双酚A对大鼠睾丸支持细胞P38丝裂原活化蛋白激酶及半胱氨酸天冬氨酸蛋白酶-3表达的影响

Effects of bisphnol A on expression of P38 MAPK and caspase 3 in cultured primary Sertoli cells of rats

目的研究双酚A(bisphenol A,BPA)对离体培养青春前期大鼠睾丸支持细胞P38丝裂原活化蛋白激酶(mitogen activated protein kinase,MAPK)、半胱氨酸天冬氨酸蛋白酶-3(caspase-3)及相关凋亡基因表达的影响。方法将睾丸支持细胞分别暴露于0(溶剂对照)、30、50、70μmol/L的BPA。采用MTT法检测离体培养细胞活力,采用RT-PCR的方法检测p38 MAPK、caspase-3、FasL、肿瘤坏死因子α(tumor necrosis factorα,TNF-α)mRNA的相对表达情况,采用western-blotting的方法检测细胞中P38 MAPK、磷酸化P38 MAPK和caspase-3酶原蛋白的表达情况。结果与溶剂对照组相比,30μmol/L BPA组支持细胞p38 MAPK mRNA的相对表达量升高,而50μmol/L BPA组p38 MAPK的表达明显降低(P<0.05);各浓度BPA染毒组caspase-3 mRNA的相对表达量均较高(P<0.05);仅50μmol/L BPA组TNF-αmRNA的相对表达量增加(P<0.05);50、70μmol/L BPA组FasL mRNA的相对表达量增加(P<0.05);各剂量BPA染毒组总P38MAPK蛋白的相对表达量无显著变化;50和70μmol/L BPA组磷酸化P38 MAPK蛋白的相对表达量均明显升高(P<0.05);仅70μmol/L BPA组caspase-3酶原蛋白的相对表达量下降(P<0.05)。结论 BPA可激活离体培养青春前期大鼠睾丸支持细胞p38 MAPK,并诱发caspase-3活化,导致细胞凋亡,这可能是通过FasL凋亡途径介导的。

Objective To study the effects of bisphenol A （BFA） on the expression of p38 MAVK,caspase-3 and apoptosis-related genes in the cultured Sertoli cells of rats. Methods Sertoli cells were collected from prepuberty rats（18-21 d）. The cuhured cells were treated with BPA at the doses of 0,30,50,70 μmol/L respectively. MTF was used to detect cell viability. The ratios of expressions of p38 MAPK,caspase-3,FasL and TNF-α mRNA were detected by RT-PCR. And western-blotting was used to investigate the protein levels of total P38 MAPK,phosphorylated P38 MAPK and pro-caspase3. Results Compared with control,the expression of p38 MAPK mRNA increased significantly after exposure for 24 h in group of 30 μmol/L BPA, whereas it decreased significantly in group of 50 μmol/L BPA;The expression of caspase-3 mRNA increased significantly in groups of all doses of BPA;The expression level of TNF-α mRNA increased significantly only in the group of 50 μ mol/L BPA;The expression of FasL mRNA increased significantly only in the groups of 50,70 μmol/L BPA;The difference of the expression of total P38 MAPK protein was not observed among all groups;The expression of phosphorylated P38 MAPK protein increased significantly in the groups of 50,70 μmol/L BPA;The expression of pro-caspase-3 protein decreased significantly in the group of 70 μmol/L BPA. Conclusion According to the results of this research, BPA may induce P38 MAPK activation in Sertoli cells and activate FasL-related caspase-3 which will result in cell apoptosis.