基于能量代谢分析益气解毒方对缺氧损伤的PC12细胞线粒体保护作用的机制

Mechanism of Yiqi Jiedu Prescriptions in Protection of Mitochondria in PC12 Cells Against Hypoxia Injury Based on Energy Metabolism

目的:采用Seahorse细胞能量代谢分析仪,通过缺氧损伤的高分化大鼠肾上腺嗜铬瘤细胞(PC12细胞)建立一种线粒体能量代谢评价方法并探究益气解毒方的线粒体保护作用。方法:应用缺氧小室体外构建PC12细胞损伤模型,实验设置空白组,模型组(Model),益气解毒方高、中、低剂量组(25,5,1μmol·L^(-1);YQ高、中、低)及阳性药曲美他嗪组(TMZ),每组3复孔,实验重复3次。采用建立的能量代谢分析方法对细胞线粒体复合物活性进行检测,筛选最佳给药浓度;随后设置益气解毒方全方及各拆方组,采用Seahorse细胞能量代谢分仪检测有氧呼吸和糖酵解功能;根据非线粒体呼吸值,质子漏值,基础呼吸值,最大呼吸值,ATP生成量,可提升呼吸能力检测值,通过主成份分析(PCA)和变量重要性指标(VIP),分析评价益气解毒方不同配伍组对缺氧损伤线粒体有氧呼吸作用效果;通过蛋白免疫印迹法(Western blot)检测细胞色素C(Cytochrome C),B细胞淋巴瘤-2(Bcl-2)和Bcl-2相关X蛋白(Bax)的表达。结果:与其他浓度组比较,FCCP的最佳给药浓度为2μmol·L^(-1);与模型组比较,5μmol·L^(-1)益气解毒方全方能提升线粒体复合物活性(P<0.05);与模型组比较,益气解毒方全方改善效果较好(P<0.01);在各拆方组中,人参皂苷和栀子苷配伍效果较好(P<0.01);VIP分析结果显示,对于线粒体呼吸功能改善,益气解毒方中栀子苷贡献度最大;与模型组比较,益气解毒方中、高剂量组线粒体细胞色素C的渗漏显著减少(P<0.01),Bax蛋白的表达显著降低(P<0.01),Bcl-2蛋白的表达明显升高(P<0.05,P<0.01)。结论:建立了一种细胞水平、高通量线粒体能量代谢多指标定量评价方法,证明了益气解毒方全方能够显著改善缺氧损伤后PC12细胞线粒体能量代谢障碍,而发挥这一作用的机制可能与线粒体凋亡保护相关。

Objective:To establish an evaluation method for mitochondrial energy metabolism with Seahorse analyzer and investigate the protective effect of Yiqi Jiedu prescriptions(YQ)on mitochondria in rat adrenal pheochromocytoma(PC12)cells against hypoxia injury.Method:The PC12 cell injury model was induced in vitro using hypoxic chambers.Five groups were set up,ie,a control group,a model group(model),high-(25μmol·L^(-1)),medium-(5μmol·L^(-1)) and low-dose(1μmol·L^(-1))YQ groups,and a positive drug trimetazidine(TMZ)group,with three replicate wells in each group.The experiment was repeated three times.The established method for energy metabolism analysis was used to assay the activity of mitochondrial complex in cells and screen the optimal dosing concentration.Subsequently,the YQ group and modified YQ groups were set up,and the aerobic respiration and glycolysis function were assayed by the Seahorse analyzer.According to the non-mitochondrial oxygen consumption,proton leakage,basal respiration,maximum respiration,ATP production,and potentially improved respiration,the effects of modified YQ groups on the aerobic respiration of mitochondria damaged by hypoxia were evaluated by principal component analysis(PCA)and variable importance in projection(VIP).The expression of cytochrome C,B-cell lymphoma-2(Bcl-2),and Bcl-2-associated X protein(Bax)was detected by Western blot.Result:Compared with the groups of other concentrations,the optimal dosing concentration of carbonyl cyanide-4(trifluoromethoxy)phenylhydrazone(FCCP)was 2μmol·L^(-1).Compared with the model group,the medium-dose YQ group showed enhanced mitochondrial complex activity(P<0.05).The YQ groups were superior to the model group in improvement(P<0.01).The combination of ginsenoside and geniposide showed the optimal effect among the modified YQ groups(P<0.01).VIP analysis revealed that for the improvement of mitochondrial respiratory function,the contribution of geniposide in YQ was the greatest.Compared with the model group,the high-dose YQ group displayed reduced leakage of mitochondrial cytochrome C(P<0.01),decreased expression of Bax protein(P<0.01),and increased expression of Bcl-2 protein(P<0.05,P<0.01).Conclusion:A cellular,high-throughput quantitative evaluation method for mitochondrial energy metabolism was established,which demonstrated that YQ could significantly improve the impaired mitochondrial energy metabolism in PC12 cells damaged by hypoxia,and the underlying mechanism might be related to the protection against mitochondrial apoptosis.