缺氧诱导因子-1α介导TGF-β/Smad通路对精索静脉曲张大鼠血睾屏障的影响

Effect of hypoxia inducible factor 1α-mediated TGF-β/Smad pathway on blood-testis barrier in rats with varicocele

目的探究缺氧诱导因子-1α(HIF-1α)介导TGF-β/Smad通路对精索静脉曲张(VC)大鼠血睾屏障(BTB)的影响机制。方法将雄性SD大鼠随机分为对照组(C组)、VC组、VC+感染慢病毒降低HIF-1α(H组)、VC+感染荧光素酶载体(L组)。采用免疫印迹与免疫组化法检测睾丸组织中转化生长因子-β1(TGF-β1)、Smad2/3、紧密连接蛋白[密封蛋白(Claudin)-11、闭合蛋白(Occludin)和闭锁连接蛋白(ZO)-1]的表达。提取大鼠原代支持细胞,将其分为常氧对照组(N组)、缺氧24 h组(24 h组)、缺氧培养24 h+感染腺病毒降低HIF-1α(H1组)、缺氧培养24 h+感染空病毒载体(L1组)、TGF-β1抑制剂组(SB组)、缺氧24 h+TGF-β1抑制剂组(24 h+SB组)。采用免疫印迹检测细胞TGF-β1、Smad2/3、Occludin与ZO-1蛋白的表达,免疫荧光法检测ZO-1蛋白表达。结果VC大鼠睾丸缺氧培养24 h后的原代支持细胞TGF-β1、Smad2/3蛋白表达含量显著上调(P<0.05),Claudin-11、Occludin、ZO-1表达含量显著下调(P<0.05);H与H1组TGF-β1、Smad2/3蛋白表达含量显著下调(P<0.05),Claudin-11、Occludin、ZO-1表达含量显著上调(P<0.05);加入TGF-β1抑制剂后的原代支持细胞TGF-β1、Smad2/3蛋白表达含量出现显著下调(P<0.05),Occludin、ZO-1蛋白表达含量上调(P<0.05)。结论VC导致睾丸缺氧,HIF-1α表达升高,激活TGF-β/Smad通路来调节BTB。

Objective To investigate the mechanism of hypoxia inducible factor 1α(HIF-1α)-mediated TGF-β/Smad pathway on blood-testis barrier(BTB)in rats with varicocele(VC).Methods Male SD rats were randomly divided into control group(C),VC group,varicocele+infection with lentivirus decreased HIF-1α(H),varicocele+infection with luciferin vector(L).Western Blotting and immunohistochemistry were used to detect the expression of TGF-β1,Smad2/3,Claudin-11,Occludin and ZO-1 proteins in testicular tissue.The primary Sertoli cells were divided into normal oxygen control group(N),24 h hypoxia group(24 h),24 h hypoxia culture+infection with adenovirus decreased HIF-1α(H1),24 h hypoxia culture+infection with empty viral vector(L1),TGF-β1 inhibitor group(SB),24 h hypoxia+TGF-β1 inhibitor group(24 h+SB).Western Blotting was used to detect the expression of TGF-β1,Smad2/3,Occludin and ZO-1 proteins,and immunofluorescence was used to detect the expression of ZO-1 proteins.Results The expression levels of TGF-β1 and Smad2/3 protein in primary serophytic cells of VC rats were significantly up-regulated(P<0.05),and the expression levels of Claudin-11,Occludin,ZO-1 were significantly down-regulated(P<0.05)after 24 h hypoxia culture.The expression of TGF-β1 and Smad2/3 protein in H and H1 groups were significantly down-regulated(P<0.05),and the expression contents of Claudin-11,Occludin,ZO-1 were significantly up-regulated(P<0.05);After the addition of TGF-β1 inhibitor,the expression of TGF-β1 and Smad2/3 protein in primary sertoli cells were significantly down-regulated(P<0.05),and the expression of Occludin and ZO-1 protein was up-regulated(P<0.05).Conclusion VC leads to testicular hypoxia,increased HIF-1a expression,and activation of TGF/Smad pathway to regulate the blood-testis barrier.