Roles Of MAPK Signal Pathways In Nickel-induced Testosterone Synthesis Disturbance In Rat Leydig Cells

Objectives:The aim of the present study is to investigate the effects of nickel sulfate（NiSO₄）on the phosphorylation levels of mitogen-activated protein kinases（MAPKs）,and explore the role of MAPK in testosterone synthesis disturbance induced by NiSO₄ in rat Leydig cells.Methods:（1）Leydig cells were prepared from the testes that were decapsulated and digested by 0.25%collagenaseⅣ.These cells were then purified by Percoll density gradient（5,30,58and 70%）centrifugation and adherent culture.The purity of Leydig cells was assessed by3β-hydroxysteroid dehydrogenase（3β-HSD）staining.（2）To determine the concentration-effect of NiSO₄ on the phosphorylation of MAPKs,Leydig cells were treated with different concentrations NiSO₄（250,500 and 1000μmol/L）.To confirm the role of MAPK in NiSO₄-induced testosterone synthesis disturbance,Leydig cells were pretreated with MAPK inhibitors（U0126,SB203580 and SP600125）for 30 min,and then treated with 1000μmol/L NiSO₄ for 24 h in the presence of 1IU/ml hCG.（3）Production of testosterone in cell culture medium was detected by enzyme linked immunosorbent assay（ELISA）.（4）RT-qPCR was performed to quantify the relative mRNA expressions of StAR,CYP11A1,3β-HSD,CYP17A1 and 17β-HSD.（5）Western blot were performed to detect the relative protein expression of testosterone synthetase and MAPK.Results:（1）After cultured with different concentrations NiSO₄,compared with the control group,the phosphorylation of MEK1/2 was significantly increased after 250μmol/L NiSO₄treatment（P<0.05）;the phosphorylations of MEK1/2,ERK1/2,MKK3,p38,MKK7 and JNK were significantly upregulated after 500μmol/L and 1000μmol/L NiSO₄ treatment（P<0.05）.（3）After pretreated with MAPK inhibitors for 30 min before treated with 1000μmol/L NiSO₄,compared with the control group,the phosphorylations of MEK1/2,ERK1/2,MKK3,p38,MKK7and JNK were significantly upregulated after 1000μmol/L NiSO₄ treatment in Leydig cells（P<0.05）;compared with 1000μmol/L NiSO₄ group,the phosphorylations of MEK1/2,ERK1/2,MKK3,p38,MKK7 and JNK were significantly suppressed by their special inhibitors,respectively（P<0.05）.（4）After pretreated with MAPK inhibitors for 30 min before treated with1000μmol/L NiSO₄,compared with the NiSO₄ group,pretreatment with U0126 and SB203580significantly increased the testosterone production（P<0.05）,while pretreatment with SP600125did not affect the testosterone production（P>0.05）.Furthermore,compared with the 1000μmol/L NiSO₄ group,pretreatment with U0126 and SB203580 significantly increased the mRNA and protein expression levels of StAR,CYP11A1,3β-HSD,CYP17A1 and 17β-HSD,while SP600125only increased StAR,CYP11A1,CYP17A1 and 17β-HSD expression levels（P<0.05）.Conclusions:NiSO₄ activated ERK1/2,p38 MAPK and JNK signal pathways in rat Leydig cells.Besides,activation of ERK1/2 and p38 MAPK induced by NiSO₄ could be inhibited by their MAPK specific inhibitors,U0126 and SB203580,respectively,as well as NiSO₄-induced downregulation of testosterone synthetase levels and decrease of testosterone production in rat Leydig cells.These indicate that ERK1/2 and p38 MAPK signal pathways play important roles in NiSO₄-induced testosterone synthesis disturbance in rat Leydig cells.