Effects Of Peroxiredoxin Ⅱ Mediated AKT/β-catenin Signaling Pathway Inhibits L02 Apoptpsis Induced By Ethanol

Peroxiredoxin II（Prx II）is a typical superoxide reductase containing two cysteine residues,which is widely expressed on the cytoplasmic and cytoplasmic membranes of mammals and has the function of eliminating intracellular reactive oxygen species（ROS）,and participates in intracellular signaling pathways to regulate cell proliferation,senescence,and apoptosis.Under pathological conditions,due to the unbalanced production and elimination of ROS,the tissues and organs were damaged by ROS.During metabolized alcohol by the liver,a large of ROS was produced in hepatocytes.Oxidative stress can down-regulation of intracellular AKT phosphorylation and induce apoptosis of hepatocytes,resulting in liver tissue damage.Our previous studies showed that Prx II can inhibit cell apoptosis through AKT signaling pathway during oxidative stress,and it is highly expressed in liver tissue.Therefore,in this study,a stable genetically L02-shPrx II human normal human liver cell was constructed in vitro.To detect the protective effect of Prx II in the process of alcohol-induced hepatocellular injury,and to study the molecular mechanisms involved in Prx II regulation of AKT signal transduction during alcohol-induced hepatocyte apoptosis,and to establish a Prx II knockout mouse alcoholic liver.The injury model confirms the protective effect of Prx II on alcoholic liver injury and provides a theoretical basis for the prevention and treatment of the alcoholic liver injury.The human Prx II gene knockdown sequences were screened by searching the literature,and the stable genetically L02-shPrx II human hepatic cell line was established after confirmation by NCBI database;The degree of knockdown detected by fluorescence microscopy,flow cytometry,and western blot.;Cell proliferation detected by MTT assay;Flow cytometry was used to detect the effect of alcohol on L02-shPrx II cell cycle;Alcohol-induced apoptosis was detected by fluorescence microscopy using Annexin V-APC staining;Fluorescent dyes were used to detect the level of reactive oxygen species in the cells after alcohol treatment by fluorescence microscopy.The signal transduction pathways related to apoptosis of alcohol-induced hepatocytes were detected by western blot and the related signal target proteins regulated by Prx II were selected.The target protein activator was added to detect the change of cell apoptosis and the level of reactive oxygen species.By adding the inhibitor of active oxygen,N-acetyl-L-cysteine,the change of cell apoptosis and the change of reactive oxygen species were detected.In vivo experiments using Prx II gene knockout mice to establish alcohol liver injury model,using PCR to identify mouse genotypes,manufacturing alcoholic liver injury mouse model,collecting mouse liver tissue to make paraffin sections,H&E staining to observe the pathological changes of liver tissue.Using the fluorescence microscope to detect the lentivirus transfection rate of more than95%,Prx II expression levels decreased significantly,and can be used for subsequent experiments;Screening the alcohol concentration of 400 mM by MTT assay,L02-shPrx II cell survival rate was significantly lower than the shCONT Cells treated with alcohol increased the number of SubG0 cells in L02-shPrx II cells at the G1 phase,and the difference was significant.The fluorescence imaging results showed that alcohol-induced L02-shPrx II cell apoptosis was significantly higher than that of shCONT cells,and alcohol treatment L02-shPrx II cells.The level of reactive oxygen species was significantly higher than that of shCONT cells.Western blot results showed that the expression levels of Cleaved-caspase12 and Cleaved-caspase3 were significantly increased in alcohol-treated L02-shPrx II cells,and the phosphorylation level of AKT was significantly decreased.Phosphorylation ofβ-catenin was significantly elevated and the expression level of HO-1 protein was decreased.Addition of AKT agonist FGF2 effectively inhibited alcohol-induced hepatocyte apoptosis and increased reactive oxygen species.In vivo results showed that alcohol-induced Prx II^-/-Mouse liver fat vacuoles were significantly redundant in wild-type mice.The results indicate that Prx II can scavenge reactive oxygen species produced during alcohol metabolism in hepatocytes and inhibit alcohol-induced L02 hepatocyte apoptosis through the regulation of intracellular reactive oxygen species mediated AKT signaling pathway,thus preventing and treating alcoholic liver injury,Provide potential molecular targets.