雷公藤甲素介导AMPK/Akt/mTOR通路对小鼠TM3睾丸间质细胞活性的影响

Effects of triptolide on the activity of TM3 Leydig cells and AMPK/Akt/mTOR pathway

目的:探究雷公藤甲素(TP)对小鼠TM3睾丸间质细胞活性及AMPK/Akt/mTOR通路的影响。方法:用不同浓度(50、100、200 nmol/L)的TP处理TM3细胞,对照组为含0.1%DMSO的等体积无血清培养液,37℃恒温孵育箱培育24h后,使用乳酸脱氢酶(LDH)活性检测和凋亡检测试剂盒测定细胞膜受损程度及凋亡率,利用Western印迹检测AMPK/Akt/mTOR通路蛋白变化情况。结果:对照组LDH的活力为(157.5±20.3)%,同时其活性随TP浓度升高显著增强[50 nmol/L:(163.4±33.6)%,100 nmol/L:(346.8±148.8)%, 200 nmol/L:(422.8±113.9)%,P<0.01]。流式细胞术检测显示,对照组凋亡细胞百分率为(6.27±1.41)%,TP各浓度组凋亡百分率与对照组相比均上调[50 nmol/L:(189.9±73.5)%,100 nmol/L:(284.0±103.5)%,200nmol/L:(419.2±155.7)%],其中200 nmol/L TP组与对照组比较差异显著(P<0.05)。Western印迹结果显示,与对照组相比,p-AMPK/AMPK比值显著降低(P<0.01),p-Akt/Akt比值升高(其中50nmol/L组,P<0.05),p-mTOR磷酸化水平升高(其中200 nmol/L组P<0.05)。结论:TP能破坏TM3细胞活性,诱导其凋亡,同时伴随着AMPK抑制、Akt/mTOR通路活化。

Objective:To investigate the effects of triptolide(TP)on the activity of TM3 Leydig cells and the AMPK/Akt/m TOR pathway.Methods:We treated TM3 Leydig cells with TP at 50,100 and 200 nmol/L,respectively,and with 0.1%DMSO as the control,and cultured them in a 37℃thermostat container for 24 hours.Then we measured the damage to the cell membrane and apoptosis of the cells using lactate dehydrogenase(LDH)activity assay and the apoptosis assay kit,and detected the changes in the AMPK/Akt/m TOR pathway-related proteins by Western blot.Results:The LDH activity was significantly increased in the 50,100 and 200 nmol/L TP groups in a dose-dependent manner compared with that in the DMSO control([163.4±33.6]%,[346.8±148.8]%and[422.8±113.9]%vs[157.5±20.3]%,P<0.01),and so was the apoptosis of the cells([189.9±73.5]%,[284±103.5]%and[419.2±155.7]%vs[6.27±1.41]%,P<0.01).The TP-treated cells showed a significantly decreased p-AMPK/AMPK ratio in comparison with the control(P<0.01),but an increased p-Akt/Akt ratio(P<0.05)and an elevated level of p-mTOR phosphorylation(P<0.05).Conclusion:Triptolide can decrease the activity and induce the apoptosis of TM3 Leydig cells,and meanwhile inhibit the AMPK and activate the Akt/m TOR pathway.