D-半乳糖对小鼠睾丸TM4支持细胞连接功能损伤及淫羊藿素的改善作用研究  

The protective effect of icaritin on D-galactose-induced TM4 cell junctional function damage

作　　者：姚志丽 赵海霞[1,3] 马小玉 付国庆 吴杰[4] 宋来新 张长城[1,2] YAO Zhi-li;ZHAO Hai-xia;MA Xiao-yu;FU Guo-qing;WU Jie;SONG Lai-xin;ZHANG Chang-cheng(Third-grade Pharmacological Laboratory on Traditional Chinese Medicine,State Administration of Traditional Chinese Medicine,China Three Gorges University,Yichang Hubei 443002,China;College of Medicine and Health Sciences,China Three Gorges University,Yichang Hubei 443002,China;College of Basic Medical Sciences,China Three Gorges University,Yichang Hubei 443002,China;Analysis and Testing Center,China Three Gorges University,Yichang Hubei 443002,China;People's Hospital of Dongxihu District,Wuhan 430040,China)

机构地区：[1]三峡大学国家中医药管理局中药药理科研三级实验室,湖北宜昌443002 [2]三峡大学健康医学院,湖北宜昌443002 [3]三峡大学基础医学院,湖北宜昌443002 [4]三峡大学分析测试中心,湖北宜昌443002 [5]武汉市东西湖区人民医院,湖北武汉430040

出　　处：《中国药理学通报》2024年第9期1634-1641,共8页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助项目(No 82074205,82274191);武汉市医学科研项目(No WZ20Q03)。

摘　　要：目的研究D-半乳糖(D-galactose,D-gal)对小鼠睾丸TM4支持细胞连接功能损伤以及淫羊藿素(icaritin,ICT)的改善作用机制。方法首先将TM4细胞分为正常对照组、不同浓度D-gal处理组,Western blot检测TM4细胞连接功能相关蛋白(ZO-1、Occludin、β-catenin和Cx43)以及ERα/FAK信号通路相关蛋白(ERα、FAK和pY397-FAK)表达水平的变化;随后MTT筛选ICT药物浓度,并将TM4细胞分为正常对照组、D-gal处理组、D-gal处理+不同浓度ICT组,Western blot检测上述蛋白表达水平变化;分子对接研究ERα与ICT之间的相互作用,并预测两者之间的亲和力;最后将TM4细胞分为正常对照组、D-gal处理组、ERα抑制剂组、D-gal+ICT组、ERα抑制剂+ICT组,Western blot检测上述蛋白表达水平的变化。结果D-gal可浓度依赖性下调连接功能相关蛋白(ZO-1、Occludin、β-catenin和Cx43)以及ERα/FAK信号通路相关蛋白(ERα、FAK和pY397-FAK)表达水平;给予ICT药物后,上述蛋白表达水平均明显上调;ERα和ICT分子对接结果显示,ERα的Asp351氨基酸残基与ICT之间形成2个距离为3.4Å和2.4Å的氢键,且两者之间的对接结合能小于-7 kcal·mol^(-1);ERα抑制剂处理TM4细胞后,上述蛋白表达水平均明显下调,给予ICT后,上述蛋白表达水平未有明显变化。结论D-gal可导致TM4细胞连接功能损伤,而ICT可改善其损伤,机制可能与上调ERα/FAK信号通路相关。Aim To investigate the mechanism of icaritin(ICT)on D-galactose(D-gal)-induced TM4 sertoli cell junctional function damage in vitro.Methods TM4 cells were divided into the normal control group and D-gal treatment group with different concentra-tions.The expression changes of TM4 cell junction function-related proteins(ZO-1,Occludin,β-catenin and Cx43)and ERα/FAK signaling pathway-related proteins(ERα,FAK and pY397-FAK)were detected by Western blot.The concentration of ICT was screened by MTT method.TM4 cells were divided into normal control group,D-gal treatment group,and D-gal treatment+different concentrations of ICT group.The expression levels of the above proteins were detected by Western blot.Molecular docking was used to study the interaction between ERαand ICT,meanwhile predict the affinity between them.Finally,TM4 cells were divided into normal control group,D-gal treatment group,ERαinhibitor group,D-gal+ICT group,and ERαinhibitor+ICT group.The expression levels of the above proteins were detected by Western blot.Results Compared with the normal control group,the expression of junctional function-related proteins(ZO-1,Occludin,β-catenin and Cx43)and ERα/FAK signaling pathway-related proteins(ERα,FAK and pY397-FAK)were significantly down-regulated.After treatment with ICT,the expression of above proteins were significantly up-regulated.The docking results of ERαand ICT molecules revealed the formation of two hydrogen bonds between Asp351 amino acid residue of ERαand ICT,with bond distances measuring 3.4Åand 2.4Å.Additionally,the docking binding energy between them was found to be lower than-7 kcal·mol^(-1).After TM4 cells were treated with ERαinhibitor,the expression of above proteins and ERα/FAK signaling pathway-related proteins were significantly down-regulated,while the expression levels of the above proteins did not change significantly after being given ICT protected group.Conclusions D-gal can cause damage to the junctional function of TM4 cells,and ICT can improve this damage,which

关 键 词：D-半乳糖 TM4细胞 淫羊藿素 雌激素受体Α 黏着斑激酶 连接功能 

分 类 号：R-332[医药卫生] R284.1R322.64R329.24R392.11R641