Protective Effects Of NAC On Ethanol-induced Injury Of Chang Liver In Vitro

Objective To investigate the protective effect of N-acetylcysteine?NAC?on ethanol-induced Chang liver injury,and use the liver enzymes,oxidative stress injury,inflammation,and related pathway protein expression to evaluate its protective effects.Methods In this study,by establishing a model of ethanol-induced liver cell injury combined with different doses of NAC pre-intervention,the protective effect of NAC on ethanol-induced liver cell injury and related mechanisms were explored.The experiment was divided into the following five groups:?1?Control group:normal control group;?2?Hurt group:ethanol injury group;?3?NAC-L group:1mM NAC+ethanol group?4?NAC-M group:2mM NAC+ethanol group?5?NAC-H group:5mM NAC+ethanol group.Furthermore,to explore whether ethanol can cause oxidative damage to Chang liver,add H₂O₂ group as a positive control group in the oxidative stress indicator part.First,the concentration of ethanol-induced Chang liver injury model was established based on cell survival rate,ethanol dehydrogenase?LDH?content and cell morphology after intervention with different ethanol concentrations.The intervention group was pre-added with different doses of NAC for 30 min,and then added with ethanol concentration to establish an ethanol-induced hepatocyte injury model and cultured for 24 hours.Each group completed the following indicators:?1?MTT method was used to detect the cell survival rate of each group;?2?Lai's method Detection of aspartate aminotransferase?AST?and alanine aminotransferase?ALT?in each group;?3?detection of lactate dehydrogenase?LDH?by microenzyme labeling method;?4?collection The cell supernatants of each group were tested for the contents of tumor necrosis factor??TNF-??in the cell supernatant of each group by Elisa method according to the kit instructions;?5?detection of cells in each group by absorbance using DCFH-DA probe method Reactive oxygen species?ROS?content;?6?Detection of malondialdehyde?MDA?,superoxide dismutase?SOD?using WST-8 method;?7?Detection of reduced glutathione?GSH?content in cells using DTNB method;?8?Observe the apoptosis by Hoechst staining;?9?Western blot was used to detect the total protein of JNK and P38 MAPK and their phosphorylation in mitogen activated protein kinase?MAPKs?pathway.Results?1?The cell survival rate measured by MTT method showed that the safe concentration range of NAC on Chang liver was 1-8 mM.?2?Among the various concentrations of ethanol-induced liver cell damage,the 400 mM group can ensure that the cell survival rate reaches about 75%and has a significant damage response to the cells.Therefore,the concentration of 400 mM ethanol and the injury for 24h were used to establish ethanol-induced liver Cell damage model.?3?The cell survival rate of each group was measured by MTT method.Compared with the ethanol-induced Chang liver injury group,the NAC pre-intervention group could improve the cell survival rate and improve the cell morphology.?4?NAC pre-intervention can reduce the content of AST,ALT and LDH in cells?P<0.05?.?5?NAC pre-intervention can reduce the contents of TNF-?in cell supernatants of each group?P<0.05?.?6?NAC pre-intervention can reduce the levels of ROS and MDA in Chang liver?P<0.05?,and increase the SOD and GSH content in each group of cells?P<0.05?.?7?The total amount of JNK and P38 MAPK proteins in each group of cells was not affected by various processing factors,but NAC pre-intervention could reduce the levels of JNK and P38 MAPK phosphorylation in damaged liver cells.Conclusion NAC can antagonize ethanol-induced Chang liver damage.This protective effect may be achieved by improving enzyme indexes,regulating inflammation levels,reducing oxidative stress injury,and regulating the activation of related proteins in the JNK and P38 MAPK pathways.These results can provide scientific basis for the role of NAC in the prevention and treatment of alcoholic liver disease.