绞股蓝不同有效成分对ox-LDL诱导的内皮细胞线粒体能量代谢相关蛋白表达的影响

Effects of different active constituents of Gynostemma pentaphyllum on mitochondrial energy metabolism-related proteins in endothelial cells induced by ox-LDL

目的:探讨绞股蓝不同成分[绞股蓝总皂苷(Gps)、绞股蓝皂苷XLIX(GpXLIX)和人参皂苷Rb3(GRb3)]对氧化型低密度脂蛋白(ox-LDL)诱导内皮细胞线粒体能量代谢相关蛋白的影响。方法:将人脐静脉内皮细胞融合细胞EA.hy926分为对照组、模型组、Gps组、GpXLIX组和GRb3组。对照组仅用DMEM完全培养基培养;模型组予以100 mg/L ox-LDL诱导内皮细胞48 h;Gps组、GpXLIX组和GRb3组在加入100 mg/L ox-LDL作用24 h后,再分别加入100 mg/L Gps、GpXLIX和GRb3处理24 h。采用ELISA法检测各组细胞ATP含量,采用Wes全自动蛋白质印迹定量分析系统及Western blot法检测线粒体能量代谢相关蛋白细胞色素C氧化酶5a亚基(Cox5a)、NADH:泛醌氧化还原酶核心亚基S1(Ndufs1)、ATP合成酶F1亚基α(ATP5a)及细胞色素C(Cyt C)的表达。结果:与对照组比较,模型组细胞ATP含量有所降低(P<0.01);与模型组相比,Gps组、GpXLIX组和GRb3组细胞ATP含量均不同程度地升高(P<0.01)。Wes全自动蛋白质印迹定量分析系统与Western blot法结果基本一致,均显示模型组细胞Cox5a、Ndufs1和ATP5a的蛋白表达与对照组比较显著减少,Cyt C的蛋白表达显著增多(P<0.01);与模型组相比,Gps、GpXLIX和GRb3均不同程度地促进Cox5a、Ndufs1和ATP5a的蛋白表达,减少Cyt C的蛋白表达(P<0.05或P<0.01)。Gps对Cox5a、Ndufs1和Cyt C蛋白表达的影响明显强于2个单体成分,在2个单体成分中则是GRb3的效果为优;而对ATP5a蛋白的影响,GpXLIX优于另外2个药物。结论:绞股蓝总皂苷及相关有效成分可以通过影响线粒体能量代谢相关蛋白发挥对ox-LDL诱导的内皮细胞的保护作用。

AIM:To investigate the effects of different components of Gynostemma pentaphyllum[gypenosides(Gps),gypenoside XLIX(GpXLIX)and ginsenoside Rb3(GRb3)]on mitochondrial energy metabolism-related proteins in endothelial cells induced by oxidized low-density lipoprotein(ox-LDL).METHODS:EA.hy926 cells were divided into control group,model group,Gps group,GpXLIX group and GRb3 group.The cells in control group were cultured only in DMEM complete medium.The cells in model group were treated with 100 mg/L ox-LDL for 48 h.The cells in Gps group,GpXLIX group and GRb3 group were treated with 100 mg/L ox-LDL for 24 h,and then treated with Gps,GpXLIX and GRb3 at 100 mg/L for another 24 h,respectively.The ATP content in each group was detected by ELISA.The expression levels of mitochondrial energy metabolism-related proteins,cytochrome C oxidase subunit 5a(Cox5a),NADH:ubiquinone oxidoreductase core subunit S1(Ndufs1),ATP synthase F1 subunit alpha(ATP5a)and cytochrome C(Cyt C),were determined by Wes automatic Western blot quantitative analysis system and Western blot.RESULTS:Compared with control group,the ATP content in model group was decreased(P<0.01).After drug intervention,the ATP content increased to different degrees in Gps group,GpXLIX group and GRb3 group(P<0.01).The results of Wes automatic Western blot quantitative analysis system were consistent with those of Western blot.These results showed that compared with control group,the protein expression of Cox5a,Ndufs1 and ATP5a in model group was decreased,and the protein expression of Cyt C was increased(P<0.01).After intervention,the protein expression of Cox5a,Ndufs1 and ATP5a was increased and the protein expression of Cyt C was decreased in Gps group,GpXLIX group and GRb3 group(P<0.05 or P<0.01).Among them,the effect of Gps on the protein expression of Cox5a,Ndufs1 and Cyt C was significantly stronger than those of the 2 monomer components,and the effect of GRb3 was found to be superior in the 2 monomer components.The effect of GpXLIX on ATP5a protein was superior to the other 2 components.CONCLUSION:Gynostemmatotal saponins and related active ingredients protect ox-LDL-induced endothelial cells by affecting mitochondrial energy metabolism-related proteins,thereby preventing and treating atherosclerosis.