Exploration of Medicine
Open Access Review

Oxidative stress and inflammation: the root causes of aging
Prasert Sobhon1* , Gavin Savedvanich2, Sawaek Weerakiet3

Department of Anatomy, Faculty of Science, Mahidol University, Bangkok 10400, Thailand
NIST International School, Bangkok 10110, Thailand
3
Department of Obstetrics and Gynecology, Bangkok Hospital Udon, Udon Thani 41000, Thailand
1
2*Correspondence: Prasert Sobhon, Department of Anatomy, Faculty of Science, Mahidol University, 272 Rama VI Rd.,
Ratchathewi District, Bangkok 10400, Thailand. prasert.sob@mahidol.ac.th; prasertsobhon@gmail.com
Academic Editor: Calogero Caruso, University of Palermo, Italy
Received: October 23, 2022 Accepted: December 16, 2022 Published: April 17, 2023

Cite this article: Sobhon P, Savedvanich G, Weerakiet S. Oxidative stress and inflammation: the root causes of aging. Explor Med.
2023;4:127–56. https://doi.org/10.37349/emed.2023.00129

Abstract
Oxygen free radicals [reactive oxygen species (ROS)] and nitrogen free radicals [reactive nitrogenspecies (RNS)] are generated by mitochondria during adenosine triphosphate synthesis, andcatalytic activities of cytochrome P450, nicotinamide adenine dinucleotide phosphate oxidases(NOXs), cyclooxygenases, and nitric oxide synthases during drug catabolism, phagocytosis, and acuteinflammation. Under normal circumstances, low levels of ROS and RNS provide redox signalings that
control many essential physiological processes. As age progresses ROS and RNS increase excessively due
to dysfunctional mitochondria, dysregulated NOX, and other free-radical generating sources, leading tooxidative stress, which causes oxidation and denaturation of key cellular components including DNA,
proteins, and lipids, which become abnormal, constituting damage-associated molecular pattern (DAMP),
recognized as ‘non-self’ by immune cells, leading to inflammation which is mediated by nuclear factor
kappa B-inflammasome, p38-c-Jun N-terminal kinase and Janus kinase-signal transducer and activator oftranscription pathways. DAMPs are continuously released from damaged and senescent cells, causing anotherwise normally transient inflammation turning into systemic chronic inflammation, the root causeof aging and age-associated diseases (AADs). Cells restore redox balance by activating the nuclear factor
erythroid 2-related factor 2 (Nrf2) pathway that induces the synthesis and release of antioxidation
molecules and enzymes including haem oxygenase-1, which also inhibits the three inflammatory pathways.
Furthermore, upregulation of autophagy (AP) can get rid of abnormal molecules, prevent the generationof DAMPs, and attenuate inflammation. Both AP and Nrf2 signalings decrease with age. The upregulationsof Nrf2, AP, and downregulation of inflammation are controlled by sensors of energy and stress levels,
i.e., adenosine monophosphate-activated protein kinase, silent information regulator 1, and Sestrins,
as well as the extracellular matrix, while mammalian targets for rapamycin complex 1, a nutrient sensor,
act in the opposite direction. If the balance of these sensor systems becomes dysregulated, aging processaccelerates, and the risk of AADs increases.
© The Author(s) 2023. This is an Open Access article licensed under a Creative Commons Attribution 4.0 International
License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, sharing, adaptation, distribution
and reproduction in any medium or format, for any purpose, even commercially, as long as you give appropriate credit to theoriginal author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

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Keywords
Oxidative stress, redox imbalance, inflammation, aging

Introduction

Demographically, the world is facing a problem of aging population: in 2022, approximately 10% of
people globally are aged 65 or over. By 2030 and 2050 the proportion of this age group is projected to be
12% and 16%, respectively. In 2022, Europe and Northern America have almost 19% of the population
aged 65 or over, followed by Australia and New Zealand with 16.6%. By 2050, one in every four
persons in these developed regions would be aged 65 years or over. For Latin America and the Caribbean,
the proportion of the population aged 65 years or over could increase from 9% in 2022 to 19% by 2050.
Additionally, the proportion of people aged 65 or over in Eastern and South-Eastern Asia, including
Thailand, is projected to double from around 13% in 2022 to 26% in 2050. Sub-Saharan Africa, despite
having a relatively young population, is still predicted to face population aging with the percentage of
people aged 65 or over being estimated to rise from 3% in 2022 to 5% in 2050 [1]. As the world progresses toa full-fledged aging society, the prevalence of age-related diseases is also rising, including metabolic-related
syndromes like obesity, dyslipidemia, nonalcoholic fatty liver, hypertension, diabetes, cardiovascular disorders,autoimmune diseases, neurodegenerative diseases (notably Alzheimer’s and Parkinson’s), various forms ofdementia, and a wide range of cancers.
The root causes of aging and age-related diseases are oxidation by free radicals and inflammation, which
are reciprocally generated. Inflammation (especially chronic inflammation) caused by oxidation, obesity,and infection induces immune cells to be continually active, resulting in chronic damage—repair cycles
whereby cells are continuously dividing, mutating, and dying. This accelerated rate of cell division has ahigh probability of DNA miscopying, causing mutations which are the immediate causes of cancer and otherage-related diseases.

Free radicals are generated by routine cellular metabolic processes: a leakage of electrons from theelectron transport chain (ETC) which is coupled with oxidative phosphorylation in mitochondria, catabolismof exogenous compounds and drugs by liver cells, and the process of fighting infection by innate immune
cells. External pollutants and contaminants in the environment, food, and the body’s immune reactions tothese challenges also generate free radicals. These results in the oxidation of lipids and proteins, the key
components that form and control the structure and functions of cell membranes, directly affecting cellular
homeostasis. Oxidation can also alter nucleotides, the building blocks of DNA, resulting in mutations at both
single nucleotides and chromosomal levels which affect cell division, cell differentiation, cellular signaling,
and general cellular functions, resulting in mutated, cancerous cells. Furthermore, free radicals can denatureor cross-link proteins causing misfolding, endoplasmic reticulum (ER) stress, mitochondrial damage, and lossof function in adenosine triphosphate (ATP) production while exacerbating the production of reactive oxygen
species (ROS). Oxidation and excess free radicals also generate inflammation. Taken together, oxidationand inflammation are the primary initiating factors that cause aging and age-related diseases. Developing
a holistic approach to mitigate the effects of aging and age-related diseases is the principal aim of a newly
emerging discipline called Geroscience.

Generation of reactive free radicals, redox signaling, oxidative stress andits consequences

Oxygen free radicals, or ROS, and the reactive nitrogen species (RNS), which provide redox signalings
at a low level and create oxidative stress (OS) at an excessively high level, are derived from exogenous aswell as endogenous sources. The exogenous sources are environmental pollution, especially PM 2.5 fromair pollution, smoking, ionizing radiation, UV radiation, household chemicals, and agricultural chemicals,
especially herbicides (e.g., paraquat and glyphosate) and insecticides which are widely used in ruralThailand. Endogenously, ROS are derived from the mitochondria, ER-associated-cytochrome P450 (CYP),
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membrane-bound nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOX 1–5), andperoxisomes. In mitochondria, the oxidative phosphorylation process consumes O2 for the synthesis of ATP
which is coupled with the ETC. However, as electrons pass through the ETC, some electrons can escape and are
captured by O2 to form the ROS. ETC sites where electron leakage occurs and ROSs are being formed include
complexes I, II, III, and IV while the terminal complex V is not involved. ROS can damage mitochondrialcomponents, affect the membrane potential, exacerbate mitochondrial dysfunction, and cause more ROS
production [2–4]. As aging progresses, mitochondria become increasingly dysfunctional and the rate ofelectron loss from ETC increases, resulting in the generation of more ROS and exacerbating deleterious effectsof OS on various tissues and organs [4].
The second major source of ROS is the mammalian CYP-dependent microsomal electron transportsystem. CYP enzymes (especially those associated with smooth ER of liver cells) catalyze the oxygenation ofexogenous and endogenous organic substrates, including drugs, pollutants, and hormones, and turn theminto more soluble and non-toxic forms, which are removed by bile and blood circulating through the kidney.
If oxygen transfer to a substrate is not tightly controlled, uncoupling of the reaction occurs, leading to theformation of ROS [5–7].

The third major source of ROS is the membrane-bound NOXs. Innate immune cells, comprisingneutrophils and macrophages, employ potent oxygen-dependent mechanisms that generate ROS to killinvading microorganisms through the catalytic activity of the membrane-bound NOX 2 [8]. In addition
to the membrane of phagocytic cells, NOX is present in cells of many tissues, as it is needed to generate
a low amount of ROS for cell signaling that governs a number of physiological processes. Aside from theabove-mentioned sources, the presence of redox-active transition metals such as Fe and Cu also contributes
to ROS generation. In the presence of Fe2+ and Fe3+, hydroxyl radical (OH•) can be generated from hydrogen
peroxide (H2O2) through the Fenton reaction [9]. Overall, the generated ROSs consist of superoxide anion(–O2•), H2O2, OH•, hydroperoxyl radical (HOO•), and peroxyl radical (ROO•). Though transient in nature, these
ROS are extremely active due to the presence of free electrons. In addition to ROS, RNSs are generated
from endogenous as well as exogenous sources. RNSs comprise nitric oxide (NO), nitrogen dioxide (NO2),
peroxynitrite (ONOO•), dinitrogen trioxide (N2O3), and nitrous acid (HNO2) [10]. NO is generated by catalytic
actions of at least three isoforms of nitric oxide synthases (NOSs) that convert L-arginine to L-citrulline
with the release of NO. If NO is generated in excessively high amount, it interacts with ROS to form many
species of RNS that contribute to the damaging OS.

ROS and RNS at low or physiological concentrations act as second messengers regulating many
redox-sensitive signaling pathways essential for controlling cell metabolism, growth, differentiation, anddeath. Among ROS H2O2 at a physiological concentration, ranging from 1–100 nmol/L, is the most prevalent
and active free radical that exercises its redox signaling by reversible oxidation of thiol groups in cysteines
within target proteins which include enzymes and transcription factors that control many essential signaling
pathways [11]. For examples, insulin signaling which controls glucose transport and utilization and anabolic
metabolism in cells; adenosine monophosphate (AMP)-activated protein kinase (AMPK) and Sirtuin signalings
which are involved in sensing energy and stress levels and maintaining energy homeostasis; nuclear factor
erythroid 2-related factor 2 (Nrf2) which senses changes in cellular redox status through its cysteine-rich
adaptor Kelch-like ECH-associated protein 1 (KEAP1) which, if oxidized by H2O2, frees up Nrf2 to enter the
nucleus and activate the production of antioxidant molecules and enzymes; the nuclear factor kappa B (NF-kB)
which mediates inflammatory responses; hypoxia-inducible factor 1α (HIF-1α) which regulates cellularresponses to decreased oxygen levels; forkhead box O (FoxO) which are critical regulators of the physiologicalstress response [12]. Other enzymes and transcription factors that are regulated by H2O2 are c-Jun N-terminal
kinase (JNK), signal transducer and activator of transcriptions (STATs) [13], and target-of-rapamycin(TOR) [14]. On the other hand, if H2O2 reaches a high concentration above 100 nmol/L, it will denature
biomacromolecules that are involved in cellular structure as well as cellular regulation [11]. Physiologically,NO, at a physiologically low level, acts as a signaling molecule that triggers many key cellular pathways
that control important homeostatic processes, including the activation of AMPK [15], the induction ofvasodilatation and angiogenesis by the catalytic action of endothelial nitric oxide synthase (eNOS) in
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Page 129endothelial cells [16], and inflammatory signaling through the catalytic action of inducible nitric oxide
synthase (iNOS) which is present in phagocytic and antigen-presenting cells (APCs) [17].

The redox balance between the generated ROS/RNS and the antioxidant defense system, especiallyantioxidant molecules and enzymes induced by Nrf2 signaling (to be discussed in details later) help
to maintain cellular redox homeostasis. On the contrary, a redox imbalance will occur if there areunphysiologically high concentrations of ROS/RNS that overwhelm the capacity of the antioxidant
system; and as a result, the excessive amounts of free radicals will cause OS, leading to the disruptionsof redox signalings and controls. Furthermore, OS is a key factor that elicits inflammation as will be
described later. Reciprocally, inflammation is also a major generator of ROS. During inflammatory phase,lipoxygenases, cyclooxygenases (COXs), xanthine oxidase, and NOXs present in phagocytic cells, such as
macrophages and neutrophils, catalyze the reactions that generate ROSs that are released locally as well
as systemically [18]. These enzymes are activated by pro-inflammatory cytokines, particularly tumor
necrosis factor α (TNFα) and interleukin 1β (IL1β), and inflammatory transcription factors including
AP-1, HIF-1α, and NF-kB that are simultaneously expressed in phagocytic cells [19]. Thus, inflammation
can induce ROS generation and contribute substantially to OS, especially if the inflammation becomeschronic [20]. Reciprocally, ROS can induce certain inflammatory signaling transductions, including p38-JNK,
and NF-kB pathways which can activate the assembly and activation of inflammasome to enhance theeffect of inflammatory process [20, 21].

The direct damaging effects of excessive ROS and RNS on biomolecules are extensive and result indeleterious outcomes. Excessive ROS/RNS can oxidize and alter nucleic acids, proteins, and lipids. The OH•
radical oxidizes DNA at purine and pyrimidine bases and the deoxyribose-phosphate chains to generate
single-strand and double-strand breaks which cause mutation. Furthermore, ROS selectively interacts
with guanine to produce 8-oxo-7,8-dihydro-deoxyguanine (8-oxo-dG) which is highly mutagenic as it
causes G:C to T:A transversion [22, 23]. On the other hand, oxidation of lipids gives rise to lipid peroxides
that generate highly toxic and mutagenic malondialdehyde (MDA) and 4-hydroxy-2-nonenal (HNE).
MDA, through its aldehyde group (-CHO), forms stable bonds with -NH2 amine groups in nucleic acids,
proteins, and phospholipids, to produce inter- and intramolecular bridges of amino-3-iminopropene.
MDA binding to nucleotide bases, especially guanine and cytosine, creates bridges between the DNA
strands that block the replication process and induce mutagenic effects. HNE reacts with the -SH groupsof cysteines, and the -NH2 groups of lysine and histidine, causing crosslinking of proteins and disrupting
their structure and functions [24, 25]. The denaturation of proteins and lipids damage cell and organelle
membranes, leading to their disruption and dysfunction and eventually cell death. As the result of DNA
oxidation and mutation, there is a mis-replication and mis-translation, leading to the production ofabnormal proteins and proteomes. Furthermore, through the process of carbonylation and crosslinking,proteins undergo abnormal folding, aggregation, or even breakdown. These result in the releases ofabnormal proteins and lipids which become parts of damage-associated molecular patterns (DAMPs)
recognized by our immune cells as non-self. Under a normal circumstance, DAMPs could be eliminatedby autophagy (AP) which comprises two processes: microautophagy by proteasome, and macroautophagy
through phagophore, a membrane-bound AP [26]. Depending on their sizes, small abnormal proteins
are labeled by ubiquitin and then transposed and digested by proteasome, whereas larger abnormal
proteins are dealt with by chaperon-mediated macroautophagy, and finally the end products arerecycled [27]. During aging, there is a decrease in activities of both proteasome and macroautophagy,
resulting in continuous release of abnormal proteins, thus increasing the quantity of DAMPs, the major
contributor to systemic chronic infection (Figure 1) [28, 29].

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Figure 1. Generation of oxygen and nitrogen free radicals and their effects on cellular constituents and activation of systemic
chronic inflammation (SCI). e: free electron; G: guanine; GPx: glutathione peroxidase; LOO•: lipid hydroperoxides; MAPK:
mitogen-activated protein kinase; →: activation;: inhibition

Inflammation: acute vs. chronic
Acute inflammation (AI) is the process whereby the body takes care of infection and injury which isevolutionarily conserved throughout the animal kingdom. AI consists of two phases which could becalled inflammatory and resolution phases, or inflammatory and anti-inflammatory phases, which are
mediated by protein and lipid mediators. When infections occur, the microbes release molecules called
pathogen-associated molecular patterns (PAMPs), which are recognized by innate immune cells including
phagocytic cells and other APCs [30, 31]. In the case of tissue injury and failure of autophagic processes
to eliminate denatured molecules arising from oxidative process, the damaged and abnormal molecules
are released from the injured cells which are collectively called DAMPs [32]. These two types of molecules
act as ligands which bind to the pattern recognition receptors (PRRs) including toll-like receptors (TLRs),
particularly TLR4, which are located in the membrane of phagocytic cells. As a result, the PRRs are activatedto undergo the process of inflammation which releases pro-inflammatory cytokines, comprising major
components such as the IL1β, IL6, IL12, IL18, TNFα and others [30]. The recruited innate immune cells,
including macrophages, neutrophils, and APC, phagocytose, digest, and clean the infection or debris fromdamaged tissues. Once the cleaning process has been completed, there are molecular cues designatedas specific proresolving mediators (SPMs) which are composed of lipids that signal the resolution of theinflammation to begin the clearance-healing phase together with the release of protein anti-inflammatory
cytokines. These include IL10, IL37, tumor growth factor β (TGFβ), and also the free receptors of TNFαand IL1R and others from macrophages which are switched from the pro-inflammatory phenotype (M1) tothe anti-inflammatory phenotype (M2) [33].
In addition to protein cytokines, the proinflammatory phase of inflammation is also mediated by lipid
mediators generated from arachidonic acid (ARA) through catalytic actions of COXs and lipoxygenases
to generate prostaglandins, thromboxane, and leukotrienes, with prostaglandin E2 being a key lipid
inflammatory cytokine. Once the proinflammatory phase is completed, there is a lipid signaling that initiates
the anti-inflammatory phase comprising lipoxins, resolvins, maresins, and protectins which are derived
from eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) [31, 33]. Hence, ARA with its derivatives
mediate proinflammation while EPA, DHA, and their derivatives mediate anti-inflammation.

The intracellular transduction pathways that lead to the synthesis and release of the above-mentioned
pro-inflammatory cytokines are chiefly controlled by the NF-kB and inflammasome. In the basal state,
NF-kB is bound to and inhibited by the inhibitor of nuclear factor kappa B (IkB) inhibitor. When a
PAMP or DAMP ligand is bound to the TLR, which is a part of PRR, there is a cascade of kinase enzymes
which phosphorylate inhibitor of nuclear factor kappa B kinase (IKK). In addition, IKK is also activated bycytosolic H2O2 generated by mitochondria. IKK itself is also a kinase that phosphorylates IkB, resulting in
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NF-kB being freed and passing into the nucleus where it turns on the genes controlling the syntheses ofthe precursors of protein and lipid pro-inflammatory cytokines and also the inflammasome receptor,
NOD-like receptor family pyrin domain containing 3 (NLRP3). NLRP3 signals the assembly and becomesa part of the inflammasome, the platform that activates Caspase1 that processes the precursors of the IL1,
IL18, and other IL to become mature peptides which are then released from innate immune cells to act aspro-inflammatory cytokines. On the other hand, there are also external signals as well as damage-signaling
molecules from mitochondria that act as NLRP3 ligands or agonists. Mitochondria, especially aging
mitochondria, release a large amount of ROS through the uncoupling of oxidative phosphorylation and theloss of electrons during ETC at complex I and II as mentioned earlier. These free radicals are very activein stimulating the assembly of NLRP3 inflammasome. At the same time the cell attempts to remove thedamaged mitochondria by initiating the process of mitophagy whereby the damaged mitochondria arephagocytosed and destroyed by the macroautophagy [30, 34].

In addition to the NF-kB pathway, there are two alternative pathways that can elicit as well as reinforce
inflammation, i.e., p38-JNK MAPK and Janus kinase (JAK)-STAT pathways. In p38-JNK MAPK pathway
proinflammatory cytokines, including TNFα, ILs, interferon (IFN)-γ, ROS, and cellular stresses, can bind tocorresponding receptors and activate receptor-associated or adaptor proteins, such as tumor necrosis factor
receptor-associated factor (TRAF) and RAC. These proteins sequentially phosphorylate upstream kinase[mitogen-activated-kinase-kinase kinase (MAP3K/MEKK)], then mid-stream kinases [mitogen-activatedkinase kinase (MAP2K/MKK)], and finally downstream kinase, p38-MAPK and JNK-MAPK, which enter the
nucleus and activate inflammatory genes which control the expressions of the proinflammatory cytokines.
The activities and intensities of both p38 and JNK could be negatively controlled by mitogen-activatedprotein kinase phosphatases (MPKs) (Figure 2) [21, 35, 36]. For JAK-STAT pathway, proinflammatory
cytokines (TNFα, ILs, IFN-γ) can bind to corresponding cytokine receptors (CTRs) which phosphorylate
receptor-associated or adaptor protein, JAK, that in turn phosphorylates STAT protein and causes dimerizationof the latter. The dimerized STAT enters the nucleus and induces genes that control the proliferation,
survival, and recruitment of immune cells, as well as the genes controlling the expressions of proinflammatory
cytokines. In turn, the activity and intensity of JAK-STAT are negatively controlled by suppressor of cytokine
signaling 1,3 (SOCS1,3) and other inhibitor proteins including protein inhibitor of STAT (PIAS) and protein
tyrosine phosphatases (PTPs) (Figure 2) [37, 38].

Figure 2. Crosstalks between inflammation and anti-oxidation pathways. The three inflammation pathways comprise NF-kB,
p38-JNK MAPK, and JAK-STAT, and the antioxidation pathway is Nrf2. Inflammation and antioxidation pathways are functionallyantagonistic towards each other (see text for description). ARE: antioxidant response element; ASC: apoptosis-associated-specklike protein containing a caspase recruitment domain or inflammasome adaptor protein; CBP: cyclic AMP response elementbinding protein or NF-kB cofactor protein; CO: carbon monoxide; CRP: C-reactive protein; HO-1: haem oxygenase-1; ICAM1:
intercellular adhesion molecule 1; LOX: lipooxygenase; IRAK: IL1 receptor-associated kinase; LT: leucotriene; MyoD88: myeloid
differentiating primary response gene 88; PA: membrane phospholipids; PGE2: prostaglandin E2; PGES: prostaglandin E synthase;
PGH2: protaglandin H2; PGI2: prostaglandin I2; PLA2: cytosolic phospholipase; Prx: peroxiredoxin; sMaf: Nrf2 cofactor protein;
TXA2: thromboxane; VCAM1: vascular cell adhesion molecule 1; →: activation;: inhibition
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In aging persons, there is an increasing number of senescent cells with dysfunctional mitochondria,
abnormal molecules including misfolded proteins and oxidized lipids which cannot be cleared due todecreased AP. These senescence cells thus become senescence-associated secretory phenotypes (SASPs)
that continuously release DAMPs and proinflammatory cytokines which generate continuous low-grade
inflammation known as age-related SCI or inflammaging [32]. The increase of ROS from dysfunctional
mitochondria during aging as mentioned above can also directly activate immune cells to induce theupregulation of inflammatory-mediating transcription factors, including AP-1, NF-kB, p38, JNK, and activate
NOX system to generate more ROS that exacerbate the effect of OS [39, 40].

Chronic inflammation, which is generated by DAMP, could also be generated by obesity. When there
are excessive intakes of carbohydrate and fat, white adipose tissue (WAT) and cells are overloaded withtriglycerides. Some overloaded adipose cells break down releasing triglyceride and free fatty acids and other
DAMPs that could attract macrophages and other immune cells into WAT and release proinflammatory
cytokines including monocyte chemoattractant protein-1 (MCP1), attracting more macrophages and otherinnate immune cells. Thus, obesity sustains inflammatory signaling contributing to SCI and OS [41, 42].

Redox homeostasis and anti-inflammation

The redox homeostasis can be restored by anti-oxidation mechanisms which include both enzymatic andnonenzymatic entities. Antioxidant enzymes consist of superoxide dismutases (SODs), GPxs, catalase (CAT),and Prxs-thioredoxin (Trx) system which cooperate in the destruction of ROS [43]. SODs in mammals
comprise two isoforms: the cytosolic CuZn-SOD localized in both extracellular and intracellular (cytoplasmic)
compartments, and mitochondrial-bound Mn-SOD. Both isoforms of SOD make up the first line of defense
against ROS and are initially activated by ROS itself; in turn, they catalyze the transformation of extracellular
as well as intracellular superoxide into H2O2 [44]. Subsequently, GPxs and CAT catalyze the breakdown of
H2O2 into water and oxygen. As mentioned earlier, mitochondria are the main source of intracellular ROS
generated from ETC and oxidative phosphorylation, especially if they become dysfunctional during cellularsenescence. Excessive amount of ROS oxidizes lipids and proteins, damaging mitochondria membranes
and eliciting the release of apoptotic signaling molecules including cytochrome c and other pro-apoptotic
factors such as Bax, which activate cytoplasmic caspases, causing cell apoptosis. Mitochondria counteract
this excessive ROS by catalyzing and transforming them into H2O2 using Mn-SOD [45].

The breakdown of H2O2, the end product of SOD catalytic activities, is further catalyzed by GPxs, CAT,and Prxs, all of which are highly conserved in mammals [46]. GPxs use glutathione (GSH), a cytoplasmic
non-enzymatic antioxidant molecule, as an electron acceptor, to turn H2O2 and simultaneously occurring
lipid peroxides into neutral water and corresponding alcohols and molecular oxygen [47]. GPxs compriseat least four isoforms localized in different cellular compartments: GPx1 in the cytoplasm and mitochondria,
GPx2 in the cytoplasm and nucleus, GPx3 in extracellular compartment, and GPx4 is membrane-boundwhich protects membranes from ROS attack [46]. Another key anti-oxidation enzyme is CAT which is present
in all cells particularly in peroxisomes. CAT catalyzes the breakdown of H2O2 derived from the conversion
of ROS by SOD as well as those generated in peroxisomes to less-reactive molecular oxygen and water, thus
preventing cellular damage [48]. Prxs, a family of peroxidases, are enzymes that hydrolyze H2O2 through theircysteine-containing active sites. Mammalian Prxs comprise six isoforms that are distributed over various
cellular compartments. The most abundant isoforms are cytosolic Prx1 and Prx2 which remove H2O2
using the two conserved Cys residues present in their catalytic sites which become oxidized [49]. The
Trx/thioredoxin reductase (TrxR) system then reduces the disulfide bond of post-catalytic Prxs to freethiol and restores their reduced state. The Trx antioxidant system consists of NADPH, TrxR, and Trx acting
in concert. TrxR is the dimeric FAD-containing enzyme that catalyzes the NADPH-dependent reduction of
the oxidized Trx (Trx-S2) to yield the reduced form, Trx-(SH)2 [50]. Thus, the GSH and Trx systems provide
additional supports to GPx and Prxs for the removal of H2O2 generated from the catalytic activity of SOD [51].
The key signaling factor which directly regulates the expressions of genes encoding antioxidant
enzymes is the Nrf2. When unactivated, Nrf2 binds with KEAP1. In their basal state, KEAP and Nrf2
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undergo degradation and turnover by proteasome. In response to rising OS, ROS, especially H2O2, can oxidize
redox-sensitive cysteine residues on KEAP1 in the Nrf2-KEAP1 complex, resulting in KEAP1 being released
from Nrf2. The freed Nrf2 then is translocated into the nucleus and forms a heterodimer with a cofactor,
a small MAF protein, to bind to a DNA sequence named AREs within the regulatory regions of multiple
antioxidant genes. Nrf2 regulates the expressions of genes encoding antioxidant proteins and enzymes,
including SODs, CAT, GPxs, Prxs, glutathione-s-transferase (GST), and HO-1 [52, 53], all of which aresynergistically counteracting OS as aforementioned.

The HO-1 system, in particular, can oxidize heme and generate biliverdin, CO, and Fe2+. CO exercises
additional beneficial action as it has an anti-inflammatory function: the inhibitions of NF-kB, iNOS, eNOS,
as well as p38-JNK, which down-regulates the production of proinflammatory cytokines, while upregulating
the anti-inflammatory cytokines [54]. It is still unclear how HO-1 inhibits p38 and JNK. One possibility
is that it may act through MPKs, a well-known p38-JNK inhibitor. However, this remains to be proven.
Biliverdin is converted to bilirubin by catalytic action of biliverdin reductase. Bilirubin can act as a strong
antioxidant, and in cooperation with CO, it can also inhibit iNOS and eNOS and attenuate the productionof NO and ONOO• (Figure 2) [55–59]. Fe2+ stimulates the production of ferritin which has antioxidant
and cytoprotective effects [55]. HO-1 can also inhibit JAK-STAT pathway by binding directly to STAT andpreventing its phosphorylation and dimerization. Thus, STAT fails to enter the nuclei of immune cells toactivate inflammatory genes [58]. Therefore, the net results of Nrf2 signaling are to provide an anti-oxidation
system for protection against OS and restoration of redox homeostasis, as well as suppressing or attenuating
inflammation via the expression of HO-1.

Aging

It has been widely accepted that the hallmarks of aging comprise genomic instability, telomere attrition,
epigenetic alterations, mitochondrial dysfunction, loss of proteostasis, deregulated nutrient-sensing, cellularsenescence, stem cell exhaustion, and altered intercellular communication [60]. Mechanistically, the mostwidely accepted theory of aging is the free radicals-OS theory first proposed by Harman [61, 62], now
considered in combination with the mitochondrial dysfunction theory proposed by Miquel et al. [63] andthe systemic chronic inflammatory theory, collectively termed oxy-inflamm-aging theory proposed by De
la Fuente [64]. This combined theory hypothesizes that mitochondria are the main generator of ROS fromthe uncoupling reactions in the ETC and the oxidative phosphorylation processes, and as aging progresses
mitochondrial DNA (mtDNA) is damaged and mutated, causing mitochondria to become more dysfunctional
and, in turn, generating more ROS. The progressive degeneration of ROS-damaged mtDNA leads to more
dysfunctional mitochondria and more ROS production which accelerates the aging process. Furthermore,
ROS/RNS that accumulate and increase over time from mitochondria, as well as from catalytic activities
of CYP, NOX, and others, can elicit inflammation which reinforces the aging process by causing additional
cellular damage and senescence, leading to tissue and organ dysfunction, as well as age-associated diseases(AADs) [65]. Additionally, OS worsens with aging since the anti-oxidation system controlled by Nrf2 signaling
is downregulated over time [66, 67].
Genomic instability and telomere attrition

ROS oxidizes both nuclear and mtDNA, particularly at guanine residue, and converts it into 8-oxo-dG,
directly causing genomic instability and telomeric attrition. These processes are exacerbated by MDA
and HNE, the metabolites from oxidation of lipids by ROS, which also crosslink DNA and proteins [25].
Oxidized nuclear DNA results in nucleotide alterations, incorrect code on, chromosomal breakages,
deletions, mutations and dysregulated translation, and abnormal proteome which disrupts various signaling
and metabolic pathways, affecting cellular signaling, metabolism, and homeostasis, culminating in cell
apoptosis (Figure 1). Oxidation of lipids and proteins also damages nuclear membranes, indirectly causing
DNA and chromosome instability, because nuclear membranes and their associated nuclear lamina serveas anchor points for chromatin and DNA, as well as controlling the nuclear shape which directly affects thetranscription and translation processes.
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Mitochondrial dysfunction
Similarly, oxidation of mtDNA results in nucleotide alterations, deletion, mutations, and syntheses ofabnormal mitochondrial proteins that cause dysfunctional mitochondria. ROS, through peroxidation of
membrane phospholipids, oxidation, and carbonylation of membrane proteins, also directly affects the
structural integrity of mitochondrial membranes, causing increased permeability and loss of membranepotential, leading to mitochondrial dysregulation, increased uncoupling of oxidative phosphorylation,
generation of additional ROS, and increased dysfunctional mitochondria [68]. Mitochondrial dysfunction
and the accumulation of ROS also lead to telomere attrition and genomic instability as mentioned above [69].
Loss of proteostasis

OS also dysregulates proteostasis which accelerates aging. ROS causes carbonylation and crosslinking
of intramembranous and cytosolic proteins, leading to their denaturation and loss of function, as well
as damaged cell organelles [70]. In normal cells, the optimal numbers of organelles and proteostasis aremaintained by AP and ubiquitin-proteasome system (UPS). These processes help cells to remove damaged
organelles as well as maintain their optimal numbers, break down denatured proteins, and recycle theirconstituents for further usage by the cells. There are three major types of AP, namely, macroautophagy,
microautophagy, and chaperone-mediated autophagy (CMA). Macroautophagy (generally referred to asAP) involves the enclosing of relatively large targeted structures, like damaged or aging cell organelles,or large aggregates of denatured molecules, by specialized membranes called phagophores which turninto autophagosomes. Finally, autophagosomes fuse with lysosomes and the sequestered materials aredigested by lysosomal acid hydrolases and their constituents recycled into the cytoplasm for reuse [71]. In
microautophagy, the lysosomal membrane is invaginated into tiny pits that pinocytose large aggregates ofabnormal molecules into the lysosomal interior where they are broken down by lysosomal enzymes [72].
In the CMA, moderately large abnormal molecules are directly transported across lysosomal membrane bychaperone proteins into the lysosomal matrix for further digestion by lysosomal enzymes [73]. The UPS
degrades small misfolded proteins, as well as regulatory and signaling proteins, to control the durationof their actions, by marking them with polymeric ubiquitin tags. These ubiquitin-labeled targets arerecognized and passed through the hollow core of cylindrical-shaped proteasomes where they are degradedby proteases [74]. Both AP and UPS are present in every kind of cell and their activities decline as aging
progresses, partly due to decreased effectiveness from oxidative actions of ROS and RNS on their lipid
and protein components [75, 76]. Hence, during aging, cells lose their regulative capacities to control
proteostasis and maintain optimal numbers of organelles. This phenomenon is most evident in the case of
mitochondria where ROS causes disruption of mitochondrial membranes and loss of membrane potential.
These dysfunctional mitochondria express PINK1 and PARKIN markers on their outer membranes that
stimulate mitophagy [77, 78], which removes damaged mitochondria and stimulates mitochondrial
biogenesis to maintain optimal numbers of fully functional mitochondria [79]. As aging progresses, thecell’s ability to control the number of mitochondria through mitophagy declines. The dysregulation as wellas the decrease of AP and UPS activities by ROS lead to diminished proteostasis, a key cause of aging andmany AADs, notably neurodegenerative diseases, such as Alzheimer disease (AD) and Parkinson disease(PD), where aging neurons with decreased AP and UPS fail to clear misfolded proteins, such as beta amyloid
in AD and alpha synuclein in PD [80]. AP plays a dual role in carcinogenesis: initially preventing normal
cells from becoming cancerous by eliminating abnormal proteins. However, if conditions destabilize and thecells become cancerous, AP is employed by definitive cancer cells to acquire extra energy for their survival
and proliferation [81].
Cellular senescence and chronic inflammation

Aging cells expressing SASP are the result of OS. ROS induces senescent cells to undergo mitotic arrest by
activating retinoblastoma protein (pRB) which upregulates cyclin-dependent kinase inhibitors (CDKIs).
In turn, CDKI inactivates cyclin-dependent kinases, leading to irreversible cell-cycle arrest. All normal
fully differentiated and aging somatic cells stop dividing after a finite number of cell divisions and enter
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Page 135a state of permanent cell cycle arrest, either in the G1 or G2/M phase, termed replicative senescence or
Hayflick limit [82]. Such replicative senescence is partly due to telomere attrition or shortening by ROS
as discussed earlier [69]. As a consequence of ROS-induced senescence, the aging cells assume SASP or
inflammatory phenotype by secreting pro-inflammatory cytokines, including IL1α, IL1β, IL6, and IL8
which, in turn, upregulate inflammatory pathways, leading to SCI and its ensuing deleterious effects asmentioned earlier. Likewise, as aging progresses, the body’s innate and adaptive immunities decline, leading
to a state known as immuno-senescence, wherein the immune system cannot readily clear senescence
cells which leads to their accumulation in various tissues and organs. This leads to further increased levelsof pro-inflammatory cytokines both locally and systemically, exacerbating SCI that is the root cause of
progressive aging as well as generation of AADs.
Stem cell exhaustion

Another hallmark of aging is stem cell exhaustion, which affects tissue regeneration and organ function
as aging progresses. Most tissues possess unipotent undifferentiated adult stem cells which undergo
self-renewal to maintain their pools, while also differentiating into definitive fully mature cells to replace
the aging and dying cells in those tissues. Additionally, it is believed that in many tissues, the tissue-specificadult stem cells are backed up by pools of mesenchymal stem cells (MSCs) that reside in the tissues
themselves, or by MSC derived from bone marrow which is the main reservoir of MSC. During aging,
the populations of these two groups of stem cells decrease due to the reduction of their self-renewal
capacities [83]. Several studies demonstrated that AP and mitophagy, together with compensating
mitochondrial biogenesis, and the ability to induce antioxidant enzymes, through Nrf2 signaling, arenecessary to maintain viability and regenerative capacities of both types of stem cells [84–86]. Furthermore,
FoxO3 is a key factor that helps maintain the pools of stem cells in mammals through its positive controls
over AP, induction of antioxidant enzymes, as well as notch-1 and notch-2 that maintain stem cells in aquiescent state, for example, muscle satellite cells [87, 88], adult neural stem cells [89], and hematopoietic
stem cells [90, 91].
Deregulation of the nutrient- and energy-sensing pathways

Finally, OS can damage and deregulate nutrient- and energy-sensing pathways (Figure 3). During nutritional
abundance (especially amino acid and glucose), nutrient-sensing pathways stimulate anabolism, i.e.,biosyntheses of proteins, carbohydrates, lipids and their storages, whereas during nutrient scarcity andenergy depletion energy-sensing pathways restore homeostasis by catabolizing internally stored nutrients
to meet energy requirements. If these nutrient- and energy-sensing pathways are deregulated, aging
accelerates and AADs occur simultaneously. The energy- and nutrient-sensing pathways are composed of
AMPK and mammalian targets for rapamycin complex 1 (mTOR1) which detect and regulate the statusesof energy and nutrient levels available to cells [92, 93]. Living cells use ATP as their primary energy source:hydrolysis of ATP to ADP, and AMP provides energy for most biological processes. The ratio of AMP to ATP
is, therefore, detected by cells as an indicator of cellular energy status, if rising will activate AMPK whichacts as a master regulator of catabolism to restore and maintain energy homeostasis [94]. Downstream,
AMPK activates AP, FoxOs, p53, Nrf2, peroxisome proliferator-activated receptor-gamma coactivator 1α(PGC-1α), and inhibits NF-kB signaling and the deleterious effects of inflammation [95]. AMPK acts insynergy with silent information regulator 1 (SIRT1) which is activated by increased nicotinamide adenine
dinucleotide (NAD+) to reduced nicotinamide adenine dinucleotide (NADH) ratio which also indicatesenergy status in the same direction as AMP to ATP ratio [93, 96, 97]. Mammalian SIRT1 is a class III protein
deacetylase targeting histones and multiple transcription factors [98]. SIRT1 restores energy depletion by
activating the same targets as AMPK, i.e., AP, FoxOs, p53, Nrf2, PGC-1α, and inhibiting NF-kB signaling [99]. It
has been demonstrated that activated AMPK stimulates SIRT1 by increasing the intracellular concentrationof NAD+ through the enhanced expression of nicotinamide phosphoribosyl transferase (Nampt) which
subsequently increases the level of NAD+ and thus potentiates SIRT1 activity [100]. Reciprocally, SIRT1
can deacetylate and activate liver kinase B1 (LKB1) kinase, an upstream activator of AMPK. Thus, SIRT1
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provides positive feedback activation of AMPK to potentiate the action of each other (Figure 3) [101].
Another positive feedback of AMPK activation is Sestrins, a stress-inducible metabolic regulator which
senses cellular stresses including OS, ER stress [unfolded protein response (UPR)], hypoxia, DNA damage,genotoxic stress, and long-term endurance exercise. The expression and activation of Sestrins are induced
by a complex interaction of FoxO and p53 [102–105]. Sestrins further enhances AMPK activity by inhibiting
mTOR1 and stimulates AP to counteract the incurring cellular stresses [106]. In addition, when stimulated
by OS, Sestrins also induce Nrf2 signaling to activate the anti-oxidation system and prevent cell apoptosis [104].
mTOR1, a conserved Ser/Thr protein kinase, counteracts against AMPK-SIRT1-Sestrins by being a potent
inhibitor of AMPK and AP. mTOR senses abundant nutrients (e.g., amino acids, glucose) and sufficientenergy status, and is synergistically connected with insulin/insulin-like growth factor (IGF) signaling (IIS)
pathways whose protein kinase B (PKB) activates mTOR1 while simultaneously represses AP and FoxOs.
AMPK inhibiting mTOR1 complex (mTORC1) via two distinct pathways, either directly by phosphorylating
the regulator-associated protein of mTOR (Raptor), an upstream regulatory component of mTORC1, or
through the phosphorylation of tuberous sclerosis protein 2 (TSC2), which subsequently suppresses theactivity of mTOR1 [107]. The IIS pathway also activates mTOR1 and consequently its downstream targets,eukaryotic initiation factor 4 (eIF4) and ribosomal protein S6 kinase 1 (S6K1), to facilitate protein synthesis.
Upregulated IIS signaling enhances mTOR1 activity via AKT/PKB kinase [108] and accelerates the aging
process due to inhibition of AMPK and AP [109], and activation of NF-B signaling [110]. In turn, AMPK can
suppress IIS pathways and indirectly inhibit mTOR1 by phosphorylating insulin receptor substrate 1 (IRS1)
at Ser794 [111]. Therefore, IIS-mTOR and AMPK-SIRT1-Sestrins signaling pathways are mutually inhibitory.
As aging progresses, AMPK signaling declines [112, 113], impairing catabolic regulation, reducing Nrf2 andAP activities, leading to increased OS and reduced autophagic clearance of abnormal molecules and damaged
organelles in cells, and allowing the release of DAMPs that activate innate immunity and cause chronic
low-grade inflammation. The Figure 3 illustrates the balanced, yet antagonistic relationships between AMPK,SIRT1, and Sestrin on one hand and IIS and mTOR on the other, their intertwining pathways in sensing
nutrient and energy statuses, their controls over the energy and nutrient homeostasis, and the outcomesof their activations. Upregulation of AMPK, SIRT1, and Sestrins activate AP, Nrf2, FoxOs while inhibit NF-kB,
leading to attenuation of aging and preventing AADs. By contrast, upregulation of IIS pathway and mTOR1
inactivate AP, Nrf2, and FoxOs, and activate NF-kB, leading to accelerated aging and occurrence of AADs.

Figure 3. Relationships and interactions between AMPK, Sirtuin 1, Sestrins, IIS, and mTOR pathways in sensing nutrient andenergy statuses, their controls over anabolic and catabolic processes, and the outcomes of their activations. AFT4: activatingtranscription factor 4; CaMKKβ: Ca2+/calmodulin-dependent protein kinase kinase β; CVD: cardiovascular disease; ERK:
extracellular signal-regulated kinase; GLUT4: glucose transporter 4; MEK: mitogen-activated protein kinase kinase; NAFLD:
nonalcoholic fatty liver disease; PIP3: phosphatidyl-inositol-3,4,5 triphosphate; SHC/SOS: Src homology 2 domain-containing
transforming protein/son of sevenless; T2DM: type 2 diabetes; ULK1,2: Unc-51-like autophagy activating kinase 1,2; →: activation;: inhibition; ↑: upregulation, ↓: downregulation

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Mechanotransduction of AP and mitochondrial function by extracellular matrix
Except highly mobile blood cells, a majority of cells constituting human body are surrounded and fixed intheir corresponding tissues by extracellular matrix (ECM) which serves to join the cells together. Furthermore,in case of epithelial and endothelial cells, in addition to binding to their neighbors through ECM on
lateral aspects while resting on the basement membrane and supporting ECM on the basal aspects, theirapical aspects, which are adorned with cilia and microvilli, are in close contact with flowing liquid orsemi-solid materials. The ECM, through its modulative action on cytoskeleton, plays important roles incontrolling cell shape, motility, proliferation, and differentiation, positioning of cell organelles, and many
intracellular processes including AP. Recently, the influence of ECM on regulation of AP is recognized as avery important process physiologically and pathologically, and this key research area is under intense
investigation. Though details on the involved mechanism are not completely known, the evidence gatheredso far indicate that this is a major regulator of AP as well as mitochondrial structure and function, which
has been intensively reviewed [114, 115]. It is well-known that AP is activated by a variety of chemicalcues and stresses, such as hypoxia, DNA damage, inflammation, and metabolic stress such as starvationand exercise. A lesser known and yet very important process is that AP is also activated by mechanicalforces and stresses exerted by surrounding ECM whose physical properties (i.e., composition, density, andrigidity) are undergoing transient changes physiologically, pathologically, and chronologically. Furthermore,
the cells’ three-dimensional parameters (e.g., size, shape, curvature) give rise to tension in the plasma
membrane and its underlying cortical zone of cytoplasm enmeshed with actin filaments. The tensive
force is also exerted by the neighboring cells’ shape, physical activities, causing cell-cell shear forces. As
well, tension is also generated by the forces from the interstitial fluids that can exert osmotic pressure toinduce cell swelling or shrinking. In addition, epithelial and endothelial cells are subjected to shear force
from the flow of liquids or semi-solid materials on their free surface in the lumen of tubular organs thatthey are lining (e.g., intestinal tract, heart and blood vessels, reproductive and urinary tracts), while cells in
extensible organs such as in the muscle, heart, blood vessels, lungs, and digestive tract, are subjected tostretching and compressing forces during expansion and contraction cycle.
Cells perceive these mechanical forces from the ECM and their immediate microenvironment bymechanoreceptors residing in their plasma membranes that are usually coupled with ECM on the externalsurface and the cytoskeleton which are anchored on the internal surface. Through this arrangement, theECM can interact with mechanosensors and exercise control over the dynamic organization of the cytoskeleton
that regulate cells’ external and internal structures, organelle distribution, intracellular transport, and theformation and distribution of autophagic organelles. These mechanosensors comprise intramembranousand interstitial protein complexes, primarily aggregates of integrin in the form of focal adhesions that bindadjacent cells, hemidesmosomes that bind epithelial and endothelial cells to basement membranes andunderlying ECM, cadherins and associated proteins and glycoproteins that bind neighboring cells throughjunctional complexes, mechanosensitive ion channels in the plasma membrane that are opened to ion influx
by changing tension in plasma membrane and associated cortical cytoplasm, and the primary cilia.

Focal adhesions are composed of clusters of transmembrane integrin proteins interacting at theinternal surface of plasma membrane with actin-binding proteins (talin, vinculin), signaling molecules [e.g.,
focal adhesion kinase (FAK), Src, phosphoinositide 3-kinase (PI3K)], adaptor proteins (e.g., paxillin) andactin linker proteins (e.g., filamin, alpha-actinin), collectively called adhesomes [116]. Externally, adhesomes
interacts with the components ECM to sense its rigidity. Thus, through adhesomes, ECM exerts its influenceon cytoskeletal organization, modulating cellular behaviors, including motility, migration, and activation of
AP which maintains cellular homeostasis as well as assists cells to face metabolic and OSs. Several studies
demonstrated how the ECM and adhesome trigger AP via FAK and integrin-linked kinase (ILK) [117, 118].
Additionally, ECM constituents can also act as activators or inhibitors of AP, for example, decorin, collagenVI, kringle 5, perlecan, and endostatin are activators [119–121], whereas laminin a2 is an inhibitor [122].
Physical properties of the ECM such as stiffness can affect the autophagic process via its influenceon cytoskeleton dynamics and actin-associated signaling proteins, such as the Rho small GTPase andExplor Med. 2023;4:127–56 | https://doi.org/10.37349/emed.2023.00129

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its downstream effector Rho-associated protein kinase (ROCK), which regulate the formation of
double-membrane phagophore and autophagosome [123], and via Yes-associated protein (YAP)/
transcriptional co-activator with PDZ-binding motif (TAZ) tension-sensing system [124, 125]. YAP and
the TAZ signaling are regulated by ECM stiffness, cell density, and cell shape. When cells are at low
density or grown on a stiff matrix, YAP and TAZ are activated and transferred from cytoplasm into the
nucleus, where they interact with the DNA-binding transcription factor TEAD to promote the expressionof several proliferation and growth-related genes that eventually induce cell division [126, 127].
Conversely, when cells are at high-cell density or grown on a soft matrix, YAP/TAZ is inactivated in thecytoplasm [126, 128] causing inhibition of cell proliferation. Such an event is a part of contact inhibition
phenomenon. YAP/TAZ directly modulates AP by controlling the transcription of actomyosin genes (MLC2,
MYH10, MYH14) essential for phagophore and autophagosome formation [124], and the expression of the
RAB7 GTPase-activating protein Armus, a factor that is essential for the autophagic pathway, especially
autophagosome-lysosome fusion [125]. Reversely, AP regulates integrin-mediated adhesion by controlling
focal adhesion turnover through a mechanism involving LC3, paxillin, and Src [129], thus directly affecting
cells’ attachment to ECM and cell migration. Despite these important observations, the exact details ofmolecular mechanisms and sequelae of how these mechanosensors control AP in response to physicalproperties of the ECM need more research.

In addition to ECM, cells in a tissue are physically adhered to other cells through junctional complexes
that are either coupling the cells electrically (e.g., gap junction, tight junction) or physically (adherens
junction and desmosomes). Among these junctional complexes, adherens junctions are shown to be theforce-sensitive complex which perceives and transmits force between the cadherin in the ECM within theintercellular space and the actin cytoskeleton. Similar to focal adhesion such a complex also comprises
structural as well as signaling proteins which is collectively termed the cadhesome [130]. Tension on
adherens junctions and its associated cadherin induces a conformational change in cadherin-associatedproteins, catenins, located on the internal face of the plasma membrane, enabling them to bind withvinculin, an actin associated protein, resulting in modulation of actin filament dynamics. Tension-induced
vinculin can activate acto-myosin interaction, resulting in localized actin polymerization [131, 132]. Thus,
vinculin functions as an integrator between external mechanical force and internal transduced signalingthat controls the cytoskeletal dynamics. It has also been demonstrated that the application of force toE-cadherin upregulated AP via LKB1 activation, which mobilizes AMPK, an AP-activating regulator, to theE-cadherin complex [133], which initiates the autophagic process. Reversely, there are turnovers of cadherin
and catenin from the cell membrane to the cytosol where they are subsequently degraded and recycled by
the autophagosome [134].
The molecular details on how the above-mentioned mechanosensors activate AP remain incomplete
and thus need more research. However, it has been shown that mechanical forces transmitted into cells
via focal adhesion and cadhesomes activate mTORC2 [135] which inhibits mTORC1 [136], which negates
the inhibition of the latter on ULK1, thus allows it to be free and initiate AP [137]. Evidently, the decreaseof mechanical forces at the focal adhesions, which may occur upon detachment of the cells from thesubstrate or due to changes in structural properties of the ECM, induces FAK dissociation from the focal
adhesion complex [138]. Soluble FAK phosphorylates and activates mTORC2 which, consequently, initiates
AP due to its inhibition of mTORC1 as mentioned. Additionally, mTORC2 can also activate AKT, which has
inhibitory action on mTORC1 [139]. Hence, the ECM can act on adhesome and cadhesome to initiate AP via
FAK, mTORC2 and AKT which inhibit mTORC1 and allows ULK1 to become active and binds to ATG13 and
FIP200 to form ULK1 complex that initiates AP. Subsequently, there is a recruitment of specific ATGs thatmobilize actin filaments to repolymerize under the membrane at the site of phagophore formation at theexisting ER or nuclear membrane to cause curving of the membrane into omegasome, the forerunner ofphagophore. Additional recruitment of membrane constituents by ATGs, particularly phospholipids, enables
the elongation of phagophore that progresses into autophagosome [140].
For epithelial and endothelial cells, shear force from the flowing fluid can induce AP by activating
the Rho GTPases (Rac1, RhoA, and Cdc42) and Bin/amphiphysin/Rvs (BAR) proteins that acts as
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tension sensors in the plasma membrane [141], to cause upregulation of Beclin-1, ATG5, ATG7 andLC3 which initiate autophagic process [142]. Another mechanism of tension sensing may be perceived
by the actin-binding protein filamin which crosslinks actin filaments together [143]. Such filamin A
crosslinks of actin can provide stability at the focal adhesions and the plasma membrane as well as theassociated cortical zone to counteract force generated by fluid flow; this action can also prevent cells from
apoptosis [144]. Stretching forces on cells can induce AP which happens during muscular activity in bothskeletal and cardiac muscles [145, 146]. The muscle-stretch sensor LIM protein [MLP; also known as cysteine
and glycine-rich protein 3 (CSRP3)] is present at the sarcomeric Z-disc, and it responds to stretching
forces by initiating AP [147].
Stretch- or tension-generated force imposed on the plasma membrane causes a conformationalchange of ion channels, particularly calcium channels, including mechano- and voltage-sensitive transient
receptor potential (TRP) channel, polycystin 1 (PC1), and pore-forming Piezo channel [148]. These channels
are opened when plasma membrane is stretched or compressed during cell migration or when the cells
are faced with shear force of fluid flow. Likewise, these channels also respond to the osmotic force which
increases membrane tension during cellular swelling. Calcium ions influx through these opened calcium
channels can initiate autophagic process via AMPK and mTOR pathway [149].

Primary cilium is present in most cell types. This cilium lacks a central microtubule doublet andis non-motile, but it can sense extracellular chemical and mechanical stimuli. Shear forces of fluid
flow cause bending of this cilium and induce calcium entry into the cell via PC2 and transient receptor
potential vanilloid 4 (TRPV4) channels [150]. The calcium influx triggers AP [151, 152] through the
LKB1-AMPK-mTOR signaling pathway [153], with LKB1 being localized in the axoneme [151]. And more
recently, folliculin (FLCN) has been demonstrated to act upstream to LKB1 in controlling AMPK-mTOR
activity, and AP [152]. Subsequently, the autophagic process is initiated and modulated by activation of
ULK1, Beclin-1 and PI3K/VPS34 signalings [154]. The generated AP is cytoprotective by inhibiting cell
apoptosis and cellular senescence, as well as inhibiting inflammation [155], which in case of endothelial cells
can contribute to the attenuation of artheroma formation and CVD complications. Reversely, the processof ciliogenesis which controls assembly and extension of primary cilium length is modulated by AP as
evident by the presence of several components of the autophagic machinery in the cilium’s basal body [156].
During aging, the ECM becomes stiffer and senescence cells also become more rigid due to lesspliable cytoskeleton and change in nucleus structure due to stretching. These changes unavoidably affectmechano-transduction. As aging progresses there is an increased synthesis of collagen fibrils [157, 158]
as well as their cross-linkings by lysyl oxidase-mediated reaction between the lysine or hydroxylysine
residues [159], resulting in ECM becoming stiffer. Similarly, fibronectin increases significantly during
aging due to overexpression of ILK, a cytoplasmic protein regulating fibronectin–matrix assembly [160],
contributing further to ECM stiffness. Senescence cells also become more rigid due to modification ofplasma membrane’s accumulations of polysaturated fatty acids and cholesterol in place of more flexible
polyunsaturated fatty acids. Such replacement of fatty acids and increased lipid peroxidation enhanceplasma membrane rigidity, resulting in a decrease in cell flexibility [161]. Cytoskeleton also becomes lesspliable, as there are increases of F-actin polymerization [162] and density of microtubules’ network. The
nuclear lamina component, especially lamin B1 (LB1), decreases dramatically as cells become senescent, andits depletion results in changes of nuclear structure and nuclear membrane architecture, causing chromatin
disorganization [163]. As there are direct connections between the nuclear lamina and the cytoskeleton
network, stiffening in the ECM and cytoskeleton causes stretching of the nucleus, resulting in chromatin
conformation turning into a decondensed (euchromatic) form that promotes gene expressions, including
the abnormal genes [164]. Thus, the stiffening of ECM coupled with the more rigid cytoskeleton andchange of nuclear shape as a result of aging disrupts the mechano-transduction pathways and causes
dysregulation of gene expressions in the cells, the details of which are intensively reviewed by Ferrari et al. [165].
In addition to the effect of ECM stiffness on disrupting mechanotransduction of AP and gene expressions,
it has been demonstrated that that ECM rigidity can affect mitochondrial morphological dynamics, their

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function and redox homeostasis in both normal and cancer cells. For example, when neonatal rat cardiac
muscle cells were cultured on fabricated stiffness-tunable fibronectin plate, the growing cells showed
increased oxygen consumption in correlation with the ECM stiffness, implying that mitochondrial function
was regulated by ECM elasticity, whereas the ability of tissues to adapt to metabolic stress is regulated
by both ECM elasticity and tissue alignment on the fibronectin substrate [166]. Through integrin receptor,
ECM stiffness also regulates mitochondrial dynamics and function, especially mitochondrial fusion andfission in response to stress, which is mediated via two distinct signaling mechanisms, namely kindlin
2-dependent up-regulation of mitochondrial fusion and PINCH-1-dependent suppression of dynamin-related
protein 1 (DRP1) expression which mediates mitochondrial fission. As such, ECM can regulate cellular energy
production, metabolism, survival, and cell behavior [167]. Furthermore, it was demonstrated that breast
cancer cells cultured on a soft ECM display increased peri-mitochondrial F-actin, promoted by Spire1C andArp2/3 nucleation factors, and increased DRP1- and MIEF1/2-dependent mitochondrial fission, leading toincreased production of mitochondrial ROS and activation of the Nrf2 antioxidant pathway, and increased
cystine uptake as well as GSH metabolism. These responses enable cancer cells to become resistant tochemotherapeutic drugs that kill cancer cells through generation of OS [168].
From above discussion, it is now clear that apart from controlling cell growth, proliferation,differentiation, and behaviors, the ECM also plays very important roles in controlling intracellular
processes, including mitochondrial dynamics and function which affect cell metabolism, and the key cellular
regulative processes, i.e., AP and possibly antioxidation pathway (Nrf2). In the near future, research on theroles of ECM in controlling the activation or inhibition of central regulators including AMPK-SIRT1-Sestrins
and mTOR should be pursued to give more insights into the working and coordination of these important
regulators in controlling cellular activities, especially with regard to aging and diseases.

Extension of longevity and attenuation of AADs

Evidently, from above discussion, OS and inflammation are the root causes of aging and AADs; therefore,a logical approach that may attenuate aging, extend longevity, and prevent AAD is the restoration of redox
homeostasis by activation of the antioxidation pathway while suppressing the inflammatory pathways.
This can be achieved by activating Nrf2 which induces the expressions of antioxidants molecules andenzymes including SODs, CAT, GPxs, Prxs, and HO-1. The latter is crucial in providing strong antioxidation
as well as inhibition of inflammation as mentioned earlier. Nrf2 signaling also upregulates AP which is
the key process that attenuates aging and extends longevity, as well as preventing AADs. The research on
the positive role of AP in anti-aging has been extensive. The findings were reported first in yeast [169],
later in mammals [170–172], and then in humans [173] while its effects on anti-AAD as well as against awide variety of other diseases have been extensively studied and recognized as described in an intensive
review [174]. By contrast, only recently have there been studies on the effects of the upregulation of
Nrf2-HO-1 in extending longevity [175], while its roles in alleviating and preventing AAD have been long
investigated and well recognized, including its ameliorating effects against inflammatory and autoimmune
diseases, such as multiple sclerosis (MS), inflammatory bowel diseases (IBDs), systemic lupus erythematosus(SLE), rheumatoid athritis [176], obesity, and metabolic diseases, including NAFLD, hypertension,arthrosclerosis, and CVD [177–180]. Furthermore, neurodegenerative diseases including AD and PD havealso been reported to be alleviated by Nrf2-HO-1 upregulation, especially with the ability of Nrf2 to activate
AP and UPS systems, which help to eliminate misfolded proteins (beta amyloid and alpha synuclein),and
attenuate the neuroinflammation caused by OS [181–184].

As mentioned above (Figure 3), the activations of Nrf2 and AP are regulated upstream by a set ofsynergistically acting energy and stress sensors, i.e., AMPK-SIRT1-Sestrins. These three sensors auto-stimulate
each other to promote catabolism to restore energy deficit and counteract OS through the activation of
the Nrf2 and AP pathways, while also directly inhibit inflammation. There have been a number of studies
indicating that activation of these central regulators leads to upregulation of their downstream targets,
especially Nrf2-HO-1 and AP, so that aging and AAD can be attenuated. The most intensively studied is theactivation of AMPK and SIRT1 which could effectively attenuate aging and extend longevity [185–189],
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as well as prevent AADs [190, 191]. AMPK and SIRT1 exert their effects through activations of AP
and Nrf2 [185, 187, 189].

Recently, research on Sestrins’ roles in anti-aging began to gather momentum [192, 193], while theirroles in attenuating and preventing AADs have long been recognized [194–197]. It has been demonstrated
that Sestrins’ signaling also upregulates Nrf2 and AP, as in the cases of AD and PD where it activates Nrf2 tocounteract OS and upregulate AP to eliminate misfolded proteins, which are the initiating causes of theseneurodegenerative diseases [195–197]. Co-activation of AMPK and Sestrins could extend longevity andprevent metabolic diseases, including obesity, NAFLD, and diabetes [194]. Activation of AMPK and Sestrins
together with the inhibition of mTOR1 can prevent neurodegenerative diseases, CVDs, cancer, as well as
musculoskeletal diseases [191]. Therefore, one key approach in extending longevity and preventing or
attenuating AADs is to find means or agents that can activate AMPK-SIRT1-Sestrin signaling, which leads tostrong upregulations of Nrf2-HO-1 and AP.

Recent studies indicated that this goal can be achieved through exercise which acts throughmitochondria by enhancing their metabolism, leading to increased AMP/ATP and NAD+/NADH ratiosand direct activation of AMPK and SIRT1 signaling [198]. Furthermore, AMPK-SIRT1 activation increases
PGC-1α gene expression which stimulates replication and transcription of mtDNA, leading to mitochondrial
biogenesis and mitochondrial oxidative phosphorylation process to satisfy the energy demand [199, 200].
Additionally, exercising muscle also releases myokines, including muscle-derived IL6 and irisin [201, 202],
as well as β-hydroxybutyrate, a ketone metabolite [203, 204], all of which synergistically suppress inflammationin muscle as well as in other organs, thus helping to extend longevity and alleviate AADs. Mechanistically,
β-hydroxybutyrate can activate AMPK and Nrf2 as well as inhibit inflammation by suppressing inflammasome
formation and ER stress [204]. Interestingly, it was recently proposed that β-hydroxybutyrate may beconsidered as a key metabolite that can extend longevity as well as attenuate AADs [205, 206]. Exercise
also activates Sestrins perhaps through the build-up of free radicals from increased metabolism ofmuscle cells [207]. Additionally, exercise induces muscle contraction and stretching which can activate
mechanoreceptor in the muscle that directly activates AP [145–147]. Thus, benefits of exercise could also be
mediated through Sestrins acting together with AMPK and SIRT1, which can extend longevity and attenuate
AADs through their combined ability to activate Nrf2-HO-1 and AP [208].
Caloric restriction (CR) with adequate nutrition can extend longevity and health span in many species
including humans, and protect against the development of AADs [209]. A key cellular response elicited by
CR is AP which is induced to compensate for nutrient deprivation and, consequently, plays beneficial rolein extending longevity and alleviating ADDs. CR, like exercise, is a mild stress that acts on mitochondria byenhancing their metabolism to meet energy demand, which leads to increased AMP/ATP and NAD+/NADH
ratios that directly activate AMPK and Sirtuin 1 signaling [210]. In turn, AMPK inhibits mTOR1, an AP
repressor. SIRT1 and AMPK auto-stimulate and enhance the activity of each other and, consequently, amplifies
the effect of CR. The net result of this signaling loop activation is the upregulation of AP [211]. Apart from
activation of AP, AMPK-SIRT1 signaling also stimulates the up-regulation of Nrf2-HO-1. The combined
effects of these two processes are the main factor through which CR extends longevity and alleviates AADs.

The other approach that can extend longevity and alleviate AADs is the intakes of natural products
as nutraceuticals, notably, polyphenols [e.g., berberine, quercetin, myricetin, caffeic acid (CA), caffeic
acid ethanolamide (CAEA), caffeic acid phenyl ester (CAPE), epigallocatechin 3-gallate (EGCG), gallic
acid (GA), curcumin], flavonoids (e.g., resveratrol), terpenoids (e.g., ginsenosides, triterpene glycosides),carotenoids (e.g., lycopene), xanthophyll (e.g., astaxanthin), spermidine, and sulforaphane, all of which
could act as CR mimetics. Most of these natural compounds have high antioxidation property, and they
can also directly stimulate AMPK (curcumin, EGCG, spermidine, GA, CAPE) or SIRT1 (berberine, EGCG,
quercetin, myricetin) or both (resveratrol, CA, CAEA), and consequently cause the upregulations of Nrf2
and AP [176, 212–216]. Some natural compounds can also suppress inflammation (curcumin, EGCG,
spermidine) [214]. However, all these natural antioxidants and free radicals, including ROS and NO,
induce hormesis or biphasic dose-responses with regard to Nrf2-HO-1 activation, where only optimally
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low doses elicit positive outcomes while high doses induce negative or even toxic outcomes [217, 218].
It has been demonstrated that hormetic activation of Nrf2 effectively extends longevity and reduce
the occurrence and severity of a wide range of human-related pathologies, including AD, PD, stroke,
age-related ocular damage, chemically induced brain damage, renal nephropathy, as well as obesity anddiabetes, indicating that activation of the Nrf2-HO-1 pathway and AP are probably the final, integrative,
and underlying mechanism that function to limit age-related damages, the progression of numerous
disease processes, and chemical- and radiation-induced toxicities [219, 220]. Furthermore, it was foundthat certain drugs (e.g., metformin, salicylate/aspirin) at low doses can activate AMPK by acting as mildly
toxic agents against mitochondria to result in the increase of AMP/ATP ratio, in a special hormesis process
called mitohormesis [213, 214]. Alternatively, certain drug like rapamycin can also hormetically inhibit
mTOR1 [212, 214], hence allowing AMPK and its ancillary pathways (SIRT1 and Sestrin) to become highly
active, which results in similar outcomes.

In summary, exercises, CR, intakes of natural products, and certain mitohormetic drugs can activate
AMPK-SIRT1-Sestrins signaling, and consequently, their downstream target genes particularly thosethat are involved in Nrf2, AP upregulations, which are the final common pathways that attenuate aging
as well as AADs. However, further investigations are needed to reveal details of molecular mechanismsof these approaches and actions of the CR mimetic natural compounds as well as their bioavailabilities,
biotransformations, hormetic properties with regard to dosage-toxicity, so that they can be developed into
efficient, non-toxic strategies or drugs that can be used alone or as adjunctive or combinatorial treatments
to extend longevity, prevent, and/or cure AADs. Stringent clinical trials in humans are also necessary
before these compounds could be declared safe for adoption as nutraceuticals.

Conclusions

Free radicals are generated in normal metabolism from mitochondria during ATP synthesis, and catalytic
activities of CYP, NOX, COXs, and NOSs during drug catabolism, phagocytosis, and AI. At low levels free radicals,
especially H2O2, elicit redox signalings that control many essential physiological processes. As age progresses
free radicals increase excessively due to dysfunctional mitochondria, dysregulated NOX and other freeradical generating sources, which overwhelm antioxidation capacity mediated by Nrf2 signaling that
also declines with aging, leading to OS, which causes oxidation and denaturation of cellular moleculesthat turn into DAMPs which stimulate SCI. Both OS and SCI contribute to the aging processes and AADs.
There may be two direct approaches that could be adopted to mitigate both aging and AAD: upregulating
the Nrf2 pathway to neutralize OS and restore redox balance, and AP to restore protein and organelles
homeostases, both of which can downregulate the inflammatory pathway to attenuate or inhibit SCI.
Alternatively, the central regulators, i.e., AMPK together with SIRT1 and Sestrin may be activated which
can lead to activations of Nrf2 signaling and AP and inhibition of inflammation, which can extend longevity
as well as prevent the occurrence of AADs. Recent studies indicate that these goals are achievable throughexercise, CR, nutraceuticals, and natural products (e.g., polyphenols, flavonoids, terpenoids, carotenoids,
xanthophyll, sulforaphane, spermidine) and certain mitohormetic drugs (e.g., metformin) which activate
AMPK-SIRT1-Sestrins and, consequently, their downstream targets including Nrf2, AP, and FoxOs which
attenuate aging and AADs. Alternatively, there are certain drugs, e.g., rapamycin, that can inhibit mTOR1
and allow activation of AMPK-SIRT1-Sestrins pathways. Natural products and chemicals possessing theseproperties are the subjects of intense investigations, and their mechanisms actions as well as clinical trials
are challenging questions that require further research and stringent tests, since they could potentially be
adopted to enhance both life and health spans.

Abbreviations

AADs: age-associated diseases
AD: Alzheimer disease

AI: acute inflammation

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AMP: adenosine monophosphate

AMPK: adenosine monophosphate-activated protein kinase
AP: autophagy

APCs: antigen-presenting cells
ARA: arachidonic acid

ATP: adenosine triphosphate
CAT: catalase

CO: carbon monoxide

COXs: cyclooxygenases
CR: caloric restriction

CTRs: cytokine receptors

CVD: cardiovascular disease
CYP: cytochrome P450

DAMP: damage-associated molecular pattern
ECM: extracellular matrix

EGCG: epigallocatechin 3-gallateeNOS: endothelial nitric oxide synthase
ER: endoplasmic reticulum

ETC: electron transport chain
FAK: focal adhesion kinase
FoxO: forkhead box O

GPx: glutathione peroxidase
GSH: glutathione

H2O2: hydrogen peroxide

HNE: 4-hydroxy-2-nonenal
HO-1: haem oxygenase-1
IFN: interferon

IIS: insulin/insulin-like growth factor signaling
IkB: inhibitor of nuclear factor kappa B

IKK: inhibitor of nuclear factor kappa B kinase
IL1β: interleukin 1β

iNOS: inducible nitric oxide synthase
JAK: Janus kinase

JNK: c-Jun N-terminal kinase

KEAP1: Kelch-like ECH-associated protein 1
LKB1: liver kinase B1

MAPK: mitogen-activated protein kinase
MDA: malondialdehyde

MSCs: mesenchymal stem cells
mtDNA: mitochondrial DNA

mTOR1: mammalian targets for rapamycin complex 1

mTORC1: mammalian targets for rapamycin complex 1 complex
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NAD+: nicotinamide adenine dinucleotide

NADH: reduced nicotinamide adenine dinucleotide

NADPH: nicotinamide adenine dinucleotide phosphate
NAFLD: nonalcoholic fatty liver disease
NF-kB: nuclear factor kappa B

NLRP3: NOD-like receptor family pyrin domain containing 3
NO: nitric oxide

NOXs: nicotinamide adenine dinucleotide phosphate oxidases
Nrf2: nuclear factor erythroid 2-related factor 2OH•: hydroxyl radical

ONOO•: peroxynitrite
OS: oxidative stress

PAMPs: pathogen-associated molecular patterns
PD: Parkinson disease

PGC-1α: peroxisome proliferator-activated receptor-gamma coactivator 1α
PKB: protein kinase B

PRRs: pattern recognition receptors
Prx: peroxiredoxin

RNS: reactive nitrogen species
ROS: reactive oxygen species

SASPs: senescence-associated secretory phenotypes
SCI: systemic chronic inflammation

SIRT1: silent information regulator 1

SOCS1,3: suppressor of cytokine signaling 1,3
SODs: superoxide dismutases

STATs: signal transducer and activator of transcriptions

TAZ: transcriptional co-activator with PDZ-binding motif
TLRs: toll-like receptors

TNFα: tumor necrosis factor αTrx: thioredoxin

TrxR: thioredoxin reductase

ULK1,2: Unc-51-like autophagy activating kinase 1,2
UPR: unfolded protein response

UPS: ubiquitin-proteasome system
YAP: Yes-associated protein

Declarations

Author contributions
PS: Conceptualization, Writing—original draft. GS: Investigation, Visualization, Writing—original draft.
SW: Writing—review & editing. All authors have read and agreed to the submitted version of the manuscript.
Conflicts of interest

The authors declare that there are no conflicts of interest.
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Ethical approval
Not applicable.

Consent to participate
Not applicable.

Consent to publication
Not applicable.

Availability of data and materials
Not applicable.
Funding

Not applicable.
Copyright© The Author(s) 2023.

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