Mechanism of Yiqi Jiedu Prescriptions in Protection of Mitochondria in PC12 Cells Against Hypoxia Injury Based on Energy Metabolism / 中国实验方剂学杂志

ABSTRACT

ObjectiveTo 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. MethodThe 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- （25 µmol·L-1）， medium- （5 µmol·L-1） and low-dose （1 µmol·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. ResultCompared with the groups of other concentrations， the optimal dosing concentration of carbonyl cyanide-4 （trifluoromethoxy）phenylhydrazone （FCCP） was 2 µmol·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）. ConclusionA 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.