Page 121 - GPD-3-3

Gene & Protein in Disease Hydrogen sulfide ameliorates NAFLD

expounded on the mechanism by which H S activates cells was determined based on the absorbance values from
2
autophagy through the AMP-activated protein kinase and different groups. For tissue samples, the target tissue weight
mechanistic target of rapamycin (AMPK-mTOR) pathway, was recorded, and the tissue was combined with pre-cooled
enhancing intracellular lipid degradation and reducing reagents in a designated grinder. Pre-cooled reagent was
lipid accumulation in hepatocytes, ultimately ameliorating added at a ratio of 1 mL/0.1 g of tissue, and the subsequent
NAFLD. steps followed the same procedure as the cytometric assay.
The resulting tissue homogenate was carefully transferred to
2. Materials and methods an Eppendorf tube and centrifuged at 8000 g for 10 min. The
2.1. Preparation of animal fat molding agent supernatant was then processed similarly to the cell lysates. 41
The edible lard (EL) was mixed with potassium hydroxide 2.5. Total cholesterol content assay
(KOH) and anhydrous ethanol in an 80°C water bath. After For cellular cholesterol measurement, the pre-cell
drying, the product was dissolved in water. Hydrochloric processing steps were consistent with those used for
acid and dilute KOH solution were added gradually, triglyceride. Cholesterol standards were diluted in
and the solution was then adjusted to the desired isopropanol to create different gradients. The volume of
volume. A modeling agent system was created with 10% the working solution was adjusted accordingly. Different
bovine serum albumin (BSA) and a free fatty acid salt solution groups were pipetted into the working solution,
concentration ranging from 5 to 6 mM. 36,37 and the absorbance was measured. Total cholesterol
2.2. Animal models content was calculated using a standard curve. For tissue
cholesterol determination, the tissue was accurately
Thirty C57BL/6J mice were randomly assigned to three weighed and ground to ensure consistency in the assay. 42
groups: the high-fat diet (HFD) group, the HFD with
NaHS treatment (HFD + NaHS) group, and the control 2.6. Glycerol content assay
group. The NAFLD animal model was induced by feeding Cellular glycerol content was assessed using a Prilosec
the HFD chow with an energy value of 5.24 kcal/g, whereas glycerol assay kit (Beijing Baiao Leibo Technology Co., LTD,
the control group received a low-fat chow with an energy China). First, the protein concentration was determined.
value of 3.85 kcal/g. During the treatment phase, the HFD The lysate was placed in a water bath and centrifuged at
+ NaHS group was administered NaHS at a concentration room temperature. The supernatant was collected and
of 50 μmol/kg/d, as per the findings from preliminary kept on ice. Glycerol standards were prepared by diluting
experiments. The HFD and control groups received them in distilled water. The working solution was prepared
daily injections of the same volume of sterile saline. All by mixing reagents R1 and R2. The blank tube, glycerol
administrations were continued until week 22, which standards, and supernatant were transferred to a cell
marked the end of the experiment. 38-40 culture plate. The working solution was added to each well
and allowed to react at 37°C. Absorbance was measured,
2.3. Cell culture condition
and a standard curve was plotted using the blank tube and
The mouse hepatocyte cell line FL83B, obtained from glycerol standards. The glycerol content in the cells was
Shanghai Jining Biological Company (China), was used in calculated based on the standard curve. 43
this study. Cells were cultured and passaged in DMEM/F12
medium supplemented with fetal bovine serum (Biological 2.7. Free fatty acid content assay
Industries, Israel). Under standard conditions, cells were Cellular-free fatty acid content was determined using the
maintained in a complete medium consisting of 90% copper fatty acid colorimetric method. The initial steps
base medium and 10% fetal bovine serum. For starvation mirrored those of the glycerol content assay. Following
culture, cells were first subjected to a medium with a serum the initial steps, a volume of supernatant was combined
content of 0.6% for 5 h, followed by a switch to a serum- with n-heptane. The mixture was vortexed, shaken, and
free medium. centrifuged to extract the free fatty acids. An oleic acid-
n-heptane solution with various oleic acid concentrations
2.4. Triglyceride content assay was prepared. The samples were treated with copper
The cell culture medium was removed, and the cells acetate solution, vortexed, shaken, and centrifuged. The
were washed using phosphate-buffered saline. Cells, resulting supernatant, which contained copper fatty acids
at a density of 5 × 10 , were harvested by scraping with in n-heptane, was measured for absorbance in a cell culture
6
a spatula. The cells were then lysed by grinding and plate. The concentration of free fatty acids in the samples
subsequently centrifuged. Triglyceride content in the was calculated using a standard curve. 44

Volume 3 Issue 3 (2024) 3 doi: 10.36922/gpd.3409