银杏叶提取物对鱼藤酮所致PC12细胞损伤的保护作用及机制研究

Neuroprotective effect and mechanism of Ginkgo biloba extract on rotenone-induced damage in PC12 cells

目的:观察银杏叶提取物(Ginkgo biloba extract,EGb)对鱼藤酮所致大鼠肾上腺嗜铬细胞瘤细胞(PC12)损伤的保护作用及机制。方法:将PC12细胞分为空白对照组、鱼藤酮模型组(0.5μmol/L鱼藤酮)和EGb761组(0.5μmol/L鱼藤酮+100 mg/L EGb761),按照分组给予药物干预后,采用JC-1探针检测各组细胞线粒体膜电位;美国海马细胞能量代谢分析仪检测线粒体能量代谢水平,Western blot检测半胱氨酸蛋白酶3(caspase-3)和裂解caspase-3(cleaved-caspase-3)、腺苷酸活化蛋白激酶(adenosine monophosphate-activated protein kinase,AMPK)、磷酸化AMPK(phosphorylated AMPK,p-AMPK)、磷脂酰肌醇-3-激酶(posphatidylinositol 3-kinase,PI3K)、磷酸化PI3K(phosphorylated PI3K,p-PI3K)、丝氨酸/苏氨酸激酶(serine/threonine-protein kinase,AKT)、磷酸化AKT(phosphorylated AKT,p-AKT)、哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)和磷酸化mTOR(phosphorylated mTOR,p-mTOR)的蛋白表达水平。使用SPSS 20.0软件进行统计分析,多组间比较采用单因素方差分析,进一步两两比较采用LSD检验。结果:(1)3组细胞的线粒体膜电位差异有统计学意义(F=34.89,P<0.05),EGb761组细胞的线粒体膜电位显著高于鱼藤酮模型组[荧光强度比值:(1.30±0.16),(0.81±0.15)](P<0.05)。(2)3组细胞的基础呼吸水平、ATP产生能力、最大呼吸能力和呼吸储备能力均差异有统计学意义(F=38.07,64.18,64.42,34.62,均P<0.05),EGb761组细胞的基础呼吸水平[(73.02±13.81)pmol/min]、ATP产生能力[(57.08±7.31)pmol/min]、最大呼吸能力[(177.70±17.14)pmol/min]和呼吸储备能力[(104.72±8.58)pmol/min]均显著高于鱼藤酮模型组[(33.69±3.91)pmol/min,(18.08±2.50)pmol/min,(113.12±13.24)pmol/min,(79.43±9.35)pmol/min](均P<0.05)。(3)3组细胞的cleaved-caspase-3/caspase-3、p-AMPK/AMPK、p-PI3K/PI3K、p-AKT/AKT、p-mTOR/mTOR比值均差异有统计学意义(F=48.05,28.68,33.73,45.81,17.39,均P<0.05),EGb761组细胞的cleaved-caspase-3/caspase-3比值[(1.95±0.36),(3.56±0.37)]和p-AMPK/AMPK比值[(1.49±0.19),(2.25±0.27)]均显著低于鱼藤酮模型组(均P<0.05);p-PI3K/PI3K比值[(0.75±0.07),(0.44±0.07)]、p-AKT/AKT比值[(0.74±0.06),(0.36±0.09)]、p-mTOR/mTOR比值[(0.90±0.06),(0.62±0.07)]均显著高于鱼藤酮模型组(均P<0.05)。结论:EGb761对鱼藤酮诱导的PC12细胞损伤具有保护作用,其机制可能与其减弱了鱼藤酮对AMPK的激活和对PI3K/AKT/mTOR通路的抑制作用、增强了线粒体的能量代谢功能并抑制细胞凋亡有关。

Objective To investigate the protective effects and mechanisms of Ginkgo biloba extract(EGb)on rotenone-induced damage in PC12 cells,a pheochromocytoma cell line derived from rat adrenal medulla.Methods PC12 cells were divided into blank control group,rotenone model group(0.5μmol/L rotenone)and EGb761 group(0.5μmol/L rotenone+100 mg/L EGb761).Mitochondrial membrane potential 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 monophosphate-activated protein kinase(AMPK),phosphorylated-AMPK(p-AMPK),phosphatidylinositol-3-kinase(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 determined by Western blot.SPSS 20.0 software was used for data analysis.One-way ANOVA was used for multiple 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 PC12 cells among the three groups(F=34.89,P<0.05).EGb761 significantly increased the mitochondrial membrane potential in PC12 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 respiration,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 PC12 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 PC12 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 PC12 cells compared to the rotenone model group(all P<0.05).Conclusion EGb761 has a protective effect on rotenone-induced PC12 cells,which was associated with the inhibition of AMPK activation and the PI3K/AKT/mTOR pathway,increasing mitochondrial activity and inhibiting apoptosis.