雌激素受体α下调导致小鼠睾丸TM4支持细胞连接功能损伤的作用机制

Role and possible mechanism of estrogen receptorαdown-regulation leading to damage of TM4 sertoli cell connectivity in mice

目的探究雌激素受体α(estrogen receptor alpha,ERα)下调导致的小鼠睾丸TM4支持细胞连接功能损伤是否与自噬相关。方法不同浓度ERα抑制剂ICI182780(ICI)处理TM4细胞,CCK-8法检测细胞增殖活性;将细胞分为正常对照组、不同浓度ICI处理组。光镜观察细胞数量及形态变化;Western blot法检测ERα蛋白、连接功能相关蛋白、自噬标志物蛋白表达变化;免疫荧光法检测缝隙连接蛋白43(Cx43)表达与定位;氯喹(chloroquine,CQ)与ICI联合作用细胞24 h,Western blot法检测自噬及连接功能相关蛋白表达水平。结果ICI 50 nmol·L^(-1)及以上浓度时,细胞活力明显下降;光镜观察ICI组细胞空泡增多;与正常对照组相比,ICI用药组ERα、闭锁小带蛋白1(ZO-1)、闭锁蛋白(occludin)、紧密连接蛋白11(claudin-11)、β-联蛋白(β-catenin)及Cx43表达水平均下降,而神经钙黏蛋白(N-cadherin)、上皮钙黏蛋白(E-cadherin)表达无明显变化;微管相关蛋白轻链3(LC3)上升,p62表达下降;免疫荧光结果显示,ICI用药组Cx43荧光表达量明显下降,位置无明显变化;与ICI组相比,CQ+ICI组LC3Ⅱ、p62、ZO-1、β-catenin、Cx43表达水平均上升。结论ERα下调可导致TM4细胞连接功能损伤,其机制可能与激活自噬有关。

Aim To investigate the role of autophagy in the dysfunction of testicular TM4 cell junction induced by ERαdown-regulation.Methods TM4 cells were treated with different concentrations of ERαinhibitor ICI182780(ICI),and the proliferative activity of TM4 cells was detected by CCK-8 method.The number and morphological changes of TM4 cells were observed by light microscope.The levels of ERα,junction function related proteins and autophagy marker proteins were detected by Western blot.The expression and localization of Cx43 were detected by immunofluorescence staining.The cells were treated with chloroquine(CQ)and ICI for 24 h.The expression levels of autophagy and junction function related proteins were detected by Western blot.Results When ICI concentration was 50 nmol·L^(-1)or above,the cell viability decreased significantly.The increase of cell vacuoles in ICI group was observed by light microscope.Compared with normal control group,the protein expression levels of ERα,ZO-1,occludin,claudin-11,β-catenin and Cx43 in ICI groups significantly dropped,while the expression levels of N-cadherin and E-cadherin had no significant changes;LC3Ⅱsignificantly rose,while p62 expression significantly fell.The results of immunofluorescence showed that the fluorescence expression of Cx43 in ICI group decreased significantly,but the position of CX43 did not change significantly.Compared with ICI group,the expression levels of LC3Ⅱ,p62,Cx43,ZO-1 andβ-Catenin significantly increased.Conclusions The down-regulation of ERαleads to damage of TM4 cell junction function,which may be related to the activation of autophagy.