Hic-5 Gene Knockdown Suppresses NF-?B/p65 Expression And Alleviates CCl₄-Induced Liver Fibrosis In Mice

Objective:To investigate the effect of Hic-5 gene knockout on the expression of NF-?B/P65 and liver fibrosis,enrich the theoretical mechanism of liver fibrosis and provide some theoretical basis for liver fibrosis treatment.Methods:Ten wild-type C57BL/6 male mice were randomly divided into two groups,the wild-type control group?WT-Control,n=5?,and the wild-type experimental group?WT-CCl₄,n=5?;ten Hic-5 knockout C57BL/6 male mice were randomly divided into two groups,the knockout control group?Hic-5KO-Control,n=5?,and the knockout experimental group?Hic-5 KO-CCl₄,n=5?.Induction of liver fibrosis in mice after one week of adaptive feeding,Mice in the experimental group were intraperitoneally injected with 20%CCl₄ solution?2.5ml/kg?,and mice in the control group were intraperitoneally injected with olive oil?2.5ml/kg?twice a week for 6 weeks,mice in each group drink freely during liver fibrosis modeling,72 hours after the last injection of CCl₄,mice in the experimental and control groups were euthanized,and liver tissue and peripheral blood samples were collected.HE staining to observe pathological changes in liver tissue;The alanine aminotransferase test kit and aspartate aminotransferase test kit detect the serum levels of ALT and AST in each group of mice,the ALT and AST levels represent liver function.Sirius red staining and Masson staining were used to observe the collagen expression of liver tissue in each group;Sirius red staining and Masson staining sections were analyzed for collagen fibers quantitatively using Image pro plus 6.0 software,and the ratio of positive signals to the total area of liver tissue in the visual field was calculated,indicating the expression of collagen fibers,and used to quantitatively evaluate the degree of liver fibers.Immunohistochemical detection of?-SMA and p65 protein expression in liver tissue,Immunohistochemical staining sections were analyzed with Image pro plus 6.0 software,and the ratio of positive signals to the total area of liver tissue in the visual field was calculated,indicating the expression of protein amount.The expressions of?-SMA,Collagen?,p65 and IL-6 mRNA in liver tissue were detected by real-time quantitative PCR.Seven-step method was used to isolate mouse primary hepatic stellate cells.After stimulation with different concentrations of TGF-b1,the total RNA was extracted by Trizol method.Real-time quantitative PCR was used to detect Hic-5,?-SMA,Collagen1,P65 and IL-6mRNA expression.The statistical description of measurement data is expressed as mean±standard deviation?x±s?,and the statistical analysis of the data uses SPSS20.0 statistical software.Measurement data were compared between multiple groups using single factor analysis of variance.Further comparison between the two groups was performed using LSD-t test.P<0.05 indicated that the difference was statistically significant.Results:The results of HE staining showed that the lobular structure of the liver was normal in the WT-Control and Hic-5 KO-Control groups.There was no obviously inflammatory cell infiltration around the portal vein and central vein.Compared with WT-CCl₄,Hic-5 KO-CCl₄ group had significantly reduced inflammatory cell infiltration around the portal vein and central vein.The serum ALT and AST test results showed that compared with the WT-CCl₄ group,the serum ALT and AST in the Hic-5 KO-CCl₄ group were reduced?P values<0.05,<0.05?,the difference was statistically significant.The quantitative analysis of Sirius red stained sections showed that the collagen fiber area ratio of the Hic-5 KO-CCl₄ group was lower than that of the WT-CCl₄ group?P value<0.001?,the difference was statistically significant;Quantitative analysis of Masson-stained sections showed that compared with the WT-CCl₄ group,the collagen fiber area ratio in the Hic-5 KO-CCl₄ group was reduced?P value<0.01?,and the difference was statistically significant.Real-time quantitative PCR results showed that Hic-5 KO-CCl₄ group had lower expressions of Collagen1,Collagen3,and?-SMA mRNA compared to WT-CCl₄ group?P values were<0.001,<0.001,and<0.05?,and the difference was statistically significant;Compared with the WT-CCl₄ group,the expression levels of P65and IL-6 in the Hic-5 KO-CCl₄ group were reduced?P values were<0.001 and<0.001?,and the difference was statistically significant.Real-time quantitative PCR results showed that 0,5,10,and 10 ng/ml TGF-?1 stimulated primary hepatic stellate cells,Hic-5 mRNA expression in Hic-5 KO group was lower than that in WT group?P values were<0.001,<0.001,and<0.001?,the expression levels of?-SMA mRNA in hepatic stellate cells in Hic-5 KO group were lower than those in WT group?P values<0.01,<0.01,<0.001?,the expression levels of Collagen1 mRNA in hepatic stellate cells in Hic-5 KO group were lower than those in WT group?P values<0.01,<0.01,<0.01?,the expression levels of P65 mRNA in hepatic stellate cells in Hic-5 KO group were lower than those in WT group?P values<0.01,<0.01,<0.01?,the expression levels of IL-6 mRNA in hepatic stellate cells in Hic-5 KO group were lower than those in WT group?P values<0.01,<0.01,<0.01?,and the differences were statistically significant.Conclusion:Hic-5 gene knockdown down-regulates P65 expression,inhibits hepatic stellate cell activation,and alleviates CCl₄-induced liver fibrosis.