氧化应激介导STAT3/p53通路调控邻苯二甲酸单(2-乙基己基)酯诱导的小鼠初级精母细胞凋亡

MEHP induced mouse primary spermatocyte apoptosis through oxidative stress mediated STAT3/p53 pathway

目的研究氧化应激在邻苯二甲酸单(2-乙基己基)酯(mono-2-ethylhexyl-phthalate,MEHP)诱导小鼠初级精母细胞(GC-2spd)毒性中的作用及机制。方法将培养的GC-2spd细胞分为对照组(含0.1%二甲基亚砜的无血清培养基)、MEHP染毒组(1、10、100μmol/L MEHP 3个不同剂量组)、N-乙酰半胱氨酸(N-acetylcysteine,NAC)预处理组(100μmol/L NAC+100μmol/L MEHP),MEHP染毒24 h,NAC预处理2 h,NAC的预处理浓度(100μmol/L)通过CCK-8实验确定。采用DCFH-DA探针法检测细胞内活性氧(reactive oxide species,ROS)水平,JC-1法检测线粒体膜电位,Western blot法检测细胞内线粒体凋亡通路相关蛋白和p-STAT3^(Tyr705)、P53蛋白表达水平。结果随着MEHP染毒剂量升高,细胞内代表活性氧水平的绿色荧光信号增多;不同剂量MEHP染毒组的线粒体膜电位均下降,表现为红绿荧光细胞比例比值减小,10和100μmol/L MEHP染毒组的红绿荧光细胞比例比值分别为5.15±1.68和4.09±1.72,明显低于对照组(7.91±1.24)(P<0.05);促凋亡蛋白Bax与抗凋亡蛋白Bcl-2表达水平比值(Bax/Bcl-2)在1、10、100μmol/L MEHP染毒组分别为1.23±0.17、2.64±0.43和4.75±0.73,与对照组(0.52±0.11)相比明显增加(P<0.05);cytochrome C蛋白表达水平在100μmol/L MEHP染毒组升高为0.83±0.09,cleaved caspase-9蛋白表达水平在10和100μmol/L MEHP染毒组升高为0.41±0.03和0.52±0.09,cleaved caspase-3蛋白表达水平在10和100μmol/L MEHP染毒组升高为0.60±0.12和0.84±0.17,与对照组相比差异均具有统计学意义(P<0.05);STAT3/p53通路中,p-STAT3^(Tyr705)蛋白表达水平在10和100μmol/L MEHP染毒组分别下降为0.70±0.14和0.41±0.04,P53蛋白表达水平在10和100μmol/L MEHP染毒组分别升高为1.32±0.05和1.66±0.22,与对照组相比差异均具有统计学意义(P<0.05);NAC预处理后,与100μmol/L MEHP组相比,NAC预处理组ROS生成减少,线粒体膜电位升高为5.92±1.64,Bax/Bcl-2下降为1.92±0.06,cytochrome C、cleaved caspase-9和cleaved caspase-3蛋白表达水平分别下降为0.57±0.07、0.35±0.04和0.53±0.06,p-STAT3^(Tyr705)蛋白表达水平升高为0.86±0.07,P53蛋白表达水平下降为1.01±0.06,差异均有统计学意义(P<0.05)。结论MEHP诱导GC-2spd细胞凋亡可能与ROS介导的STAT3/p53通路调控线粒体凋亡通路有关。

OBJECTIVE To investigate the role and possible mechanism of oxidative stress on mouse primary spermatocyte(GC-2 spd)toxicity induced by mono-2-ethylhexyl-phthalate(MEHP).METHODS GC-2 spd cells were cultured in vitro and divided into control group(serum free medium containing 0.1%DMSO),MEHP-treated group(including 1,10,100μmol/L MEHP three different dose groups),N-acetylcysteine(NAC)pretreatment group(100μmol/L NAC+100μmol/L MEHP).GC-2 spd cells were treated with MEHP 24 h,and pretreated with NAC 2 h.The concentration of NAC(100μmol/L)was determined by CCK-8 experiment.The levels of intracellular reactive oxide species(ROS)were detected by DCFH-DA probe,mitochondrial membrane potential was detected by JC-1 method,and the expression levels of proteins in mitochondrial apoptosis pathway,p-STAT3^(Tyr705) and P53 proteins were detected by Western blot.RESULTS With the increase of MEHP dose,the level of intracellular green fluorescence signal representing ROS increased.The mitochondrial membrane potential in cells exposed to different doses of MEHP decreased,which showed that the ratio of red-green fluorescent cells decreased,and the ratio of red-green fluorescent cells in 10 and 100μmol/L groups were 5.15±1.68 and 4.09±1.72,respectively,which were significantly lower than that in the control group(7.91±1.24)(P<0.05).The ratio of expression levels of pro-apoptotic protein Bax to anti-apoptotic protein Bcl-2(Bax/Bcl-2)in 1,10 and 100μmol/L MEHP groups were 1.23±0.17,2.64±0.43 and 4.75±0.73,respectively,which were significantly increased compared with the control group(0.52±0.11)(P<0.05).The expression levels of cytochrome C protein increased to 0.83±0.09 in the 100μmol/L group,the cleaved caspase-9 protein increased to 0.41±0.03 and 0.52±0.09 in the 10 and 100μmol/L groups,the cleaved caspase-3 protein increased to 0.60±0.12 and 0.84±0.17 in the 10 and 100μmol/L groups,and the differences were statistically significant compared with the control group(P<0.05).In STAT3/p53 pathway,the expression levels of p-STAT3^(Tyr705) protein decreased to 0.70±0.14 and 0.41±0.04 in 10 and 100μmol/L groups,the P53 increased to 1.32±0.05 and 1.66±0.22 in 10 and 100μmol/L groups,and the differences were statistically significant compared with the control group(P<0.05).Compared with 100μmol/L MEHP group,ROS production decreased,mitochondrial membrane potential increased to 5.92±1.64,Bax/Bcl-2 decreased to 1.92±0.06,the expression levels of cytochrome C,cleaved caspase-9,and cleaved caspase-3 proteins decreased to 0.57±0.07,0.35±0.04 and 0.53±0.06,respectively,the expression level of p-STAT3^(Tyr705) protein increased to 0.86±0.07,the P53 protein decreased to 1.01±0.06 in NAC pretreatment group,and the differences were statistically significant(P<0.05).CONCLUSION MEHP induces apoptosis of GC-2 spd cells,which may be related to the mitochondrial apoptosis pathway regulated by the ROS-mediated STAT3/p53 pathway.