自噬在镉致小鼠睾丸血-睾屏障损伤中的作用

Role of autophagy in cadmium-induced damage to the blood-testis barrier in mice

目的:探究自噬在氯化镉致小鼠睾丸血-睾屏障(BTB)损伤中的作用。方法:将20只4周龄雄性C57BL/6小鼠随机分为对照组[0 mg/(kg·d)]、低剂量组[0.5 mg/(kg·d)]、中剂量组[1.0 mg/(kg·d)]和高剂量组[2.0 mg/(kg·d)],腹腔注射氯化镉,连续28 d。采用HE染色法分析睾丸组织形态学变化,生物示踪法观察BTB的完整性,Western印迹检测BTB组分ZO-1和N-Cadherin蛋白的表达。采用0、2.5、5和10μmol/L CdCl_(2)处理睾丸支持细胞株(TM4细胞)24 h,Western印迹检测ZO-1和N-Cadherin蛋白,以及自噬相关蛋白LC3II和p62的变化情况。在含CdCl_(2)(10μmol/L)的细胞培养液中分别加入自噬抑制剂氯喹(CQ)(5μmol/L)或自噬激动剂雷帕霉素(Rap)(50 nmol/L)处理细胞24 h,Western印迹检测LC3II、p62、ZO-1和N-Cadherin蛋白表达。结果:与对照组小鼠相比,镉染毒组小鼠的生精小管间隙增大、生精细胞内空洞形成,层次减少、排列紊乱,BTB完整性受到破坏;中、高剂量CdCl_(2)组小鼠的睾丸组织中ZO-1和N-Cadherin蛋白表达含量显著下调(P<0.05)。与对照组细胞相比,CdCl_(2)染毒组细胞中ZO-1和N-Cadherin蛋白表达显著下调(P<0.01),而LC3II和p62蛋白表达则显著上调(P<0.05)。与CdCl_(2)组细胞相比,自噬抑制剂CQ和CdCl_(2)共处理组细胞中ZO-1、N-Cadherin、LC3II和p62蛋白表达均显著上调(P<0.01);自噬激动剂Rap和CdCl_(2)共处理组细胞中ZO-1、N-Cadherin和p62蛋白表达显著下调(P<0.05),而LC3II蛋白表达上调(P<0.05)。结论:氯化镉可破坏小鼠BTB的完整性,其机制可能与镉能够增强睾丸支持细胞中自噬水平,调控BTB蛋白表达含量相关。

Objective:To investigate the role of autophagy in cadmium chloride(CdCl_(2))-induced damage to the blood-testis barrier(BTB)in mice.Methods:Twenty four-week-old male C57BL/6 mice were randomly divided into four groups and intraperitoneally injected with CdCl_(2) at 0 mg/kg/d(the control),0.5 mg/kg/d(low-dose),1.0 mg/kg/d(medium-dose)and 2.0 mg/kg/d(high-dose)respectively for 28 consecutive days.Then the morphological changes of the testis tissue was observed by HE staining,the integrity of BTB measured with the biotracer,and the expressions of the BTB components ZO-1 and N-Cadherin proteins detected by Western blot.The TM4 Sertoli cells were treated with CdCl_(2) at 0,2.5,5 and 10μmol/L respectively for 24 hours,followed by determination of the expression levels of ZO-1 and N-Cadherin as well as the autophagy-related proteins LC3II and p62.Then the cells were again treated with CdCl_(2) in the presence of the autophagy inhibitor chloroquine(CQ)at 5μmol/L or the autophagy inducer rapamycin(Rap)at 50 nmol/L for 24 hours,followed by measurement of the expressions of LC3II,p62,ZO-1 and N-Cadherin by Western blot.Results:Compared with the control group,the cadmium-exposed mice showed increased interstitial space in the seminiferous tubules,formation of intracellular cavitation in the germ cells with decreased layers and disordered arrangement,and damaged integrity of the BTB.The expressions of the ZO-1 and N-Cadherin proteins were significantly down-regulated in the testis tissue of the mice in the medium-and high-dose CdCl_(2) groups(P<0.05),and even more significantly in the CdCl_(2)-exposed cells in comparison with those in the control mice(P<0.01),while the expressions of the LC3II and p62 proteins were remarkably up-regulated(P<0.05).The expressions of ZO-1,N-Cadherin,LC3II and p62 were also up-regulated in the cells co-treated with CQ and CdCl_(2)(P<0.01),those of ZO-1,N-Cadherin and p62 down-regulated(P<0.05)and that of LC3II up-regulated(P<0.05)in the cells co-treated with Rap and CdCl_(2).Conclusion:CdCl_(2) can damage the integrity of the mouse BTB,which may be attributed to its ability to enhance the autophagy in Sertoli cells and regulate the expressions of BTB proteins.