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Microbes & Immunity Hydrogen alleviates non-alcoholic fatty liver disease

predominant cause of morbidity and mortality among solution (E607318-0200, Sangon Biotech, Shanghai,
liver-related diseases, particularly due to the significantly China); Oil Red O solution (C0157S, Beyotime Technology,
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growing prevalence of diabetes-related metabolic diseases Shanghai, China); Mouse Lipopolysaccharides (LPSs),
and cardiovascular disease in NAFLD patients. Currently, enzyme-linked immunosorbent assay (ELISA) kit (CSB-
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no approved drugs that are specific for NAFLD are E13066m, Cusabio, Wuhan, China); Mouse TNF-α,
available. 4 TNF-α ELISA Kit (CSB-E04741m, Cusabio, Wuhan,
Intestinal microorganisms are involved in the China); SpectraMax M5 Reader (Molecular Devices,
development of NAFLD through the gut-liver axis. USA); genomic DNA extraction kit for soil and fecal
An imbalance in the intestinal flora causes changes samples (CW2091S, Novogene, Tianjing, China); GeneJET
in intestinal permeability, resulting in the release of Gel Recovery Kit (K0691, Thermo Scientific, USA);
TruSeq DNA polymerase chain reaction (PCR)-Free
metabolites produced by the intestinal flora into the Library Preparation Kit (20000902, Illumina, USA); Qubit
blood, thus activating the inflammatory pathway and
releasing pro-inflammatory factors (e.g., tumor necrosis Instrument (Novogene, Tianjing, China); and NovaSeq
factor-alpha [TNF-α] and interleukin [IL]-6) into 6000 sequencing system (Novogene, Tianjing, China).
the liver. Elevated serum concentrations of TNF-α in 2.2. Mouse model and H intervention
NAFLD patients correlate with the severity of liver tissue 2
destruction. Moreover, previous studies have shown Male C57BL/6 mice (aged 6 weeks) were housed in
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that the intestinal bacterium Akkermansia muciniphila an animal facility under standard specific pathogen-
is negatively associated with metabolic disorders such as free conditions at 22°C on a 12-h light/dark schedule.
obesity, diabetes, and inflammation. Oral administration All animal studies were approved by the Institutional
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of A. muciniphila was found to increase the expression Animal Care and Use Committee of the Tongji University
of intestinal tight junction proteins such as zonula Affiliated East Hospital (T3LAC-015-038). After 1 week of
occludens-1 and occludin in the intestines of mice with acclimatization to a normal diet, the mice were divided into
NAFLD. Therefore, A. muciniphila is thought to reduce three groups: the normal control group, the HFD group,
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endotoxemia levels and alleviate local inflammation by and the HFD + H group, with five mice in each group. The
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reducing intestinal permeability and strengthening the mice of the control group were fed with ordinary diet. The
intestinal barrier. 8,9 model was induced by feeding the animal with HFD diet,
which contained 20% protein, 20% carbohydrates, and
Hydrogen (H ) is a novel method for treating 60% fat (Figure 1A). The experiment of the mouse model
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inflammatory diseases. Recent studies have shown lasted for 11 weeks.
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that H plays a promising role in antioxidation, weight
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reduction and Kawasaki disease treatment. H can also Mice in the HFD + H group were subjected to H
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12,
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2
2
th
alleviate NAFLD-related symptoms. Although a few inhalation for 11 weeks starting at the 12 week (Figure 1B).
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reports have suggested that the development of NAFLD H was provided by our self-developed device (AMS-H-
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is related to changes in intestinal microflora, whether H 01 H /oxygen nebulizer), which consists of a gas source
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alleviates NAFLD patients’ symptoms through alterations device, a gas mixing device, a carbon dioxide absorption
in certain intestinal microflora remains unclear. 15,16 To device, and a H treatment chamber. The gas source unit
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explore the therapeutic effect and mechanism of H in produces a H -oxygen gas mixture by electrolyzing water.
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NAFLD, in our study, we investigated changes in the gut The flow rate of the gas mixture was adjusted through
microbiota in NAFLD model mice treated with 30% H . the gas mixing unit. The carbon dioxide absorption unit
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This study provides a theoretical basis for the treatment of is mainly composed of soda lime and can absorb carbon
NAFLD with H . dioxide produced by the animals in the chamber. For the
2 H absorption treatment, the mouse cage box was placed in
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2. Materials and methods the H treatment chamber and the H -oxygen gas mixture
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(containing 33.3% H ) was inhaled with a total flow rate of
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2.1. Materials and instrument 3 L/min for 8 h, once daily.
Male C57BL/6 mice (aged 6 weeks; SCXK (su)
2018 – 0008) were procured from GemPharma Tech LLC 2.3. Blood glucose and body weight measurements
(China). Materials and instruments used in this study are Blood glucose measurement was performed as follows: the
listed as follows: High-fat diet (HFD; 60% fat diet, XTHF60, tail of each mouse was pierced by a needle, and a drop of
XIETONG Biology, Nanjing, China); AMS-H-01 H /oxygen venous blood was collected to measure blood glucose levels
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nebulizer (Asclepius, Shanghai, China); glucometer (GA-6, with a glucose meter once every 2 weeks. The weights of
Sanocare, Shenzhen, China); hematoxylin-eosin (H&E) mice were also measured once every 2 weeks.
Volume 1 Issue 2 (2024) 71 doi: 10.36922/mi.3896