Hydrogen-Rich Water Ameliorates Metabolic Disorder via Modifying Gut Microbiota in Impaired Fasting Glucose Patients: A Randomized Controlled Study

Objective: Molecular hydrogen (H2) exhibits antioxidant, anti-inflammatory and anti-apoptotic effects, and has shown benefits in glucose and lipid metabolism in certain animal metabolic disorder models. However, the potential benefits of H2 treatment in individuals with impaired fasting glucose (IFG) has seldom been studied. This randomized controlled study (RCT) aims to investigate the effects of hydrogen-rich water (HRW) on IFG subjects and explore the underlying mechanism involved. Methods: Seventy-three patients with IFG were enrolled in a randomized, double-blind, placebo-controlled clinical study. These patients were assigned to receive either 1000 mL per day of HRW or placebo pure water (no H2 infusion) for a duration of eight weeks. Metabolic parameters and fecal gut microbiota were assessed at baseline (week 0) and at week 8. A combined analysis of metabolomics and intestinal microbiota was conducted to investigate the correlation between the effect of H2 on the metabolisms and the diversity of intestinal flora in the IGF patients. Results: Both pure water and HRW demonstrated a significant reduction in fasting blood glucose in IFG patients, with a significant difference between pure water and HRW after eight weeks. Among IFG patients with abnormal pre-experimental fatty liver, 62.5% (10/16) in the HRW group and 31.6% (6/19) in the pure water group achieved remission. Furthermore, 16S RNA analysis revealed HRW-modified gut microbiota dysbiosis in the fecal samples of IGF patients. Through Pearson correlation analysis, the differential gut microbiota obtained by 16S analysis was found to be highly correlated with nine metabolites. Conclusion: H2 slightly improved metabolic abnormalities and gut microbiota dysbiosis, providing a novel target and theoretical basis for the prevention and treatment of blood glucose regulation in patients with IFG.

UPLC/MS-Based Metabolomics Investigation of the Protective Effect of Hydrogen Gas Inhalation on Mice with Calcium Oxalate-Induced Renal Injury

Hydrogen has a significant protective effect on calcium oxalate-induced renal injury, but its effect on metabolic profiles is unknown. This study showed the effects of hydrogen on serum and urine metabolites in a renal injury model. Ultra-HPLC quadrupole time-of-flight-MS-based metabolomics was used to characterise metabolic variations. Twenty-five serum metabolites and 14 urine metabolites showed differences in the the nitrogen and oxygen inhalation (NO), nitrogen and oxygen inhalation combined with calcium oxalate induction (CaOx), and hydrogen inhalation combined with calcium oxalate induction (HO+CaOx) groups. Nineteen serum metabolites and 7 urine metabolites showed significant restoration to normal levels after hydrogen gas (H2) treatment. These metabolites are primarily related to amino acid metabolism, fatty acid metabolism, and phospholipid metabolism. This study showed that a comprehensive metabolomics approach is an effective strategy to elucidate the mechanisms underlying the effects of hydrogen treatment on calcium oxalate-induced renal injury.

Magnesium Hydride Ameliorates Endotoxin-Induced Acute Respiratory Distress Syndrome by Inhibiting Inflammation, Oxidative Stress, and Cell Apoptosis

Acute respiratory distress syndrome (ARDS) causes uncontrolled pulmonary inflammation, resulting in high morbidity and mortality in severe cases. Given the antioxidative effect of molecular hydrogen, some recent studies suggest the potential use of molecular hydrogen as a biomedicine for the treatment of ARDS. In this study, we aimed to explore the protective effects of magnesium hydride (MgH2) on two types of ARDS models and its underlying mechanism in a lipopolysaccharide (LPS)-induced ARDS model of the A549 cell line. The results showed that LPS successfully induced oxidative stress, inflammatory reaction, apoptosis, and barrier breakdown in alveolar epithelial cells (AEC). MgH2 can exert an anti-inflammatory effect by down-regulating the expressions of inflammatory cytokines (IL-1β, IL-6, and TNF-α). In addition, MgH2 decreased oxidative stress by eliminating intracellular ROS, inhibited apoptosis by regulating the expressions of cytochrome c, Bax, and Bcl-2, and suppressed barrier breakdown by up-regulating the expression of ZO-1 and occludin. Mechanistically, the expressions of p-AKT, p-mTOR, p-P65, NLRP3, and cleaved-caspase-1 were decreased after MgH2 treatment, indicating that AKT/mTOR and NF-κB/NLRP3/IL-1β pathways participated in the protective effects of MgH2. Furthermore, the in vivo study also demonstrated that MgH2-treated mice had a better survival rate and weaker pathological damage. All these findings demonstrated that MgH2 could exert an ARDS-protective effect by regulating the AKT/mTOR and NF-κB/NLRP3/IL-1β pathways to suppress LPS-induced inflammatory reaction, oxidative stress injury, apoptosis, and barrier breakdown, which may provide a potential strategy for the prevention and treatment of ARDS.

Oral Hydrogen-Rich Water Alleviates Oxalate-Induced Kidney Injury by Suppressing Oxidative Stress, Inflammation, and Fibrosis

Objective: To explore the theraputic effects and potential mechanisms of hydrogen-rich water (HRW) against oxalate-induced kidney injury. Methods: The mouse model of Calcium oxalate (CaOx) crystallization was established by feeding a soluble oxalate diet. Crystal deposition, tubular injury, fibrosis and reactive oxygen species (ROS) production in kidneys were examined by histology. Serum indexes of renal injury, inflammation and oxidative stress were detected by commercial kits. RNA sequencing (RNA-seq) was performed to screen potential pathways and the expressions of key molecules in these pathways were determined by western blotting and immunohistochemistry. Results: Crystal deposition, tubular injury, fibrosis and increased ROS production in kidneys of mice induced by oxalate diet were improved with HRW administration. The indexes of renal injury, inflammation and oxidative stress in serum of mice were upregulated by oxalate diet, which were reduced by HRW. A total of 3,566 differential genes were screened by RNA-seq and these genes were analyzed by pathway enrichment and PI3K/AKT, NF-κB, and TGF-β pathways were selected for further verification. The expressions of molecules related to PI3K-AKT pathway (PI3K, AKT, and p-AKT), NF-κB pathway (NF-κB p65, p- NF-κB p65, NLRP3, and IL-1β) and TGF-β pathway (TGF-β, TGF-βRI, TGF-βRII, p-Smad2, and p-Smad3) in renal tissues were increased by oxalate diet, which were reduced by HRW administration. Conclusion: HRW may alleviate oxalate-induced kidney injury with its anti-oxidative, anti-inflammatory and anti-fibrotic effects via inhibiting PI3K/AKT, NF-κB, and TGF-β pathways.

Hydrogen therapy reduces apoptosis in neonatal hypoxia-ischemia rat model

Hypoxia-ischemia (HI) brain injury is a major cause of neuronal cell death especially apoptosis in the perinatal period. This study was designated to examine the effect of hydrogen therapy on apoptosis in an established neonatal HI rat pup model. Seven-day-old rat pups were subjected to left common carotid artery ligation and then 90 min hypoxia (8% oxygen at 37 C). Immediately after HI insult, pups were placed into a chamber filled with 2% H(2) for 30 min, 60 min, or 120 min, respectively. 24 h after 2% H2 therapy, the pups were decapitated and brain injury was assessed by 2,3,5-triphenyltetrazoliumchloride (TTC), Nissl, and TUNEL staining, as well as caspase-3, caspase-12 activities in the cortex and hippocampus. H(2) treatment in a duration-dependent manner significantly reduced the number of positive TUNEL cells and suppressed caspase-3 and -12 activities. These results indicated H(2) administration after HI appeared to provide brain protection via inhibition of neuronal apoptosis. (C) 2008 Elsevier Ireland Ltd. All rights reserved.

Hydrogen-rich saline protects against intestinal ischemia/reperfusion injury in rats

Hydrogen gas was reported to reduce reactive oxygen species and alleviate cerebral, myocardial and hepatic ischemia/reperfusion (I/R) injuries. This paper studied the effect of hydrogen-rich saline, which was easier for clinical application, on the intestinal I/R injury. Model of intestinal I/R injury was induced in male Sprague-Dawley rats. Physiological saline, hydrogen-rich saline or nitrogen-rich saline (5 ml/kg) was administered via intravenous infusion at 10 min before reperfusion, respectively. The intestine damage was detected microscopically and was assessed by Chiu score system after I/R injury. In addition, serum DAO activity, TNF-alpha, IL-1beta and IL-6 levels, tissue MDA, protein carbonyl and MPO activity were all increased significantly by I/R injury. Hydrogen-rich saline reduced these markers and relieved morphological intestinal injury, while no significant reduction was observed in the nitrogen-rich saline-treated animals. In conclusion, hydrogen-rich saline protected the small intestine against I/R injury, possibly by reduction of inflammation and oxidative stress.

Hydrogen gas is ineffective in moderate and severe neonatal hypoxia-ischemia rat models

Hydrogen gas (H(2)) has been shown to ameliorate brain injury in experimental adult rat focal ischemia and in a mild neonatal hypoxia-ischemia (HI, 90 min hypoxia) rat model. In this study we tested H(2) in moderate (120 min hypoxia) and severe (150 min hypoxia) neonatal HI rat models. We hypothesized that H(2) would improve outcomes after neonatal HI by scavenging free radicals. Two hundred (200) unsexed Sprague-Dawley rats at day 10 of life (p10) underwent neonatal HI with the Rice-Vannucci model. Multiple treatment protocols were studied, including pre-ischemic treatment, intra-ischemic treatment, and post-ischemic treatment (Sham n=32, HI n=82, HI+H(2)n=86). We also tested H(2) in middle cerebral artery occlusion (MCAO) in adult rats (MCAO n=9, MCAO+H(2)n=7) for comparison. Analysis at 24 h included infarction volume, measurement of brain concentration of malondialdehyde (MDA) (an end-product of lipid peroxidation), daily weight, Nissl histology, and mortality. In moderate and severe neonatal HI models, hydrogen gas therapy (2.9% concentration H(2)) was not associated with decreased volume of infarction or decreased concentration of MDA. H(2) gas pretreatment (2.9%) was associated with increased infarction volume in neonatal HI. In MCAO in adult rats, H(2) gas therapy demonstrated a trend of beneficial effect. Exposure of H(2) gas to non-ischemic neonates resulted in a significant increase in brain concentration of MDA. We conclude that 2.9% H(2) gas therapy does not ameliorate moderate to severe ischemic damage in neonatal hypoxia-ischemia.

Hydrogen-rich medium protects human skin fibroblasts from high glucose or mannitol induced oxidative damage

Reactive oxygen species (ROS) are an important factor in the development of skin lesions in diabetes. A new antioxidant, hydrogen, can selectively neutralize hydroxyl radicals (OH) and peroxynitrite (ONOO−) in cell-free systems, whereas it seldom reacts with other ROS. Fibroblasts are a key component of skin. In the present study, we investigated the protective effects of hydrogen-rich medium on human skin fibroblasts (HSFs) under oxidative stress. Confocal microscopy was used to assay both the intracellular superoxide anion () concentration and the mitochondrial membrane potential (ΔΨ). Cell viability was determined using the Cell Counting Kit-8 (CCK-8). The concentrations of cellular malonaldehyde (MDA), superoxide dismutase (SOD), glutathione (GSH), 8-hydroxy-2′-deoxyguanosine (8-OHdG) and 3-nitrotyrosine (3-NT) were also measured. The results revealed that both mannitol and high glucose could cause oxidative stress in HSFs. Interestingly, the use of a hydrogen-rich medium significantly reduced the level of intracellular , stabilized the ΔΨ and attenuated production of MDA, 8-OHdG and 3-NT which efficiently enhanced the antioxidative defense system and protected the HSFs from subsequent oxidative stress damage. In other words, hydrogen decreased the excessive generation of intracellular and elevated the cellular antioxidative defense. Based on our results, hydrogen may have applications in the treatment of skin diseases caused by diabetes.

The Protective Role of Hydrogen -Rich Saline in Experimental Liver Injury in Mice

Reactive oxygen species (ROS) are considered to play a prominent causative role in the development of various hepatic disorders. Antioxidants have been effectively demonstrated to protect against hepatic damage. Hydrogen (H(2)), a new antioxidant, was reported to selectively reduce the strongest oxidants, such as hydroxyl radicals (·OH) and peroxynitrite (ONOO(-)), without disturbing metabolic oxidation-reduction reactions or disrupting ROS involved in cell signaling. In place of H(2) gas, hydrogen-rich saline (HS) may be more suitable for clinical application. We herein aim to verify its protective effects in experimental models of liver injury. H(2) concentration in vivo was detected by hydrogen microelectrode for the first time. Liver damage, ROS accumulation, cytokine levels, and apoptotic protein expression were, respectively, evaluated after GalN/LPS, CCl(4), and DEN challenge. Simultaneously, CCl(4)-induced hepatic cirrhosis and DEN-induced hepatocyte proliferation were measured. HS significantly increased hydrogen concentration in liver and kidney tissues. As a result, acute liver injury, hepatic cirrhosis, and hepatocyte proliferation were reduced through the quenching of detrimental ROS. Activity of pro-apoptotic players, such as JNK and caspase-3, were also inhibited. HS could protect against liver injury and also inhibit the processes leading to liver cirrhosis and hepatocyte compensatory proliferation.

H2 inhibits TNF-α-induced lectin-like oxidized LDL receptor-1 expression by inhibiting nuclear factor κB activation in endothelial cells

H(2) is a therapeutic antioxidant that can reduce oxidative stress. Oxidized low-density lipoprotein, which plays roles in atherosclerosis, may promote endothelial dysfunction by binding the cell-surface receptor LOX-1. LOX-1 expression can be upregulated by various stimuli, including TNF-α. Thus, we aimed to examine whether the upregulation of LOX-1 by different stimuli could be blocked by H(2) in endothelial cells. H(2) significantly abolished the upregulation of LOX-1 by different stimuli, including TNF-α, at the protein and mRNA levels. The TNF-α-induced upregulation of LOX-1 was also attenuated by the NF-κB inhibitor N-acetyl-L-cysteine. H(2) inhibited the TNF-α-induced activation of NF-κB and the phosphorylation of IκB-α. Furthermore, H(2) inhibited the expression of LOX-1 and the activation of NF-κB in apolipoprotein E knockout mice, an animal model of atherosclerosis. Thus, H(2) probably inhibits cytokine-induced LOX-1 gene expression by suppressing NF-κB activation.