COX-2shRNA Improve Hepatic Fibrosis By Inducing Hepatic Stellate Cell Senescence

ObjectiveConstruction of COX-2shRNA lentivirus vector and study of the effect and mechanism on hepatic stellate cells senescence of hepatic fibrosis induced by high fat diet and CCL4.Methods1.Three COX-2shRNA sequences were constructed into lentivirus vector GV248 and infected rat HSC-T6 cells.COX-2-shRNA sequences with the highest interference efficiency were screened by real-time fluorescence quantitative PCR.2.The COX-2-shRNA lentivirus plasmid,which had the highest interference efficiency,was injected into the tail vein of hepatic fibrosis rats induced by high fat diet and CCL4 for 12 weeks.Oil red,HE and Masson staining were used to analyze the degree of hepatic steatosis and fibrous lesion,?-galactosidase staining was used to detect cell senescence.The expression of COX-2,a-SMAmRNA and p53mRNA in liver homogenate was detected by real-time fluorescence quantitative PCR,and the contents of serum total cholesterol,triglyceride,alanine aminotransferase and alanine aminotransferase were also detected.3.The role of p53 pathway in HSC-T6 senescence induced by COX-2shRNA was studied by using PFT-a inhibition of p53 activity in rat HSC-T6 cell lines.?-galactosidase staining was used to detect cell senescence.The cell proliferation was detected by CCK8.The expression of p53 and p21 was detected by flow cytometry.Results1.Compared with normal control group,COX-2 expression of COX-2shRNA-1,2,3 groups decreased 84.7%,86.3%and 91.8%,respectively.2.The animal experiment indicated that:(1)HE staining showed that the hepatic steatosis was reduced in the COX-2shRNA group compared with the model group of hepatic fibrosis.(2)Oil red O staining showed that compared with the model group of hepatic fibrosis,the fatty acreage of liver tissue in the COX-2shRNA group was significantly reduced(P<0.01).(3)Masson staining showed that the area of collagen in liver tissue in COX-2shRNA group was significantly lower than that in liver fibrosis model group(P<0.01).(4)The results of liver function showed that compared with the model group of hepatic fibrosis,the AST,ALT,TC and TG serum levels of rats in COX-2shRNA group decreased significantly(P<0.05).(5)The results of QPCR showed that compared with the model group of hepatic fibrosis,the expression of a-SMA and COX-2 in liver tissue of rats with COX-2shRNA was significantly down-regulated(P<0.01),and the expression of p53mRNA was enhanced significantly(P<0.01).(6)The results of liver beta-galactoidase staining showed that compared with the model group of hepatic fibrosis,the aging index of COX2shRNA group increased significantly(P<0.05).3.Cell experiments show that:(1)The transfection efficiency of COX-2shRNA in HSC-T6 cells was more than 90%.(2)The activity of ?-galactosidase was increased after transfection of HSC-T6 cells with COX-2shRNA vector,Cell division arrest in G0/G1 phase,aging related molecules p53mRNA and p21mRNA expression increased.(3)CCK8 detection showed that PFT-a had a promoting effect on proliferation of HSC-T6 cells.(4)With the inhibition of p53 activity by PFT-alpha,the activity of beta galactosidase(SA-beta-gal)of HSC-T6 cells transfected with COX2-shRNA virus vector weakened.Conclusions1.COX-2shRNA is effective for hepatic fibrosis induced by high fat diet and ccl4,it can improve the metabolism of serum lipids,liver function and liver pathological indexes.2.The liver protective effect of COX-2shRNA on hepatic fibrosis induced by high-fat diet and ccl4 in rats is related to cutting with COX-2 gene expression,promoting hepatocytes cell senescence.3.The hepatic stellate cells senescence involved in the occurrence and development of hepatic fibrosis may be induced by COX-2shRNA via P53 signal.