GnRH类似物对大鼠睾丸间质细胞MAPK的影响

Effects of GnRH analogues on MAPK pathway in rat Leydig cells

目的:研究大鼠睾丸间质细胞中GnRH激动剂(GnRHa)和拮抗剂(GnRHant)对促分裂原活化蛋白激酶(MAPK)途径的影响。方法:原代培养大鼠睾丸间质细胞24h后,血清饥饿2h。GnRHa(10-7mol/L)或Gn-RHant(10-6mol/L)处理大鼠睾丸间质细胞不同时间(0、5、15、30、60、90min)后,Western印迹检测磷酸化ERK(p-ERK)和磷酸化p38(p-p38)蛋白水平以0min组为对照。此外,不同终浓度(1、5、10、20μmol/L)的PKC抑制剂GF109203X和GnRHa或GnRHant共同作用后,Western印迹检测p-ERK蛋白水平。结果:GnRHa或GnRHant刺激间质细胞不同时间后,p-p38蛋白水平与对照组比较均无显著性差异(P>0.05);加入不同终浓度的PKC抑制剂GF109203X处理细胞20min后,再用GnRHa刺激细胞10min,发现GF109203X在终浓度为10μmol/L和20μmol/L时p-ERK水平显著下降(P<0.05)。GnRHant刺激间质细胞后,p-ERK水平在15min时升高了约65%(P<0.05);30min时升高了约81%,达到最高水平(P<0.05);60min时开始下降,90min时恢复到基础水平。不同浓度GF109203X处理细胞20min后,再用GnRHant刺激细胞30min,p-ERK水平与对照组比较无显著性差异(P>0.05)。结论:GnRHa可能通过PKC途径来诱导ERKMAPK信号通路的活化,而GnRHant对ERKMAPK信号通路的激活作用并非通过PKC途径来实现的;p38可能不参与间质细胞中GnRH类似物作用的MAPK分子信号通路。

Objective：To investigate the effects of GnRH analogues GnRHa and GnRHant on the MAPK pathway in rat Leydig cells.Methods：Rat Leydig cells were primarily cultured for 24 hours in vitro and serum-starved for 2 hours,followed by treatment with GnRHa（10-7 mol/L）or GnRHant（10-6 mol/L）for 0,5,15,30,60 and 90 minutes,with the 0 min group as the control.Then the protein levels of phosphorylated ERK（p-ERK）and phosphorylated p38（p-p38）were detected by Western blot,and that of p-ERK determined by the same means after co-incubation of GnRHa or GnRHant with the PKC inhibitor GF109203X at 1,5,10 and 20 μmol/L.Results：After stimulation of the Leydig cells with GnRHa or GnRHant for different times,the protein level of p-p38 showed no significant difference from that of the control group（P 0.05）.Then the Leydig cells were treated with GF109203X at different concentrations for 20 minutes and with addition of GnRHa for another 10 minutes.The level of p-ERK was significantly decreased（P 0.05）by GF109203X at 10 and 20 μmol/L.Compared with the control,the p-ERK expression was increased by 65% at 15 minutes（P 0.05）in the GnRHant stimulation group,by 81%（to the peak）at 30 minutes（P 0.05）,began to fall at 60 minutes,and returned to the base level at 90 minutes.The p-ERK level exhibited no significant difference from that of the control（P 0.05）after treatment of the Leydig cells with different concentrations of GF109203X for 20 minutes and then with GnRHant for 30 minutes.Conclusion：The ERK MAPK activation induced by GnRHa depends on the PKC pathway,but not that induced by GnRHant.The p-38 MAPK pathway may not be involved in the effect of GnRH analogues on rat Leydig cells.