邻苯二甲酸单丁酯通过Caspase-3/GSDME通路诱导睾丸间质细胞焦亡

Monobutyl Phthalate Induced Pyroptosis of Testicular Interstitial Cells Through Caspase-3/GSDME Pathway

目的探讨邻苯二甲酸单丁酯(monobutyl phthalate,MBP)诱导睾丸间质细胞(TM-3细胞)焦亡及其可能的发生机制。方法用浓度为0、2.5、5、10 mmol·L^(-1) MBP(对照组,低剂量组,中剂量组,高剂量组)染毒对数生长期细胞24 h后,光学显微镜观察细胞形态学变化;微量酶标法检测乳酸脱氢酶(lactate dehydrogenase,LDH)的释放率;荧光显微镜检测PI阳性细胞比例;Western blot检测焦亡相关蛋白Caspase-3、GSDME的相对表达水平。将对数生长期的TM-3细胞暴露于浓度梯度(0、2.5、5、10、20μmol·L^(-1))的Caspase-3抑制剂Ac-DEVD-CHO中24 h,CCK-8法检测细胞活力,确定Ac-DEVD-CHO染毒剂量。Ac-DEVD-CHO作用于细胞验证MBP诱导TM-3细胞焦亡的机制。结果与对照组相比,各染毒组细胞LDH的释放率、PI阳性细胞比例以及Caspase-3和GSDME蛋白的相对表达水平均升高(P均<0.05)。当Ac-DEVD-CHO浓度为5.0μmol·L^(-1)时,细胞存活率为99.17%(P>0.05),并且中剂量+抑制剂组的细胞存活率较中剂量组升高(P<0.01)。确定抑制剂的干预浓度为5.0μmol·L^(-1)。并且中剂量+抑制剂组的LDH的释放率、PI阳性细胞比例以及Caspase-3和GSDME蛋白的相对表达水平较中剂量组下降(P均<0.05)。结论MBP可以通过Caspase-3/GSDME通路诱导睾丸间质细胞焦亡。

Objective To investigate whether monobutyl phthalate(MBP)induced pyroptosis in testicular stromal cells(TM-3 cells)and its possible pyroptosis mechanism.Methods After 24 h of exposure to cells in the logarithmic growth phase with concentrations of 0,2.5,5,and 10 mmol·L^(-1) MBP,the morphological changes of the cells were observed under an optical microscope.The release rate of lactate dehydrogenase(LDH)was measured by microenzyme.Fluorescence microscopy was used to detect the proportion of PI positive cells.Western blot was used to detect the relative expression levels of apoptosis related proteins Caspase-3 and GSDME.TM-3 cells in the logarithmic growth phase were exposed to the Caspase-3 inhibitor,Ac-DEVD-CHO in a concentration gradient(0,2.5,5,10,20μmol·L^(-1))for 24 h.Cell viability was measured by CCK-8 method to determine the toxic dose of Ac-DEVD-CHO.The Ac-DEVD-CHO was applied to the cells to verify the mechanism of MBP-induced pyroptosis in TM-3 cells.Results The level of LDH release rate,proportion of PI positive cells and relative expression levels of Caspase-3 and GSDME protein were increased in each infected group compared with the control group(P all<0.05).When Ac-DEVD-CHO concentration was 5.0μmol·L^(-1),cell viability was 99.17%(P>0.05)and the middle dose add inhibitor group increased than the middle dose group(P<0.01).The intervention concentration of the inhibitor was later determined to be 5.0μmol·L^(-1).Moreover,the level of LDH release rate,the proportion of PI positive cells,and the relative expression level of Caspase-3 and GSDME proteins in the middle dose add inhibitor group decreased compared with the middle dose group(P all<0.05).Conclusion MBP can induce cell pyroptosis in interstitial cells of testis by Caspase-3/GSDME pathway.