整合素α4在美洲大蠊提取物黏糖氨酸抗肝纤维化中的作用  

The role of integrinα4 in the anti-liver fibrosis effect of the sticky sugar amino acid extract of Periplaneta americana

作　　者：鲁杰 周义霞 刘叶 高雅[1] 陈科璇 李顶春 陈一晖[1] 刘怀鄂[1] 王宏图[1] 李武[1] LU Jie;ZHOU Yixia;LIU Ye;GAO Ya;CHEN Kexuan;LI Dingchun;CHEN Yihui;LIU Huaie;WANG Hongtu;LI Wu(Department of Infection and Liver Diseases,The First Affiliated Hospital of Kunming Medical University,Kunming 650032,China)

机构地区：[1]昆明医科大学第一附属医院感染与肝病科,昆明650032

出　　处：《临床肝胆病杂志》2022年第9期2027-2033,共7页Journal of Clinical Hepatology

基　　金：国家自然科学基金(82160801);云南省“万人计划”名医专项(云人卫发〔2020〕20号,RLMY20200015);云南省科技厅-应用基础研究重点项目[2017FE468(-173)];云南省科技厅-应用基础研究联合专项资金项目[2018FE001(-214)]。

摘　　要：目的本研究基于黏糖氨酸(SSAA)在大鼠中的抗肝纤维化作用,研究整合素α4(ITGA4)在肝纤维化中的作用机制。方法大鼠腹腔内注射CCl_(4)诱导肝纤维化模型,在此基础上使用秋水仙碱或SSAA低、中、高剂量进行干预,以空白对照组、SSAA组作为对照。实验干预12周后,收集大鼠血清、肝脏标本,检测大鼠血清ALT、AST水平,HE染色、天蓝猩红染色观察肝组织病理情况;实时荧光定量PCR检测肝组织ITGA4、整合素β1(ITGB1)、TGFβ1、α-SMA、TIMP2的转录水平;Western Blot检测ITGA4、ITGB1、TGFβ1、α-SMA、MMP2、TIMP1、TIMP2的相对蛋白表达;免疫组化观察TGFβ1、α-SMA蛋白表达。计量资料多组间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CCl_(4)模型组中AST、ALT显著升高,秋水仙碱或SSAA低、中、高剂量干预后能降低AST、ALT水平,各组与CCl_(4)模型组相比差异均有统计学意义(P值均<0.05)。CCl_(4)模型组中的HE染色和天蓝猩红染色结果显示肝小叶结构紊乱、胶原纤维增多,秋水仙碱或SSAA低、中、高剂量干预后肝小叶结构得到改善。CCl_(4)模型组中的ITGA4、TGFβ1、α-SMA和TIMP2各指标转录水平均显著高于其余各组,秋水仙碱或SSAA干预后能降低各因子转录水平,与CCl_(4)模型组相比差异均有统计学意义(P值均<0.05)。CCl_(4)模型组中的ITGA4、TGFβ1、α-SMA、TIMP2和TIMP1蛋白表达高于其余各组,MMP2蛋白表达低于其余各组,在秋水仙碱或SSAA干预后均能抑制ITGA4、TGFβ1、α-SMA、TIMP2、TIMP1表达,促进MMP2表达。免疫组化结果提示CCl_(4)模型组的TGFβ1、α-SMA的表达显著高于其余各组,秋水仙碱或SSAA干预后均能抑制表达。SSAA高剂量组在降低转氨酶、改善肝小叶结构和抑制致肝纤维化因子蛋白表达上效果最显著。结论肝脏中ITGA4的高表达与肝纤维化的发生相关,与TGFβ1、α-SMA表达升高一致,抑制ITGA4表达可为抗肝纤维化Objective To investigate the mechanism of action of integrinα4(ITGA4)in liver fibrosis based on the anti-liver fibrosis effect of sticky sugar amino acid(SSAA)in rats.Methods A rat model of liver fibrosis was induced by intraperitoneal injection of CCl_(4),and then colchicine and low-,middle-,and high-dose SSAA were used for intervention,with blank control group and SSAA group as control.After 12 weeks of experimental intervention,serum and liver samples were collected to measure the serum levels of alanine aminotransferase(ALT)and aspartate aminotransferase(AST),and HE staining and Sirius Red staining were used to observe the pathological conditions of liver tissue;quantitative real-time PCR was used to measure the transcriptional level of ITGA4,integrinβ1(ITGB1),transforming growth factor-β1(TGFβ1),alpha-smooth muscle actin(α-SMA),and TIMP2 in liver tissue;Western blot was used to measure the relative protein expression levels of ITGA4,ITGB1,TGFβ1,α-SMA,MMP2,TIMP1,and TIMP2;immunohistochemistry was used to observe the protein expression of TGFβ1 andα-SMA.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 comparison between two groups.Results There were significant increases in AST and ALT in the CCl_(4) model group,and intervention with colchicine or low-,middle-,and high-dose SSAA reduced the levels of AST and ALT,with a significant difference between the CCl_(4) model group and the other groups(all P<0.05).HE staining and Sirius Red staining showed disordered structure of hepatic lobules and an increase in collagen fibers in the CCl_(4) model group,and the structure of hepatic lobules was improved after intervention with colchicine or low-,middle-,and high-dose SSAA.The CCl_(4) model group had significantly higher transcriptional levels of ITGA4,TGFβ1,α-SMA,and TIMP2 than the other groups,and there were significant reductions in the transcriptional levels of each factor after intervention with co

关 键 词：肝硬化 黏糖氨酸 整合素类 大鼠 Sprague-Dawley 

分 类 号：R575.2[医药卫生—消化系统]