The Roles Of Oxidative Stress And Endoplasmic Reticulum Stress On Cadmium-inhibited Testosterone Synthesis In Mouse Leydig Cells

Objective Antioxidant NAC and chemical small chaperone 4-PBA were used to inhibit oxidative stress and endoplasmic reticulum stress induced by cadmium in TM3cells to investigate the roles of oxidative stress and endoplasmic reticulum stress in cadmium-inhibited testosterone synthesis in TM3 cells.Methods（1）To elucidate the effects of oxidative stress on cadmium-inhibited testosterone synthesis in TM3 cells,TM3 cells were divided into four groups:negative control group,NAC group,CdCl₂ group and NAC+CdCl₂ group.The supernatants of each experimental group were extracted,and the levels of testosterone in each group were determined by ELISA.Extract mRNA and protein from each group of cells,and the expressions of key enzymes in testosterone synthesis and the changes of oxidative stress were detected by RT-PCR and western blot.（2）To elucidate the effects of endoplasmic reticulum stress on cadmium-inhibited testosterone synthesis in TM3 cells,TM3 cells were divided into four groups:negative control group,4-PBA group,CdCl₂ group,and 4-PBA+CdCl₂ group.The mRNA and protein of each group were extracted,and the changes of oxidative stress index in different groups were detected by RT-PCR and western blot.The supernatant of each group was collected,and the testosterone levels of each group were determined by ELISA.The expressions of key enzymes in testosterone synthesis after endoplasmic reticulum stress inhibited in TM3 cells was detected.（3）To study the effects of oxidative stress on endoplasmic reticulum stress induced by cadmium in TM3 cells,TM3 cells were divided into four groups:negative control group,NAC group,CdCl₂ group and NAC+CdCl₂ group.The mRNA and protein of each group were extracted,and the effects on endoplasmic reticulum stress after using antioxidant NAC were detected by RT-PCR and western blot.Results（1）Compared with the negative control group,the secretion of testosterone in TM3 cells in the CdCl₂ group was significantly reduced,while the NAC intervention significantly antagonized the inhibitory effect of CdCl₂ on the secretion of testosterone in TM3 cells（p<0.01）.In addition,CdCl₂ significantly reduced the expression of mRNA and protein of StAR,P450scc,3β-HSD and 17β-HSD in TM3 cells.NAC can protect the expression of mRNA and protein of key enzymes involved in testosterone synthesis inhibited by CdCl2.At the same time,it was found that the expression of HO-1 mRNA and protein and antioxidant enzymes SOD1,SOD2,SOD3,GSH-Px and CAT mRNA in CdCl₂ group were significantly decreased（p<0.01）,and NAC pretreatment could restore CdCl₂-inhibited the expression of oxidative stress related mRNAs and proteins.（2）Compared with the negative control group,CdCl₂ significantly up-regulated the expression of mRNA and protein related to PERK,ATF6 and IRE1 signaling pathways in endoplasmic reticulum stress.4-PBA pretreatment could alleviate CdCl₂-induced the expression of endoplasmic reticulum stress related mRNAs and protein in TM3 cells.At the same time,4-PBA pretreatment was found to restore partial testosterone secretion in TM3 cells inhibited by CdCl₂（p<0.01）.Further studies showed that 4-PBA has a protective effect on the expression of mRNA and protein of key enzymes in testosterone synthesis in cadmium-inhibited TM3 cells.（3）The results showed that NAC pretreatment significantly down-regulated the expression of endoplasmic reticulum stress protein GPR94,GPR78 and CHOP mRNA induced by CdCl₂（p<0.01）,and significantly attenuated CdCl₂-induced the expression of endoplasmic reticulum stress protein GPR78,p-PERK,p-eIF2α,CHOP and p-IRE1（p<0.01）.Conclusions（1）NAC can attenuate CdCl₂-induced oxidative stress and restore part of testosterone secretion by down-regulating the expression of oxidative stress-related mRNA and protein.CdCl₂ inhibits testosterone synthesis by inducing stress in the endoplasmic reticulum of TM3 cells,and 4-PBA can alleviate CdCl₂-induced endoplasmic reticulum stress and restore testosterone secretion by protecting the expression of key enzymes mRNA and protein in testosterone synthesis（2）NAC can attenuate the expression of stress-related proteins and mRNA in endoplasmic reticulum,which suggesting that oxidative stress may inhibit the synthesis and secretion of testosterone in TM3 cells through endoplasmic reticulum stress.