Hydrogen: An Endogenous Regulator of Liver Homeostasis

Basic and clinical studies have shown that hydrogen (H2), the lightest gas in the air, has significant biological effects of anti-oxidation, anti-inflammation, and anti-apoptosis. The mammalian cells have no abilities to produce H2 due to lack of the expression of hydrogenase. The endogenous H2 in human body is mainly produced by anaerobic bacteria, such as Firmicutes and Bacteroides, in gut and other organs through the reversible oxidation reaction of 2 H+ + 2 e- ⇌ H2. Supplement of exogenous H2 can improve many kinds of liver injuries, modulate glucose and lipids metabolism in animal models or in human beings. Moreover, hepatic glycogen has strong ability to accumulate H2, thus, among the organs examined, liver has the highest concentration of H2 after supplement of exogenous H2 by various strategies in vivo. The inadequate production of endogenous H2 play essential roles in brain, heart, and liver disorders, while enhanced endogenous H2 production may improve hepatitis, hepatic ischemia and reperfusion injury, liver regeneration, and hepatic steatosis. Therefore, the endogenous H2 may play essential roles in maintaining liver homeostasis.


INTRODUCTION
Hydrogen (H 2 ) is the lightest and diffusible gas molecule. H 2 is produced as an endproduct of carbohydrate fermentation, and is reoxidized primarily by sulfate-reduction, methanogenesis, and acetogenesis (Wolf et al., 2016). However, due to lack of the functional hydrogenase genes, mammalian cells fail to produce H 2 themselves; the endogenous H 2 in mammalian is mainly produced by hydrogenases-containing bacterial species present in human gastrointestinal tract, the respiratory system, mouth and pharynx, vagina, and skin (Zhang et al., 2018b;Tan et al., 2019). Over 200 pathogens and pathobionts, and 70% of microbial species in gastrointestinal tract listed in the Human Microbiome Project encode genes for hydrogenases (Wolf et al., 2016;Benoit et al., 2020). Early in 1975, Malcolm Dole et al. firstly reported that exposed hairless albino mice with squamous cell carcinoma to a mixture of 97.5 percent H 2 and 2.5 percent oxygen (O 2 ) at a total pressure of 8 atmospheres for periods up to 2 weeks would cause a marked regression of the skin tumors possibly by neutralizing toxic free radicals (Dole et al., 1975). In 2007, the milestone publication in Nature Medicine by Ikuroh Ohsawa et al. had confirmed that H 2 acted as a therapeutic antioxidant by selectively reducing the cytotoxic hydroxyl radicals in PC12 cells, however, H 2 did not react with other reactive oxygen species (ROS), which possess physiological roles . Thus, inhalation of H 2 gas markedly suppressed focal ischemia and reperfusion (I/R)-induced brain injury in rats by buffering the effects of oxidative stress. From then on, researchers have extensively investigated the functions and mechanisms of H 2 , studies indicated that supplement of exogenous H 2 has the potential abilities to protect against acute or chronic damage of tissues or organs, including brain, heart, blood vessel, lung, stomach, intestine, pancreas, liver, gallbladder, kidney, testis, ovary, breast, eye, ear, bones, skin, et al. (Guo et al., 2013;Sun et al., 2013;Zhang et al., 2016a;Zhang et al., 2016b;Ge et al., 2017;Zhang et al., 2017;Frajese et al., 2018;Zhang et al., 2018b;Chen et al., 2019;Tan et al., 2019;Tao et al., 2019). H 2 dissolved in medium protected PC12 cells against cell death in a dose-dependent manner, as that H 2 > 25 mM had significant anti-oxidative effect . The concentrations of H 2 is about 168 mM in small intestine and 43 mM in the stomach of mice, with similar levels predicted in humans (Benoit et al., 2020), this indicates that the concentration of endogenous H 2 in human body is significantly higher than that needed for anti-oxidative effect.

THE EXOGENOUS HYDROGEN IN LIVER DISEASES
Liver has strong ability to increase and accumulate H 2 after supplement (Kamimura et al., 2011;Sun et al., 2011;Sobue et al., 2015;Iketani et al., 2017;Yamamoto et al., 2019). Among the organs examined in vivo, liver has the highest mean maximum concentration (Cmax, 29.0 ± 2.6 mmol/L) in rats by continuous inhalation of 3% H 2 (Yamamoto et al., 2019). The concentration of H 2 in liver peaked approximately 5 min following intraperitoneal injection of 8 ml/kg H 2 rich saline in mice, and returned to the normal levels 40 min later (Sun et al., 2011). Oral intake of H 2 rich water rapidly but transiently increased H 2 concentrations in liver and atrial blood, while H 2 concentrations in arterial blood and kidney were one-tenth of those in rat liver and atrial blood (Sobue et al., 2015). Mechanistically, hepatic glycogen accumulated H 2 after oral administration of H 2 rich water in vivo, and the in vitro experiment also confirmed that glycogen solution maintained H 2 , explaining why consumption of even a small amount of H 2 over a short span time efficiently improved various liver diseases in animal models (Kamimura et al., 2011;Ohta, 2014).
The liver is a central hub for lipids metabolism, with uptake, esterification, oxidation and secretion of fatty acids (FAs) all occurring in hepatocytes (Chen, 2015;Gluchowski et al., 2017). Hepatic FAs originate from three sources, plasma non-esterified free FAs (lipolysis in adipocytes and unabsorbed portions of lipoproteins after lipoprotein lipase hydrolysis in peripheral tissues), de novo biosynthesis from acetyl CoA derived from different sources, and lipoproteins such as chylomicron remnants (leftover of triacylglycerol (TAGs) from the dietary source) (Chen, 2015). FAs in hepatocytes are esterified with glycerol 3-phosphate to generate TAG or with cholesterol to produce cholesterol esters, which are either stored in hepatic lipid droplets or secreted into the circulation in the forms of very low-density lipoprotein (VLDL) particles (Rui, 2014;Chen, 2015). FAs are also incorporated into phospholipids, which are an essential component of cell membranes, and the surface layer of lipid droplets, VLDL, and bile particles (Rui, 2014). During fasted state, FAs are transported into mitochondria for boxidation to generate acetyl CoA, which in mitochondria can be used for the production of ketone bodies (Chen, 2015). H 2 has the abilities to modulate lipids profiles and functions in vivo. H 2 rich saline decreased plasma total cholesterol (TC) and lowdensity lipoprotein (LDL) cholesterol levels, and reduced the levels of apolipoprotein (apo) B100 in LDL and apo E in VLDL, improved hyperlipidemia-injured high-density lipoprotein (HDL) functions, including the capacity of enhancing cellular cholesterol efflux and protecting against LDL oxidation, in highfat diet (HFD)-fed Syrian golden hamsters (Zong et al., 2012). In a before-after self-controlled study, patients with potential metabolic syndrome consuming H 2 rich water for 10 weeks resulted in decreased serum TC and LDL-cholesterol levels, and apo B100 and apo E levels, improved dyslipidemia-injured HDL functions, including the ability to inhibit LDL oxidation, the ability to suppress TNF-a-induced monocyte adhesion to endothelial cells (ECs) and TNF-a-induced ECs apoptosis, and the ability to stimulate cholesterol efflux from macrophage foam cells (Song et al., 2013). These were further confirmed in patients with hypercholesterolemia in a double-blinded, randomized, and placebo-controlled trial . They found that H 2 treatment increased plasma HDL3-mediated cholesterol efflux via ATP-binding cassette transporter A1 from macrophages ex vivo; enhanced HDL antiatherosclerotic functions as that of suppressing LDL oxidation, oxidized-LDL-induced THP-1 monocytes adhesion to ECs, ox-LDL-induced ICAM-1, VCAM-1, and IL-6 expression in ECs, and oxidized-LDLinduced ECs apoptosis; decreased plasma levels of TC and LDL cholesterol, apo B100; and decreased plasma levels of malondialdehyde (MDA), interleukin-6 (IL-6) and TNF-a, increased the activity of superoxide dismutase (SOD) in plasma, and increased the activity of paraoxonase-1 (PON-1), an antioxidant enzyme associated with HDL, in both plasma and HDL3 fractions . Using cigarette smoke exposure mice model, Qin Shucun et al. found that H 2 saturated saline minimized the impaired plasma lipid profiles and HDL functionalities, moreover, improved the impaired process of reverse cholesterol transport (RCT), in which it promoted the efflux of excess cholesterol from peripheral tissues and returned it to the liver for utilization, direct secretion into bile and feces disposal (Zong et al., 2015). Therefore, H 2 is an essential regulator of lipids profiles, HDL functions and RCT et al.
Hepatic glucose production accounts for~90% of endogenous glucose production, and it is crucial for systemic glucose homeostasis, and the net hepatic glucose production is the summation of glucose fluxes from gluconeogenesis, glycogenolysis, glycogen synthesis, glycolysis, and other pathways (Petersen et al., 2017). H 2 has been shown to maintain the glucose homeostasis, improve fatty liver diseases in animal models and in human beings. Drinking H 2 rich water reduced obesity, decreased levels of plasma glucose, insulin, and triglyceride, and improved hepatic oxidative stress in db/db mice, and alleviated fatty liver in db/db mice and HFD-fed wild-type mice (Kamimura et al., 2011;Jackson et al., 2018). Mechanistically, H 2 increased O 2 consumption and CO 2 production without influencing movement activities, and enhanced the expression of hepatic fibroblast growth factor 21 (FGF21), which functioned to improve carbohydrate and lipid homeostasis (Kamimura et al., 2011;BonDurant and Potthoff, 2018). H 2 rich saline alleviated streptozotocin (STZ) and HFDinduced nonalcoholic fatty liver disease (NAFLD) in rats, decreased fasting blood glucose and insulin levels, improved insulin sensitivity and glucose tolerance, lowered hepatic TNF-a, IL-1b, 8-hydroxy-2′-deoxyguanosine (8-OHdG), 3 −nitrotyrosine levels, and Caspase-3 activity, increased hepatic expression of PPARa, which induced the expression of mediumchain acyl-CoA dehydrogenase and acyl-CoA oxidase 1, the ratelimiting enzymes in mitochondrial and peroxisomal fatty acids b-oxidation, respectively, and PPARg, which contributed to hepatic steatosis (Zhai et al., 2017;Wang et al., 2020). Intraperitoneal injection of H 2 gas had the therapeutic effect on methionine-choline-deficient (MCD) diet-induced NAFLD in mice via inhibiting hepatic MDA levels and JNK phosphorylation (Zhou et al., 2020). Daisuke Kawai et al. revealed that H 2 rich water improved MCD diet-induced nonalcoholic steatohepatitis (NASH) in mice by decreasing plasma ALT levels, hepatic TNF-a and IL-6, oxidative stress and apoptosis related markers, free fatty acid (FFA) uptakerelated gene fatty acid translocase (FAT) (Kawai et al., 2012). H 2 rich water also reduced tumor numbers and maximum tumor size in STZ-induced NASH-related hepatocarcinogenic mice model (Kawai et al., 2012). However, they found that H 2 decreased hepatic PPARa and its targeted gene FFA boxidation-related gene acyl-CoA oxidase expression in MCD diet-induced NASH mice model (Kawai et al., 2012). Therefore, the regulated effects of H 2 on PPARa might be dependent on the animal models examined, it is possible that H 2 regulates hepatic lipid metabolism via maintaining the balance of hepatic de novo lipogenesis/FFAs uptake and b-oxidation. H 2 also had the protective effects on chronic-plus-binge ethanol (EtOH) feeding-induced liver injury, possibly by inducing acyl ghrelin to suppress the expression of pro-inflammatory cytokines TNF-a and IL-6 and induce the expression of IL-10 and IL-22, thus activating antioxidant enzymes against oxidative stress . In human beings, drinking H 2 rich water improved lipids and glucose metabolism in patients with type 2 diabetes or impaired glucose tolerance (Kajiyama et al., 2008), reduced liver fat accumulation in overweight patients suffering from mild-to-moderate NAFLD (Korovljev et al., 2019), improved liver function and reduced viral load in patients with chronic hepatitis B (Xia et al., 2013), attenuated biological reaction to radiation-induced oxidative stress without compromising antitumor effects in patients with liver tumors (Kang et al., 2011), and alleviated liver injury of colorectal cancer patients treated with mFOLFOX6 chemotherapy . Therefore, exogenous H 2 has the abilities to regulate hepatic glucose and lipids metabolism, attenuate virus and chemotherapy related liver injuries, and improve I/R or drugs-induced hepatic inflammation and oxidative stresses.

THE ENDOGENOUS HYDROGEN IN LIVER HOMEOSTASIS
H 2 has antioxidant activity and, in the healthy colon, physiological concentrations of H 2 might protect the mucosa against oxidative insults, whereas an impaired H 2 economy might facilitate inflammation or carcinogenesis (Carbonero et al., 2012). Moreover, the decreased endogenous H 2 levels might also play essential roles in Parkinson's disease, cerebral and myocardial I/R injuries, and chronic heart failure pathogenesis, while supplement of exogenous H 2 may act as a possible therapy for these brain and heart diseases (Fu et al., 2009;Fujita et al., 2009;Shinbo et al., 2013;Yoritaka et al., 2013;Zhai et al., 2013;Hasegawa et al., 2015;Ostojic, 2018;Shibata et al., 2018;Suzuki et al., 2018). In liver, the endogenous H 2 produced by intestinal flora had the ability to improve Concanavalin A (Con A)-induced hepatitis by decreasing serum TNF-a and IFN-g, while inhibition of intestinal flora by antibiotics aggravated Con A-induced hepatitis (Kajiya et al., 2009). Feeding diet with 20% high amylose cornstarch enhanced H 2 generation in intestine, and subsequently alleviated hepatic I/R injury in rats (Tanabe et al., 2012). Lactulose accelerated liver regeneration after hepatectomy in rats by inducing endogenous H 2 production, which may increase hepatic SOD expression and activity, decrease hepatic MDA, IL-6 and TNF-a levels . Supplement of exogenous H 2 by H 2 rich saline had a similar protective effect as lactulose, in contrast, the antibiotics inhibited the regeneration-promoting effect of lactulose by reducing H 2 production . L-arabinose, a naturally occurring plant pentose, elicited gut-derived endogenous H 2 production and alleviated HFD-induced metabolic syndrome, including reduced body weight gain especially fat weight, alleviated liver steatosis, improved glucose homeostasis, reduced systemic dyslipidemia and inflammation in mice . Mechanistically, L-arabinose modulated gene-expressions involved in lipid metabolism and mitochondrial function in key metabolic tissues . Therefore, endogenous H 2 is an essential regulator of liver homeostass, such as improving hepatitis, hepatic I/R injury, liver regeneration, hepatic steatosis as well as glucose and lipids homeostasis.

DISCUSSION
The total H 2 levels in mammals are dependent on the balance between H 2 -producing fermentative bacteria, such as colonic Firmicutes and Bacteroidetes et al., and H 2 consumers, H 2 acts as a substrate for acetic acid producing bacteria, methanogenic bacteria, and sulfate reducing bacteria to utilize and support their energy metabolism (Nakamura et al., 2010;Carbonero et al., 2012;Wolf et al., 2016). The H 2 cycling is central to microbial composition and metabolic homeostasis in the human gastrointestinal tract (Wolf et al., 2016). The gastrointestinal tract-products such as host and/or microbial metabolites (including H 2 ) and pathogen-associated molecular patterns translocate to the liver via the portal vein or by free diffusion and influence liver functions (Tripathi et al., 2018). In contrast, liver transports bile salts, antimicrobial molecules as well as other liver metabolites to the intestinal lumen through the biliary tract and systemic circulation, some of which maintain microecology balance by controlling unrestricted bacterial overgrowth (Tripathi et al., 2018). Therefore, H 2 might be as a novel bridge between gut and liver, and play an important role in gut-liver axis (Figure 1).
Colonic gases, including H 2 , CO 2 , methane (CH 4 ), nitrogen and O 2 as well as other trace gases including volatile amines, NH 3 , mercaptans, and sulfur-containing gases, such as hydrogen sulphide (H 2 S), which is synthesized by cystathionine g-lyase (CSE), cystathioine b-synthase (CBS), and 3mercaptopyruvate sulfurtransferase (3-MST) in concert with cysteine aminotransferase (CAT) in mammalian cells, and is also a by-product of H 2 metabolism by sulphate-reducing bacteria, are among the most tangible features of digestion, clinically, changes in volume or composition of colonic gases have linked with bowel disorders, including lactose and glucose intolerance, small intestinal bacterial overgrowth (SIBO), irritable bowel syndrome (IBS), inflammatory bowel diseases, constipation, colorectal cancer et al., and measurement of H 2 and CH 4 by breath can indicate lactose and glucose intolerance, SIBO and IBS (Nakamura et al., 2010;Carbonero et al., 2012;Mani et al., 2014). It should be noticed that both probiotics and harmful bacteria (such as carcinogenic strains of Helicobacter pylori) can produce H 2 (Olson and Maier, 2002;Benoit et al., 2020), therefore, during H 2 breath test, the relative ratio and balance of these two kinds of bacteria in human body should be taken into consideration for evaluating the long term benefits or harms of H 2 breath in health.
Similar to the protective effects of endogenous H 2 on hepatic I/R injury, H 2 S can also act as an endogenous gas molecule that protects against hepatic I/R injury (Hine et al., 2015). Through knockdown or knockout (KO) of H 2 S-generating enzymes in cells or in animals, endogenous H 2 S has been shown to have essential roles in affecting glucose and lipids metabolism, insulin sensitivity, hepatic oxidative FIGURE 1 | The model of endogenous H 2 in modulating liver homeostasis. The endogenous H 2 is primarily produced by hydrogenase-expressing fermentative bacteria in the gastrointestinal tract. Moreover, H 2 S and CH 4 are by-products of H 2 metabolism derived from sulphate-reducing bacteria and methanogenic bacteria, respectively. It should be noticed that hepatocyte can also produce H 2 S and CH 4 . The colonic gas mixture, including H 2, H 2 S, CH 4 , and other bioactive gas molecules produced by gut microbiota, can arrive at the liver by free diffusion or by blood circulation. These gas molecules may influence hepatic redox homeostasis, glucose and lipids homeostasis, immune and inflammatory homeostasis, respectively, together by another one or more. Therefore, H 2 may act as a novel bridge between gut and liver, and play a central role among the colonic gas mixture in modulating liver homeostasis. stress, hepatic mitochondrial bioenergetics (modulating mitochondrial structure and function, respiratory chain, and cellular bioenergetics), hepatic fibrosis and autophagy et al. (Mani et al., 2014;Sun et al., 2015;Ci et al., 2017;Wang et al., 2017;Wenzhong et al., 2017;Untereiner and Wu, 2018;Wu et al., 2019). However, genetically manipulating the H 2 S-producing enzymes in mammalian cells could not exclude the biological effects of endogenous H 2 S produced by sulphate-reducing bacteria in animals. In addition, CH 4 is another by-product of H 2 metabolism derived from methanogenic bacteria, and Mihaĺy Boros group found that CH 4 can also be produced by rat liver mitochondria, it has hepatic protective effects by exogenous supplement (Ghyczy et al., 2008;Ye et al., 2015;He et al., 2016;Strifler et al., 2016;Yao et al., 2017;Feng et al., 2019;Li et al., 2019a;Li et al., 2019b), however, the hepatic functions of endogenous CH 4 produced by methanogenic bacteria or by hepatocytes are not clear. As that H 2 , H 2 S, and CH 4 are endogenous gas molecules, they may exist as colonic gas mixture and transport into liver by blood circulation or free diffusion, H 2 S and CH 4 can also be produced in liver. Therefore, the functional crosstalk among H 2 , H 2 S and CH 4 in liver, and the influences of these gas mixture on hepatic homeostasis are interesting topics for further investigation. It seems that H 2 might have a central role among these gases in regulating liver homeostasis (Figure 1). The temporal and spatial metabolism of microbial H 2 in human body is relevant to health status, modulating endogenous H 2 metabolism either by diminished utilization or enhanced production, and the strategies such as developing personalize dietary supplementation and precision medicine based on an individual's H 2 -producing or consuming microbiome, might provide a novel means of regulating liver homeostasis.

ACKNOWLEDGMENTS
We apologize to all of the authors whose invaluable work we could not discuss or cite in this review due to space constraints. We should thank Bi Mingmin for the technical preparation of figure.