基于PINK1/Parkin信号通路研究MEHP对小鼠睾丸间质细胞线粒体自噬的影响

Effects of MEHP on Mitochondrial Autophagy in Mouse Leydig Cells Based on PINK1/Parkin Signaling Pathway

目的 探讨邻苯二甲酸单乙基己基酯[mono(2-ethylhexyl)phthalate,MEHP]对小鼠睾丸间质(TM-3)细胞线粒体自噬的影响。方法 将对数生长期的TM-3细胞分别暴露于自噬抑制剂3-甲基腺嘌呤(3-MA)(0、0.25、0.5、1.0、1.25、1.5 mmol·L^(-1))24 h后,采用CCK-8法检测细胞活力,确定后续3-MA干预组染毒剂量;培养TM-3细胞,设置对照组、MEHP低、中、高剂量组(200、400、800μmol·L^(-1))和抑制剂组(MEHP中剂量+3-MA 1.0 mmol·L^(-1))。化学发光法检测细胞腺嘌呤核苷三磷酸(ATP)水平;荧光探针JC-1法检测细胞线粒体膜电位(MMP)水平;免疫印迹法(Western blot)检测抗增殖蛋白2(PHB2)、同源性磷酸酶张力蛋白诱导激酶1(PINK1)、帕金森蛋白(Parkin)、P-Parkin蛋白表达水平。结果 与对照组相比,3-MA浓度为1.0 mmol·L^(-1)时,细胞存活率为(99.56±2.51)%(P>0.05),确定抑制剂组3-MA干预浓度为1.0 mmol·L^(-1)。与对照组相比,MEHP中剂量组细胞存活率降低,各染毒组TM-3细胞ATP水平均下降(P均<0.05),中、高剂量组TM-3细胞MMP水平、Parkin蛋白表达水平均下降(P均<0.05),各染毒组PHB2、PINK1、P-Parkin蛋白表达均增高(P均<0.05);与低剂量组相比,高剂量组TM-3细胞的ATP水平下降(P<0.05),中、高剂量组TM-3细胞线粒体膜电位水平下降,PHB2、PINK1、P-Parkin蛋白相对表达水平均上升(P均<0.05),Parkin蛋白相对表达水平下降(P均<0.05);与中剂量组相比,抑制剂组细胞存活率降低(P<0.05),高剂量组、抑制剂组细胞ATP水平、线粒体膜电位水平均降低(P均<0.05),高剂量组TM-3细胞中PHB2、PINK1、P-Parkin蛋白相对表达水平均上升(P均<0.05),Parkin蛋白相对表达水平下降(P<0.05);抑制剂组PHB2、PINK1、P-Parkin蛋白的相对表达水平均低于中剂量组(P均<0.05),Parkin蛋白的表达水平高于中剂量组(P<0.05)。结论 MEHP可通过激活PINK1/Parkin信号通路来诱导TM-3细胞线粒体自噬。

Objective To investigate the effects of mono-(2-ethylhexyl)phthalate(MEHP)on mitochondrial autophagy in mouse leydig cells(TM-3).Methods TM-3 cells in logarithmic growth phase were exposed to autophagy inhibitor 3-methyladenine(3-MA)(0,0.25,0.5,1.0,1.25,1.5 mmol·L^(-1))for 24 hours.Then,the cell viability was detected by CCK-8 method,and the exposure dose of the subsequent 3-MA intervention group was determined;TM-3 cells were cultured and control,low,medium and high MEHP doses(200,400,800μmol·L^(-1))and inhibitor groups(medium dose of MEHP+3-MA 1.0 mmol·L^(-1))were set.Chemiluminescence method was used to detect the level of adenosine triphosphate(ATP)in cells;fluorescent probe JC-1 method was used to detect the level of mitochondrial membrane potential(MMP);Western blot method was used to detect antiproliferative protein 2(PHB2),and The expression levels of derived phosphatase tensin-inducible kinase 1(PINK1),Parkinson protein(Parkin)and P-Parkin protein.Results Compared with the control group,the cell survival rate was(99.56±2.51)%when the concentration of 3-MA was 1.0 mmol·L^(-1)(P>0.05),and the intervention concentration of 3-MA in the inhibitor group was determined to be 1.0 mmol·L^(-1).Compared with the control group,the survival rate of cells in the MEHP medium-dose exposure group decreased,and the ATP levels of TM-3 cells in each exposure group both decreased(P all<0.05),the level of MMP and the expression of Parkin protein expression both decreased in TM-3 cells in the medium and high-dose group(P all<0.05).The expressions of PHB2,PINK1 and P-Parkin proteins in each exposure group both increased(P all<0.05).Compared with the low-dose group,the ATP level of TM-3 cells in the high-dose group decreased(P<0.05),the mitochondrial membrane potential level of the TM-3 cells in the medium and high-dose groups decreased,and the relative expression levels of PHB2,PINK1 and P-Parkin proteins all increased(P all<0.05),while the relative expression levels of Parkin proteins decreased(P all<0.05).Compared with the medium-dose group,the cell survival rate of inhibitor group decreased(P<0.05),and the high-dose group,inhibitor group cell ATP level and mitochondrial membrane potential level all decreased(P all<0.05),and the relative expression levels of PHB2,PINK1,P-Parkin protein in TM-3 cells of mice in high-dose group both increased(P all<0.05),The relative expression level of Parkin protein decreased(P<0.05);The relative expression levels of PHB2,PINK1 and P-Parkin proteins in the inhibitor group were lower than those in the medium-dose group(P all<0.05),the expression level of Parkin protein was higher than that in the medium-dose group(P<0.05).Conclusion MEHP can induce mitochondrial autophagy in mouse TM-3 cells by activating PINK1/Parkin signaling pathway.