Hydrogen Sulfide Ameliorates High Fat Diet-induced Nonalcoholic Fatty Liver Disease

Background:With the prevalence of obesity and type 2 diabetes,NAFLD has developed into the most common chronic liver disease worldwide,and its prevalence has increased year by year.At present,about one-fourth of ordinary adults suffer from NAFLD,which is constantly becoming younger.NAFLD has its own specific histological features and the risk of progression of liver disease,which can lead to hepatitis,decompensated liver cirrhosis,liver fibrosis,and even development of hepatocellular carcinoma.The clinical burden of NAFLD,the health risks to patients and the economic pressures are still increasing.Therefore,finding ways to alleviate NAFLD is particularly important in the field of contemporary medicine.Hydrogen sulfide?H2S?is an important gas signal molecule in mammals.It is involved in the regulation of various physiological and pathological processes in the body.It plays an important regulatory role in lipid metabolism,myocardial cell injury,oxidative stress response,and inflammatory response.Usually in mammals,H2S uses cysteine as a substrate,cystathionine?-lyase?CSE?,cystathionine?-synthase?CBS?and 3-mercapto Produced by the catalysis of 3-mercaptopyruvatesulfurtransferase?3-MST?,it has an important anti-inflammatory effect.A large number of recent studies have also confirmed that H2S has the effects of alleviating lipid accumulation and alleviating cell damage.However,whether H2S can alleviate the mechanism of action of NAFLD has rarely been reported.Objective:Using OA to induce steatosis in human normal liver cells QSG-7701 and L02 cells,and feeding mice with high-fat diet to construct a non-alcoholic fatty liver mouse model.To investigate the effects and mechanisms of H₂S on non-alcoholic fatty liver disease and its mechanism.Methods:1.in vitro experimentQSG-7701 and L02 cells were incubated with OA for 24 hours to induce Hepatotoxicity.The experiment was divided into three groups:control group,OA group,OA+H₂S group.The OA+H₂S group was treated with 100?mol/L NaHS and the control and OA group were treated with PBS.PBS and H₂S were added to the medium in equal volumes,and the relevant indicators were detected 24 hours later.1.1 MTT and CCK-8 cell viability testExogenous MTT can be reduced to succinate dehydrogenase in living cell mitochondria to blue-violet crystalline formazan and deposited in cells.While dimethyl sulfoxide?DMSO?dissolves the formazan in the cells,the OD value is measured at a wavelength of 490 nm by a microplate reader,and the larger the OD value,the stronger the cell activity.1.2 EdU cell proliferation testEdU?5-Ethynyl-2'-deoxyuridine?is very similar to the structure of thymidine,and its attached alkyne group replaces thymine?T?into the DNA molecule during DNA replication.Based on the specific reaction with Apollo?fluorescent dye,the replication activity of cellular DNA was accurately detected to determine the effect of H₂S on cell proliferation and cell differentiation.1.3 Detection intracellular reactive oxygen species ROSThe detection of ROS?Reactive Oxygen Species?is based on the intracellular reactive oxygen probe DCFH-DA,which is free to enter the cell.It is hydrolyzed by esterase into DCFH which cannot penetrate the cell membrane,so that DCFH-DA is loaded in the cell.When reactive oxygen species are produced in the cells,the non-fluorescent DCFH is oxidized to the strong green fluorescent substance DCF,and the more green fluorescence of the cells,the more ROS are produced.Therefore,the amount of DCF green fluorescence is measured to determine the effect of H₂S on intracellular reactive oxygen species levels.1.4 Antioxidant enzyme assaySuperoxide Dismutase?SOD?is capable of scavenging superoxide anions and generating hydrogen peroxide,which generates superoxide anion?O₂-.?by catalytic reaction of xanthine oxidase and xanthine.O₂-.Reducing nitroblue tetrazolium?NBT?is blue formazan,and SOD inhibits the production of formazan by removing O₂-.Therefore,the detection of the depth of the blue of the formazan indicates the activity of the SOD.Glutathione Peroxidase?GSH-PX?catalyzes the decomposition of hydrogen peroxide and specifically acts on the reaction of reducing glutathione?GSH?to hydrogen peroxide.The activity of GSH-PX is related to its enzymatic reaction.The activity of GSH-PX is obtained by measuring the rate of enzymatic reaction to determine the consumption of GSH.GSH is reacted with dithiodinitrobenzoic acid to form a yellow 5-dithiodinitrobenzoic acid anion,and the OD value is detected.Catalyst?Micro Catalase,CAT?can decompose hydrogen peroxide,and the molybdenum acid is added to rapidly terminate the reaction.Hydrogen peroxide that is not involved in the reaction interacts with ammonium molybdate to form a pale yellow substance,and its OD value is measured toCalculate CAT activity.The SOD activity test kit,the GSH-PX activity test kit,and the CAT activity test kit were used in accordance with the instructions.Through calculation and analysis,the effect of hydrogen sulfide on oxidase activity was judged.1.5Western BlotDetection of protein by Western Blot:expression of endogenous H₂S synthase-related protein ?CBS,CSE,3-MST?;expression of apoptosis-related proteins?Bax,Bcl-2,Bax/Bcl-2,Bad,Bcl-xl,Bad/Bcl-xl,Cleved caspase-3,Cleved caspase-9,Cleved-PARP?;Expression of phage-associated proteins?LC3,Beclin-1,p62?;expression of proteins associated with the PI3K/AKT/mTOR signaling pathway?PI3K,p-PI3K,AKT,p-AKT,mTOR,p-mTOR?.2 In vivo experiment2.1 Establishment and grouping of C57BL/6 mouse liver injury modelEight-week-old C57BL/6 mice were purchased and grouped for one week.They were divided into three groups:low-fat group,high-fat group,and hydrogen sulfide treatment group.Modeling:High-fat group and hydrogen sulfide treatment group were fed with high-fat diet for 8 weeks to establish a non-alcoholic fatty liver model.Low-fat mice were fed a low-fat diet for 8 weeks.Treatment:Mice in the hydrogen sulfide treatment group were given 100?M/kg/day of sodium hydrogen sulfide daily for 4 weeks.The low-fat group and the high-fat group were given the same volume of normal saline daily for 4 weeks.2.2 Measurement of mouse body weight and dietary waterAfter the mice were grouped,the body weight was measured weekly.During the intraperitoneal injection of hydrogen sulfide,the mice were weighed weekly.The amount of food and the amount of water consumed by the mice were recorded for 24 hours.After the end of the in vivo experiment,blood was collected from the carotid artery of the mouse,and each tissue was collected and stored frozen or fixed.2.3 Detection of blood biochemical indicators in miceWhole blood was centrifuged to measure the contents of total cholesterol?TC?,triglyceride?TG?,aspartate aminotransferase?AST?and alanine aminotransferase?ALT?in plasma.Used to assess the relief of H₂S on nonalcoholic fatty liver disease.2.4 Detection of biochemical indicators of mouse liver tissueThe liver tissue is taken into small pieces for grinding and centrifugation.The contents of total cholesterol?TC?,triglyceride?TG?,and free fatty acid?NEFA?were measured.2.5 Histological observation and analysisThe liver tissue of the mouse was fixed,embedded,and sliced.Hematoxylin-eosin staining?HE? was performed,and antibodies Cleved caspase-3,Ki67,and Beclin-1 were incubated,liver tissue damage and changes were observed.Results:1.OA induces steatosis in QSG-7701 and L02 cells and decreases the expression levels of CBS, CSE and 3-MST in cells?P<0.05?.2.H₂S reduces the levels of reactive oxygen species?ROS?in OA-induced QSG-7701 and L02 cells and reduces apoptosis.Compared with the OA group,the cell proliferation ability of the OA H₂S group was significantly enhanced,and the cell viability level was significantly increased?P<0.05?.3.Western-Blot results of apoptosis-related proteins show:compared with the OA group,the expression of Bax/Bcl-2,Bad/Bcl-xl,Cleved caspase-3,Cleved caspase-9,and Cleved-PARP was decreased in the OA+H₂S group?P<0.05?.4.The expression of PI3K/AKT/mTOR signaling pathway-related proteins was detected by Western Blot.The expression levels of p-PI3K,p-AKT and p-mTOR in OA group were increased.Compared with OA group,the expression of OA+H₂S group was significantly higher?P<0.05?.5.Expression of autophagy-related proteins:LC3A/LC3B,Beclin-1 protein expression was significantly decreased in OA group,p62 protein expression was significantly increased;compared with OA group,OA+H₂S group LC3A/LC3B,Beclin-1 protein expression was significantly increased,p62protein expression was significantly decreased?P<0.05?.6.Animal experiments showed that H₂S reduced the contents of TG,TC and NEFA in liver tissue of mice,and also decreased the contents of TG,TC,ALT and AST in plasma.7.Histologically,the results of immunohistochemical Cleved caspase-3,Ki37 and Beclin-1 showed that compared with the HFD group,the liver tissue proliferative capacity and autophagy level were significantly increased and the apoptosis was inhibited in the HFD+H₂S group?P<0.05?.Conclusion1.1.OA can induce steatosis in human normal liver cells and reduce the expression levels ofH₂S synthetase CSE,CBS and 3-MST in cells.2.H₂S can significantly enhance the viability and cell proliferation of OA-induced steat cells3.H₂S can significantly inhibit OA-induced adipose tissue apoptosis4.H₂S inhibits apoptosis through the PI3K/AKT/mTOR signaling pathway5.H₂S improves non-alcoholic fatty liver by enhancing autophagy...