杞归八味口服液对鱼藤酮诱导PC12细胞损伤的防护作用

Protective Effect of Qigui Bawei Oral Liquid on PC12 Cell Injury Induced by Rotenone

目的探讨杞归八味口服液对鱼藤酮诱导PC12细胞损伤的防护作用及作用机制。方法通过4μmol/L鱼藤酮作用PC12细胞24 h构建细胞损伤模型,将建模成功的细胞分为杞归1组、2组、3组、4组、5组(相当于杞归八味口服液原液的0.1%,0.2%,0.4%,0.6%,0.8%),鱼藤酮1组、2组、3组、4组、5组(相当于鱼藤酮0.5,1.0,2.0,4.0,8.0μmol/L),另取正常细胞作为空白对照组(水),各组细胞均予相应药物。采用CCK-8检测细胞活性,并计算细胞存活率;采用AnnexinV-FITC/PI双染及流式细胞术检测细胞凋亡率;采用蛋白质印迹(Western blotting)法检测聚腺苷二磷酸-核糖多聚酶(PARP)、cleaved-Caspase3(c-Caspase3)、蛋白激酶B(AKT)、磷酸化AKT(p-AKT)、哺乳动物雷帕素靶蛋白(m TOR)、磷酸化m TOR(p-m TOR)、c-Jun氨基端蛋白激酶(JNK)、磷酸化JNK(p-JNK)、磷酸甘油醛脱氢酶(GAPDH)细胞的蛋白表达水平。结果与空白对照组比较,杞归3组、4组、5组的细胞存活率均显著升高,鱼藤酮1组、2组、3组、4组、5组的细胞存活率均显著降低,且均呈量效依赖趋势(P<0.05);与鱼藤酮4组比较,杞归3,4,5+鱼藤酮4组的细胞存活率均显著升高,且呈量效依赖趋势(P<0.01);与空白对照组比较,鱼藤酮4组的细胞凋亡率、c-Caspase3和p-JNK蛋白表达水平均显著升高,PARP,p-AKT,p-m TOR蛋白表达水平均显著降低(P<0.05);与鱼藤酮4组比较,杞归4+鱼藤酮4组的细胞凋亡率、c-Caspase3和p-JNK蛋白表达水平均显著降低,PARP,p-AKT,p-m TOR蛋白表达水平均显著升高(P<0.05)。结论杞归八味口服液能减轻鱼藤酮对PC12细胞的损伤作用,可能与减弱了鱼藤酮对AKT/m TOR及JNK信号通路的影响有关。

Objective To investigate the protective effect and mechanism of Qigui Bawei Oral Liquid on PC12 cell injury induced by rotenone.Methods PC12 cells were induced by 4μmol/L rotenone for 24 h to construct the cell injury model.The cells successfully modeled were divided into Qigui groups 1,2,3,4,5(equivalent to 0.1%,0.2%,0.4%,0.6%and 0.8%of the original solution of Qigui Bawei Oral Liquid),rotenone groups 1,2,3,4,5(equivalent to 0.5,1.0,2.0,4.0,8.0μmol/L of rotenone),and the normal cells were taken as the blank control group(water),all the cells in each group were given corresponding drugs.CCK-8 assay was used to detect cell viability and calculate the cell survival rate.AnnexinV-FITC/PI double staining and flow cytometry were used to detect the apoptosis rate.Western blotting was used to detect the expression levels of poly adenosine diphosphate ribose polymerase(PARP),cleaved Caspase3(c-Caspase3),protein kinase B(AKT),phosphorylated AKT(p-AKT),mammalian target of rapamycin(m TOR),phosphorylated m TOR(p-m TOR),c-Jun NH2-terminalprotein kinase(JNK),phosphorylated JNK(p-JNK)cells and glyceraldehyde-3-phosphate dehydrogenase(GAPDH).Results Compared with those in the blank control group,the cell survival rates of Qigui groups 3,4 and 5 significantly increased in a dose-dependent manner,and the cell survival rates of rotenone groups 1,2,3,4 and 5 significantly decreased in a dose-dependent manner(P<0.05).Compared with those in the rotenone group 4,the cell survival rates of Qigui groups 3,4 and 5+rotenone group 4 significantly increased in a dose-dependent manner(P<0.01).Compared with those in the blank control group,the apoptosis rate,the expression levels of c-Caspase3 and p-JNK protein significantly increased,while the expression levels of PARP,p-AKT and p-m TOR protein significantly decreased in the rotenone group 4(P<0.05).Compared with those in the rotenone group 4,the apoptosis rate,the expression levels of c-Caspase3 and p-JNK protein significantly decreased,while the expression levels of PARP,p-AKT and p-m TOR protein significantly increased in Qigui group 4+rotenone group 4(P<0.05).Conclusion Qigui Bawei Oral Liquid can reduce the PC12 cell injury induced by rotenone,which may be related to reducing the effects of rotenone on AKT/m TOR and JNK signal pathway.