来氟米特通过PI3K/Akt/mTOR信号通路诱导大鼠系膜细胞凋亡的作用

Effect of leflunomide on rat mesangial cells apoptosis by PI3K/Akt/mTOR signaling pathway

目的探讨来氟米特(A771726)对大鼠肾小球系膜细胞凋亡的影响及可能机制。方法体外培养大鼠肾小球系膜细胞,分为正常对照组(加入含5%胎牛血清的DMEM培养液)、A771726组(在对照组基础上加入A771726,使其终浓度为50μg/mL)、LY294002组(在正常对照组基础上加入LY294002,使其终浓度为2μg/mL),LY294002+A771726组(先加2μg/mL的LY294002干预系膜细胞4h后,加入50μg/mL的A771726),采用流式细胞术检测各组干预48h后对大鼠肾小球系膜细胞凋亡的影响,采用免疫荧光法检测各组干预48h对大鼠系膜细胞中mTOR蛋白表达的影响,采用免疫印迹法检测各组干预48h后对大鼠系膜细胞中Caspase-3表达的影响。结果与正常对照组比较,A771726、LY294002、LY294002+A771726组大鼠肾小球系膜细胞的凋亡率均明显升高,mTOR蛋白表达均明显降低,Caspase-3蛋白表达均明显增加;与A771726组比较,LY294002、LY294002+A771726组大鼠肾小球系膜细胞的凋亡率均明显升高,mTOR蛋白表达均明显降低,Caspase-3蛋白表达均明显增加;与LY294002组比较,LY294002+A771726组大鼠肾小球系膜细胞的凋亡率明显升高,mTOR蛋白表达明显降低,Caspase-3蛋白表达明显增加。结论来氟米特可能通过下调PI3K/Akt/mTOR信号通路而诱导大鼠肾小球系膜细胞的凋亡,且来氟米特可协同LY294002共同诱导大鼠系膜细胞的凋亡。

Objective To investigate the effects of leflunomide（A771726）on the apoptosis of the rat glomerular mesangial cells and its possible mechanism.Methods The cultured rat glomerular mesangial cells were divided into the normal control group（adding DMEM culture solution containing 5%fetal calf serum）,A771726group（adding 50A771726 on the basis of control group,making it a concentration as 50μg/mL）,LY294002group（adding LY294002 on the basis of normal control group,making its final concentration as 2μg/mL）and the LY294002＋A771726group（first adding 2μg/mL LY294002 to interfere the mesangial cells for2 h,then adding A771726 50μg/mL）.After 48 hintervention,its influence on mesangial cell apoptosis in each group were measured by flow cytometry.The expression of mTOR was measured by immunofluorescence.The expression of Caspase-3was measured by Western-blot.Results Compared with the normal control group,the apoptosis rate of rat glomerular mesangial cells in the A771726 group,LY294002group and LY294002＋A771726group were significantly increased,the expression of mTOR was decreased,the expression of Caspase-3was increased;compared with the A771726 group,the apoptosis rates of rat glomerular mesangial cells in the LY294002 group and LY294002＋A771726group were significantly increased,the expression of mTOR was significantly decreased,the expression of Caspase-3was significantly increased;compared with the LY294002 group,the apoptosis rate of rat glomerular mesangial cells in the LY294002＋A771726group was significantly increased,the expression of mTOR was significantly decreased,while the expression of Caspase-3 was significantly increased.Conclusion Leflunomide may induce the rat mesangial cells apoptosis through down-regulating the PI3K/Akt/mTOR signaling pathway,moreover,leflunomide may synergize with LY294002 to induce rat mesangial cells apoptosis.