A randomized, placebo-controlled clinical trial of hydrogen/oxygen inhalation for non-alcoholic fatty liver disease

Abstract

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease worldwide with increasing incidence consistent with obesity, type 2 diabetes and cardiovascular diseases. No approved medication was currently available for NAFLD treatment. Molecular hydrogen (H₂ ), an anti-oxidative, anti-inflammatory biomedical agent is proved to exhibit therapeutic and preventive effect in various diseases. The purpose of this study was to investigate the effect of hydrogen/oxygen inhalation on NAFLD subjects and explore the mechanism from the perspective of hepatocyte autophagy. We conducted a randomized, placebo-controlled clinical trial of 13-week hydrogen/oxygen inhalation (China Clinical Trial Registry [#ChiCTR-IIR-16009114]) including 43 subjects. We found that inhalation of hydrogen/oxygen improved serum lipid and liver enzymes. Significantly improved liver fat content detected by ultrasound and CT scans after hydrogen/oxygen inhalation was observed in moderate-severe cases. We also performed an animal experiment based on methionine and choline-deficient (MCD) diet-induced mice model to investigate effect of hydrogen on mouse NASH. Hydrogen/oxygen inhalation improved systemic inflammation and liver histology. Promoted autophagy was observed in mice inhaled hydrogen/oxygen and treatment with chloroquine blocked the beneficial effect of hydrogen. Moreover, molecular hydrogen inhibited lipid accumulation in AML-12 cells. Autophagy induced by palmitic acid (PA) incubation was further promoted by 20% hydrogen incubation. Addition of 3-methyladenine (3-MA) partially blocked the inhibitory effect of hydrogen on intracellular lipid accumulation. Collectively, hydrogen/oxygen inhalation alleviated NAFLD in moderate-severe patients. This protective effect of hydrogen was possibly by activating hepatic autophagy.

Keywords: MCD-induced NASH; NAFLD; autophagy; clinical trial; molecular hydrogen.

FIGURE 1

Serum biological, oxidative and immunological index of subjects before and after the trial. Serum samples of subjects in hydrogen/oxygen and placebo groups were collected before and after the trial and analysed at the Second Affiliated Hospital of Shandong First Medical University by automatic biochemical analyser (HITACHI 7080). Serum MDA, SOD, TNF‐α and IL‐6 were quantified using commercial kits. N: placebo group, H₂: hydrogen/oxygen group. Student's t‐test was applied and asterisk * indicates significant difference (p < 0.05)

FIGURE 2

Representative liver CT scan of patients from hydrogen/oxygen and placebo groups. CT scans were conducted in Shandong Provincial Coal Taishan Sanatorium before and after the trial. Images were analysed using Onis Viewer 2.6. Hounsfield units were obtained from 2 region of interest (ROI) in the right lobe of liver and 1 site of the spleen. Liver/spleen CT ratio (CT _L/S ) was obtained by calculation of L/S

FIGURE 3

Hydrogen inhalation ameliorates MCD diet‐induced NASH model in C57B6/J mice. Serum ALT, AST (A) and cytokines (C), liver redox status were measured (B). (D) Liver H&E staining was performed, and representative images were shown. (E) Autophagy of mice liver from Chow, MCD + N₂ and MCD + H₂ groups were assessed by Western blot. Comparisons were conducted between 2 groups using Students' t‐test. * indicates p < 0.05, ** indicates p < 0.01, ***p < 0.0001

FIGURE 4

Hydrogen decreased lipid accumulation in AML‐12 cells partially depending on promotion of autophagy. AML‐12 cells were incubated with FFA or PA as indicated and maintained either in conventional CO₂ incubator (mock) or H₂ incubator (H₂) for 24 h. Quantification of TG was normalized to soluble protein by BCA kits (A) and oil red O staining (B). (C) Autophagy of AML‐12 cells incubated with FFA or PA for 12 h was detected by Western blot. (D) Transmission electron microscopy (TEM) of AML‐12 cells exposed to 0.5 mmol/L PA maintained in either CO₂ incubator or H₂ incubator for 12 h was conducted. Green, red and blue arrows represent mitochondria, double layered autophagosomes and autolysosomes, respectively. Quantification of autophagic vesicles was shown. Scale bars =1 μm. (E) Intracellular TG quantification of cells incubated with 0.5 mmol/L PA either in CO₂ incubator or H₂ incubator for 24 h with additional 3 mmol/L 3‐methyladenine (3‐MA). Statistical analysis was performed using student’s t‐test. Asterisk * indicates p < 0.05