Role of Molecular Hydrogen in Ageing and Ageing-Related Diseases

Ageing is a physiological process of progressive decline in the organism function over time. It affects every organ in the body and is a significant risk for chronic diseases. Molecular hydrogen has therapeutic and preventive effects on various organs. It has antioxidative properties as it directly neutralizes hydroxyl radicals and reduces peroxynitrite level. It also activates Nrf2 and HO-1, which regulate many antioxidant enzymes and proteasomes. Through its antioxidative effect, hydrogen maintains genomic stability, mitigates cellular senescence, and takes part in histone modification, telomere maintenance, and proteostasis. In addition, hydrogen may prevent inflammation and regulate the nutrient-sensing mTOR system, autophagy, apoptosis, and mitochondria, which are all factors related to ageing. Hydrogen can also be used for prevention and treatment of various ageing-related diseases, such as neurodegenerative disorders, cardiovascular disease, pulmonary disease, diabetes, and cancer. This paper reviews the basic research and recent application of hydrogen in order to support hydrogen use in medicine for ageing prevention and ageing-related disease therapy.


Introduction
Ageing is a physiological process of progressive decline in an organism's functional reserve. It is almost universal throughout the living world [1]. Researchers have focused on exploring the underlying cellular mechanisms of ageing for decades [2] and have found that a variety of metabolic, biochemical, and molecular alterations that occur at a cellular level contribute to functional losses during the ageing process [3]. Nine candidate pathways contributing to the process of ageing have been identified and categorized as the "hallmarks of ageing" [4] (Figure 1).
Ageing represents a continuous risk of chronic noncommunicable diseases, such as neurodegenerative diseases, cardiovascular diseases (CVDs), diabetes, and cancer [5], although it is not the only factor. Over the past decades, the average human life expectancy has become substantially longer [6]. In particular, the absolute number of elderly people has increased in many countries [7]. Understanding the ageing mechanism and then further delaying the ageing process and the onset of age-related pathologies are of great importance.
Molecular hydrogen (H 2 ) is a colorless, odorless gas and is the lightest among all gas molecules. Its therapeutic effect was first demonstrated in skin squamous carcinoma treatment [8]. In some bacteria, H 2 can be enzymatically catabolized as an electron source. It can also be a product of anaerobic metabolism. In mammalian cells that have no functional hydrogenase genes, it was determined to be an inert gas that does not react with any biological compounds [9]. However, in 2007, investigators have discovered that H 2 has antioxidant properties after selectively neutralizing hydroxyl radicals (•OH) and peroxynitrite (ONOO − ) in cultured cells. It also prevented ischemia-reperfusion (I/R) injury and stroke in a rat model [10]. To date, prosurvival properties of some antioxidants have been demonstrated in some disease models [11]. H 2 has been shown to improve lipid and glucose metabolism in patients with mild type 2 diabetes mellitus or impaired glucose tolerance [12]. Moreover, a recent study has shown that hydrogen-rich water (HRW) intake favorably affected several ageing-related features in healthy elderly, including extended mean telomere length, and tended to improve DNA methylation [13]. This review discusses the possible underlying mechanisms of H 2 acting against ageing and its potential preventive and therapeutic applications in ageing-related diseases.

Potential Mechanisms of Molecular H 2
Acting against Ageing 2.1. Antioxidation 2.1.1. Oxidative Stress. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are reactive radical and nonradical derivatives of oxygen and nitrogen, respectively [14]. They are produced by all aerobic cells and play critical roles in both normal physiological and pathological conditions. ROS and RNS are generated through endogenous and exogenous routes. Endogenous routes include ROS generated in mitochondria nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, lipoxygenase, and angiotensin II. Exogenous routes include air and water pollution, tobacco, alcohol, heavy metals, industrial solvents, cooking, and radiation, which are metabolized into free radicals inside the body [14,15]. The oxidative stress occurs when there is an imbalance in formation and removal of ROS and RNS due to metabolic and pathophysiological changes and environmental stress exposure [16].Oxidative stress can cause accumulative oxidative damage in macromolecules (lipids, DNA, and proteins) and eventually lead to age-associated functional losses [14,17,18]. Genomic instability is a common denominator of ageing. In vitro studies have shown that ROS can induce DNA damage by directly oxidizing nucleoside bases and inducing replication stress [19]. They also cause mitochondrial DNA (mtDNA) strand breaks and degradation [20] in vivo, while ionization radiation and ultraviolet light exposure may also be associated with DNA damage. However, it may not be a key species participating in endogenous oxidative DNA damage [21]. Moreover, researchers in recent years have unexpectedly observed that increasing ROS does not accelerate ageing, while decreasing ROS levels by increasing antioxidant defenses may result in shortened lifespan [17]. Nevertheless, ROS and RNS may play a critical role in the ageing process, and the relationship between ROS/RNS and ageing is complex. ROS and RNS can both be beneficial and detrimental depending on the species and conditions. 2.1.2. Characteristics of Antioxidative Effect due to H 2 . The antioxidant activity of H 2 is the basis of its preventive and therapeutic effects. H 2 has been shown to exert its beneficial effects in various pathological conditions that involve free radicals and oxidative stress [22][23][24], as reflected by a reduction in malondialdehyde (MDA), 8-hydroxy-2 ′ -deoxyguanosine (8-OHdG), myeloperoxidase (MPO), and 4hydroxynonenal (4-HNE).
The mechanism of antioxidative effect due to H 2 involves the following aspects ( Figure 2): (1) H 2 Directly Neutralizes •OH. The •OH is produced by the Fenton reaction and Haber-Weiss reaction [25,26], and •OH formed in vivo reacts with biomolecules present at its formation site, making it difficult to trap •OH and directly demonstrate its formation in the biological systems [25]. H 2 can accumulate in the lipid phase more than in the aqueous phase, especially in the unsaturated lipid region, which is the main location for the primary free radical chain reactions [27]. Therefore, H 2 may have an advantage in suppressing these reactions.
(2) H 2 Directly Scavenges ONOO − . Compared to •OH, the half-life of ONOO − is long, which has a greater chance to react with H 2 at the lesion site [28,29]. In addition, H 2 inhibits the generation of nitrotyrosine, which reflects the generation of ONOO − [30]. However, there is a controversy regarding the direct reaction of H 2 with ONOO − and its influence on tyrosine nitration by ONOOH [31]. This discrepancy may be caused by different experimental conditions and investigators and requires further study.
(3) H 2 Indirectly Reduces Nitric Oxide (NO) Production. NO is produced by nitric oxide synthase (NOS). High amounts of NO resulting from inducible NOS (iNOS) can trigger the inflammatory process, which is associated with ageing and inflammatory conditions, such as type 2 diabetes and Alzheimer's disease (AD) [32]. H 2 does not scavenge NO. However, it inhibits iNOS expression [33,34], decreasing its related NO production. Additionally, H 2 may eliminate the NO-derived ONOO − , which is formed through a reaction between superoxide anion (O 2 • ) and NO. This may consume NO and indirectly decrease its quantity [35].
(4) H 2 Inhibits NADPH Oxidase Activity. NADPH oxidase is a prooxidative enzyme that transfers electrons from NADPH to oxygen to generate O 2 • and other downstream ROS [36]. Several homologs of the cytochrome NADPH oxidase subunit have been found, including NOX1-5, DUOX1, and DUOX2 [36]. H 2 suppresses the NADPH oxidase activity and downregulates NOX2 and NOX4 expression, which are notably relevant to cardiac pathophysiology, such as cardiac hypertrophy and interstitial fibrosis [37,38]. Further Antagonistic hallmarks exert beneficial effects at low levels but become harmful at high levels. Integrative hallmarks are results of previous two categories, directly influencing tissue homeostasis and function. 2 Oxidative Medicine and Cellular Longevity study has shown that H 2 decreased the levels of NADPH oxidase subunits, including p40 phox, p47 phox, and p67 phox in the cell membrane, but increased their levels in the cytoplasm. By limiting the translocation of these molecules to the cell membrane, H 2 reduces the NADPH oxidase activity [39].
(5) H 2 Decreases Mitochondrial ROS. ROS are mainly generated in the mitochondria [40]. H 2 is the smallest molecule and therefore capable of passing through the mitochondrial membrane to neutralize •OH and ONOO − [41]. In addition, H 2 suppresses electron leakage in the electron transport chain (ETC), prevents superoxide generation in the mitochondrial complex I, rectifies the electron flow, and thus suppresses oxidative damage in the mitochondria [42].
(6) H 2 Induces Antioxidant Gene Expression and Increases Antioxidant Enzyme Activity. In addition to directly reducing oxidative stress, H 2 can trigger the antioxidation systems. The NF-E2-related factor 2 (Nrf2) functions as an important defense system against oxidative stress by inducing expression of various genes, such as heme oxygenase1 (HO-1). H 2 can activate Nrf2 and induce its translocation into the nucleus, enhancing the transcription of catalase (CAT) and glutathione 1 (GPX1) [43].
(7) Neutrophil Activity Action. Neutrophils are great producers of ROS and play a role in ageing [44]. H 2 reduces neutrophil infiltration in the injured tissue [45], potentially decreasing the generation of ROS. MPO is a hemecontaining peroxidase that is mainly expressed in neutrophils. It plays an important role in microbial killing by neutrophils but is also a local mediator of tissue damage and the resulting inflammation in various inflammatory diseases [46]. As discussed above, H 2 can decrease the amount of MPO [47], which may be associated with inhibition of its release by neutrophils.

Impact of H 2 on Ageing Hallmarks via antioxidative Effect
(1) Maintaining Genome Stability. As mentioned above, ROS contribute to accumulative DNA damage, which is one of the common denominators of ageing. H 2 protects against DNA damage caused by various stimulations through its antioxidative effect. In radiation-caused DNA damage, H 2 alleviated nucleobase DNA damage in aerated aqueous solutions [48] and reversed exhausted cellular endogenous antioxidants [49]. In ultraviolet A-(UVA-) induced skin damage, H 2 significantly alleviated nuclear condensation and DNA fragmentation of keratinocytes [50]. Similarly, in cigarette smoke-(CS-) induced emphysema, H 2 significantly decreased phosphorylated histone H2AX and 8-OHdG levels, which are markers of oxidative DNA damage [51]. Oral administration of water containing hydrogen-rich saline (HRS) prepared in alternating current electrolysis was effective for preventing systemic oxidative DNA injuries and for clinical diabetes treatment [52]. These findings suggest that H 2 can potentially intervene in accumulation of genetic damage in the living body caused by oxidative stress and alleviate the ageing process.
(2) Modulating Cellular Senescence. Cellular senescence is a stress response characterized by arrested cell proliferation and resistance to apoptosis [53]. It takes part in tumor suppression and ageing process in vertebrates and plays an important role in maintaining body homeostasis [54,55]. However, senescent cells are also drivers of ageing that contribute to a series of age-related pathologies [55].
H 2 modulates cell senescence in multiple cell types. When human umbilical vein endothelial cells were induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin, which can strongly induce cellular senescence, the cells exhibited increased expression of 8-OHdG and acetyl-p53, decreased the ratio of NAD (+) to NADPH, impaired Sirt1 activity, and activated senescence-associated protein β-galactosidase. H 2 inhibited these senescence-related changes by activating the Nrf2 pathway [56]. When H 2 was produced in nanoparticles that do not easily disappear and collapse after a long period of time under water, it inhibited the accumulation of β-galactosidase in hydroxyurea-induced oxidative stress and protected against senescence and death in murine embryonic fibroblasts [57]. In a pyocyanin-stimulated cyto •OH-induced cellular senescence model, supersaturated concentrations of H 2 added into the cell culture medium suppressed cyto •OH-mediated lipid peroxide formation and cellular senescence induction, and the investigator speculated that H 2 generated in human gut bacteria may be involved in the suppression of aging [58].
For histone modification, manipulations of histonemodifying enzymes may influence the ageing process [4]. Studies have shown that H 2 can modulate histone methylation and acetylation.

Oxidative Medicine and Cellular Longevity
In the liver of mice and rats, H 2 treatment changed the H3K27 methylation status and induced H3K27 demethylase, which can activate mitochondrial unfolded protein response-related genes to protect the mitochondrial function. It also activated the expression of a series of genes regulated by the histone H3K27 methylation status [59].
Sirtuins are NAD (+)-dependent histone deacetylases that regulate various physiological functions. Human sirtuin isoform Sirt1-7 is considered an attractive therapeutic target for aging-related diseases [60]. Studies have shown that H 2 can modulate the sirtuin family via its antioxidative effect. In the kidneys, H 2 suppressed the downregulated Sirt3 expression, which is the most abundant member of the sirtuin family, by reducing oxidative stress reactions [61]. In the liver, H 2 elevated HO-1 to induce Sirt1 expression, inhibited the inflammatory response and apoptosis, and suppressed palmitatemediated abnormal fat metabolism [62,63]. In the blood vessels, H 2 inhibited oxidized low-density lipoprotein and induced inflammatory cytokine expression via Sirt1mediated autophagy, potentially inhibiting the progression of atherosclerosis [64].
The effects of H 2 on DNA methylation and chromatin remodeling remain unclear.
(4) Effect on Telomere Attrition. Telomeres are particularly susceptible to age-related deterioration. Physically, ageing in mammals is accompanied by a progressive loss of telomere length and function due to normal replication [65,66]. The telomere shortening rate may be accelerated by oxidative stress [67]. It can thus be inferred that H 2 can alleviate telomere shortening via its action on inflammation and oxidative stress. However, studies that have specifically explored the effect of H 2 on telomere maintenance are limited. Recently, a randomized controlled pilot trial showed that HRW intake for six months extended mean telomere length by~4% [13]. More studies are still needed to determine the intervention effect of H 2 on telomere-lengthening and to identify its potential mechanism.
Collectively, these multiple lines of inquiry indicate that by modulating ROS and reducing oxidative stress, H 2 holds a great promise to maintain DNA stability, modulate cell senescence, alleviate epigenetic alterations and telomere attrition, and extend a healthy lifespan [68].

Anti-inflammation
2.2.1. Inflammation and Inflamm-Ageing. Inflammation is a protective life process that repairs damaged lesions and restores homeostasis by inhibiting injurious activators. It is a dynamic and continuous remodeling network as a result of the interaction among genes, lifestyles, and environments [69,70]. However, it is not always helpful and may even be harmful when it persists and becomes chronic [71,72]. It is now increasingly recognized that inflammation is the common molecular pathway that underlies the pathogenesis of diverse diseases ranging from infection to chronic ageingrelated diseases and ageing itself [73]. The so-called "inflamm-ageing" is a chronic subclinical systemic progres-sive increase in inflammation and is an important characteristic of the ageing process [74]. The extended lifespan may be a consequence of pro-and anti-inflammatory process fine-tuning [75]. Thus, imbalance in pro-and antiinflammatory cytokines may take part in the process of inflamm-ageing. In addition, imbalance in age-related redox, DNA damage, decreased autophagy activity, and increased senescent cell numbers, especially in the immune system with ageing, also play important roles in the process of inflamm-ageing [72,76].

Anti-inflammatory Effect of H 2 and Its Impact on
Ageing Hallmarks. The mechanism for the antiinflammatory effects of H 2 involves several aspects.
It also increases the level of anti-inflammatory cytokines, such as IL-4, IL-10, and IL-13 [63,80] (2) H 2 promotes macrophage polarization from proinflammatory M1 type to anti-inflammatory M2 type, which in turn generates additional antiinflammatory cytokines, such as IL-10 and transforming growth factor-(TGF-) β [80] (3) H 2 reduces the aggregation and infiltration of macrophages and neutrophils [81,82] (4) The anti-inflammatory effect of H 2 may involve inhibiting several inflammatory pathways. (1) NF-κB pathway: H 2 inhibits the NF-κB pathway in various disease conditions. It is the most common inflammatory pathway that takes part in a variety of pathological models, including the ageing process [67,83]. (2) NLRP3 pathway: H 2 inhibits NLRP3, which fuels both chronic and acute inflammation and contributes to inflamm-ageing [84,85]. (3) Toll-like receptor (TLR) 4-mediated inflammatory pathway: H 2 inhibits TLR4, which involves hyperglycemia in type 2 diabetes mellitus [86] Inflammation is a prominent ageing-related process that alters intercellular communication. H 2 also inhibits chronic inflammation, which may contribute to inflamm-ageing. For example, it improved inflammation biomarkers in patients with metabolic syndrome [87] and attenuated inflammatory airway status in patients with asthma and chronic obstructive pulmonary disease (COPD), especially tobacco smoke-induced COPD [88]. In the brain, H 2 can inhibit neuroinflammation caused by a variety of pathological conditions, such as cerebrovascular disease, neonatal brain disorders, and neurodegenerative disease [89]. Therefore, H 2 can effectively attenuate the inflammation process in diverse pathological conditions, slow down the inflammageing process, and prevent ageing-related diseases. Further studies are needed to investigate how H 2 regulates the physiological process of ageing via its anti-inflammatory effects. 4 Oxidative Medicine and Cellular Longevity 2.3. Regulating mTOR and Autophagy 2.3.1. mTOR, Autophagy, and Ageing. mTOR is a multifunction protein that can integrate signals based on nutrient availability, energy status, growth factors, and various stressors and regulate key cellular processes, including mRNA translation, protein synthesis, autophagy, transcription, and mitochondrial function. All of these functions are involved in maintaining cellular homeostasis and modulating extended lifespan [90,91]. Therefore, mTOR is a key modulator of ageing and age-related disease [92]. Autophagy is an evolutionarily ancient and highly conserved catabolic process that involves a series of evolutionarily conserved autophagy-related genes (Atg) [93,94]. mTOR is a primordial negative modulator of human autophagy and is inhibited under fasting conditions by activating mTOR targets ULK1, ULK2, and Atg13 [95]. A previous study has shown that increased autophagy delayed ageing and extended longevity while decreasing autophagy by mutating essential Atg genes that inhibit longevity [96].

Modulatory Effect of H 2 on mTOR and Autophagy and
Its Impact on Ageing Hallmarks. Deregulated nutrientsensing and loss of proteostasis are two other ageing hallmarks. mTOR belongs to one of the nutrient-sensing systems. Dysregulation of mTOR signaling can result in metabolic disorders, neurodegeneration, cancer, and ageing [97]. For example, the activity of mTOR increases during ageing and contributes to age-related obesity. This can be reversed by directly infusing rapamycin to the hypothalamus [98]. Impaired proteostasis, such as misfolded or aggregated proteins, contributes to the development of AD, Parkinson's disease (PD), and cataracts. Proteostasis is maintained by stabilizing correctly folded proteins and by degrading proteins through the proteasome or lysosome [4,99]. The autophagy-lysosomal system often experiences an ageingassociated decline [100]. Therefore, measurements targeting autophagy can potentially improve proteostasis and delay the ageing process.
H 2 modulates mTOR and autophagy in multiple diseases and conditions. For example, H 2 inhibits mTOR, activates autophagy, and alleviates cognitive impairment resulting from sepsis [101]. It inhibits the activation of the PTEN/ AKT/mTOR pathway and alleviates peritoneal fibrosis [102]. The activated mTOR/TFEB autophagy alleviates the LPS-induced endothelial damage [103].
Most of the studies have focused on the pathological conditions. At present, there is no direct evidence that H 2 administration delays the normal ageing process through autophagy. However, it is conceivable that long-term administration of H 2 can modulate mTOR and autophagy to help remove aggregated or misfolded proteins or defective organelles, subsequently maintaining proteostasis and cellular homeostasis and potentially delaying the ageing process and ageing-related diseases.
Autophagy is a two-edged sword, as its excess may cause cell death and have other harmful effects on the body. Nonetheless, H 2 can harness autophagy to achieve the ultimate goal of maintaining homeostasis in the body.

Regulating Mitochondria
2.4.1. Mitochondria and Ageing. Mitochondria are cellular powerhouses for producing ATP required by the cell [108]. In addition, emerging investigations have focused on their role in ageing. As cells and organisms age, the efficacy of the respiratory chain tends to decrease, leading to an increase in electron leakage and a reduction in ATP generation [109]. The mechanisms involved in mitochondrial ageing include mtDNA damage, oxidation of mitochondrial protein, dysregulation of mitochondrial dynamics, and impaired mitophagy that causes the accumulation of aberrant mitochondria as demonstrated in cardiovascular, metabolic, and neurodegenerative disorders [110][111][112][113].Therefore, mitochondria are promising therapeutic targets for influencing specific age-related disorders [111].

Protective
Effect of H 2 on Mitochondria and Its Impact on Ageing Hallmarks. Mitochondrial dysfunction is one of the ageing hallmarks. Improving mitochondrial function may delay the ageing process and extend lifespan.
As mentioned above, H 2 prevents mitochondrial oxidative stress by directly neutralizing ROS in mitochondria and suppresses the electron leakage in ETC. In addition, H 2 can improve mitochondrial function represented by the following mechanism: (1) H 2 can block the opening of the mitochondrial permeability transition pores and restore mitochondrial construction and function in the cell [114]; (2) H 2 regulates mitochondrial dynamics by increasing the levels of MFN2 and decreasing Drp1 [115]; (3) H 2 modulates mitophagy, which is an important mitochondrial quality control mechanism, and alleviates inflammation and apoptosis in tissue injury [116,117]; (4) H 2 can target mitochondria to improve the energy metabolism. It stimulates mitochondrial ETC function and increased levels of ATP production by complex I and II substrates [118]. (5) H 2 modulates mitohormesis, a process in which low and noncytotoxic concentrations of ROS promote mitochondrial homeostasis [119], as manifested by enhanced mitochondrial activities with an elevated level of oxidative stress, and then increases expression of antioxidative enzymes [43].
These findings outline the possibilities that H 2 targets mitochondria to prevent ageing-related injury, providing a new way to delay ageing and ageing-related disorders.
2.5. Regulating Apoptosis 2.5.1. Apoptosis and Ageing. Apoptosis is a canonical form of programmed cell death [120]. It plays an indispensable role in both physiological and pathological conditions. For example, it is involved in developmental processes, including cell differentiation and tissue remodeling, it provides an important anticancer mechanism, and the p53 pathway is a vital modulator in this response [121]. Abnormal regulation of apoptosis is associated with a variety of human diseases, including developmental disorders, neurodegeneration, and cancer [122]. Ageing is associated with decreased apoptosis and increased cell senescence. Increased resistance to apoptosis in the ageing process can lead to the survival of postmitotic cells but at the price of damaging housekeeping functions [123].

Effect of H 2 on Apoptosis and Its Impact on Ageing
Hallmarks. H 2 can modulate apoptosis in various disease models. In most cases, H 2 protects tissue from injury through antiapoptotic effects, such as inhibiting the expression of proapoptotic factors Bax, caspase-3, caspase-8, and caspase-12, inhibiting p53 signaling, and upregulating antiapoptotic factors, such as Bcl-2 and Bcl-xl [124][125][126]. However, it may promote apoptosis in some conditions. For example, apoptosis evasion is a prominent hallmark of cancer that is closely associated with ageing, where H 2 increases rates of early and late apoptosis in lung cancer [127,128], facilitates scavenging of carcinoma cells in the body, and reduces proliferation of cancer cells. This proapoptotic effect in cancer cells indicates that H 2 can modulate cell death to protect the body against harmful attacks and maintain homeostasis in the body. Whether H 2 can affect ageing hallmarks through apoptosis remains unknown and requires further studies.
The antiageing mechanism of H 2 and the influence on ageing hallmarks are summarized in Figure 3.

Prevention and Therapy Using H 2 in Ageing-Related Diseases
As many infectious diseases can be cured, more and more people now die of noncommunicative diseases, although these types of illnesses cannot be simply attributed to ageing alone. Efforts to delay the onset of diseases have been made in the past decades, but most diseases still maintain a significant impact on the population [129]. The studies on H 2 in the areas of prevention and therapy in ageing-related diseases may provide some information for treating these conditions in human beings.

Effects of H 2 on AD.
In AD, Aβ accumulation stimulates a proinflammatory response in resident immune cells, microglia, and astrocytes in the brain, leading to plaque phagocytosis, as well as their proteolytic degradation. In addition, the aggravated proinflammatory state occurring during the process of disease can trigger the hyperphosphorylation of tau [130]. Furthermore, microglia, which produce excessive Aβ and become senescent in the progression of AD, continue to produce proinflammatory, microgliarecruiting mediators, including cytokines and chemokines. This results in them becoming overactive in neurodegeneration, eventually leading to more microglia becoming senescent [131].
Animal studies have shown that H 2 can alleviate AD by inhibiting the inflammatory response and oxidative stress. In a rat model utilizing intracerebroventricular injection of Aβ, intracerebroventricular injection of hydrogen saline (HS) prevented Aβ-induced neuroinflammation and oxidative stress, significantly suppressed inflammatory cytokines (IL-6, TNFα, and IL-1β), MDA, and 8-OHdG, and improved memory dysfunction [132]. A further study has demonstrated that H 2 attenuates the activation of c-Jun NH₂-terminal kinase (JNK) and nuclear NF-κB, which are involved in neuroinjury [133]. HRW can also upregulate Sirt1-Forkhead box protein O3a (FOXO3a) by stimulating AMP-activated protein kinase to alleviate potential Aβ-induced mitochondrial loss and oxidative stress [134]. In addition to suppressing memory impairment and neurodegeneration, drinking hydrogen water (HW) directly extended the mean lifespan in a dementia rat model. Interestingly, in a transgenic AD mouse model, investigators found that three months of HRW treatment more profoundly ameliorated oxidative stress and inflammatory responses in the brains of female transgenic AD mice than in those of males. This sex-specific beneficial effect of H 2 was associated with estrogen and brain ERβ-BDNF signaling in AD pathogenesis [135].
In clinical human research, a previous study has found that H 2 administration did not change the Alzheimer's Disease Assessment Scale-cognitive subscale (ADAS-cog) scores after one year in patients with mild cognitive impairment. However, in the H 2 group of apolipoprotein E4 genotype carriers, six and five out of seven subjects had improved ADAS-cog and word recall task scores [136].

Effects of H 2 on PD.
In animal experiments, 6hydroxydopamine (6-OHDA) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyrine (MPTP) are neurotoxic by generating ROS and are therefore often used to produce models of PD [137]. In a 6-OHDA-induced PD model, drinking 50% saturated HW before or after stereotactic surgery was found to prevent development and progression of the nigrostriatal degeneration, effectively preventing the dopaminergic neuron loss [138]. In MPTP-induced (including acute and chronic) PD, drinking HW significantly reduced the loss of dopaminergic neurons. This effect was independent of H 2 concentration in water, such that H 2 significantly decreased MPTP-induced accumulation of cellular 8-oxoguanine (marker of DNA damage) and 4-HNE (marker of lipid peroxidation) and reduced oxidative stress in the brain [139]. Photobiomodulation (PBM) is an effective method to alleviate PD symptoms by enhancing mitochondrial function and boosting ATP production, although it is often accompanied by increased ROS production. Concomitant treatment with H 2 and PBM for a week significantly improved the Unified Parkinson's Disease Rating Scale (UPDRS) scores and eliminated the adverse effect of PBM [140]. Brenner et al. have found that PD may be caused by melanin in the substantia nigra, which fails to produce molecular H 2 from water dissociation and subsequently cannot protect the brain from oxidative stress. Therefore, restoring melanin function or providing supplemental H 2 might be a potential therapy for PD [141]. 6 Oxidative Medicine and Cellular Longevity A randomized clinical pilot study and a later multicenter study showed that drinking HW improved the total UPDRS scores, while placebo worsened them [142,143]. However, a pilot study carried out by the same team has revealed that the inhalation of molecular H 2 gas was safe but did not show any beneficial effects in patients with PD [144]. Another study has shown that inhaling a 1.2-1.4% H 2 -air mixture for 10 min twice a day for four weeks did not significantly influence the clinical PD parameters but increased urinary 8-OHdG levels. Researchers explained that the increased ROS levels are not always associated with toxicity and disease. They also have essential roles in modulating the cellular adaptation process known as hormesis, which exerts a cytoprotective effect. This beneficial increase in oxidative stress effect of H 2 is partly mediated by hormetic mechanisms [145].

Effects of H 2 on CVDs.
Ageing has a prominent effect on the cardiovascular system, leading to an increase in incidence of CVDs, such as atherosclerosis, myocardial infarction, hypertension, and stroke [146,147]. H 2 can protect the heart and blood vessels from ageing-related degeneration.
3.2.1. Effect of H 2 on the Heart. H 2 can protect the heart from myocardial infarct injuries and alleviate cardiohypertrophy and heart failure. HRS significantly alleviated the inflammation and apoptosis induced by myocardial I/R injury by activating PINK1/Parkin-mediated mitophagy [116]. In a swine model, inhalation of 2% H 2 gas improved myocardial stunning. When the inhalation concentration was increased to 4%, H 2 gas significantly reduced myocardial infarct size [148]. In humans, oxidative stress and inflammation are the primary risk factors in hypertension-caused left ventricular hypertrophy [149][150][151]. Chronic treatment with HRS effectively attenuated left ventricular hypertrophy in rats, restored the activity of antioxidant enzymes, suppressed NADPH oxidase activity, inhibited NF-κB activation and proinflammatory cytokines, and alleviated pressure overload-induced interstitial fibrosis and cardiac dysfunction in rats [38,152]. H 2 can especially alleviate mitochondrial dysfunction in hypertensive cardiac hypertrophy by restoring ETC enzyme activity and increasing levels of ATP production in the left ventricle [152].
In addition, H 2 improved interstitial fibrosis in the heart. In pressure-overloaded heart injury, H 2 suppressed TGF-β1 signaling, effectively preventing heart failure [38,153]. Moreover, H 2 inhibited p53-mediated apoptosis and alleviated progression of chronic heart failure [154].
So far, the evidence for the protective effect of H 2 on the heart has been restricted to animal experiments and human studies remain limited. Interestingly, a prior study has found that a decrease in exhaled H 2 during night sleep was associated with congestive heart failure (CHF) severity and can be used as a marker of CHF [155].

Effect of H 2 on Blood Vessels.
The vasculature is composed of endothelial cells, vascular smooth muscle cells (VSMCs), and fibroblasts. These components influence each other in an autocrine or paracrine manner [156]. Vascular ageing is a progressive decline of vascular function, including endothelial dysfunction, inflammation, proliferation, fibrosis, and calcification in VSMCs [157,158]. Therefore, it is one of the major risk factors of ageing-related CVDs.
HRW intake decreased serum concentrations of oxidized low-density lipoprotein (LDL) and free fatty acids and improved high-density lipoprotein (HDL) function and glucose metabolism [12,159,160]. In an apolipoprotein E knockout mouse model of spontaneous atherosclerosis development, drinking HW for four months significantly reduced atherosclerotic lesions and decreased oxidative stress level in the aorta   Oxidative Medicine and Cellular Longevity [161]. H 2 can also stimulate Sirt1-mediated autophagy and attenuate oxidized LDL-induced inflammation [64]. Treatment with HRS in hypertensive rats markedly alleviated vascular dysfunction, restored baroreflex function, and modulated NO bioavailability by abating oxidative stress, suppressing inflammation, and preserving mitochondrial function [152].

Effect of H 2 on
Ageing-Related Pulmonary Disease. COPD and idiopathic pulmonary fibrosis are regarded as lung diseases related to accelerated ageing, which exhibit all of the hallmarks of ageing [162]. COPD is the fourth leading cause of death in the world, with a particularly increasing prevalence in the elderly people [163]. It is an abnormal response to chronic inflammation and injury with excessive activation of macrophages, neutrophils, lymphocytes, and fibroblasts in the lungs, leading to breathlessness and reduction in exercise tolerance [164]. The etiology of COPD involves exposure to external noxious particles or gases, particularly during CS and indoor cooking [163]. Pulmonary fibrosis is one of the major causes of morbidity, and there is still no effective treatment to abate the aberrant repair [165]. Research evidence has shown that ROS and inflammation play a crucial role in inducing a fibrotic response in the lungs by modulating extracellular matrix deposition [166,167].
3.3.1. Effect of H 2 on COPD. H 2 therapy may be a novel and effective treatment for COPD [164] with anti-inflammatory, antioxidant, and antiapoptotic effects [168].
In animal experiments, HRS significantly alleviated CS exposure caused by COPD, alleviated small-airway remodeling and goblet-cell hyperplasia in the tracheal epithelium, and reduced the number of inflammatory cells in the bronchoalveolar lavage fluid (BALF) [169,170]. In addition, HRW treatment significantly reduced the mean linear intercept, restored static lung compliance, decreased the levels of oxidative DNA damage and senescence markers, and attenuated emphysema [51].
In clinical studies, inhalation of 2.4% H 2 -containing steam mixed with gas for 45 min in patients with asthma and COPD significantly attenuated the inflammatory status in the airways [88]. Similarly, a recent randomized multicenter clinical trial showed that combination therapy of H 2 and oxygen was superior compared to single oxygen therapy in improving symptoms in patients with acute exacerbation of COPD (AECOPD). As a result, breathlessness, cough, and sputum scale scores were improved in the combination group [171]. This may provide a feasible alternative emergency management strategy for patients with AECOPD.

Effect of H 2 on Pulmonary Fibrosis.
In bleomycin-induced pulmonary fibrosis, H 2 inhalation reduced the ROS content. It specifically inhibited TGF-β1, decreased the expression level of mesenchymal cell marker vimentin, and increased the expression level of the epithelial cell marker E-cadherin, therefore inhibiting bleomycin-induced epithelial-to-mesenchymal transition (EMT) [172]. In a rheumatoid arthritis-(RA-) associated interstitial lung disease model, H 2 decreased the levels of proinflammatory factors, apoptosis, and extracellular matrix molecules associated with RA pathogenesis and fibrosis. It also ameliorated oxidative stress by decreasing serum levels of lipid peroxide and 8-OHdG-positive cell numbers and alleviating RA-associated lung fibrosis [173].
So far, human studies on the action of H 2 in pulmonary fibrosis are still lacking.

Effect of H 2 on Metabolic Diseases.
Ageing is associated with body composition changes that cause glucose intolerance and increase the risk of diabetes mellitus (DM). The incidence of DM increases with age as the general population's life expectancy also increases [174]. Type 2 diabetes mellitus (T2DM) is characterized by insulin resistance, hyperglycemia, and relative impairment in insulin secretion. Both genetic and environmental factors, such as obesity and ageing, play key roles in its pathogenesis [175]. Long-term HW drinking significantly improved obesity, hyperglycemia, and plasma triglyceride levels in genetically diabetic male db/db mice. This effect of H 2 on hyperglycemia was similar to a diet restriction. H 2 improved the expression of hepatic fibroblast growth factor 21 (HFGF21), which has the function of enhancing fatty acid and glucose expenditure [176]. By reducing oxidative stress and enhancing the antioxidative system, H 2 may improve insulin resistance and alleviate the symptoms of DM [177].
In patients with T2DM or impaired glucose tolerance, consuming pure HRS for 8 weeks significantly improved lipid and glucose metabolism [12]. Another study found that after a single dose of acarbose in patients with T2DM, H 2 gas production was inversely associated with a reduction in the peripheral blood IL-1β mRNA level [178]. Therefore, H 2 potentially inhibited the inflammatory process in T2DM.

Effects of H 2 on Cancer.
There is no doubt that there is a link between ageing and cancer, where the incidence of cancer increases with age [179]. Although the molecular mechanisms underlying the association of ageing and cancer remain unknown, increased ROS levels, products of oxidative stress and mitochondrial dysfunction that occur in ageing and ageing-related disorders, have also been found in cancer [179].
Studies on H 2 as an anticancer therapy can be traced back to 1975, when a two-week hyperbaric administration of H 2 gas caused a marked regression in skin tumors [8]. Since then, mounting evidence has shown that H 2 has an anticancer effect in various types of cancer via diverse mechanisms.
By reducing hepatic oxidative stress, apoptosis, and inflammation, H 2 prevents progression of nonalcoholic steatohepatitis-related hepatocarcinogenesis [180]. However, a previous study has found that combining H 2 with platinum nanocolloids exerts carcinostatic and carcinocidal effects by increasing H 2 peroxide generation and cell death in a human gastric cancer cell line NUGC-4 [181]. It can also be inferred that H 2 had an enhancing ROS effect in cancer cells but protected normal cells by inhibiting ROS. By downregulating chromosome 3, which is a regulator of chromosome condensation, H 2 inhibits lung cancer progression [127].
H 2 can also enhance the anticancer effects when combined with other therapies. HW combined with 5fluorouracil enhanced cell apoptosis in colon cancer cells [182]. A recent study has found that hydrogenated palladium nanocrystals used as multifunctional H 2 carriers 8 Oxidative Medicine and Cellular Longevity together with near-infrared irradiation caused a higher initial ROS loss, more apoptosis, and severe mitochondrial metabolism inhibition in cancer cells, significantly enhancing the anticancer efficacy of thermal therapy [183].
In addition, H 2 can alleviate the side effects of other anticancer therapies, such as chemotherapy and radiotherapy, improving quality of life in cancer patients. For example, H 2 protected irradiated cells from oxidative damage and consequent apoptosis by reducing oxidative stress and inflammation [184] and attenuated gefitinib-induced exacerbation of naphthalene-evoked acute lung injury while not impairing antitumor activity [185]. A previous study has found that intraperitoneal injection of HRS ameliorated mortality, cardiac dysfunction, and histopathological changes caused by doxorubicin in a rat model [186].
In patients with advanced non-small-cell lung cancer, two weeks of H 2 inhalation can significantly reverse adaptive and innate immune system senescence [187]. H 2 therapy can decrease tumor progression and alleviate the adverse events of medications [188]. In patients with advanced colorectal cancer, H 2 restored the exhausted cluster of differentiated (CD)8+ T cells and improved prognosis [189].
H 2 therapy in ageing-related diseases is summarized in Table 1.

Administration Routs of H 2
H 2 can be easily administered in multiple ways, including inhalation, injection of HRS, drinking HRW, and bathing in HW (Table 2). There are several factors that may limit   [190].

Conclusion and Perspectives
Although modern medicine has evolved rapidly in the 21 st century, many significant questions still need to be addressed and many diseases still cannot be cured. As a "philosophical molecule," H 2 may overcome intractable diseases and ageing [41] and solve various problems via its use alone or synergistically with other therapies. Moreover, H 2 gas has demonstrated a safety profile in a number of research studies, which is pivotal for clinical trials. H 2 modulates ageing mainly via antioxidative and antiinflammatory effects. In addition, it can regulate autophagy, mTOR, mitochondria, and apoptosis. All of these factors contribute to the ageing process and may take part in ageing-related diseases. However, the details of specific molecular mechanisms for the antiageing H 2 effects still need further investigation, especially because ageing is a complex and multifactor process. To date, nine ageing hallmarks have been identified. In addition to the hallmarks discussed above, the influence of H 2 on other hallmarks needs further study. For example, proteostasis can be destroyed by ROS and lead to protein oxidation. Protein oxidation can be divided into reversible and irreversible modifications [191], in addition to counteracting protein damage by proteolysis and autophagy. Whether H 2 can repair the reversible protein oxidation through its antioxidative effect is unclear. Stem cell exhaustion is another ageing hallmark. Different ROS doses have different roles in regulating stem cells. Low ROS levels are regulated by intrinsic factors (cell respiration or NADPH oxidase activity) and extrinsic factors (stem cell factors or prostaglandin E2) to maintain stem cell self-renewal. However, high ROS levels due to stress and inflammation may cause stem cell exhaustion, induce stem cell differentiation, and enhance motility [192]. Whether H 2 can modulate and maintain ROS at a suitable level and facilitate stem cell metabolism requires further study. In addition to the nine hallmarks above, circadian clocks modulate various biological processes and are progressively lost during the ageing process. Disruption of the circadian clock may influence the ageing process and pathogenesis of agerelated diseases. Progressive loss of the circadian clock is also categorized as the common hallmark of ageing [193]. Studies have found that there is a connection between the circadian clock and oxidative stress [194,195]. Interestingly, intestinal microbiota that regularly produce H 2 gas also undergo diurnal oscillations in function and composition, and the amount of H 2 generated varies depending on the individual and time of day. Therefore, there may be some interconnectedness between H 2 and circadian rhythms [190], and this mechanism still needs to be elucidated. In addition, recent investigations about reductive stress, the counterpart of oxidative stress, which is defined as a condition of excess accumulation of reducing equivalents [196], have shown that overexpression of antioxidant enzymatic systems can lead to excess reducing equivalents and deplete ROS. Furthermore, feedback regulation establishment in which chronic reductive stress induces oxidative stress, in turn stimulates reductive stress [197]. Whether a long-term H 2 administration elicits reductive stress and influences ageing and ageing-related diseases requires further study in the future. Finally, many of the studies on H 2 have been limited to the topics of ageing-related diseases and may not be directly related to ageing under normal physiological conditions. The majority of the studies on H 2 have been performed using in vivo animal and in vitro cell models. Therefore, its applications in humans remain unknown and require clinical studies to validate. Therefore, further long-term studies are needed to investigate the influence of H 2 on the process of physiological ageing. Nevertheless, we believe that H 2 plays a critical role in the ageing process and ageingrelated diseases, providing optimistic prospects for therapy in this area.

Conflicts of Interest
The authors declare that they have no conflicts of interest