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Microbes & Immunity Hydrogen alleviates non-alcoholic fatty liver disease

Fresh mouse feces were collected, and DNA was extracted blood glucose levels of the HFD group gradually increased
from the samples using a genomic DNA extraction kit compared to those of the control group at the 22 week
nd
with magnetic beads for soil and fecal samples. The PCR (P < 0.01) (Figure 2G and E). However, the blood glucose
amplification procedure was as follows: PCR reaction system levels in the HFD + H group were lower than those in the
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(30 µL): Phusion Master Mix (×2) 15 µL, primer (2 µM) 3 HFD group (P < 0.05), while there was no difference in
µL (6 µM), fecal gDNA (1 ng/µL) 10 µL (5 – 10 ng), and the blood glucose levels between the HFD+H group and
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water 2 µL. The PCR procedure encompasses the following the control group at the 22 week (P > 0.05) (Figure 2F).
nd
stages: Pre-denaturation at 98°C for 1 min; followed by These data suggest that H inhalation ameliorated
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30 cycles of 98°C for 10 min, 50°C for 30 min, and 72°C for hyperglycemia in NAFLD mice. After the experiment
30 min; and 72°C for 5 min. PCR products were detected finished, the mice were fasted overnight, and the OGTT
by electrophoresis using 2% agarose gel. Then, the PCR was performed. The fasting blood glucose level of the HFD
products were mixed in equal concentrations according group was significantly higher than that of the control group
to the PCR product concentration. After mixing, the PCR (P < 0.0001). The fasting blood glucose level of the HFD
products were purified by electrophoresis using 1 × TAE + H group was significantly lower than that of the HFD
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agarose gel at 2% concentration, and the target bands were group (P < 0.01) but still higher than that of the control
recovered by the GeneJET Gel Recovery Kit. The TruSeq group (P < 0.01) (Figure 2I). The blood glucose level in the
DNA PCR-free Library Preparation Kit was subsequently HFD + H group decreased to the same level as that in the
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used for library construction. After the constructed library control group 2 h after glucose administration, but the blood
was quantified and analyzed with a Qubit instrument, the glucose in the HFD group was significantly greater than that
NovaSeq 6000 sequencing system was used for sequencing. in the control group 2 h after glucose gavage (Figure 2H).
These results suggest that an HFD induces abnormalities in
2.8. Statistical analysis
glucose tolerance in mice and that H inhalation can reverse
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The values in each group were normally distributed, and HFD-induced abnormalities in glucose tolerance.
the overall variance was homogeneous in the three groups. Compared with those in the control group, the levels
All values are presented as mean ± standard deviation for of TC (P < 0.05) and TG (P < 0.0001) in the HFD group
each group. One-way analysis of variance and LSD test were significantly higher. However, the levels of TC
were used to compare the statistical differences among (P < 0.001) and TG (P < 0.05) in the HFD + H group were
multiple groups. P < 0.05 was considered significantly significantly lower than those in the HFD group. There was
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different. GraphPad Prism (GraphPad Software, USA) no significant difference in TG level between the HFD + H
7was used for statistical analysis. 2
group and the control group (P > 0.05), although the TC
3. Results levels in the HFD + H group were still higher than those in
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the control group (P < 0.0001) (Figure 3B and C).
3.1. Effect of H on the body weight of NAFLD mice
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We successfully established a NAFLD mouse model that was 3.3. H administration alleviates liver damage and
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improves liver function in NAFLD mice
characterized by typical obesity through an HFD feeding
protocol. Compared to those in the control group, the mice Compared with those in the control group, the livers of
in the HFD and HFD + H groups stored more fat, with the mice in the HFD group were significantly larger, with a
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knotted and glossy fur, as well as a large accumulation of tense and smooth envelope, blunted edges, a yellow color,
fat in the abdominal cavity, as was observed after dissection scattered yellow fat spots, a soft texture, and a greasy feeling.
(Figure 2A). The body weights of the mice in both the HFD The livers of mice in the HFD + H group were also enlarged
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group and the HFD + H group were significantly greater in size, with a reddish color but fewer scattered yellow fat
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than those of the mice in the control group (P < 0.0001), but spots than those in the HFD group. H&E staining revealed
there was no significant difference between the HFD group that the liver structure of HFD group mice was abnormal,
and the HFD + H group (P > 0.05) (Figure 2B and C). with incomplete hepatic lobules and dense voids. Compared
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with those in the HFD group, the liver morphology and
3.2. H administration improves blood glucose levels structure in the HFD+H group were greatly restored, and
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and lipid metabolism in mice with NAFLD the number and area of cavities were reduced, suggesting
Given that NAFLD is strongly associated with impaired that H alleviated the structural abnormalities of the liver
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levels of TGs and glucose metabolism, we monitored blood in NAFLD mice. Compared with those in the control
glucose levels every 2 weeks. There was no significant group, liver lipid droplets in the HFD group were densely
difference in random blood glucose levels among the distributed, and the lipid droplets were larger according to
control mice at weeks 1, 11, and 22 (P > 0.05). The random Oil Red O staining. In the HFD+H group, the distribution
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Volume 1 Issue 2 (2024) 73 doi: 10.36922/mi.3896