Hydrogen gas alleviates acute ethanol-induced hepatotoxicity in mice via modulating TLR4/9 innate immune signaling and pyroptosis

Alcoholic liver disease (ALD), which is induced by chronic heavy alcohol consumption, accompanies complicated pathological mechanisms, including oxidative stress, inflammation, cell death, epigenetic changes and acetaldehyde-mediated toxicity. Hydrogen (H2) is the lightest gas with multiple biological effects such as high selective anti-oxidation, anti-inflammation and anti-apoptosis. However, the dose effects and innate immune mechanisms of intraperitoneal injection of H2 on ALD are limited. Here, we used acute ethanol-induced hepatotoxicity mice models to estimate the actions of intraperitoneal injection of H2 on ALD. The effects of H2 on acute ethanol-induced liver damage were examined by hepatic oil red O staining, quantitative PCR (qPCR) for lipid metabolic genes, hepatic triglyceride (TG) and serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels. Hepatic mitochondrial superoxide (MitoSOX), 3-nitrotyrosine (3-NT), malondialdehyde (MDA), and glutathione (GSH) levels were examined to evaluate oxidative stress. Immunoblot, and immunofluorescence staining were used to further confirm the innate immune molecular targets of H2. Our results showed that intraperitoneal injection of H2 improved acute ethanol-induced liver injury in mice in a dose dependent manner, as indicated by decreasing serum ALT and AST levels, hepatic TG levels, and increasing lipid export genes (Mttp and Apob) mRNA levels and reducing fatty acid uptake gene (CD36) mRNA levels. Mechanistically, H2 inhibited hepatic oxidative stress as indicated by reducing reactive oxygen species (ROS), 3-NT, and MDA levels in the liver, while increasing hepatic GSH levels; inhibited the overactived TLR4/9-NF-κB-TNF-α/IL-1β/IL-18 innate immune signaling; suppressed the canonical Caspase-1-GSDMD pyroptosis signaling, and the non-canonical pyroptosis signaling, such as Caspase-11-GSDMD, Caspase-8-GSDMD and Caspase-3-GSDME signaling. Therefore, our study highlights that intraperitoneal injection of H2 may represent a novel therapeutic and safe strategy for ALD via modulating oxidative stress, innate immunity and pyroptosis.

Hydrogen (H2) Inhibits Isoproterenol-Induced Cardiac Hypertrophy via Antioxidative Pathways

Background and Purpose: Hydrogen (H2) has been shown to have a strong antioxidant effect on preventing oxidative stress-related diseases. The goal of the present study is to determine the pharmacodynamics of H2 in a model of isoproterenol (ISO)-induced cardiac hypertrophy. Methods: Mice (C57BL/6J; 8–10 weeks of age) were randomly assigned to four groups: Control group (n = 10), ISO group (n = 12), ISO plus H2 group (n = 12), and H2 group (n = 12). Mice received H2 (1 ml/100g/day, intraperitoneal injection) for 7 days before ISO (0.5 mg/100g/day, subcutaneous injection) infusion, and then received ISO with or without H2 for another 7 days. Then, cardiac function was evaluated by echocardiography. Cardiac hypertrophy was reflected by heart weight/body weight, gross morphology of hearts, and heart sections stained with hematoxylin and eosin, and relative atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) mRNA levels. Cardiac reactive oxygen species (ROS), 3-nitrotyrosine and p67 (phox) levels were analyzed by dihydroethidium staining, immunohistochemistry and Western blotting, respectively. For in vitro study, H9c2 cardiomyocytes were pretreated with H2-rich medium for 30 min, and then treated with ISO (10 μM) for the indicated time. The medium and ISO were re-changed every 24 h. Cardiomyocyte surface areas, relative ANP and BNP mRNA levels, the expression of 3-nitrotyrosine, and the dissipation of mitochondrial membrane potential (MMP) were examined. Moreover, the expression of extracellular signal-regulated kinase1/2 (ERK1/2), p-ERK1/2, p38, p-p38, c-Jun NH2-terminal kinase (JNK), and p-JNK were measured by Western blotting both in vivo and in vitro. Results: Intraperitoneal injection of H2 prevented cardiac hypertrophy and improved cardiac function in ISO-infused mice. H2-rich medium blocked ISO-mediated cardiomyocytes hypertrophy in vitro. H2 blocked the excessive expression of NADPH oxidase and the accumulation of ROS, attenuated the decrease of MMP, and inhibited ROS-sensitive ERK1/2, p38, and JNK signaling pathways. Conclusion: H2 inhibits ISO-induced cardiac/cardiomyocytes hypertrophy both in vivo and in vitro, and improves the impaired left ventricular function. H2 exerts its protective effects partially through blocking ROS-sensitive ERK1/2, p38, and JNK signaling pathways.

Hydrogen inhibits isoproterenol‑induced autophagy in cardiomyocytes in vitro and in vivo

A previous study from our group has demonstrated that hydrogen administration can attenuate cardiovascular hypertrophy in vivo by targeting reactive oxygen species‑dependent mitogen‑activated protein kinase signaling. The aim of the present study is to determine the effect of hydrogen on cardiomyocyte autophagy during β‑adrenoceptor activation in vivo and in vitro. We prepared hydrogen‑rich medium, and the concentration of hydrogen was measured by using the MB‑Pt reagent method. For the in vitro study, H9c2 cardiomyocytes were stimulated with isoproterenol (ISO; 10 µM) for 5, 15 and 30 min, and then the protein expression levels of the autophagy marker microtubule‑associated protein 1 light chain 3β II (LC3B II) were examined by western blotting. The effect of hydrogen‑rich medium was then tested by pretreating the H9c2 cardiomyocytes with hydrogen‑rich medium for 30 min, then stimulating with ISO, and examining the protein expression levels of the autophagy marker LC3B II. For the in vivo study, mice received hydrogen (1 ml/100 g/day, by intraperitoneal injection) for 7 days prior to ISO administration (0.5 mg/100 g/day, by subcutaneous injection), and subsequently received hydrogen with or without ISO for another 7 days. Hypertrophic responses were examined by heart weight (HW) and heart weight/body weight (HW/BW) measurements. The protein expression of autophagy markers Beclin1, autophagy‑related protein 7 (Atg7) and LC3B II were examined. The results demonstrated that excessive autophagy occurred following 5 min of ISO stimulation in vitro. This enhanced autophagy was blocked by pretreatment with hydrogen‑rich medium. Furthermore, hydrogen improved the deteriorated hypertrophic responses and inhibited the enhanced autophagic activity mediated by ISO administration in vivo, as indicated by decreasing HW and HW/BW, and suppressing the protein expression levels of Beclin1, Atg7 and LC3B II. Therefore, the results of the present study demonstrated that hydrogen inhibited ISO‑induced excessive autophagy in cardiomyocyte hypertrophy models in vitro and in vivo.

H2 Protects Against Lipopolysaccharide-Induced Cardiac Dysfunction via Blocking TLR4-Mediated Cytokines Expression

Background and Purpose: Septic cardiomyopathy, which is one of the features of multi-organ dysfunction in sepsis, is characterized by ventricular dilatation, reduced ventricular contractility, and reduction in ejection fraction and, if severe, can lead to death. To date, there is no specific therapy that exists, and its treatment represents a large unmet clinical need. Herein, we investigated the effects and underlying anti-inflammatory mechanisms of hydrogen gas in the setting of lipopolysaccharide (LPS)-induced cardiomyocytes injury. Experimental Approach: Hydrogen gas was intraperitoneally injected to mice in LPS plus hydrogen group and hydrogen group for 4 days. On fourth, LPS was given by intraperitoneal injection to mice in LPS group and to mice in LPS plus hydrogen group. In addition, H9c2 cardiomyocytes were treated with hydrogen-rich medium for 30 min before LPS. The transthoracic echocardiography was performed at 6 h post‐LPS to assess left ventricular end-systolic diameter (LVESD), left ventricular end-diastolic diameter (LVEDD), left ventricular ejection fraction (EF%), fractional shortening (FS%), left ventricular mass average weight (LV mass AW), and LV mass AW (Corrected). The histological and morphological analyses of left ventricular were performed by hematoxylin and eosin (H&E) staining and Masson’s trichrome staining. The mRNA levels of ANP and BNP were examined by PCR in vitro. The expression of cytokines were assayed by Enzyme Linked Immunosorbent Assay (ELISA) and PCR. Moreover, Western blotting was performed to examine the expression of TLR4, the activation of ERK1/2, p38, JNK, and the expression of NF-κB in nucleus after 6 h of LPS challenge in vivo and in vitro. Key Results: LPS induced cardiac dysfunction; hydrogen therapy improved cardiac function after LPS challenge. Furthermore, pretreatment with hydrogen resulted in cardioprotection during septic cardiomyopathy via inhibiting the expression of pro-inflammatory cytokines TNFα, IL-1β, and IL-18; suppressing the phosphorylation of ERK1/2, p38, and JNK; and reducing the nuclear translocation of NF-κB and the expression of TLR4 by LPS. Conclusion and Implications: Hydrogen therapy prevents LPS-induced cardiac dysfunction in part via downregulation of TLR4-mediated pro-inflammatory cytokines expression.

Hydrogen gas alleviates acute alcohol-induced liver injury by inhibiting JNK activation

Binge alcohol drinking is fast becoming a global health concern, with the liver among the first organ involved and the one afflicted with the greatest degree of injury. Oxidative stress, alterations in hepatic metabolism, immunity and inflammation have all been reported to contribute to the development of alcoholic liver disease (ALD). Hydrogen gas (H2) serves a key role in the modulation of hepatic redox, immune and inflammatory homeostasis. However, the effects of treatment using intraperitoneal injection of H2 on ALD remain unexplored. Therefore, the aim of the present study was to investigate the effects and underlying mechanism of intraperitoneal injection of H2 on acute alcohol-induced liver injury in a mouse model. H2 was administered by daily intraperitoneal injections (1.0 ml/100 g) for 4 days. On day 4, the mice received H2 after fasting for 5.5 h. After 30 min, the mice were administered with 33% (v/v) ethanol at a cumulative dose of 4.5 g/kg body weight by four equally divided gavages at 20-min intervals. Blood and liver tissues were collected at 16 h after the first ethanol gavage. Subsequently, serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride and total cholesterol (TC) levels were analyzed using an Automatic Clinical Analyzer. Hepatic JNK activity and GAPDH levels were examined by western blotting. It was observed that acute ethanol gavage induced liver injury, as indicated by significantly increased serum ALT and AST levels, which were effectively decreased by H2 at 16 h after the first ethanol gavage. In addition, H2 treatment reduced serum TC levels in the Alcohol+H2 group when compared with those in Alcohol group. Mechanistically, H2 attenuated hepatic JNK phosphorylation induced by acute ethanol gavage. Therefore, the results of the present study demonstrated that treatment with exogenous H2 by intraperitoneal injection may alleviate acute alcohol-induced liver injury by inhibiting hepatic JNK activation, which may represent a novel therapeutic strategy for ALD.