Smac参与雷公藤甲素对TGF-β1作用的大鼠系膜细胞凋亡的研究

Smac Involved in Promoting TGF-β1 Treated Mesangial Cells Apoptosis Induced by Triptolide

目的探讨雷公藤甲素(Triptolide)对转化生长因子(TGF)-β1作用下的大鼠系膜细胞凋亡及Smac表达的影响。方法大鼠HBZY-1系膜细胞株分为对照组、TGF-β1组和低、中、高Triptolide组。低、中、高Triptolide组加入含不同浓度Triptolide(0.4、2、10μg/L)预处理24 h后再加TGF-β1(10μg/L)培养24 h。TUNEL法检测细胞凋亡;Real-time PCR法检测Smac mRNA表达;Western blot检测Smac蛋白表达;激光共聚焦荧光显微镜检测Smac蛋白亚细胞定位。结果与对照组相比,TGF-β1组系膜细胞凋亡减少,且Smac mRNA及蛋白表达降低(P<0.05);与TGF-β1组相比,Triptolide能够促进系膜细胞凋亡,且高Triptolide组Smac mRNA及蛋白表达量最高(P<0.05)。对照组及TGF-β1组Smac蛋白呈点状分布的线粒体定位,而经过Triptolide处理后Smac蛋白呈胞浆溶解状及染色体浓集。结论Triptolide可通过上调大鼠系膜细胞Smac表达,促进其由线粒体释放至细胞质及细胞核,从而促进TGF-β1作用下的大鼠系膜细胞凋亡。

Objective To investigate the effects of Triptolide on apoptosis of cultured rat mesangial cells treated by TGF-β1 and the role of Smac in this process. Methods The mesangial cells were pre-treated with different concentrations of Triptolide for 24 hours, then stimulated with TGF-β1 for 24 hours. Apoptotic cells were detected by TUNEL assay. Smac transcription level was determined by Real time-PCR analyses. Smac expression level was assessed using Western blot anal-yses. Localization of Smac was shown by confocal fluorescence microscopy. Results Compared with control group, TGF-β1 inhibited apoptosis and Smac transcription and expression in rat mesangial cells. By contrast, Triptolide promoted mesangial cells apoptosis. In Triptolide groups, Smac mRNA and protein levels were up-regulated. Additionally, in normal and TGF-β1 groups Smac protein was mainly localized in mitochondriawhile in Triptolide groupit was mainly localized in cytoplasm and nucleus with increased fluorescence intensity. Conclusion Triptolide could promote the effect that TGF-β1 inhibited apop-tosis of mesangial cells, through both up-regulation the expression of Smac and stimulating it translocation from mitochon-dria to cytoplasm and nucleus.