Our previous study reported that HI can induce significant decrease in BW of rats, while HRW did not21. Consistent with previous study, the results of this study also showed markedly decrease in BW in HI group, while no obvious effect was observed in HRW group. The metabolic cage experiments showed that HI could significantly decrease the food intake, water intake, defecation and urination. The decreased metabolic function may contributed to the reduction of BW in HI group. Our previous study also showed that both HRW intake and HI can induce significant changes in several serum biochemical parameters in normal rats21. To obtain a more comprehensive understanding of the metabolic alterations in response to HRW intake or HI, LC–MS based pseudotargeted metabolomics analysis was performed. The OPLS-DA models indicated clear separations between any two of the three groups based on their metabolomic responses. Compared with the control group, 14 and 10 DMs were identified in HRW and HI group, respectively. It is worth noting that all the DMs in HRW group were down-regulated, while all the DMs were up-regulated in HI group, 22 DMs were identified between HRW and HI groups, indicating that the modulatory effects of HRW intake on metabolism differ markedly from HI. Further functional enrichment analysis suggested the DMs were mainly involved in starch and sucrose metabolism and arginine biosynthesis in HRW and HI group, respectively.
Previous study showed that 4 weeks of HRW intake could significantly decrease the levels of blood glucose, lactate, and blood urea nitrogen (BUN) and exert antifatigue effects in chronic forced swimming mice22. It has also been reported that 3 months of HRW intake could markedly decrease the blood uric acid levels in male patients with hyperuricemia23. Consistent with these findings, our results also showed that 6 months of HRW intake could down-regulate plasma levels of d-glucose 6-phosphate, l-lactic acid, and uric acid. In addition, the plasma levels of nucleotides and their derivatives were also reduced after HRW intake, indicating the regulatory effects of HRW intake on nucleotides metabolism. Notably, all the changed nucleotides derivatives, including m6A, pseudouridine, and N2,N2-dimethylguanosine, were belong to RNA modifications. Among them, m6A is the most widespread epigenetic modification on mammalian mRNA and has been shown to act as a key regulator of numerous important biological processes in normal physiology and in disease, including cancer, heart failure, viral infection, and type 2 diabetes24,25. Pseudouridine was reported to undergo dynamic changes in response to serum starvation, hydrogen peroxide and heat shock in mammalian cells24. It has previously been reported that H2 administration could regulate expression of diverse genes26, our results suggest that molecular hydrogen may regulate gene expression by affecting epigenetic modifications. In addition, the plasma levels of N1-methyl-2-pyridone-5-carboxamide was significantly decreased by HRW intake. Nicotinamide adenine dinucleotide (NAD+) is an important coenzyme for redox reactions, making it central to energy metabolism27. Nicotinamide mononucleotide (NMN) is one of the substrates for NAD+ synthesis, which can be further metabolized to N1-methyl-2-pyridone-5-carboxamide. Thus, HRW intake may regulate energy metabolism via affecting NAD+ synthesis.
Unlike HRW intake, HI had an up-regulatory effects on plasma DMs. Six of those DMs, including l-citrulline, l-leucine, sarcosine, l-glutamic acid, N-carbamoyl-l-aspartic acid, and NAAG, were belong to amino acids and their derivatives. Among them, the plasma levels of l-citrulline showed the most considerable increase in HI group compared to the controls. It has been showed in rats that only the intestine produced circulating citrulline, and the increased citrulline levels may be caused by either increased production or decreased utilization28. The decreased utilization could arise from a decrease in clearance, i.e. renal failure. However, no significant change of creatinine plasma levels was observed in our study, indicating that the increased citrulline levels was not caused by the impairment of renal function. Previous study showed that the increase in plasma citrulline was correlated with protein absorption improvement in patients with short bowel syndrome (SBS) followed in the first year after resection29. We supposed that the increased citrulline plasma levels may be associated with the improvement of enterocyte function, although this need to be further investigated. HI also induced a significant increase in NAAG plasma levels. NAAG is the most prevalent and widely distributed dipeptide in the mammalian nervous system30. The levels of NAAG in plasma and cerebrospinal fluid (CSF) were much lower than those in brain tissues31. It is well established that the increase in NAAG is neuroprotective against N-methyl-d-aspartate (NMDA) receptor-mediated neurotoxicity, including ischemic brain injury30. HI was first reported to exert neuroprotective effects on ischemic stroke, and further studies also found its protective effects on other neurological impairment, including traumatic brain injury, subarachnoid hemorrhage, and neurodegenerative diseases32. Consistent with our results, the HI induced increase in NAAG was also observed in cortex tissues of mice with ischemic stroke33. Although the ability of molecular hydrogen to scavenge hydroxyl radicals may partly explain its neuroprotective effects, the regulatory effects on NAAG may also responsible for its protective benefits. In addition, the significant increase in plasma levels of two citric acid cycle intermediates, cis-aconitic acid and malic acid, were also observed in our study, indicating that HI may accelerate mitochondrial energy metabolism. N-carbamoyl-l-aspartic acid and inosine are intermediates of pyrimidine and purine metabolism, respectively. The increase in the two metabolites suggest that HI may have modulatory effects on nucleoside metabolism. The plasma levels of other DMs, including l-leucine, sarcosine, l-glutamic acid, and l-lactic acid, varied slightly by HI.
Recent studies have provided evidence that modulation of host gut microbiota may be one of the mechanisms contributing to the biological effects of exogenous hydrogen consumption. Qiu et al. showed that saturated hydrogen saline treatment could modulate the abundance of Bacteroides, Bifidobacteria, and Lactobacillus in feces, which may responsible for the improvement of lipid metabolism disorders in high-fat diet mice34. Jin et al. reported that sustained H2 release in the gut by hydrogen nanocapsule could increase the abundance of Akkermansia muciniphila and attenuate metabolic dysfunction-associated fatty liver disease35. In this study, HRW intake induced significant changes in the structure of gut microbiota, while no marked bacterial community differences was observed in HI group. Previous study showed that the peak of the hydrogen concentration in small intestine after oral intake of 5 ppm of HRW was approximately 20 times higher than that after inhalation of 4% hydrogen gas36. The significant difference in hydrogen concentration in intestine between HRW intake and HI may contribute to the different effects on microbiota composition.
In our study, HRW intake induced significant increase in the proportion of Lactobacillus, Ruminococcus, Clostridium XI, Elusimicrobium, Barnesiella, and Aquabacterium, and decrease in Bacteroides, Anaerotruncus, Desulfovibrio, Mucispirillum, and Bifidobacterium. Lactobacillus and Bifidobacterium are the most common probiotic bacteria with the reported beneficial effects including aid digestion, reduce constipation, resist infections, prevent traveler’s diarrhea and ameliorate intestinal bowel disease (IBD)37. In our study, HRW intake induced a significant increase in the abundance of Lactobacillus. The increased abundance of Lactobacillus induced by HRW intake may contribute to the beneficial effects of HRW. Although HRW also induced a marked decrease in Bifidobacterium, the relative abundances of Bifidobacterium is very low. A recent clinical study showed that drinking hydrogen-dissolved alkaline electrolyzed water (AEW) for two weeks induced an increase in Bifidobacterium in healthy volunteers38. The different pH values (HRW 7.5 vs. AEW 9.5) or duration of hydrogen treatment (HRW 6 months vs. AEW 2 weeks) may contribute to the different effects on the levels of Bifidobacterium. It has been shown that supplementation with Ruminococcus flavefaciens could attenuate the antidepressant effects of duloxetine on depressive-like behavior39, although the increased Ruminococcus has also been shown to be beneficial regarding antidepressant-induced constipation39. Previous study showed that 4 weeks of HRW intake could exert beneficial effects on depressive-like behavior in mice via suppression of the inflammasome activation40, the antidepressive effects of HRW may be diminished by long-term HRW intake induced increase in Ruminococcus according to our study. Clostridium XI belongs to class Clostridia, which have been reported to attenuate inflammation and allergic diseases41. It has also been demonstrated that Clostridium species can utilize indigestible polysaccharide and produce lots of short-chain fatty acids (SCFAs), which are now considered as key players in the interactions with the host that impact on health and disease, especially given recent evidence for their capacity to modify the epigenome and effects on tissues and organs beyond the gut42. The increase in Clostridium XI derived SCFAs may also contributed to the effects of HRW. In a study of 345 Chinese individuals, members of the genera Bacteroides has been shown to be more abundant in type II diabetic subjects compared to controls with normal glucose metabolism43. The improved glucose tolerance and hyperglycemia lowering effect of HRW intake have been previously reported9,44, which may be attributed by the HRW-induced decrease in Bacteroides levels. The results of the Spearman correlation analyses revealed a great number of significant correlations between the abundant of Bacteroides and DMs, including urocanic acid, l-lactic acid, N-acetyl-d-glucosamine, and N-Acetyl-l-tyrosine, however, the causal relationships between alterations in Bacteroides abundance and plasma DMs need to be further investigated. Although the levels of other genera, including Elusimicrobium, Barnesiella, Aquabacterium, Anaerotruncus, Desulfovibrio, Mucispirillum, and Bifidobacterium changed significantly, their relative abundance was very low.
Compared with HRW group, the changes in fecal microbiota were found to be much less in HI group. Among these changed genera, the abundances of Blautia and Paraprevotella were significantly increased. Blautia has been considered as a probiotic bacterium that occur widely in mammalian feces and intestines45. The increased abundances of Blautia may contributed to the effects of HI. The Paraprevotella genus has been found to be negatively correlated with the BMI index46. Consistently, the negative correlation was also observed between the abundance of Paraprevotella genus and the BW of rats. Spearman correlation analyses revealed a great number of significant correlations between the abundant of Blautia and DMs, Correlation analyses revealed significant negative correlations between the abundance of Blautia and DMs, including l-leucine and malic acid. The abundance of Paraprevotella was negatively correlated with DMs, including inosine, cis-aconitic acid, and N-carbamoyl-l-aspartic acid. The other changed genera, including Elusimicrobium, Propionibacterium, Porphyrobacter, Methanosphaera, and Bifidobacterium, all had relatively low abundance.
Although both HRW and HI have beneficial effects in various diseases, the underlying mechanisms of them may be different. For HRW intake, it has been reported that many kinds of diseases, especially gastrointestinal symptoms, such as constipation and diarrhea, could benefit from HRW. It is known that the gut microbiota plays an important role in health and disease, the modulatory effect of HRW on gut microbiota may significantly contribute to the improvement of these diseases. In our study, the results of metabolic cage experiments showed that HI induced significant changes in food/water intake of rats, which positively correlates with changes in defecation/urination, while no obvious effect was observed in HRW group. The reasons for the decline in food intake are multifactorial and involve both peripheral and central mechanisms47. Hyspler et al. used deuterium gas as a metabolic tracer to quantify hydrogen metabolism in the mammalian body and found that the deuterium can be oxidized to water48. In our study, the inhaled hydrogen gas may probably be oxidized to water, providing an endogenous supply of H2O, which might explain the decrease in the water intake of rats. Moreover, in our previous studies we have provided preliminary evidence that eukaryocytes have the ability of hydrogen metabolism, which may affect its metabolic activities49,50. It is, therefore, possible to hypothesize that HI may exert its biologic role through modulation of metabolic function of mitochondria, although this needs to be further investigated.
One limitation of the present study is that research on the effects of hydrogen intervention on faecal microbiota profiles was focused on the genus level, and did not conduct in-depth studies at the species or even strain levels. Another limitation is the lack of proven causal relationships between alterations in microbiota profiles and plasma metabolites. In addition, further study should also evaluate the effects of hydrogen intervention on the production of SFCAs, which play a key role in microbiota-host interactions.
Collectively, the results of this study could provide basic data for further research on hydrogen medicine. Our results also shed light on the effects of different routes of hydrogen intervention on microbiota profiles, which may significantly contributed to the therapeutic effects of hydrogen in various diseases.
