Neuroprotective effect and mechanism of Ginkgo biloba extract on rotenone-induced damage in PC12 cells
Objective To investigate the protective effects and mechanisms of Ginkgo biloba extract(EGb)on rotenone-induced damage in PC 12 cells,a pheochromocytoma cell line derived from rat adrenal medulla.Methods PC12 cells were divided into blank control group,rotenone model group(0.5 µmol/L rotenone)and EGb761 group(0.5 μmol/L rotenone+100 mg/L EGb761).Mitochondrial membrane poten-tial was detected by JC-1 probe.Mitochondrial energy metabolism levels were measured using a Seahorse XFe24 analyzer.Furthermore,protein expression levels of caspase-3,cleaved-caspase-3,adenosine mono-phosphate-activated protein kinase(AMPK),phosphorylated-AMPK(p-AMPK),phosphatidylinositol-3-ki-nase(PI3K),phosphorylated-PI3K(p-PI3K),serine/threonine-protein kinase(AKT),phosphorylated-AKT(p-AKT),mammalian target of rapamycin(mTOR),and phosphorylated-mTOR(p-mTOR)were deter-mined by Western blot.SPSS 20.0 software was used for data analysis.One-way ANOVA was used for multi-ple group comparisons and LSD test was used for further pairwise comparisons.Results(1)JC-1 staining showed statistically significant differences in the mitochondrial membrane potential of PC 12 cells among the three groups(F=34.89,P＜0.05).EGb761 significantly increased the mitochondrial membrane potential in PC 12 cells compared to the rotenone model group(fluorescence intensity ratio:(1.30±0.16),(0.81± 0.15),P＜0.05).(2)Seahorse experiments showed statistically significant differences in the basal respira-tion,ATP production,maximal respiration and spare respiratory capacity of PC12 cells among the three groups(F=38.07,64.18,64.42,34.62,all P＜0.05).EGb761 significantly increased the basal respiration((73.02±13.81)pmol/min,(33.69±3.91)pmol/min),ATP production((57.08±7.31)pmol/min,(18.08±2.50)pmol/min),maximal respiration((177.70±17.14)pmol/min,(113.12± 13.24)pmol/min),and spare respiratory capacity((104.72±8.58)pmol/min,(79.43±9.35)pmol/min)of PC 12 cells compared to the rotenone model group(all P＜0.05).(3)Western blot showed statistically significant differences in the ratio of cleaved-caspase-3/caspase-3、p-AMPK/AMPK、p-PI3K/PI3K、p-AKT/AKT、p-mTOR/mTOR in PC 12 cells among the three groups(F=48.05,28.68,33.73,45.81,17.39,all P＜0.05).EGb761 significantly decreased the ratio of cleaved-caspase-3/caspase-3((1.95±0.36),(3.56± 0.37))and p-AMPK/AMPK((1.49±0.19),(2.25±0.27))and increased the ratio of p-PI3K/PI3K((0.75±0.07),(0.44±0.07)),p-AKT/AKT((0.74±0.06),(0.36±0.09)),and p-mTOR/mTOR((0.90±0.06),(0.62±0.07))in PC 12 cells compared to the rotenone model group(all P＜0.05).Con-clusion EGb761 has a protective effect on rotenone-induced PC 12 cells,which was associated with the inhi-bition of AMPK activation and the PI3K/AKT/mTOR pathway,increasing mitochondrial activity and inhibi-ting apoptosis.
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