Hic-5基因敲除对NF-κB/p65表达及CCl4诱导的肝纤维化小鼠模型的影响

杨大银; 杜毅超; 谭鹏; 陈浩; 钱保林; 王安康; 黄诗尧; 付文广; 夏先明

doi:10.3969/j.issn.1001-5256.2019.11.019

Hic-5基因敲除对NF-κB/p65表达及CCl4诱导的肝纤维化小鼠模型的影响

DOI: 10.3969/j.issn.1001-5256.2019.11.019

1. 西南医科大学附属医院肝胆外科
2. 西南医科大学附属医院院士工作站

基金项目:

四川省科技计划-重点研发计划(重大科技专项)(2017SZYZF0015)；

详细信息

中图分类号: R-332;R575.2
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出版历程
- 收稿日期: 2019-07-10
- 出版日期: 2019-11-20

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

Department of Hepatobiliary Surgery,The Affiliated Hospital of Southwest Medical University

Research funding:

摘要

摘要:
目的探讨Hic-5基因敲除对NF-κB/p65表达及肝纤维化的影响。方法野生型C57BL/6雄性小鼠10只,随机分为2组,野生型对照组（WT-Control,n=5）、野生型实验组（WT-CCl4,n=5）; Hic-5基因敲除C57BL/6雄性小鼠10只,随机分为2组,敲除对照组（Hic-5 KO-Control,n=5）、敲除实验组（Hic-5 KO-CCl4,n=5）。检测血清ALT和AST;天狼猩红染色观察肝组织胶原沉积;免疫组化检测α平滑肌肌动蛋白（α-SMA）、p65蛋白表达;定量PCR检测肝组织中α-SMA、Collagen1、p65 mRNA表达。分离小鼠原代肝星状细胞,在予以不同浓度TGFβ1刺激后定量PCR检测肝星状细胞中α-SMA、Collagen1、p65 mRNA的表达。多组间计量资料比较采用单因素方差分析,进一步两两比较采用LSD-t检验。结果天狼猩红染色显示,与WT-CCl4组相比较Hic-5 KO-CCl4组肝组织胶原纤维减少（P值<0. 001）。血清ALT和AST检测结果提示WT-Control组、WT-CCl4组、Hic-5 KO-Control组、Hic-5 KO-CCl4组间ALT、AST比较差异均有统计学意义（F值分别为22.85、25.15，P值均＜0.001），Hic-5 KO-CCl4组血清ALT和AST水平均低于WT-CCl4组（P值均＜0.05）；免疫组化显示WT-Control组、WT-CCl4组、Hic-5 KO-Control组、Hic-5 KO-CCl4组4组间肝组织α-SMA、p65蛋白表达水平比较差异均有统计学意义(F值分别为207.10、9816，P值均＜0.001），Hic-5 KO-CCl4组肝组织α-SMA、p65蛋白表达量低于WT-CCl4组（P值均＜0.01）；定量PCR结果示WT-Control组、WT-CCl4组、Hic-5 KO-Control组、Hic-5 KO-CCl4组4组间肝组织α-SMA、Collagen1、p65 mRNA相对表达量比较差异均有统计学意义（F值分别为41.62、13.93、98.16，P值均＜0.001），Hic-5 KO-CCl4组肝组织α-SMA、Collagen1、p65 mRNA相对表达量低于WT-CCl4组（P值均＜0.05）；0 ng/ml、5 ng/ml、10 ng/ml TGFβ1刺激原代肝星状细胞，WT(0 ng/ml、5 ng/ml、10 ng/ml)组及KO(0 ng/ml、5 ng/ml、10 ng/ml)组间α-SMA、Collagen1、p65 mRNA相对表达量比较差异均有统计学意义（F值分别为53.90、75.82、52.41，P值均＜0.001），不同浓度TGFβ1刺激原代肝星状细胞Hic-5 KO组α-SMA、Collagen1、p65 mRNA相对表达量均低于WT组（P值均＜0.01）。结论Hic-5基因敲除抑制NF-κB/p65表达，抑制肝星状细胞活化，缓解CCl4诱导的肝纤维化。
- 肝硬化 /
- 肝星状细胞 /
- 小鼠,基因敲除 /
- 小鼠,近交C57BL
Abstract:
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.
- liver cirrhosis /
- hepatic stellate cells /
- mice,knockout /
- mice,inbred C57BL

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