Effect of Hic-5 gene knockout on NF-κB/p65 expression and CCl4-induced liver fibrosis degree in mice

Objective To investigate the effect of Hic-5 gene knockout on NF-κB/p65 expression and liver fibrosis. Methods Ten wild-type male C57 BL/6 mice were randomly divided into wild-type control group( WT-Control group with 5 mice) and wild-type experimental group( WT-CCl4 group with 5 mice),and ten male C57 BL/6 mice with Hic-5 gene knockout were randomly divided into Hic-5 knockout control group( Hic-5 KO-Control group with 5 mice) and Hic-5 knockout experimental group( Hic-5 KO-CCl4 group with5 mice). The serum levels of alanine aminotransferase( ALT) and aspartate aminotransferase( AST) were measured. Picrosirius red staining was used to observe collagen deposition in liver tissue. Immunohistochemistry was used to measure the expression of alpha-smooth muscle actin( α-SMA) and p65 protein,and real-time quantitative PCR was used to measure the mRNA expression of α-SMA,collagen1,and p65 in liver tissue. The primary hepatic stellate cells of mice were isolated and stimulated with different concentrations of TGF-β1,and then real-time quantitative PCR was used to measure the mRNA expression of α-SMA,collagen 1,and p65 in primary hepatic stellate cells. A one-way analysis of variance was used for comparison of continuous data between multiple groups,and the least significant difference t-test was used for further comparison between two groups. Results Picrosirius red staining showed that compared with the WT-CCl4 group,the Hic-5 KO-CCl4 group had a significant reduction in collagen fibers in liver tissue( P < 0. 001). Measurement of serum ALT and AST showed that there were significant differences in ALT and AST between the WT-Control group,the WT-CCl4 group,the Hic-5 KO-Control group,and the Hic-5 KO-CCl4 group( F = 22. 85 and 25. 15,both P < 0. 001),and the Hic-5 KO-CCl4 group had significantly lower serum levels of ALT and AST than the WT-CCl4 group( both P < 0. 05). Immunohistochemistry showed that there were significant differences in the expression levels of α-SMA and p65 protein in liver tissue between the WT-Control group,the WT-CCl4 group,the Hic-5 KO-Control group,and the Hic-5 KO-CCl4 group( F = 207. 10 and 98. 16,both P < 0. 001),and the Hic-5 KO-CCl4 group had significantly lower expression of α-SMA and p65 protein in liver tissue than the WT-CCl4 group( both P < 0. 01).The results of real-time quantitative PCR showed that there were significant differences in the relative mR NA expression of α-SMA,collagen 1,and p65 in liver tissue between the WT-Control group,the WT-CCl4 group,the Hic-5 KO-Control group,and the Hic-5 KO-CCl4 group( F = 41. 62,13. 93,and 98. 16,all P < 0. 001),and the Hic-5 KO-CCl4 group had significantly lower relative mR NA expression of α-SMA,collagen 1,and p65 in liver tissue than the WT-CCl4 group( all P < 0. 05). After the primary hepatic stellate cells were stimulated by TGF-β1 at concentrations of 0,5,and 10 ng/ml,there were significant differences in the relative mR NA expression ofα-SMA,collagen 1,and p65 between the WT 0 ng/ml group,the WT 5 ng/ml group,the WT 10 ng/ml group,the KO 0 ng/ml group,the KO 5 ng/ml group,and the KO 10 ng/ml group( F = 53. 9,75. 82,and 52. 41,all P < 0. 001),and the Hic-5 KO group had significantly lower relative mR NA expression of α-SMA,collagen 1,and p65 than the WT group( all P < 0. 01). Conclusion Hic-5 knockout inhibits NF-κB/p65 expression and hepatic stellate cell activation and alleviates CCl4-induced liver fibrosis.