p38/JNK MAPK信号通路对MEHP致小鼠睾丸间质细胞凋亡的影响

Study on the Effects of Leydig Cells Apoptosis in Mice Induced by Mono-(2-ethylhexyl)Phthalate Based on p38/JNK MAPK Signaling Pathway

目的探讨p38/c-Jun氨基末端激酶(JNK)丝裂原活化蛋白激酶(MAPK)信号通路对邻苯二甲酸单(2-乙基己基)酯[mono(2-ethylhexyl)phthalate,MEHP]致小鼠睾丸间质(TM-3)细胞凋亡的影响及其作用机制。方法将对数生长期的TM-3细胞分别暴露于p38抑制剂SB203580(5、10、20、40μmol·L^(-1))、JNK抑制剂SP600125(2.5、5、10、20μmol·L^(-1))24 h后,采用CCK-8法检测细胞活性,确定后续SB203580及SP600125干预组染毒剂量;将TM-3细胞分别暴露于0、200、400、800μmol·L^(-1) MEHP、10μmol·L^(-1) SB203580+400μmol·L^(-1) MEHP和10μmol·L^(-1) SP600125+400μmol·L^(-1) MEHP溶液24 h后,光镜下观察细胞形态;CCK-8法检测细胞活性;Annexin V-FITC/PI法检测细胞凋亡情况;荧光探针DCFH-DA法检测细胞活性氧(ROS)水平;免疫印迹法(Western blot)检测细胞p38、p-p38、JNK、p-JNK、Caspase-3和Caspase-9蛋白表达水平。结果与对照组相比,200、400、800μmol·L^(-1) MEHP剂量组ROS水平、凋亡率均升高(P均<0.05),细胞内p38和JNK蛋白表达水平差异均无统计学意义(P均>0.05),蛋白p-p38、p-JNK、Caspase-9和Caspase-3表达均增高(P均<0.05),且随着MEHP染毒剂量的升高,增高越明显(P均<0.05)。与400μmol·L^(-1) MEHP剂量组相比,SB203580干预组和SP600125干预组贴壁细胞数量增多,细胞间隙减小,细胞增殖率上升,细胞凋亡率和ROS水平均降低(P均<0.05),p38和JNK蛋白表达差异均无统计学意义(P均>0.05),p-p38、p-JNK、Caspase-9和Caspase-3蛋白表达均减少(P均<0.05)。结论MEHP可通过激活p38/JNK MAPK信号通路诱导TM-3细胞凋亡。

Objective To investigate the effect of p38/JNK MAPK signaling pathway on apoptosis of mice leydig cells(TM-3)induced by mono-(2-ethylhexyl)phthalate(MEHP)and its mechanism.Methods Mice leydig cells in logarithmic growth stage were exposed to p38 inhibitor SB203580(5,10,20,40μmol·L^(-1)),JNK inhibitor sp600125(2.5,5,10,20μmol·L^(-1))for 24 hours,then CCK-8 method was used to detect the changes of cell viability and determine the exposure doses of SB203580 and SP600125 intervention groups;mice leydig cells in logarithmic growth stage were exposed to 0,200,400,800,10μmol·L^(-1) SB203580+400μmol·L^(-1) MEHP,10μmol·L^(-1) SP600125+400μmol·L^(-1) MEHP solution for 24 h,then the morphology of mice leydig cells was observed under light microscope.The viability of mice leydig cells was detected by CCK-8 method,and the apoptosis was detected by Annexin V-FITC/PI method.The level of reactive oxygen species(ROS)was detected by fluorescent probe DCFH-DA.The expression levels of p38,p-p38,JNK,p-JNK,Caspase-3 and Caspase-9 were detected by Western blot.Results Compared with the control group,200,400,800μmol·L^(-1) MEHP exposed group,ROS level and apoptosis rate of mice leydig cells increased significantly(P all<0.05).There was no significant difference in the expression levels of p38 and JNK proteins in cells(P all>0.05),but the expression levels of p-p38,p-JNK,Caspase-9 and Caspase-3 proteins increased significantly with the increase of exposure doses(P all<0.05).Compared with 400μmol·L^(-1) MEHP group,both SB203580 and SP600125 intervention groups showed an increase in the number of adherent cells,the cell gap decreased,the cell viability increased,the apoptosis rate and ROS level decreased(P all<0.05),the expression of p38 and JNK protein were not obvious(P all>0.05),and the expression of p-p38,p-JNK,Caspase-9 and Caspase-3 protein decreased(P all<0.05).Conclusion MEHP can induce apoptosis of mice Leydig cells by activating p38/JNK MAPK signaling pathway.