in ratio does not lead to significant abnormalities in cells. However, it can weaken the intracellular availability of NAD+, resulting in the accumulation of NADH, which is released to the outside in the form of lactic acid. This process not only increases the demand for NAD+ in cells but also leads to immune suppression of senescent cell clearance triggered by lactic acid, and further results in the paralysis of the CD38-NAD+-AMPK-dependent inflammatory signal reversal mechanism.
4.6.2.1 CD38-NAD⁺-AMPK axis: a cell-autonomous inflammatory signaling reversal mechanismAging is a physiological process accompanied by the gradual aggravation of low-grade chronic inflammation (inflammaging) [1]. The initiation, amplification, and loss of control of inflammation have been widely described in various diseases and tissue functional decline [2,3]. However, the body has also developed multiple layers of autonomous negative feedback mechanisms aimed at limiting the excessive spread of inflammatory signals and maintaining cellular homeostasis. The CD38-NAD⁺-AMPK axis has been proposed as a fundamental "inflammatory signal reversal pathway" present in almost all cell types, with its core logic lying in the active reversal of pro-inflammatory states through metabolic-signal coupling [8,41].
Although this axis was initially elucidated in the functional maintenance of iNKT cells [8], literature [41] indicates that CD38, as an inflammation-sensitive molecule, can be directly induced by classic inflammatory factors such as TNF-α and IL-6, thereby rapidly mediating the regulation of NAD⁺ levels under inflammatory conditions. This discovery provides direct evidence for explaining the link between aging-related chronic inflammation and NAD⁺ baseline depletion.
4.6.2.2 Consumption of NAD⁺ by CD38 and Allosteric Activation of AMPK
This pathway is initiated by the induction of CD38 expression [41]. CD38 is a transmembrane protein with an extracellular enzyme domain exhibiting NAD⁺ glycosidase activity. Under the stimulation of inflammatory cytokines (such as TNF-α and IL-6), CD38 expression is significantly upregulated [41], catalyzing the hydrolysis of NAD⁺ to generate ADP-ribose (ADPR) and cyclic ADP-ribose (cADPR) [8]. This process leads to a rapid depletion of the cellular NAD⁺ pool, triggering transient perturbations in energy response signaling. The decrease in NAD⁺ elevates the AMP/ATP ratio, and this metabolic crisis signal is captured by the central energy sensor AMPK [14,15]. AMPK achieves full activation through the phosphorylation of Thr172 site in coordination with its upstream kinase LKB1 [14].
In this process, cADPR, as a byproduct of CD38, may also participate in the regulation of intracellular calcium homeostasis by modulating calcium signaling, providing a molecular basis for subsequent metabolic-inflammatory integration [11]. Furthermore, the activation of AMPK is closely related to mitochondrial energy status, and it can regulate mitochondrial function, ROS levels, and autophagy activity through a feedback mechanism, forming a dual metabolic-signal regulation [2,3]. This mechanism explains why the CD38-NAD⁺-AMPK axis is not only a trigger for inflammation reversal but also a core hub for maintaining cellular homeostasis.
4.6.2.3 Multidimensional inhibition of NF-κB transcriptional activity by AMPK
Activated AMPK is the core executor of this reverse pathway, which inhibits the pro-inflammatory transcription factor NF-κB through multiple mechanisms:
Direct phosphorylation of p65 subunit: AMPK can directly phosphorylate the p65 (RelA) subunit of NF-κB, such as at the Ser536 site. This modification not only does not enhance transcriptional activity but instead weakens the binding ability of p65 to DNA response elements and promotes its binding to nuclear export proteins, accelerating its extranuclear transport, thereby directly reducing the transcriptional activity of NF-κB [8].
Inhibiting the activity of the IKK complex: AMPK activation indirectly inhibits the IκB kinase (IKK) complex, reducing the phosphorylation and degradation of IκBα. This results in more NF-κB being anchored in the cytoplasm, preventing it from entering the nucleus to initiate the transcription of inflammatory genes [8].
Synergistic inhibition with SIRT1: Despite an overall decrease in NAD⁺ levels, AMPK activation can promote NAMPT expression, maintaining or re-establishing NAD⁺ supply in local microdomains, thereby supporting the activity of the deacetylase SIRT1. SIRT1 deacetylates the p65 Lys310 site, significantly inhibiting its transcriptional ability [8]. The synergistic action of AMPK and SIRT1 forms a "molecular clamp" on NF-κB activity, achieving a potent reversal of pro-inflammatory signaling.
This multi-layered inhibitory mechanism indicates that the CD38-NAD⁺-AMPK axis not only serves as a sensor for metabolic energy sensing, but also plays a "gatekeeper" role in the molecular regulation of inflammatory signaling, providing a reliable mechanism for cells to actively maintain homeostasis.
4.6.2.4 Closed-loop feedback and homeostasis remodeling of inflammatory signaling
The CD38-NAD⁺-AMPK axis establishes a self-limiting negative feedback loop:
Inflammatory signals ↑ → CD38 ↑ → NAD⁺ ↓ → AMPK ↑ → NF-κB ↓ → Decreased inflammatory signals
cADPR byproducts may further participate in cell repair, metabolic adaptation, and mitochondrial function regulation by modulating calcium signaling [11]. This closed-loop mechanism effectively terminates the malignant spread of inflammation and plays a central role in maintaining cellular metabolic-immune homeostasis.
During the aging process, persistent inflammatory stress and baseline depletion of NAD⁺ impair the function of this axis, preventing cells from effectively terminating the inflammatory program activated by NF-κB, leading to chronic low-grade inflammation and accelerating tissue and systemic decline [10,41]. Therefore, the CD38-NAD⁺-AMPK axis is regarded as the cell's own "inflammatory signal reset program," and maintaining its full functionality may be key to delaying aging and various aging-related diseases.
4.6.3 The increase in the proportion of glycolysis metabolism through metabolic switching further promotes replicative senescence of stem cells through TIN2.
In the previous section, 2.5, it has been demonstrated that hypoxia-induced sustained glycolysis can trigger TIN2 by inhibiting telomere regeneration of TERT [131][132]. However, in 2.5, the cause of glycolysis is only the hypoxic effect of microcirculation disorders, while the alteration of energy supply ratio in the metabolic model further intensifies the severity of stem cell replicative senescence, forming the second core contributing factor to stem cell replicative senescence, which collaboratively inhibits the self-renewal function of stem cells.
4.7 Skeletal Muscle Decline: From Muscle Loss to Uncontrolled Systemic Inflammation
Skeletal muscle is not only the largest protein reserve and energy metabolism organ of the body, but also an important endocrine and immune regulatory tissue [83,84]. In the context of GH-IGF-1 axis decline (the third factor), the decline in skeletal muscle mass has become a long-neglected yet crucial pathological link [80,81,83]. This hypothesis proposes that decorin, as a muscle factor directly regulated by GH, plays a pivotal role in this process [83].
4.7.1 Research on the direct induction of Decorin expression by GH and its anti-atrophy mechanism
has found that GH can directly induce the expression of Decorin in skeletal muscle without relying on IGF-1 (validated in mouse models) [83]. As a small leucine-rich proteoglycan, Decorin's key function lies in its potent inhibition of Myostatin, a key factor that negatively regulates muscle growth [83]. Myostatin significantly limits muscle mass by inhibiting myoblast differentiation and protein synthesis. Therefore, by upregulating Decorin, GH relieves the inhibition of muscle growth by Myostatin, representing an important pathway independent of IGF-1 for maintaining skeletal muscle mass [83].
4.7.2 Loss of skeletal muscle function as an anti-inflammatory endocrine organ
During contraction and metabolism, skeletal muscle can secrete a series of myokines with anti-inflammatory and immune regulatory functions, such as IL-6 (exhibiting anti-inflammatory effects during exercise), IL-10, IL-1ra, etc. [84]. These factors collectively constitute a "muscle-derived anti-inflammatory network" that systematically suppresses systemic low-grade inflammation [84].
When GH levels decline, leading to reduced expression of decorin and release of myostatin inhibition, skeletal muscle begins to undergo progressive atrophy [80,81,83]. As muscle mass decreases, the total amount of anti-inflammatory myokines secreted also significantly diminishes, resulting in insufficient systemic anti-inflammatory signaling input [84].
4.7.3 Vicious cycle of muscle loss and uncontrolled inflammation
Muscle atrophy and uncontrolled inflammation constitute a typical positive feedback vicious cycle:
Muscle loss → reduced anti-inflammatory secretions: The decrease in muscle mass directly leads to a reduction in the secretion of anti-inflammatory factors such as IL-10, weakening the body's buffering capacity against inflammation [84].
Inflammatory factors activate Myostatin: Elevated inflammatory factors in circulation, such as TNF-α and IL-1β, can further upregulate Myostatin expression, exacerbating muscle breakdown [83,84].
Lipid-muscle axis imbalance: Muscle loss is often accompanied by the proliferation of adipose tissue, and adipose tissue (especially visceral fat) is the main source of pro-inflammatory factors (such as leptin and TNF-α). With this give-and-take, the endocrine balance of the body tilts from an "anti-inflammatory state" to a "pro-inflammatory state" [84].
Systemic inflammatory storm: Following the depletion of anti-inflammatory factors derived from muscle, the pro-inflammatory signals originating from sources such as senescent cells (SASP) and adipose tissue lose their balance. This leads to an excessive activation of the inflammation-glucocorticoid axis, ultimately resulting in a sustained increase in cortisol levels, triggering widespread immunosuppression and metabolic disorders [84].
Therefore, the decline of skeletal muscle is not only a loss of muscle function, but also a crucial link in the collapse of systemic anti-inflammatory defense lines [83,84]. As a direct effector and amplifier downstream of the GH-IGF-1 axis, it converts the attenuation of hormone signals into actual uncontrolled inflammation and systemic collapse [80,81,83].
4.8 Failure of conventional anti-inflammatory pathways in growth hormone.
In young individuals, the GH-JAK2-MAPK (JNK)-c-Jun-ST6GAL1 axis [100,101,104] maintains a low cellular responsiveness to inflammatory signals under steady-state conditions. Growth hormone (GH) promotes the moderate phosphorylation of the transcription factor c-Jun by activating the MAPK pathway, thereby driving the continuous expression of ST6GAL1. As a sialyltransferase, ST6GAL1 plays a crucial role in glycosylation modification of the cell membrane. Its upregulation reduces the affinity of membrane receptors for inflammatory ligands (such as TNF-α, IL-6), thus achieving a "feedforward inhibition" of inflammatory signals. This axis constitutes the anti-inflammatory steady-state pathway of GH.
However, as growth hormone (GH) secretion decreases during the aging process, the GH-MAPK-c-Jun-ST6GAL1 axis gradually becomes inactivated, and the cellular inflammatory response to stress is no longer controlled. At this point, excessive inflammatory factors induce the activation of the hypothalamic-pituitary-adrenal axis (HPA axis), promoting the release of glucocorticoids. Glucocorticoids induce the expression of MKP-1 through their receptor GR, leading to the dephosphorylation and temporary inactivation of MAPK family members. However, at the same time, GR itself activates the Ras → Raf → MEK → ERK pathway through Src and PI3K kinase signaling. This pathway cross-activates upstream kinases of JNK, resulting in the phosphorylation of JNK. Due to the extremely short half-life of MKP-1 protein, it is quickly degraded, while JNK activation persists, forming a "continuously activated but out-of-control JNK state".
Continuously activated JNK enters the nucleus, phosphorylates the Ser63 and Ser73 residues of c-Jun, and enhances the transcriptional activity of the AP-1 complex formed by the binding of c-Jun and c-Fos. The AP-1 complex binds to the TPA response element (TRE) of the ST6GAL1 gene promoter, thereby re-driving the expression of ST6GAL1. This process forms a "glucocorticoid-ERK-JNK-c-Jun-ST6GAL1 compensatory pathway", which temporarily maintains the expression of ST6GAL1 after the collapse of the GH axis, serving to buffer the inflammatory response. [102,103,104]
However, this compensatory pathway exhibits high stress-dependency and instability. Its continuous activation often accompanies increased oxidative stress and energy burden, ultimately leading to the formation of a persistent inflammatory state, known as "inflammaging" of aging tissues.
4.8 Co-regulation of immune aging by GH signaling and glucocorticoids
4.8.1 GH decline directly leads to thymus atrophy and T-cell output failure
Growth hormone (GH) is a core nutritional signal that maintains the structure and function of the immune center, the thymus. Thymic epithelial cells highly express GH receptors, and GH, by directly activating these receptors and in conjunction with locally produced insulin-like growth factor-1 (IGF-1), constitutes a key signaling axis that supports the proliferation of thymic epithelial cells and inhibits their apoptosis. This mechanism is crucial for maintaining the homeostasis of the thymic microenvironment and ensuring the continuous generation and output of naïve T cells. With the attenuation of GH pulsed secretion, this powerful nutritional and regenerative signal is withdrawn, leading to progressive atrophy and fat infiltration of the thymic epithelial network, and its ability to support T cell development declines sharply. Therefore, the decline of GH directly disrupts the "cradle" of the adaptive immune system, resulting in reduced output of naïve T cells and atrophy of T cell receptor diversity, which becomes the upstream driving force of immune aging. [107]
4.8.2 GH deficiency and cortisol synergistically drive abnormal fat distribution and immunosuppression
In the hormonal reprogramming of aging, the decline of growth hormone (GH) and the concomitant changes in glucocorticoid levels jointly constitute a synergistic axis that drives metabolic-immune imbalance. GH is a potent lipolytic hormone, and its decreased levels directly impair the mobilization capacity of limb and subcutaneous fat. Simultaneously, the compensatory increase in cortisol levels activates glucocorticoid receptors on visceral adipose tissue, strongly driving fat to accumulate centrifugally. This visceral obesity, shaped by the combination of "low GH-high cortisol", has profound pathological implications. Accumulated visceral fat is not only a manifestation of metabolic disorders but also an active inflammatory endocrine organ. It creates a systemic environment that continuously damages immune cells by releasing excessive free fatty acids and pro-inflammatory factors. These factors are directly toxic to lymphatic organs such as the thymus and can further disrupt immune homeostasis. Consequently, a vicious cycle driven by hormonal imbalance is established: decreased GH and increased cortisol → synergistically leading to visceral fat accumulation → accumulated fat creating an inflammatory microenvironment → directly inhibiting thymic function and systemic immune surveillance. This leaves the body not only deprived of the direct nutritional support of GH to the immune system but also trapped in a persistent state of immune suppression derived from abnormal fat distribution. [108]
4.8.3 Cortisol-induced fat distribution: from visceral infiltration to "occupying lesions" in lymphatic organsIn the context of the third cause of aging (decline of the GH-IGF-1 axis), the metabolic effect of cortisol is not only to promote the centripetal accumulation of fat, but also to systematically guide fat to infiltrate abnormally into immune-privileged areas, especially central lymphoid organs. This is a "space-occupying lesion" of the immune space directed by hormones.
1. Common molecular target: The glucocorticoid receptor (GR) orchestrates a potent adipogenic program by activating glucocorticoid receptors (GR) distributed throughout visceral adipose stromal cells and preadipocytes.
Notably, stromal cells in lymphatic organs such as the thymus and bone marrow also highly express GR. When cortisol levels persistently rise, the differentiation fate of these "soil" cells, which are supposed to support the development of immune cells, is forcibly reversed, transitioning from stromal cells supporting immunity to lipid-storing adipocytes.
2. Thymic fat infiltration: Physical collapse of the immune "cradle"
The thymus is the most typical victim of this process. With the attenuation of GH signaling, the intrinsic anti-adipogenic barrier of thymic epithelial cells becomes fragile. At this point, the continuous influx of cortisol acts as a direct "adipogenic switch," driving the transformation of endogenous stromal cells in the thymus into adipocytes. Simultaneously, the systemic metabolic environment shaped by cortisol, particularly high levels of free fatty acids, also floods into the thymus through blood circulation. This leads to abnormal lipid deposition within the thymic parenchyma, forming the typical "thymic fat infiltration." This is not simply a spatial occupation but a complete reconstruction of the thymic microenvironment: adipocytes replace epithelial cells, which are unable to provide cytokines such as IL-7 necessary for T cell development, ultimately resulting in the abrupt cessation of thymic output function. [109,110]
3. Bone marrow adiposification: A similar process of soil degradation at the "root" of the immune system
also occurs in the bone marrow, which is the origin of the immune system. Cortisol is the strongest inducer that drives mesenchymal stem cells in the bone marrow to differentiate into adipocytes rather than osteoblasts. An increase in its level directly leads to the expansion of bone marrow adipose tissue. These ectopically accumulated adipocytes not only occupy the physical space of hematopoietic stem cells and lymphocyte precursors, but also create an adverse microenvironment that inhibits hematopoiesis and lymphopoiesis by secreting specific adipokines (such as adiponectin and resistin), thereby fundamentally weakening the regenerative potential of the immune system. [111,112]
In summary, cortisol plays a role as a "spatial reshaper" in the third factor of aging. By collaborating with GH decline, it not only creates a pro-inflammatory "encirclement" around visceral fat, but also directly infiltrates the interior of immune organs. By initiating the adipogenesis process, it "hollows out" the thymus and bone marrow from within. This dual fat attack, from the outside to the inside and from function to structure, is the core foundation leading to the collapse of the immune system accompanied by metabolic decline.
4.9 Imbalance in GH/IGF-1 signaling ratio: From physiological synergy to pathological divergenceIn the context of the overall decline of the GH-IGF-1 axis (the third factor), a key pathological feature is the dissociation of GH and IGF-1 signals, which should normally vary in concert. As mentioned earlier (4.4, 4.6, 4.7), GH plays an irreplaceable direct role in ketone body production, skeletal muscle maintenance (via Decorin), and an inflammation-suppressing pathway independent of IGF-1 (the GH-MAPK-ST6GAL1 axis). The execution of these functions is neither dependent on IGF-1 nor achieved through its dominant mTOR pathway.
However, during the aging process, the levels of IGF-1 are maintained by various factors such as nutritional signals (e.g., high insulin), and their decline rate and extent often lag behind that of GH. This results in an imbalanced pattern where a "weak GH signal" coexists with a "relatively strong IGF-1 signal." Under this pattern, IGF-1-mTOR signaling, which is guided by the rhythmicity of GH pulses, transitions from a controlled, intermittent anabolic drive to a persistent low-level background activation. This rhythmicity-disrupted chronic mTOR activity, instead of effectively driving youthful anabolism, becomes one of the key factors exacerbating the accumulation of senescent cells by continuously inhibiting autophagy, leading to the chronic accumulation of senescent cells. [113]
4.10 The decrease in GH pulse triggers a reduction in IGF-1 signaling in dermal fibroblasts, leading to a decrease in collagen, with inflammatory factors collaborating to damage the skin and driving the expression of transforming growth factor to protect blood vessels.
Growth hormone (GH) alters the conformation of the fibroblast growth hormone receptor (GHR) through high pulses. This conformational change leads to a decrease in the local signaling of IGF-1 in fibroblasts. This signaling is independent of the background level of insulin-like growth factor (IGF-1) in the blood and is solely related to the local IGF-1 signaling dominated by GH. This process results in a decline in the elasticity and water-locking functions dominated by fibroblasts, due to the inhibition of collagen and elastin regeneration.
4.10.1 Systemic compensation under the principle of survival priority: from co-injury of skin and blood vessels to the inevitable fate of fibrosis
In the previous context, the decline of the GH-JAK2-MAPK (JNK)-c-Jun-ST6GAL1 axis can be regarded as a positive negative feedback mechanism aimed at maintaining survival.
When the inhibitory response of the GH-JAK2-MAPK (JNK)-c-Jun-ST6GAL1 axis to inflammatory factors in senescent cells decreases, it triggers an increase in the secretion of SASP levels from senescent cells.
The SASP contains various inflammatory factors that lead to a synchronized attack on structural proteins across tissues: collagen and elastin in the skin, as well as collagen and elastin in the vascular wall, are simultaneously affected and damaged ([50] Li et al., 2023). The former is related to the maintenance of appearance, while the latter directly sustains blood circulation, a fundamental life activity. However, in this process, inflammatory factors not only disrupt signals but also serve as regenerative signals. Interleukin-11 (IL-11), one of the SASP secreted by senescent cells, promotes the expression of collagen and elastin by fibroblasts and erroneously promotes fibrosis. At the same time, in the face of survival crisis, vascular damage initiates the secretion of stress signals that trigger the secretion of transforming growth factors (TGFs), which in turn further elevates the level of IL-11, forming a negative cycle.
4.10.2 Identification of survival crisis and initiation of dual compensatory pathwaysThe integrity of blood vessels is the bottom line for life sustenance. When inflammatory factors erode the vascular matrix, leading to relaxation of its collagen framework and rupture of the elastic network, the body recognizes this as the highest level of survival threat. To this end, it activates two fundamental compensatory pathways almost simultaneously:
Emergency anti-inflammation: By upregulating the secretion of glucocorticoids, systemic suppression of excessive inflammatory responses aims to reduce the continuous attack on vascular (and skin) structural proteins from the source ([50] Li et al., 2023). This is the "throttling" strategy.
Forced repair: Simultaneously, it significantly enhances the expression level of transforming growth factor-β (TGF-β), strongly driving fibroblasts to synthesize new collagen and elastin ([51] Ren et al., 2023; [52] Kuang et al., 2007), aiming to urgently repair the damaged vascular network and ensure structural support for the circulatory system. This is an "open source" strategy.
This "one suppression, one enhancement" response mechanism constitutes a protective loop aimed at maintaining vascular function in the short term, but it promotes vascular sclerosis during the aging process.
4.10.3 The Cost of Compensation and the Vicious Cycle of Fibrosis
However, under the continuous pressure of aging, this compensatory mechanism intended for emergency rescue loses precise regulation. The abundantly secreted TGF-β cannot distinguish between repair targets, and while attempting to "strengthen" blood vessels, it also acts indiscriminately on fibroblasts throughout the body, including the dermis of the skin ([51] Ren et al., 2023; [52] Kuang et al., 2007). This process leads to:
The mandatory repair procedure initiated to salvage the vital blood vessels simultaneously leads to excessive fibrosis and sclerosis of the skin, manifesting as loss of elasticity and deepening of wrinkles.
A vicious cycle of self-reinforcement is formed: inflammation damages blood vessels and skin → the body increases glucocorticoids and TGF-β to protect blood vessels → excessive activation of TGF-β leads to fibrosis of skin and multiple tissues → the microcirculation of fibrotic tissues is obstructed, and sclerosis intensifies → local hypoxia and metabolic disorders → triggering stronger inflammation → further damage to blood vessels and skin matrix.
Therefore, visible fibrosis and wrinkles in the skin, at a deeper level, are the direct cost paid by the body to delay the more fatal threat of vascular collapse ([50] Li et al., 2023; [51] Ren et al., 2023; [52] Kuang et al., 2007). This positioning reveals the common driving factors of skin aging and cardiovascular aging at the molecular level, and explains the "inflammation-fibrosis transition" as a typical example initiated by survival instincts but ultimately leading to common decline due to systemic dysregulation.
5. The fourth cause of aging: collapse of the antioxidant network
Mitochondria are the primary source of intracellular reactive oxygen species (ROS), and their functional decline inevitably leads to exacerbation of oxidative stress [68,69]. Simultaneously, the cell's own antioxidant defense network gradually collapses due to aging, rendering it unable to effectively eliminate excessive ROS, resulting in cumulative damage [70,71].
5.1 Functional imbalance between NRF1 and NRF2 and chronic oxidative stress
NRF1 and NRF2 are pivotal in maintaining the antioxidant network [[62,63,68,69]. Under normal conditions, NRF1 catalyzes the oxidation-reduction of oxidized glutathione (GSH) through glutathione reductase. Glutathione (GSH): GSH is the most important antioxidant in cells, with its core mechanism being its ability to directly antioxidantize the electron leakage site of mitochondrial complex III. However, during aging, due to decreased GH levels, central imbalance, and other factors, NRF1 expression continuously declines, leading to a decrease in glutathione reductase expression. GSSG cannot be effectively oxidized and reduced, weakening the antioxidant capacity of cells [65,70]. To compensate, cells tend to rely excessively on the stress-induced activation of NRF2 [65,71]. One of the downstream target genes of NRF2 is heme oxygenase-1 (HO-1), which releases free iron during heme decomposition [69,70]. Continuous activation of NRF2 leads to an increase in intracellular free iron levels, and iron is a key catalyst for catalyzing the Fenton reaction to generate highly toxic hydroxyl radicals (·OH) [66,67]. Therefore, long-term non-rhythmic high expression of NRF2 may, after reaching a critical point, exacerbate the collapse of the antioxidant network due to chronic oxidative stress downregulating NRF1, leading to a sharp decline in antioxidant function and forming a state of "chronic oxidative stress," further damaging mitochondria and other organelles [68,70]. From this point on, cause becomes effect, and effect becomes cause, with chronic oxidative effects forming a self-sustaining, self-cycling, and self-reinforcing steady state.
5.2 Chronic oxidative stress states lead to a state of low antioxidant capacity in the antioxidant network.
The antioxidant defense of cells constitutes a complex network, in which vitamin C (VC), vitamin E (VE), and glutathione (GSH) serve as key nodes [68,69,71]. This network is extremely fragile and susceptible to disruption by chronic oxidative stress.
VC is an important water-soluble antioxidant both inside and outside cells, and its function relies on the cycle between reduced ascorbic acid and oxidized dehydroascorbic acid [69]. During the aging process, due to the decline in GSH regeneration led by NRF1 and the oxidative stress caused by the atypical pathway of NRF2, the proportion of oxidized VC in cells increases [70]. This makes it difficult for exogenous reduced VC to enter cells and exert its function, while the endogenous antioxidant cycle is also hindered [68,71]. At the same time, NRF2 discharges oxidized glutathione (GSSG) into the blood, a process that requires VC in the blood to provide electrons to oxidize and reduce GSSG, thereby recycling the raw materials for glutathione production. If the reduced form in the blood is insufficient, this pathway is blocked.
During the aging process, the excessive expression of NRF2 leads to a decline in the regeneration of GSH through the NRF1-dominated pathway, resulting in the blockage of the NRF1-mediated GSSG regeneration pathway. NRF2 recovers raw materials through the oxidation-reduction process by releasing GSSG into the bloodstream via VC, which increases the oxidation ratio of VC in the blood. This change affects the ratio between vitamin C oxidized forms (dehydroascorbic acid and reduced ascorbic acid) in cells. This imbalance not only further weakens the VE regeneration function but also reduces the efficiency of the electron bridge maintained by lipoic acid, which mediates the transfer of electrons from VC to GSSG and oxidized VE [68,71], leading to a decrease in the stability of the entire antioxidant network [68,69,71]. Just as when reduced VC is abundant, glutathione can still promote glutathione regeneration through the mediation of lipoic acid even in the face of sudden oxidative stress, forming a backup mechanism. However, the increase in oxidized vitamin C in cells not only weakens the backup mechanism achieved by the electron transport mediated by lipoic acid but also requires glutathione to spend some resources to reduce oxidized vitamin C, leading to a weakening of the basic antioxidant function of cells.
5.3 The malfunction of the antioxidant network and the accelerated accumulation of senescent cells.
VC is not only an electron donor, but also the core regulatory hub and electron reservoir of the cellular antioxidant defense system [68,69]. Its function relies on the precise cascade reaction initiated by the abundance of reduced VC.
When the intracellular environment is in a highly reduced state, the concentration of reduced vitamin C (VC) is abundant. At this time, antioxidant resources are optimally allocated: glutathione (GSH) is released from other antioxidant tasks and more specifically enriched towards mitochondria, ensuring the antioxidant needs of this core energy organ [70].
In the cellular antioxidant network, when the proportion of reduced vitamin C (VC) is high, intracellular glutathione (GSH) no longer needs to participate significantly in the regeneration of VC, resulting in a "surplus" state of the antioxidant system [68,69,70]. At this time, the NADPH in the cell can direct more reducing power resources to the thioredoxin (Trx) system, which is used to maintain the reduced state of key signaling proteins, including PTEN [68,70,71,78].
PTEN, as a crucial phosphatase, relies heavily on the sulfhydryl (-SH) group in its active center being in a reduced state for its activity. During the aging process, due to the functional collapse of the upstream antioxidant network (especially the NRF1/2 axis and glutathione system), the level of intracellular oxidative stress rises sharply. This leads to the oxidation of the sulfhydryl group at the active site of PTEN, thereby causing it to lose its phosphatase activity [38].
One of the core functions of PTEN is to degrade phosphatidylinositol (3,4,5)-trisphosphate (PIP3), thereby negatively regulating the PI3K-AKT-mTOR signaling axis. Once PTEN is inactivated due to oxidation, the degradation of PIP3 is weakened, leading to the continuous activation of AKT, which in turn triggers the excessive activation of the mTORC1 signaling pathway [38,39].
The overactivated mTORC1 pathway not only drives abnormal anabolism but also exerts a strong suppression on the cell apoptosis program. Its core action lies in inhibiting the activation and oligomerization of BAX protein [40]. BAX is the core executor of the mitochondrial apoptosis pathway. Upon receiving pro-apoptotic signals such as p53, BAX translocates from the cytoplasm to the outer mitochondrial membrane and undergoes oligomerization, forming pores that lead to the release of cytochrome c, thereby initiating the caspase apoptosis cascade. However, the active mTORC1 pathway can interfere with this process through various mechanisms:
Indirect inhibition: mTORC1 can promote the synthesis of anti-apoptotic proteins (such as Mcl-1, Bcl-2), which can bind to BAX and trap it in an inactive state [40].
Direct regulation: The mTOR pathway can influence the phosphorylation status of BAX through downstream targets such as S6K1, directly hindering its translocation to the mitochondria and pore formation ability [40].
Thus, under severe injury signals that should trigger apoptosis, the excessive activation of mTOR inhibits the function of BAX, leading to the failure of the formation of the mitochondrial membrane's apoptosis pore. Cytochrome c cannot be effectively released, the caspase cascade reaction cannot be initiated, and damaged cells cannot be cleared through the normal apoptosis pathway [38-40]. Consequently, cells are forced into an abnormal survival state. They are affected by erroneous signals and exit the normal cell cycle, but their metabolism remains abnormally active, and they secrete a large amount of SASP factors, becoming "zombie cells" that continuously damage the tissue microenvironment. Since then, the accumulation of senescent cells has accelerated.
5.4 The dual regulatory mechanism of vitamin A (VA) and the inverse regulation of the PTEN-MTOR pathway.
Vitamin A (VA) plays a complex and crucial role in aging regulation, primarily through its two main forms: reduced retinol and oxidized retinoic acid. These two forms activate distinct signaling pathways, exerting bidirectional regulation on cell growth and apoptosis, and can be regarded as the "switch" of cell fate.
When vitamin A is maintained in its reduced state, it binds to the membrane receptor STRA6 through plasma retinol-binding protein (holo-RBP), triggering STRA6-dependent membrane signaling. This complex, upon binding, initiates the recruitment and activation of the receptor's intracellular domain JAK2, which in turn induces tyrosine phosphorylation of the STAT family transcription factor STAT5. The tyrosine-phosphorylated STAT5 can directly bind to the core component of mTORC1, Raptor. This binding contributes to the stabilization, assembly, and localization of the mTORC1 complex, thereby directly promoting its kinase activity and inhibiting the AMPK pathway. When vitamin A can maintain its reduced state, PTEN inhibits the level of mTOR due to sufficient redox conditions, forming an opposite negative regulation between the two to avoid excessive inhibition of mTOR in cells.
5.5 Oxidized VA (retinoic acid) promotes apoptosis through the RAR-p53/Bax pathway
In the central axis model, vitamin A (VA) in its reduced form has played a negative regulatory role in the PTEN-inhibited MTOR pathway. When cells are under oxidative stress and lack antioxidant resources, intracellular reduced vitamin A (retinol) is more prone to be oxidized into retinoic acid (RA). RA, as an active ligand, enters the nucleus and forms heterodimers with retinoic acid receptors (RAR)/RXR, binding to retinoic acid response elements (RARE) on target genes to induce a series of transcriptional programs related to cell cycle arrest and programmed cell death. The RA-RAR signaling pathway can upregulate tumor suppressor factors such as p19^ARF (human counterpart is p14^ARF), releasing the inhibition on MDM2, thereby stabilizing and accumulating p53 protein. Simultaneously, the RAR complex can recruit transcriptional coactivators (such as p300/CBP) to promote the acetylation and transcriptional activation of p53, further triggering the expression of p53 downstream pro-apoptotic genes (such as PUMA, NOXA, BAX). Activated p53 collaborates with upregulated BH3-only proteins (PUMA/NOXA) to trigger changes in mitochondrial outer membrane permeability, cytochrome c release, and the caspase cascade, thereby initiating mitochondrial pathway-mediated apoptosis. Therefore, under oxidative conditions, the molecular chain of RA-RAR → p19^ARF/MDM2 → p53 → PUMA/NOXA/BAX forms a clear apoptotic pathway from nuclear receptor signaling to mitochondrial execution. [27]
This pathway will form a reverse regulation of the PTEN oxidative modification and MTOR high expression pathway, maintaining the cell's self-apoptotic function through the mechanism of increasing P53 expression. However, this overall low antioxidant capacity state of cells not only affects VA but also VE status, and VE status can indirectly influence BAX activation by regulating the membrane oxidative environment and ROS signaling, thereby adjusting the apoptosis threshold: reduced VE helps maintain the cell repair window, allowing cells to have repair opportunities even under high P53 expression conditions; while an increased proportion of oxidized VE enhances ROS signaling, making BAX more easily activated, lowering the apoptosis threshold, and promoting cells to enter a senescent state more easily [70][71][72]. Such changes in oxidation status lead to increased P53 expression and weakened BAX inhibition.
This results in a cost for this reverse regulation: normal cells that have not yet reached the stage of apoptosis experience an increase in P53 levels, leading to the failure of VE, which inhibits BAX function, making the cells more prone to entering the apoptosis program rather than undergoing self-repair through P53.
VE is in an oxidized state, unable to maintain the fluidity of the cell membrane, and even more incapable of inhibiting the cellular bax pathway through its reduced state, leading to cells being more prone to entering the apoptosis program.
6. The fifth cause of aging: inflammatory factors hijack the glucocorticoid axis, leading to desensitization of signaling and decreased regulatory effects, thereby amplifying the negative effects of immunosuppression.
The chronic inflammatory state during aging (inammaging) is a key factor driving various age-related diseases. This article posits that the hijacking of the intrinsic anti-inflammatory mechanism, the glucocorticoid axis, by chronic inflammation will be the core mechanism that pushes chronic inflammation to its highest level of harmfulness.
6.1 Collapse of senescent cell clearance: failure of the glucocorticoid-senescent cell apoptosis axis
After the blockade of the p53-BAX main apoptosis pathway, the collapse of the glucocorticoid-senescent cell apoptosis axis, the body's backup pathway for clearing senescent cells, is not simply a functional decline, but rather a "malignant hijacking" by a persistent high-inflammatory environment. Driven by inflammatory factors, senescent cells reverse from a "state pending clearance" to a "pro-survival state", actively resisting and disrupting their own clearance program.
6.1.1 Construction and Startup Logic of Backup Paths
The physiological role of this pathway is to enforce the clearance program of senescent cells when the main pathway fails: ketone bodies inhibit HDAC through Kbhb modification [114], thereby enhancing the transcription of glucocorticoid receptors (GR) [115]. Cortisol can induce apoptosis through the mitochondrial pathway, with the mechanisms primarily involving Bax/Bcl-2 imbalance and the caspase cascade. It can initiate the reverse regulation of mTOR during the mTOR-inhibited apoptosis process. [116,117]
6.1.2 The collapse mechanism of this axis: High-concentration inflammation hijacks and collaborates with mTOR to orchestrate the collapse of this axis.
During the aging process, chronic inflammation is triggered by multiple factors throughout the body. As previously mentioned, factors such as the imbalance of T4/T3 ratio, changes in the nuclear factor kappa B (NF-κB) expression pathway, and disorders in glucose and lipid metabolism, as well as the senescence-associated stemness phenotype (SASP) released by senescent cells themselves, and the disruption of the antioxidant network leading to the oxidation of PTEN and increased expression of Mtor, will collectively act on this axis, becoming the fundamental force that disrupts this axis, rendering it into an inefficient working state.
Continuous stimulation by high concentrations of inflammatory factors, such as TNF-α, activates potent pro-survival signaling pathways like NF-κB. The initiation of this survival program directly leads to the disintegration of the clearance axis.
Core mechanism: The NF-κB pathway inversely suppresses the transcription and expression of the glucocorticoid receptor (GR). This implies that senescent cells downregulate their sensitivity to Mtor-negative regulatory hormones, thereby reducing cortisol's effects on the secretion of inflammatory factors and the inhibition of Mtor expression in senescent cells. [118]
Secondary mechanism: Simultaneously, the high inflammatory cytokine environment also upregulates key anti-apoptotic proteins such as c-FLIP and Bcl-2 through the TNFR2-TRAF2-NF-κB (and parallel PI3K-Akt) axis; this step not only blocks the mitochondrial apoptosis pathway of glucocorticoids but also inhibits the TNFR1-mediated death receptor apoptosis pathway, preventing the apoptosis of senescent cells from multiple pathways. [119]
When these two situations occur simultaneously, it inherently implies that the inhibitory effect of glucocorticoids on mTOR is suppressed, the damage caused by inflammation will be exacerbated, the beneficial effects of glucocorticoids will decrease, and the harmful effects of glucocorticoids will be intensified.
6.2 Inhibitory effects of elevated glucocorticoid levels on immunity and metabolism
Chronic inflammation, resulting from elevated inflammatory levels, prompts the organism to increase the secretion of glucocorticoids for survival purposes, thereby suppressing the level of inflammation within the organism. However, glucocorticoids themselves have inhibitory effects on the immune system, such as suppressing the normal immune functions of T cells and NK cells. In section 6.1, glucocorticoids have been hijacked by inflammatory factors. Considering the situation described in the second factor mentioned earlier, the glucocorticoid receptor (GR) has experienced a decline in transcription. At this point, the organism will further elevate the level of glucocorticoids to counteract inflammatory factors, amplifying the inhibitory effect on the immune system. However, due to the already decreased transcription of the glucocorticoid receptor GR, this regulation itself forms a weak and vicious cycle.
6.3 The glucocorticoid axis is hijacked by inflammatory factors, which trigger the collapse of calcium oscillation rhythms through the continuous activation of the CD150-IP3 pathway.
Chronic inflammation induces high expression of CD150, which, through the action of IP3, leads to continuous emptying of the endoplasmic reticulum calcium stores. This not only depletes calcium reserves but also alters the dynamics of calcium ions: the duration of calcium release is shortened, and the refilling time is prolonged [28][31]. This disorder directly inhibits the frequency and intensity of mitochondrial flash [2][3][4].
Mitochondrial flashes, as transient, high-energy ROS pulses, serve as crucial physiological signals for resetting local epigenetic modifications such as histone acetylation and phosphorylation, and constitute the energy basis for maintaining the "plasticity" and "youthfulness" of the cellular epigenome [1][3][4]. Flash failure implies the loss of driving energy required to maintain dynamic renewal in the epigenetic landscape. The final pathway for cells to maintain epigenetic homeostasis declines, further promoting epigenetic gene disorder based on decreased AKG levels and acetyl-CoA levels [1][2][6][7].
6.3.2 Transcriptional inhibition of non-essential amino acid synthesis by epigenetic disordersThe most direct consequence of epigenetic disorders is the complete loss of control over gene expression programs, with difficulties in demethylation of key enzyme genes involved in non-essential amino acid synthesis, linking epigenetic dysregulation to the collapse of downstream antioxidant systems [1][22].
Glycine metabolic pathway blockade: Glycine synthesis relies on serine hydroxymethyltransferase. Epigenetic disorders can lead to hypermethylation of its promoter and continuous inhibition of gene expression, disrupting the glycine-serine-cysteine-glutathione antioxidant raw material supply chain [22].
Taurine synthesis pathway dysfunction: The genes of key enzymes such as cysteine dioxygenase and sulfinate decarboxylase are silenced, leading to reduced taurine levels, weakened antioxidant stress resistance, and compromised mitochondrial membrane stability [1][22].
Imbalance in arginine metabolism: Abnormal expression of enzymes related to arginine metabolism, such as argininosuccinate synthase, affects nitric oxide metabolism and polyamine synthesis, exacerbating vascular endothelial dysfunction and cellular regeneration decline [1][22]. This process further intensifies microcirculatory disorders, making the first factor, which may not be a major factor affecting mitochondrial functionality during metabolic instability in some individuals, become a prevalent factor in the fifth factor.
6.4 Endogenous retrovirus release and autoimmune attack
Upon the malfunction of the epigenetic maintenance system, derepression of endogenous retroviruses (ERVs) occurs [10][11]. ERV RNAs and virus-like proteins form a "viral mimicry" in the cytoplasm, which is recognized as a foreign invasion by the cGAS-STING pathway, triggering a type I interferon response and an inflammatory cytokine storm [10][11].
This process is essentially an "autoimmune" clearance of the body's own cells, where sustained high levels of interferon signaling induce cell senescence or apoptosis, further occupying depleted transcription and translation resources, and stifling the potential for anabolism and repair [1][10][11].
6.4.1 Endogenous retrovirus release and CD150-inflammatory self-locking cycle
Epigenetic disturbances can trigger the derepression of transcription of endogenous retroviruses (ERVs) that should be methylated. These remnants of ancient viruses, which were integrated into the human genome during evolution, originally had their promoter regions tightly "sealed" by high DNA methylation. However, due to the combined effects of TET enzyme inactivation caused by AKG deficiency and abnormal methylation donor metabolism, the epigenetic seal on ERV elements is largely released. [6]
Subsequently, there is a significant accumulation of ERV RNA and virus-like proteins in the cytoplasm, forming a strong "viral mimicry" scenario. This state is recognized by the innate immune system within the cell, particularly the cGAS-STING pathway, as viral invasion. The type I interferon response and inflammatory cytokine storm triggered by this, which are far more intense than conventional chronic inflammation, essentially constitute an "autoimmune" attack by the body against its own cells.
At this point, a self-driven and self-locking inflammatory cycle has officially formed:
1. ERV release drives inflammation: The transcription of ERV and viral mimicry signals, through pathways such as cGAS-STING, lead to the production of a large amount of type I interferons (such as IFN-α/β) and pro-inflammatory factors (such as TNF-α, IL-6).
2. Inflammation enhances CD150 expression [28]: These high-intensity inflammatory signals, as the strongest stressors, further strongly and continuously upregulate the expression of CD150. As an "amplifier" and "actuator" of inflammatory signals, the expression level of CD150 is directly positively correlated with the intensity of inflammation.
3. CD150 exacerbates epigenetic collapse: Highly expressed CD150 leads to continuous emptying of the endoplasmic reticulum calcium store through the IP3 pathway, completely disrupting the rhythm of calcium oscillations. Disordered calcium signaling inhibits mitochondrial flicks, which are energy-critical pathways for resetting the epigenome.
Meanwhile, persistent calcium disorder itself can also directly interfere with the activity of epigenetic modification enzymes.
4. Epigenetic collapse leads to increased ERV release: The secondary collapse of the epigenetic maintenance system (initially caused by AKG deficiency and calcium flash inhibition) results in permanent loss of transcriptional control over ERVs and other repetitive elements. More ERVs are activated and transcribed, releasing stronger viral mimic signals.
5. Cyclic closure and system lockdown: stronger virus mimic signals → more severe inflammatory storms → higher levels of CD150 expression → more thorough calcium oscillations and epigenetic collapse → more extensive ERV release.
The positive feedback loop consisting of "ERV release - virus simulation - inflammatory storm - CD150 upregulation - epigenetic collapse" is a typical self-driven and self-amplifying malignant closed loop. It locks the system in a malignant state characterized by high inflammation, high interferon levels, and complete epigenetic deregulation. [10][11]
6.5 Calcium ions and stem cell functionCalcium ions (Ca²⁺) are one of the most important second messengers within cells. The dynamic changes in their concentration, known as calcium oscillations, play a central role in regulating the proliferation, differentiation, and apoptosis of stem cells. For adult stem cells, precise regulation of calcium signaling is crucial for maintaining self-renewal capacity and multilineage differentiation potential ([31]).
6.5.1 Regulation of endoplasmic reticulum calcium levels by the CD150-IP3 pathway
CD150 (also known as SLAMF1) is a receptor molecule expressed on the surface of hematopoietic stem cells (HSCs) and various immune cells ([28],[29]). Studies have shown that CD150 can indirectly affect endoplasmic reticulum (ER) calcium storage and release by regulating inositol 1,4,5-trisphosphate (IP3) levels ([30]). When CD150 expression is elevated, it promotes IP3 production, and IP3 binds to IP3 receptors (IP3R) on the ER membrane, leading to the release of calcium ions into the cytoplasm and thus reducing ER calcium levels.
Endoplasmic reticulum (ER) calcium ions constitute the primary calcium storage compartment within cells, and their stability is crucial for protein folding, lipid synthesis, and mitochondrial function maintenance. A sustained decrease in ER calcium can disrupt these vital activities and potentially activate the unfolded protein response (UPR), triggering cellular stress ([28],[30]).
Early differentiation-promoting stage
In the early stages of chronic inflammation, CD150 is induced to express, leading to a moderate increase in IP3 levels and a transient and limited release of calcium from the endoplasmic reticulum (ER). This calcium signal can serve as a "stress signal" to drive stem cells to undergo symmetric or asymmetric differentiation, replenishing damaged cells and manifesting as a compensatory regenerative response ([28],[31]). During this stage, the frequency and amplitude of calcium oscillations remain within an acceptable range for stem cells, supporting energy supply and mitochondrial flickering activity, thereby ensuring the maintenance of stem cell pluripotency.
Late suppression phaseWith the persistence of chronic inflammation, the CD150-IP3 pathway is chronically activated, leading to the continuous depletion of the endoplasmic reticulum (ER) calcium pool. The calcium oscillation pattern undergoes fundamental changes, including a sustained shortening of calcium release and a prolonged refilling time. Abnormal calcium signaling inhibits mitochondrial fission, which is a crucial energy event for resetting the epigenome and maintaining stem cell pluripotency. Ultimately, this disorder leads to a decrease in the self-renewal ability of stem cells, favoring symmetric differentiation, that is, the generation of two daughter cells that both proceed towards differentiation, resulting in the depletion of the stem cell pool and the loss of differentiation potential ([31]).
Regulation of CD150 expression and stem cell functionIn aging mouse models, CD150^high HSCs exhibit a differentiation bias towards the myeloid lineage and a decreased self-renewal capacity, whereas CD150^low HSCs are closer to the functional level of young HSCs. By reducing CD150^high HSCs or inhibiting CD150 expression, HSC function can be improved in aging mice, prolonging lifespan and restoring stem cell pluripotency ([13]). This discovery provides a potential strategy for reversing the decline in stem cell function and further supports the critical role of the CD150-IP3-Ca²⁺ pathway in stem cell aging.
6.5.2 Negative regulation mechanism and failure of vitamin K-CD150-IP3 axis
Vitamin K (VK)-dependent carboxylation of endoplasmic reticulum Gla proteins (ERGP) is a crucial mechanism for maintaining cellular calcium homeostasis [123]. Carboxylated ERGP serves as a calcium buffer within the endoplasmic reticulum, finely regulating store-operated calcium entry (SOCE) by limiting STIM1-Orai1 interactions, thereby preventing cytoplasmic calcium overload. However, inflammatory factors are stimulatory signals for CD150 [28,29,30]. During the aging process, as aging progresses, the concentration of inflammatory factors gradually increases, leading to a higher stimulatory signal for CD150 than its inhibitory signal. Ultimately, this results in an increased expression of CD150 during the aging process.
In the fifth factor of the inflammatory cytokine hijacking of the cortisol axis, inflammatory cytokines significantly promote stem cell senescence and inhibit its self-renewal function through the CD150-IP3 pathway.
7. The sixth cause of aging: accumulation of senescent cells and impaired immune clearance
After experiencing the oxidation of the PTEN pathway due to the fourth factor and the dysregulation of the glucocorticoid counter-regulation axis due to the fifth factor, the sixth factor acquires sufficient initiation conditions, becoming the primary driving factor of current aging, and reinforces the other four aging factors mentioned earlier through the fifth factor.
7.1 Theoretical construction of the clearance axis: A dynamic clearance model driven by NAD⁺ levels, integrating metabolic sensing and fate decision-making layers (NAD⁺-ketone body-P53 axis)
This hypothesis proposes that the clearance of senescent cells by the body is not a singular pathway, but rather an elaborate defense system that relies on cellular metabolic status and exhibits a strict hierarchical relationship and feedback logic. The effective operation of this system is central to maintaining tissue renewal and immune homeostasis; whereas its systemic collapse serves as a pivotal point for the accumulation of senescent cells and the driving of inflammaging.
The fate of senescent cells is primarily determined by their intracellular metabolic state, particularly the abundance of nicotinamide adenine dinucleotide (NAD⁺). NAD⁺ is a core coenzyme involved in energy metabolism and DNA repair, and its level constitutes a "metabolic switch" that determines the cell's fate.
NAD⁺ depletion and the "silent program": When senescent cells activate consumption pathways such as poly(ADP-ribose) polymerase 1 (PARP1) due to continuous DNA damage, leading to severe depletion of NAD⁺ [53, 54], although mitochondria maintain relatively normal function due to severe NAD⁺ depletion, the tricarboxylic acid (TCA) cycle enters a 'substrate-locked' state. At this point, β-hydroxybutyrate (BHB) entering the cell cannot enter and advance the TCA cycle due to the lack of the key coenzyme NAD⁺, and therefore cannot be effectively oxidized to provide energy for the cell.
Accumulates in the cytoplasm. High concentrations of β-hydroxybutyrate (BHB) directly inhibit the transcriptional activity of p53 through β-hydroxybutyrylation of Kbhb modification [57]. Downregulation of upstream signaling p53 leads to downregulation of the pro-apoptotic effector protein Bax expression, and the cell autonomous apoptosis program is silenced. Simultaneously, the expression of immune checkpoint molecules also decreases due to the downregulation of P53 expression, resulting in the loss of upstream signaling and entering a state of metabolic quiescence and low inflammatory signaling, termed as "immune-visible quiescence state". At this time, if the activity of immune cells, such as NK cells and T cells, is sufficient and their functional status is normal, achieving a balance where the "eat me" signal outweighs the "don't eat me" signal, these quiescent senescent cells can be normally killed.
NAD⁺ peak state and "activation/clearance program": As senescent cells enter a quiescent state and P53 levels decline, the expression of DNA repair enzymes, such as poly(ADP-ribose) polymerase 1 (PARP1), which primarily metabolizes NAD+, decreases. The level of NAD+ in senescent cells gradually recovers over time. The inability of β-hydroxybutyrate (BHB) to enter and progress through the tricarboxylic acid cycle due to the lack of NAD+ is reversed, allowing BHB to re-enter and oxidize in the tricarboxylic acid cycle. One of the core signaling effects of this metabolic flow is a significant increase in the level of the key intermediate metabolite succinyl-CoA. As a substrate, succinyl-CoA drives the succinylation modification of the γ regulatory subunit of the AMPK complex. This post-translational modification directly alters the conformation of AMPK, enabling it to effectively activate AMPK even in the context of metabolic signaling disorder in senescent cells and in the absence of classical AMP/ATP ratio signaling, which would otherwise induce AMPK expression [56]. Activated AMPK directly stabilizes and enhances the activity of p53 through phosphorylation [55]. Activated p53 initiates a decisive positive feedback loop: on the one hand, it promotes the transcription of repair genes (temporarily consuming energy), and on the other hand, it strongly upregulates Bax, driving the cell towards apoptosis via the mitochondrial pathway.
If the anti-apoptotic mechanisms within senescent cells (such as high expression of Bcl-2 and mTOR inhibition of Bax oligomerization) lead to "punch-out failure" of apoptosis, and the cells fail to self-destruct, invariant natural killer T cells (iNKT cells) will undergo immune replenishment. They enhance the oxidative metabolism of senescent cells through the tricarboxylic acid cycle, rapidly increase the presentation of CD1d antigens on senescent cells by driving the AMPK process, and recruit iNKT cells for immune clearance, thereby eliminating the senescent cells that have entered an active state. [32]
7.2 Eliminating systematic collapse of the axis: From dynamic equilibrium to dual-line collapse
In the systemic pathological environment of aging, the aforementioned sophisticated hierarchical defense system undergoes a comprehensive breakdown.
1. Depletion of power sources and paralysis of professional forces
The chronic inflammation associated with aging leads to the continuous negative feedback of the CD38 molecule-NAD+-AMPK pathway on the nuclear factor kappa B (NF-κB) pathway in cells throughout the body. That is, while inflammatory factors activate NF-κB, they also activate CD38, which enhances the AMPK pathway by hydrolyzing NAD+ and inhibits the activation of downstream pathways of NF-κB. However, this also leads to a decrease in NAD+ levels [8, 41, 53].
Metabolic exhaustion of iNKT cells: The proliferation and functional maintenance of iNKT cells rely on the activation of inflammatory cytokines (such as IL-6, TNF-α). Inflammatory cytokines can upregulate the expression of the differentiation cluster 38 (CD38) molecule on the surface of iNKT cells. CD38 generates ADPR and cADPR by consuming NAD+, thereby activating the AMPK pathway and promoting the proliferation and activity of iNKT cells.
This is a compensatory mechanism aimed at enhancing immune function by consuming energy molecules.
However, during the aging process, due to the decline in mitochondrial function, the rate of NAD+ regeneration slows down, and the continuous consumption of CD38 rapidly reduces the NAD+ pool to a critical point. Once NAD+ is depleted, this compensatory pathway becomes ineffective, leading to a decrease in the activity of iNKT cells and a weakened ability to eliminate senescent cells. [8]
2. Instability of silent window and malfunction of backup mechanism
Dysregulation of glycolipid metabolism leads to unstable and overall decreased ketone body levels, disrupting the stability of the "silent state". During the decline in GH levels, the metabolic switch in somatic cells results in a decreased proportion of oxidative phosphorylation and an increased proportion of glycolysis, leading to elevated blood lactate levels. Both ketone bodies and lactate can enter cells through monocarboxylate transporters (MCTs). Ketone bodies directly inhibit the transcriptional activity of p53 through β-hydroxybutyrylation (Kbhb) modification, while lactate, on the contrary, inhibits immune clearance by affecting the expression of immune checkpoint molecules, resulting in the inhibition of T cells and NK cells by immune checkpoint inhibitors. This leads to a decline in the clearance function of senescent cells, where the "don't eat me" signal outweighs the "eat me" signal.
7.2 The interplay between NAD⁺ levels and the apoptosis program: pathway selection in the PARP1-AMPK axis
The determination of cell fate depends on the precise interplay between its internal energy and signaling pathways. This hypothesis reveals that upon receiving severe DNA damage signals, the intrinsic apoptosis program of senescent cells initiates the vigorous activation of the PARP1 gene. As a "molecular sensor" of DNA damage, the extraordinary activity of PARP1 rapidly depletes the intracellular NAD⁺ pool, aiming to label and initiate repair by synthesizing poly-ADP ribose chains [53][54].
This seemingly repair-oriented process indirectly initiates a crucial "backup plan": the sharp decline in NAD⁺ triggers a surge in the AMP/ATP ratio, thereby allostatically activating AMPK. The deep strategic purpose of this temporary AMPK activation in the context of apoptosis is to assist the p53-Bax axis in fulfilling its core apoptotic mission - that is, the energy crisis signal provided by AMPK can enhance the activity of p53 and promote the effective oligomerization and "punching" of Bax protein on the mitochondrial membrane, initiating programmed cell clearance and achieving "self-termination" of cells [55].
However, this precise apoptosis preparation program is easily disrupted in the macroscopic context of aging. The core obstacle stems from the abnormally high expression of the mTOR pathway. The overactive mTORC1 stifles this apoptosis initiation through the following dual mechanisms [55]:
Indirect blockade: mTORC1, as the master switch of protein synthesis, significantly drives the synthesis of anti-apoptotic proteins such as Mcl-1 and Bcl-2. These proteins can bind to Bax, firmly sequestering it and preventing its translocation to the mitochondria and oligomerization.
Direct interference: The downstream signaling of mTOR may directly affect the phosphorylation status of Bax, thereby directly hindering its pore-forming ability.
"Bax puncture failure" implies that the apoptosis program, meticulously orchestrated by the PARP1-NAD⁺-AMPK axis, is disrupted by mTOR at the final execution stage. Instead of undergoing apoptosis as intended, the cell becomes trapped in an abnormal survival state, with intracellular NAD⁺ being depleted.
Here exists a fundamental metabolic pathway bifurcation: in cells with normal NAD⁺ levels, incoming ketone bodies (β-hydroxybutyrate, BHB) can smoothly enter mitochondrial metabolism, which increases the AMP/ATP ratio, thereby promoting the continuous activation of AMPK [56]. The activated AMPK can synergistically enhance the p53 signaling pathway, significantly increasing the sensitivity of cells to internal damage signals, and precisely preparing for subsequent possible repair or clearance decisions.
However, in senescent cells, this pathway is completely shut down due to the depletion of the NAD⁺ pool by PARP1. When ketone bodies enter such cells, they cannot enter the tricarboxylic acid cycle due to the lack of NAD⁺, a crucial coenzyme, and thus accumulate abnormally in the cytoplasm [58]. The accumulated ketone bodies then enter the epigenetic regulatory pathway and strongly inhibit the transcriptional activity of p53 through lysine β-hydroxybutyrylation modification [57].
This modification reverses the cell fate from a stress state of "attempting to initiate apoptosis but failing" to a "silent" state where p53 activity is suppressed and metabolism is quiescent. Therefore, from the initiation of NAD⁺ depletion by PARP1 to the accumulation of ketone bodies triggering Kbhb modification of silent p53, a passive stabilization mechanism is formed in senescent cells after the apoptosis program is blocked by mTOR. This also constitutes the complete molecular pathway of the "silent mechanism of senescent cells" in this theory [53-58].
8.1 Gender-specific hormone network connectivity as the second and third factorsThis model reveals that sex hormones constitute a key differential physiological background connecting the second and third causes of aging. The core lies in the differential regulation of fat distribution, inflammation initiation, and the growth hormone axis by androgens and estrogens, ultimately leading to significant temporal and phenotypic differences in the aging pathways of both sexes [42].
Male pathway: vicious cycle of testosterone-abdominal fat accumulation-leptin resistance axis
Under the dominance of androgens, fat distribution exhibits a typical visceral centripetal accumulation trend [42]. When the mitochondrial fatty acid oxidation capacity driven by secondary factors decreases, the influx of plasma free fatty acids into abdominal fat cannot be effectively cleared, resulting in functional accumulation within the abdominal cavity. This accumulated adipose tissue is not an inert energy depot, but rather an active endocrine organ that continuously releases pro-inflammatory factors such as TNF-α and IL-6, initiating local and systemic low-grade inflammation [44][].
This inflammatory environment performs a triple destructive task:
Localized lipolysis amplification: Within abdominal adipose tissue, inflammatory signals exacerbate localized lipolysis by activating hormone-sensitive lipase, generating more free fatty acids and releasing them into the bloodstream, forming a futile cycle of lipolysis-fatty acid spillover-re-esterification, further elevating systemic free fatty acid levels [44][47].
GH axis suppression: Elevated plasma free fatty acids (FFAs) and the inflammatory cytokines derived from them jointly act on the hypothalamic arcuate nucleus, directly suppressing the amplitude and frequency of pulsatile growth hormone secretion from the anterior pituitary gland by inhibiting GHRH and stimulating somatostatin (SST) release [43].
Subcutaneous fat lipolysis and cortisol compensation: Inflammatory factors in the circulation simultaneously induce lipolysis in relatively healthy subcutaneous adipose tissue. This process activates the hypothalamic-pituitary-adrenal axis, compensatorily increasing cortisol secretion in an attempt to control uncontrolled inflammation, but cortisol also promotes lipolysis [44][47][48][49].
Meanwhile, the continuous presence of androgens continues to reinforce the physiological tendency of fat transfer to the abdomen. This process exacerbates systemic aging from two dimensions:
Decreased adiponectin and impaired skin barrier: It reduces the secretion of adiponectin, which has beneficial metabolic effects, by weakening the total amount and function of subcutaneous adipose tissue [45]. Adiponectin positively promotes the synthesis of ceramide in the stratum corneum of the skin, and its decreased level directly impairs the integrity and moisturizing ability of the skin lipid barrier.
Elevated oxidative stress: The persistent inflammatory burden of abdominal fat, coupled with the decreased buffering capacity of the subcutaneous layer to inflammatory factors due to fat reduction, collectively amplifies the body's oxidative stress index [44].
A more critical turning point lies in the fact that excessive transfer and accumulation of adipose tissue in the abdomen can lead to excessive and non-rhythmic secretion of leptin [43]. At this point, the body's energy regulation logic underwent a fundamental paradigm shift: from the negative feedback mode of "prototype fat - moderate leptin - stimulating GH secretion to promote fat oxidation" in a healthy young state, it reversed to the leptin resistance mode of "pathological abdominal fat - excessive leptin - inhibiting GH secretion by continuously activating hypothalamic SOCS3 signaling and stimulating SST expression". This hormone network reconstruction, jointly grounded by the background of androgen and the decline in mitochondrial lipid metabolism, precisely connects the second and third factors in males into a self-driven malignant pathway [42][43][44][45].
Female pathway: Epigenetic sheltering of the growth hormone axis by estrogen
In stark contrast to the male pathway, females are effectively shielded by estrogen during their reproductive years. Estrogen, by maintaining a healthier subcutaneous fat distribution and its inherent anti-inflammatory properties, significantly shields the GH secretion axis from the direct impact of early lipid metabolism disorders and inflammatory factors [42][46].
Therefore, in early aging, the connection between the second and third factors in females relies more on a more direct energy crisis signal - the decrease in ATP production rate directly caused by mitochondrial dysfunction, which activates AMPK by increasing the AMP/ATP ratio. Although AMPK can indirectly affect mTOR and autophagy in energy sensing, it has not yet exerted strong epigenetic silencing pressure on the transcription promoter region of the GH gene [46].
This implies that in females, the initiation of the third factor is more focused on a global decline in energy availability, rather than the early and intense epigenetic silencing of the GH gene and systemic upregulation of SST levels, which are mediated by the inflammation-leptin axis, as observed in males [46].
Self-locking mechanism of the aging pathway: interlocking cascade amplificationThe aging pathway depicted in this hypothesis is internally driven by a series of closely interconnected and self-reinforcing positive feedback loops. Once the aging process is initiated, these loops act like a series of carefully arranged dominoes, with each "result" precisely transforming into the "cause" for the next step, continuously amplifying the upstream initial signal in reverse, ultimately forming an unbreakable and continuously deteriorating system state. This precise self-locking mechanism is the core driving force that propels bodily functions from initial decline towards sustained collapse.
Self-locking mechanism of aging pathway: interlocking cascade amplification
The aging pathway depicted in this hypothesis is driven by a series of self-reinforcing positive feedback loops. This intricate self-locking mechanism ensures that once the aging process is initiated, it is akin to toppling a series of dominoes, where each "result" precisely becomes the "cause" for the next step, continuously amplifying the upstream initial signal in reverse, ultimately driving the body's functional decline from an initial decline to a sustained collapse.
The first link: Establishment of initial driving force - decline in mitochondrial functionality
The aging process is initiated by a fundamental internal driving force, namely the decline in mitochondrial function (the primary cause). Its core lies in the "epigenetic inertia" of key genes such as PPARα, leading to reduced oxidative phosphorylation efficiency and an imbalance in ROS production and clearance. This positioning as the initial event of the upstream "primary cause" creates the necessary pathophysiological conditions for all subsequent cascade reactions.
Second link: Energy metabolic collapse and initiation of systemic disorder
As the second causative factor of aging, dysregulation of glucose and lipid metabolism is directly triggered by the first causative factor. The decline in mitochondrial fatty acid oxidation capacity leads to reduced lipid metabolism, resulting in decreased free carnitine levels and the accumulation of cytotoxic acylcarnitines. Simultaneously, the energy supply is compelled to shift towards glucose oxidative phosphorylation, leading to an increased proportion of glucose metabolism and directly resulting in the accumulation of advanced glycation end products (AGEs).
AGEs activate the NF-κB pathway through their receptor RAGE, initiating chronic mild inflammation. Simultaneously, the oxidative stress generated during this stage disrupts vitamin E homeostasis, and the increased proportion of its oxidized state competitively inhibits the synthesis of coenzyme Q10 via the SREBP pathway. Thus, the driving factors of aging have evolved from a simple "decline in mitochondrial function" to a complex pathological state of "decline in mitochondrial function + impaired synthesis of coenzyme Q10 + chronic mild inflammation".
Key Advancements: The chronic mild inflammation generated during this stage is sufficient as an early signal to initiate the upregulation of CD150 expression [28]. CD150, through the IP3 pathway, leads to continuous calcium efflux from the endoplasmic reticulum, initially disrupting the calcium oscillations in stem cells.
This results in mild inhibition of stem cell self-renewal and asymmetric differentiation at an early stage, with the telomere length of newly generated daughter cells already showing a downward trend. This indicates that the regenerative potential of the system has been subtly compromised before significant decline in the hormone axis. [12][13]
Third ring: The loss of regulatory center and global deterioration
The chronic mild inflammation and metabolic disorder environment (decreased ketone bodies and accumulation of AGEs) shaped by the second factor begin to systematically impact the core regulatory hubs, leading to the decline of the GH-IGF-1 axis (the third factor). The deficiency of AKG caused by decreased ketone bodies may trigger epigenetic silencing of the GH gene itself. The decline in GH levels produces a catastrophic cascade of events:
The reduction in the expression of its downstream key target NRF1 directly leads to a decrease in the number of mitochondria, exacerbating the energy crisis of the primary factor from the "hardware" level.
By attenuating the activity of deiodinase, it co-regulates the decrease in the conversion ratio of T4 to active T3. The deficiency of active T3 not only exacerbates the carnitine synthesis disorder (the second factor of reverse deterioration), but also leads to the decline in its ability to regulate protein quality control through the FOXO3a-ULK1 autophagy axis, resulting in the accumulation of misfolded proteins and protein aggregates.
Thus, the first vicious cycle across multiple links is formed: metabolic disorders and mild inflammation (the second factor) not only solidify endocrine decline (the third factor), but the latter's functional decline, in turn, exacerbates the upstream crisis by impairing mitochondrial biosynthesis, protein quality control, and intensifying the decline in antioxidant substrate synthesis triggered by CD150-mediated stem cell function inhibition and epigenetic gene disorder, as well as reducing the expression of inflammation-suppressing related genes, precisely reinforcing and exacerbating the upstream crisis.
Meanwhile, the release of endogenous retroviruses exacerbates the epigenetic gene disorder induced by CD150, forming a self-reinforcing and self-sustaining trend.
Male-specific self-locking mechanism: mandatory suppression of growth hormone by the abdominal fat-leptin axis
In the malignant transformation from the second cause to the third cause, as described in Section 8.2.7 "The Male Pathway", the background of male hormones and the decline in mitochondrial lipid metabolism jointly drive the transfer of fat to the abdomen and functional accumulation. The "male hormone-abdominal fat accumulation-leptin resistance axis" triggered by this process constitutes a powerful self-locking cycle.
Specifically, as described in the previous mechanism, the persistent inflammatory burden and excessive lipolysis of abdominal fat lead to excessive and non-rhythmic secretion of leptin. This causes a fundamental paradigm shift in the body's energy regulation logic: from a negative feedback mode in a healthy state to a leptin resistance mode characterized by "pathological abdominal fat - excessive leptin - inhibition of GH secretion through continuous activation of hypothalamic SOCS3 signaling and stimulation of SST expression".
This mechanism converts the metabolic disorder of the second cause into a precise and sustained suppression of the third cause (GH-IGF-1 axis) without any loss, perfectly explaining why the aging process in males often exhibits a "cliff-like" characteristic earlier, and closely connects the second and third causes of males into a self-driven malignant pathway.
The fourth link: collapse of the defense system and imbalance of the inflammation-immune axis
With the decline of the GH-IGF-1 axis (the third factor), leading to a continuous decrease in NRF1 expression, the antioxidant network of cells collapses (the fourth factor).
To compensate, cells non-rhythmically overactivate NRF2, which instead exacerbates oxidative stress by elevating free iron levels. The sharply increased oxidative stress directly oxidizes and inhibits PTEN protein, thereby releasing the brake on the mTOR pathway. Overactivation of mTOR inhibits autophagy. Simultaneously, the depletion of the NAD+ pool leads to the functional paralysis of iNKT cells, allowing senescent cells to accumulate and secrete a large amount of SASP factors, escalating mild inflammation to high-intensity inflammation. The body's compensatory increase in cortisol ultimately transforms into a broad-spectrum inhibitor of immunity and metabolism, forming a second cross-link vicious cycle.
The fifth circle: The steady-state lock of system collapse
is trapped in the high inflammation and high cortisol internal environment created by the immune collapse triggered by the inflammation-glucocorticoid axis (the fifth factor). The regeneration and repair processes of various tissues throughout the body are strongly inhibited, and systemic phenotypes such as muscle loss and skin aging begin to emerge. At this point, all the damages in the first four circles - energy crisis, metabolic disorder, hormone decline, oxidative damage, and immune suppression - are continuously amplified against this adverse background. The entire body system gradually enters a state of "steady-state lock": that is, all internal environmental parameters are maintained at a new equilibrium point of low function, high consumption, and high inflammation, and they are interdependent and mutually supportive, jointly resisting the system's return to a young, healthy steady state.
In summary, the essence of this self-locking mechanism lies in its ability to weave five independent "aging factors" into a dynamic, self-energizing network. From the initial decline in mitochondrial function to the ultimate collapse of systemic functions, each step reinforces and exacerbates the previous one through precise reverse amplification, ultimately driving the entire living system along a predetermined path, steadily and irreversibly sliding towards decline. Understanding this self-locking mechanism provides a crucial theoretical map for developing systemic anti-aging strategies that can simultaneously interrupt multiple key links.
10 Synthesis and Outlook: Potential Targets for Anti-Aging Strategies
Intervention Strategies: From Theory to Practice
Based on the aging pathway model proposed in this article, we can identify a series of potential anti-aging intervention targets. These strategies aim to block or reverse the decline in mitochondrial function and the cascade reactions it triggers from upstream, thereby achieving the goal of systematically delaying aging.
10.1 Core metabolic intervention: Precision protein ketogenic diet in synergy with circadian rhythm
Based on this theory, a potent intervention strategy is to construct a dietary structure of "precise protein-moderate fat-very low carbohydrate" and precisely synchronize it with circadian rhythms. The core lies in concentrating adequate but not excessive protein intake during the nighttime window.
The underlying mechanism of this strategy lies in its clever avoidance of the continuous activation burden of traditional "high-protein" diets on the mTOR pathway, and instead, it utilizes the inherent physiological rhythms of the human body to perform multi-target resetting on the aging cascade:
Nighttime precision protein intake in synergy with GH rhythm: Growth hormone (GH) secretion peaks during deep sleep at night. Intake of precise and adequate protein (such as high-quality protein rich in lysine and arginine) during this period aims to provide the most critical substrate and signal for the pulsatile secretion of GH, while avoiding the sustained, non-rhythmic activation pressure of excessive amino acids on the mTOR pathway. This approach can most effectively combat the third cause of aging (decline of the GH-IGF-1 axis), providing a powerful and clean hormonal driving force for upstream reactivation of NRF1 expression and mitochondrial biosynthesis.
Multi-target regulation of extremely low carbohydrate intake: Maintaining an extremely low carbohydrate intake throughout the day, its core function lies in:
Initiating fat metabolism: The generated ketone bodies (especially β-hydroxybutyrate) can directly "silence" senescent cells through epigenetic modification, inhibiting their secretion of SASP, thereby reducing the inflammatory storm caused by the sixth factor of aging (accumulation of senescent cells) from its root.
Eliminating lipid inflammation drive: Extremely low insulin levels can eliminate its continuous activation of Δ-6 desaturase, thereby blocking the upstream conversion of linoleic acid to a series of strong pro-inflammatory metabolites such as arachidonic acid, precisely cutting off a major class of key lipid inflammation sources in glucose-lipid metabolic disorders (the second factor).
Metabolic Switching in Chronological Fasting: The combination of a prolonged fasting window during the day and precise nutritional supplementation at night forms a powerful metabolic rhythm. This not only periodically activates AMPK, elevates NAD+ levels, and stimulates autophagy, but more importantly, it forcibly switches the systemic metabolic pattern from a pro-inflammatory mode reliant on glucose metabolism to a repair and cleansing mode reliant on fatty acid oxidation and ketone body metabolism.
Cold stimulation: Promotes the conversion of T4 to T3, improves the ratio of pro-inflammatory and anti-inflammatory factors in the gene profile downstream of nuclear factor kappa B (NF-κB), inhibits inflammation, and enhances stem cell function. This pathway can lead to axis deviation, resulting in the formation of a cold-induced mitochondrial complex III electron flow leakage. When NRF2 expression inhibits the oxidative damage caused by electron flow leakage, it simultaneously suppresses the expression of MTOR through redox PTEN, alleviating the inhibition of AMPK by MTOR. However, this process can trigger axis deviation. This negative effect can be eliminated by drug-induced reverse regulation of the axis, which can simultaneously correct the axis. Due to cold stimulation, the direction of axis deviation gradually becomes unified, while the gap is caused by abnormal self-maintenance mechanisms due to cold, resulting in weak expression of PPARα.
Since then, intervention strategies have promoted an increase in the proportion of lipid metabolism by inducing hunger during a precise protein diet, thereby improving the second, third, fourth, and fifth factors. Combined with cold exposure, the third and fifth factors are further improved. At this point, by increasing the activity of the immune system through oxygen inhalation and prolonging the quiescent state of senescent cells through ketone bodies generated by hunger, the "eat me" signal becomes stronger than the "don't eat me" signal, thus improving the sixth factor.
10.2 Four-axis regulatory drugs.
Conjugated linoleic acid enhances PPARα, lipoic acid enhances PGC-1α, leucine enhances MTOR, and medium-chain triglycerides enhance AMPK. The regulatory drugs selected for the tetraple axis are all weak regulators. However, strong AMPK activators such as metformin are not suitable as micro-regulators due to their low tolerable doses, excessive efficacy, and relatively high side effects.
The descending pathway of the four-axis system can be restored to its central axis rhythm through the mutual regulatory mechanism within the four axes, by using weak activators to exert inverse regulation.
Research hypothesis: Confirm the initiation of the primary cause of aging by verifying "structural oxygen supply deficiency"
To verify the hypothesis that "mitochondrial dysfunction is the primary cause of aging" at minimal cost, we should not delve into the downstream complex signaling pathway network, but rather directly address the root of the problem.
Preliminary theoretical verification - constructing a logical chain based on widely recognized insights and classical researchThis approach aims to construct a complete causal logic chain by connecting knowledge recognized across multiple disciplines and supported by classic literature.
The logical argumentation chain and the known evidence are as follows:
Red blood cell deformability is a prerequisite for unobstructed microcirculation
Known evidence: Red blood cells (RBCs) must deform in order to pass through capillaries smaller in diameter than themselves, which is a fundamental principle of microcirculatory physiology. Fedosov et al. revealed through multiscale modeling that RBCs rely on the coordination of highly elastic membrane structures and fluid dynamics to maintain normal fluidity when passing through narrow capillaries [20]. Musielak's review of measurement techniques for RBC deformability further confirmed the crucial role of this characteristic in maintaining blood flow patency [21].
Capillary size constitutes a physical constraint
Known evidence: The diameter of capillaries in specific areas of the human body (such as the choroid plexus of the eye and the glomerulus) is indeed 4–6 μm, while the diameter of red blood cells is approximately 7–8 μm. This size difference determines the necessity for red blood cell deformation [20,21]. This physical structural difference implies that a decrease in the deformability of red blood cells will directly lead to an increase in microcirculatory perfusion resistance.
Reduced erythrocyte deformability leads to microcirculatory disorders and hypoxia
Known evidence: Under pathological conditions such as diabetes and metabolic syndrome, it has been clearly observed that decreased erythrocyte deformability is directly related to microcirculatory blood flow stasis and reduced tissue oxygen partial pressure. Ebenuwa et al. found in clinical studies that the deformability of red blood cells in diabetic patients significantly decreased, and was positively correlated with increased blood viscosity and limited tissue perfusion [17]. Meanwhile, Williams et al. reviewed and pointed out that diabetes-related oxidative stress and glycation reactions can lead to cross-linking of erythrocyte membrane skeleton proteins, thereby exacerbating erythrocyte dysfunction and local hypoxia [16]. This establishes a causal relationship of "erythrocyte function → microcirculatory perfusion → oxygen supply".
Hypoxia can selectively and rapidly inhibit mitochondrial function
Known evidence: Numerous cell biology studies have confirmed that hypoxia can significantly reduce the efficiency of oxidative phosphorylation (OXPHOS) within minutes to hours through mechanisms such as disrupting the chemical gradient of the mitochondrial electron transport chain and inhibiting ATP synthase activity. In a classic study, Gnaiger et al. found that under hypoxic conditions, mitochondrial phosphorylation efficiency decreases and respiratory rate significantly weakens, and this process occurs before structural damage [18]. This indicates that hypoxia has a rapid and selective inhibitory effect on mitochondrial function.
Orbital ecchymosis is a visible indication of microcirculatory disturbance
Known evidence: The lightness or darkness of skin color depends on the oxygenation status of blood flow in capillaries. Blood flow stasis and increased deoxyhemoglobin can cause the skin to appear bluish-purple, which is a known sign in diagnostics (such as cyanosis). Hartley et al.'s review of "spontaneous periorbital ecchymosis" points out that the skin in the orbital region is thin and rich in capillaries, and abnormal blood flow or local capillary rupture can cause persistent bluish-purple manifestations [19]. Therefore, persistent orbital ecchymosis can be reasonably inferred as a poor local microcirculation and oxygenation status.
Argumentation Summary
Thus, we have completed a logical chain supported by public knowledge and classic research:
erythrocyte deformability (known function) → passage through capillaries (known structural constraints) → impact on microcirculatory perfusion and oxygen supply (known pathophysiology) → inhibition of mitochondrial OXPHOS efficiency (known cellular effect).
Orbital ecchymosis provides a visible and logical clinical anchor for the initiation of this chain. This path, where "structural oxygen supply deficiency is sufficient to drive mitochondrial functional decline," is highly reasonable.
However, the existing orbital bruising fails to accurately describe the subject. Is it caused by abnormalities in nitric oxide, red blood cell count, red blood cell deformation, or red blood cell oxygenation driven by the mTOR pathway, or is it due to weak expression of the AMPK pathway, reduced ATP energy production, and comprehensive decline in cellular motility → microcirculatory failure → venous congestion and pigment deposition, or is it a result of other pathways? The verification of the existence of recessive hypoxia itself only demonstrates the existence of a necessary precursor for mitochondrial dysfunction, but it fails to accurately analyze the underlying mechanism. Follow-up research is needed to further investigate.
Empirical verification: Conduct a rigorous controlled experiment based on visible signs of microcirculatory disorders such as scleral hypoxia and orbital ecchymosis to demonstrate whether microcirculatory disorders can spread to multiple locations throughout the body.
As previously mentioned, one of the earliest visible indicators of microcirculatory stasis caused by decreased erythrocyte deformability occurs in the eye. The oxygen supply to the sclera (the white part of the eye) primarily relies on the capillary network on its surface. When microcirculation is obstructed, the scleral tissue enters a state of chronic hypoxia, and the scleral hypoxia theory is the core mechanism underlying the accumulation of myopia.
Meanwhile, the skin around the orbit is one of the thinnest in the human body, with an extremely rich and superficial subcutaneous capillary network. The stasis of microcirculation in this area can lead to an increased proportion of deoxyhemoglobin in the blood, manifesting as a bluish-purple, non-fading dullness or patchy bruising on the skin. This is highly consistent with the pathological sign of "spontaneous periorbital bruising" observed in patients with metabolic diseases such as diabetes in experimental studies.
Therefore, persistent orbital ecchymosis and scleral hypoxia jointly constitute a pair of highly indicative clinical anchor points, indicating significant microcirculation and oxygen supply disorders in the craniofacial region.
Inference and verification of systemic microcirculatory disorders
According to the logic of this hypothesis, microcirculatory dysfunction triggered by red blood cell dysfunction should not be a local exception, but rather a systemic phenomenon. Its impact will extend to all organs and tissues that rely on efficient capillary perfusion:
Joint function: Articular cartilage lacks a direct blood supply, and its nutrition and oxygen supply are entirely dependent on the diffusion of synovial fluid. Microcirculatory disorders will lead to a decrease in the partial pressure of oxygen in synovial fluid, placing chondrocytes in an hypoxic environment, impairing mitochondrial function, and reducing the ability to synthesize and repair extracellular matrix. This, in turn, accelerates joint degeneration, manifesting as decreased mobility, stiffness, and pain.
Nail morphology: The capillary plexus of the nail bed serves as a classic window for observing microcirculation. Insufficient oxygen supply can lead to morphological abnormalities and blood flow stasis in the capillary plexus of the nail bed, subsequently affecting the normal growth and keratinization processes of nail matrix cells. This manifests as slow nail growth, loss of luster, the appearance of longitudinal ridges, or thinning and brittleness of the nail.
Neurological function: Neurons are the cells with the highest oxygen consumption in the body and are extremely sensitive to hypoxia. Even minor impairments in cerebral microcirculation can lead to insufficient energy supply to cortical neurons, initially manifesting as a decrease in the efficiency of higher-order cognitive abilities, such as difficulty concentrating, sluggish thinking, reduced short-term memory, and increased mental fatigue.
Validation scheme: Systemic microcirculation assessment based on a control group
To empirically test this systematic inference, a controlled study can be designed as follows:
Individuals exhibiting typical orbital ecchymosis/scleral hypoxia were selected as the experimental group, while age-matched healthy individuals aged 10-18 years without these signs were chosen as the control group.
Subsequently, multi-site and multi-technique microcirculation and functional assessments were conducted on both groups of subjects:
Nailfold Microcirculation Microscopy: Direct observation of the morphology, density, and blood flow dynamics of the nail bed capillaries, along with quantitative analysis of blood flow velocity and red blood cell aggregation.
Near-infrared spectroscopy technology for joints: Non-invasive measurement of blood oxygen saturation in tissues surrounding the joints to assess local oxygenation levels.
Transcutaneous oxygen pressure measurement: Measuring the oxygen pressure diffusing through the skin at specific locations on the limb (such as the dorsum pedis) reflects the oxygen supply status of subcutaneous tissue.
Cognitive function scales and neuroelectrophysiological examinations: Standardized scales are used to assess attention and memory functions, and combined with examinations such as event-related potentials, to objectively reflect the speed of brain information processing.
Expected results and theoretical value: If the experimental group consistently demonstrates more significant microcirculatory disorders and functional decline than the control group across multiple indicators mentioned above, it will strongly prove that "structural oxygen supply deficiency" is a systemic and fundamental pathophysiological state. This not only provides a systematic evidence chain for "orbital bruising" as a marker of early aging, but also validates the pathway of "microcirculatory disorder - tissue hypoxia - mitochondrial dysfunction" from a spatial dimension as a common mechanism driving parallel decline in multiple systemic functions of the body. This places the "first cause" hypothesis on a solid empirical foundation that can be observed and verified, and lays a solid foundation for future epigenetic verification of the pathways leading to aging caused by the 80 non-steady states of the first cause. Conversely, if orbital bruising is not found in individuals aged 10-18, or if individuals aged 10-18 only exhibit orbital bruising, scleral hypoxia, abnormal nailfold microcirculation, and decreased transcutaneous oxygen partial pressure measurement, with one of these conditions present, it indicates that the theoretical first cause has been falsified.
If local microcirculatory disorders are not reflected in systemic microcirculatory disorders, there must be an error in the mechanism by which systemic microcirculatory disorders lead to a decrease in cellular ATP levels throughout the body.
If both conditions coexist, such as the presence of orbital bruising, scleral hypoxia, and abnormal nailfold microcirculation in some individuals, coupled with a decrease in transcutaneous oxygen partial pressure measurements, while others only exhibit one of these conditions, it can be concluded that these variations are due to accidental factors resulting from human developmental differences.
Note: The essence of the primary cause is the non-steady-state decline in gene expression, leading to reduced ATP levels in most cells throughout the body. What the hypoxia model can demonstrate is merely the existence of this mechanism, rather than the model itself dominating the entire process of the primary cause. The entire primary cause is akin to a pendulum. Before humans have a clear understanding of it, even if the issue of latent hypoxia is improved, this pendulum will arbitrarily enter another non-steady-state state, maintaining a state of reduced mitochondrial functionality. The amplitude and size of the pendulum's swing determine, to some extent, the intensity of promoting physical aging.
Conclusion: Mitochondrial function serves as the core hub of anti-aging research
In summary, this article systematically expounds the theoretical framework of mitochondrial dysfunction as the primary cause of aging. I believe that aging is not a passive, random process of damage accumulation, but rather a programmed process actively driven by mitochondrial dysfunction, with inherent logic and causal chains. Starting from the point of mitochondrial dysfunction, we sequentially deduce a series of interrelated "aging factors" such as glycolipid metabolism disorders, decline of the GH-IGF-1 axis, collapse of the antioxidant network, imbalance of the inflammation-glucocorticoid hormone axis, and accumulation of senescent cells. This model not only integrates various existing theories of aging but also reveals the inherent connections between them, providing a new perspective for understanding the complexity of aging.
More importantly, this theoretical framework provides direction for anti-aging research and intervention. It tells us that, rather than focusing on "symptomatic treatment" of various aging-related diseases downstream, we should concentrate our efforts upstream, with mitochondrial function as the core hub, and carry out systematic intervention. Whether through nutritional supplementation, lifestyle adjustments, or future drug and gene therapies, the ultimate goal should be to restore and maintain the health and high-efficiency function of mitochondria. By breaking the vicious cycle of aging, we may truly achieve "healthy aging," extend the healthy lifespan of humans, and allow life to retain vitality and dignity for a longer period of time.
11. Author's preface: The derivation from "mTOR culprit" to "mitochondrial primary cause", and its simplified version of the evolution model of major factors contributing to aging.
As we embark on this systematic analysis of aging, the author wishes to share with readers the thought process that led to the formation of this theory. This was not a sudden flash of inspiration, but rather a scientific research process that spanned several years and involved continuous upstream exploration of its origins.
Starting Point: Identifying the Root Causes of "Cell Zombies" My research began with the intuitive phenomenon of senescent cell accumulation. The SASP factors secreted by these "zombie cells" are key elements that promote tissue inflammation and functional decline. At that time, I clearly recognized that the excessive activation of the mTOR pathway was the core root cause of these cells' "survival and retention" and obstacles to clearance. This formed my "first version" of the aging model.
First traceability: The collapse of the antioxidant network However, the author immediately raised a question: why does mTOR become out of control? The answer points to its upstream regulatory factor, PTEN. When PTEN loses its activity due to oxidation, its inhibitory function on mTOR is lost. So, why is PTEN oxidized? This leads the focus of the author's research to a more upstream event - the overall collapse of the intracellular antioxidant network. At this point, the author's research focus shifts to the balance between the core transcription factors NRF1 and NRF2.
Secondary traceability: The decline of the hormone axis and energy pulses After conducting in-depth research on the antioxidant network, a more fundamental question emerged: why does NRF1, which dominates basic antioxidant defense, decline first? The evidence chain points to the decline in growth hormone (GH) levels. GH is a key signal for maintaining NRF1 expression. The secretion of GH relies on the precise pulsing of the hypothalamic pulse generator for energy metabolism, a process highly dependent on mitochondrial functions such as coenzyme Q10. At this point, the core of the problem is close to the mitochondria.
Reaching the Source: Following the "Pulse Energy Metabolic Decline" and tracing back further, all clues ultimately converge on one point: the functional decline of mitochondria themselves. Whether it is the insufficient synthesis of CoQ10 or the reduced efficiency of oxidative phosphorylation, the root cause is the dysfunction of mitochondria as the "energy-signal center" of cells. At this point, the author has completed this traceable exploration: senescent cell accumulation ← excessive activation of mTOR ← PTEN oxidation ← disruption of the antioxidant network ← decrease in NRF1 ← decline in GH pulse ← mitochondrial functional decline. This complete causal chain has convinced the author that mitochondrial functional decline is not just one of the manifestations of aging, but rather the most upstream, endogenous "primary cause" that initiates the entire aging process.
From the first version to the current version: Theoretical development and unification The systematic model you are currently viewing can be referred to as the "current version" of the theory. The model starting with mTOR is the valuable "first version". They are not mutually exclusive, but rather represent an enhancement in cognitive depth. The "first version" accurately elucidates the key events in mid-aging, while the "current version" reveals the initial cause that triggers the entire chain reaction.
Simplified version of the model depicting the evolution of key factors in aging.
Latent period (adolescence): The "primary cause" of aging has already been initiated, and its early manifestations may be "four-axis" dysregulation, leading to mitochondrial functional decline through one of the eight unstable states, such as triggering latent tissue hypoxia and reducing mitochondrial ATP production.
However, this stage is strongly compensated by extremely vigorous growth hormone, and the high mitochondrial biosynthesis and quality maintenance capabilities mask the decline in mitochondrial functionality, temporarily preventing the system from collapsing downstream. This would be the earliest form of aging, sub-health.
Adolescence (approximately 18-25 years old): The growth hormone plateau ends and begins to decline. The metabolic disorders of glucose and lipids triggered by sub-optimal health formally establish themselves as the dominant feature of this stage, leading to a decrease in growth hormone levels. This weakens the compensatory advantage of anabolism, and the expression of the NRF1 gene gradually decreases due to the decline in growth hormone levels. Growth hormone falls into a vicious cycle of decreased NRF1 gene expression, decreased mitochondrial quality, decreased quantity, and induced ATP energy decline, which again triggers a decline in growth hormone levels.
Adulthood (approximately 25-34 years old): Persistent metabolic stress pushes the antioxidant compensatory mechanism of NRF2 to its limit, leading to the collapse of the antioxidant network. This is typically marked by an increased proportion of oxidized vitamin E and a feedback inhibition of coenzyme Q10 synthesis, resulting in an accelerated accumulation of senescent cells for the first time. By approximately 34 years old, the antioxidant network resembles a ruptured spring, creating a situation where chronic oxidative stress inhibits the recovery of antioxidant function. This, in turn, suppresses the state where PTEN enhances MTOR, accelerating the accumulation of senescent cells and leading the body to enter the next steady state of aging.
Middle age (after approximately 45 years old): Against the backdrop of the decline of the GH-IGF-1 axis in youth, coupled with the secondary impact of energy depletion brought about by the steep decline in sex hormone levels, the body's long-term reliance on the cellular strategy of "compensatory diversity in stem cell number" is further exacerbated. Due to the decrease in ATP energy, GH signaling weakens again, inflammatory factor concentrations increase once more, and inflammatory factors hijack the glucocorticoid axis, triggering reverse regulatory factors of the CD150 pathway. The mitochondrial flicker rhythm is further disrupted, leading to a second steep decline in tissue repair capacity.
In old age (after approximately 70 years old): the inflammation-glucocorticoid axis (the fifth factor) persists due to the hijacking of inflammatory factors. The stem cell reservoir continuously affects calcium homeostasis through the CD150 pathway, preventing self-renewal and leading to exhaustion. The system falls into a vicious cycle of "inflammatory factors-inflammatory factors", gradually heading towards complete collapse.
In the terminal stage (after approximately 105 years of age): the aging process ceases, and the increased risk of death due to aging in the body ceases as well. The core mechanism underlying this cessation of increased mortality risk is that all positive mechanisms in the human body that maintain antioxidant, anti-inflammatory, and energy generation pathways have collapsed to the extreme. The human body is no longer capable of collapsing towards the next unstable steady state, akin to the true vacuum state of vacuum decay.
13. Limitations of the study and future prospects
The fundamental limitation of this study lies in the fact that its logical starting point, the core assertion that "mitochondrial dysfunction" is the "primary cause" of aging, has not been experimentally verified in the most direct and cost-effective manner.
Specifically, the early initiation pathway deduced from this theory, namely "structural oxygen supply deficiency → decreased mitochondrial OXPHOS efficiency", is currently primarily based on the logical chain supported by literature and the author's own clinical observations (such as persistent orbital bruising). It urgently requires a well-designed, cost-controllable controlled study to provide crucial empirical support.
Therefore, the primary and most urgent verification task entrusted to the scientific community by this theory is not the downstream complex molecular network, but the upstream clear physical hypothesis:
Through a rigorously controlled clinical study, it has been confirmed that visible signs of microcirculatory disorders such as "orbital bruising/scleral hypoxia" exhibit systematic and systemic covariation with biomarkers of nailfold microcirculatory abnormalities, decreased transcutaneous oxygen partial pressure, and early mitochondrial dysfunction (such as plasma acylcarnitine profile and platelet oxygen consumption rate).
The completion of this verification will lay an unshakable foundation for the entire theoretical edifice and greatly optimize the resource allocation for all subsequent intervention studies.
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