宽缨酮减轻顺铂诱导的NRK-52E细胞损伤的作用研究

Study of eurycomanone in alleviating cisplatin-induced injury in NRK-52E cells

目的探讨宽缨酮(EN)对顺铂诱导的大鼠近端肾小管上皮细胞NRK-52E细胞损伤的作用,为宽缨酮防治顺铂所致急性肾损伤提供实验依据。方法体外培养NRK-52E细胞,随机分为对照组(Control)、顺铂组(Cisplatin)、宽缨酮干预组(EN+Cisplatin)。Annexin V-APC/7-ADD双染色结合流式细胞术检测细胞凋亡率;采用ATP检测试剂盒检测细胞ATP含量;采用MitoSOX荧光探针结合流式细胞术检测线粒体活性氧(ROS)改变;实时定量PCR检测细胞促炎因子(TNF-α、IL-6),PGC-1αmRNA的表达水平以及线粒体DNA(mtDNA)拷贝数的变化情况;Western blot检测细胞中促凋亡蛋白Cleaved Caspase-3和线粒体膜蛋白Tom20的表达水平。结果与对照组相比,顺铂组Cleaved Caspase-3的蛋白表达及细胞凋亡率均升高(P<0.05);TNF-α、IL-6的mRNA表达升高(P<0.05);ATP含量及mtDNA拷贝数降低,线粒体ROS含量增高,PGC-1α的mRNA表达及Tom20的蛋白表达均降低(P<0.05);与顺铂组相比,宽缨酮干预组细胞Cleaved Caspase-3的蛋白表达及细胞凋亡率均降低(P<0.05);TNF-α、IL-6的mRNA表达降低(P<0.05);ATP含量及mtDNA拷贝数增多,线粒体ROS含量降低,PGC-1α的mRNA表达及Tom20的蛋白表达均升高(P<0.05)。结论宽缨酮减轻顺铂诱导的NRK-52E细胞损伤,其机制可能与改善线粒体损伤、抑制细胞凋亡及炎症反应有关。

Objective To investigate the effect of eurycomanone(EN)on cisplatin-induced NRK-52E cells injury of proximal renal tubular epithelial cells in rats,and to provide experimental basis for the prevention and treatment of cisplatin-induced AKI.Methods NRK-52E cells were cultured in vitro and randomly divided into Control group(Control),Cisplatin group(Cisplatin)and EN intrvention group(EN+Cisplatin).Annexin V-APC/7-ADD double staining and flow cytometry were used to detect the apoptosis rate;ATP detection kit was used to detect cell ATP content.Changes of mitochondrial reactive oxygen species(ROS)were detected by MitoSOX fluorescence probe combined with flow cytometry.The expression levels of pro-inflammatory factors(TNF-α,IL-6),PGC-1αmRNA and the copy number of mitochondrial DNA(mtDNA)were detected by real-time quantitative PCR.The expression levels of pro-apoptotic protein Cleaved Caspase-3 and mitochondrial membrane protein Tom20 were detected by Western blot.Results Compared with the control group,in the cisplatin group,cleaved Caspase-3 protein expression and apoptosis rate were increased(P<0.05)and the mRNA expressions of TNF-αand IL-6 were increased(P<0.05);ATP content and mtDNA copy number decreased,mitochondrial ROS content increased,PGC-1αmRNA expression and Tom20 protein expression decreased(P<0.05).Compared with the cisplatin group,in the EN+cisplatin group,the cleaved Caspase-3 protein expression and apoptosis rate were decreased(P<0.05)and the mRNA expressions of TNF-αand IL-6 were decreased(P<0.05);ATP content and mtDNA copy number increased,mitochondrial ROS content decreased,while the PGC-1αmRNA expression and Tom20 protein expression increased(P<0.05).Conclusion The mechanism of eurycomanone in alleviating cisplatin-induced NRK-52E cells injury may be related to improving mitochondrial damage,inhibiting apoptosis and inflammatory response.