脱敏煎通过TGF-β1介导的p38MAPK信号途径对大鼠睾丸支持细胞紧密连接的调节作用

Effect of Tuominjian in TGF-β1 Mediated P38MAPK Pathway on Sertoli Cell’s Tight Junctions

[目的]探讨转化生长因子-β1(transforming growth factor-β1,TGF-β1)介导的p38丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)信号途径在睾丸支持细胞紧密连接中的作用以及脱敏煎含药血清对该信号途径的影响。[方法]体外分离培养大鼠睾丸支持细胞,分为空白对照组、阴性对照组、p38MAPK抑制剂组、TGF-β1刺激组、TGF-β1+p38MAPK抑制剂组和TGF-β1+脱敏煎组共6组。建立支持细胞原代双室培养模型,应用跨膜上皮电阻测定法检测细胞两侧跨膜上皮电阻值(trans-epithelial electrical resistance,TER);分别采用Real-time PCR和Western blot检测支持细胞闭合蛋白(Occludin)、p38MAPK的mRNA和蛋白表达量。[结果]与空白对照组比较,阴性对照组TER、Occludin和p38MAPK mRNA和蛋白表达差异均无统计学意义(P>0.05);与空白对照组及阴性对照组比较,p38MAPK抑制剂组TER、Occludin和p38MAPK mRNA和蛋白表达差异均无统计学意义(P>0.05);而TGF-β1刺激组TER和Occludin mRNA和蛋白表达均显著降低(P<0.01),p38MAPK mRNA和蛋白表达明显升高(P<0.01)。与TGF-β1刺激组比较,TGF-β1+p38MAPK抑制剂组与TGF-β1+脱敏煎组细胞TER明显升高(P<0.01),Occludin mRNA和蛋白相对表达量明显升高(P<0.01),p38MAPK mRNA和蛋白相对表达量明显降低(P<0.05或0.01)。[结论]TGF-β1可通过p38MAPK信号途径抑制Occludin蛋白表达,进而影响血睾屏障(blood-testis barrier,BTB)开闭,脱敏煎能够有效抑制p38MAPK表达,促进Occludin蛋白的表达。

[Objective]To explore the role of p38 mitogen-activated protein kinase(MAPK)pathway mediated by transforming growth factor-β1(TGF-β1)on sertoli cell’s tight junction(TJs)and the effect of Tuominjian in this process.[Methods]The rats sertoli cells were separated in vitro and primarily cultured,and then divided into blank control group,negative control group,p38MAPK inhibitor group,TGF-β1 stimulation group,TGF-β1+p38MAPK inhibitor group and TGF-β1+Tuominjian group.The trans-epithelial electrical resistance(TER)value was detected.The relative expression quantity of Occludin and p38MAPK were detected by Real-time PCR and Western blot.[Results]Compared with blank control group,there were no significant difference in TER value,Occludin and p38MAPK mRNA and protein expression in negative control group(P>0.05);compared with the two control groups,there were no significant difference in TER values,Occludin and p38MAPK mRNA and protein expression in p38MAPK inhibitor group(P>0.05),while TER value and mRNA and protein expression of Occludin in TGF-β1 stimulation group’s decreased(P<0.01),mRNA and protein expression of p38MAPK increased(P<0.01).Compared with TGF-β1 stimulation group,TER value and Occludin mRNA and protein expression increased(P<0.01),while p38MAPK mRNA and protein expression decreased in TGF-β1+p38MAPK inhibitor group and TGF-β1+Tuominjian group(P<0.05,P<0.01).[Conclusion]TGF-β1 can inhibit Occludin expression through p38MAPK pathway,and affect the function of blood-testis barrier.The therapeutic effect of Tuominjian may be correlated to reduce p38MAPK expression and enhance Occludin expression.