芍药苷对MPP^(+)/MPTP诱导的帕金森病模型小鼠的保护作用

Protective effect of paeoniflorin on MPP^(+)/MPTP-induced Parkinsonian mice

帕金森病(Parkinson’s disease,PD)是常见的神经退行性疾病,已严重影响老年人的身体健康。为探究芍药苷(peoniflorin)抗帕金森病作用的活性及作用机制,本试验首先建立了1-甲基-4-苯基-吡啶离子(1-methyl-4-phenylpyridinium,MPP^(+))诱导PC12帕金森细胞模型。通过细胞形态学观察、检测细胞活性、乳酸脱氢酶(LDH)的释放量、细胞内Ca^(2+)浓度、活性氧(ROS)水平以及细胞凋亡检测,对芍药苷的神经保护作用进行初步的活性评价。再利用1-甲基-4-苯基-1,2,3,6-四氢吡啶(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine,MPTP)致PD小鼠模型为研究对象,通过检测自主活动和滚轴能力表现,对芍药苷抗PD的作用进一步验证。在此基础上,通过Western blot检测相关凋亡蛋白Bcl-2、Bax、Caspase-3的表达水平研究其作用机制。结果显示,与正常对照组比较,模型组细胞数量减少,细胞体积明显缩小;细胞活性显著降低(P<0.01);MPP^(+)显著增加PC12细胞LDH释放率(P<0.01)、ROS水平(P<0.01)和Ca^(2+)浓度(P<0.01),同时细胞凋亡率显著增加(P<0.01)。与模型组比较,药物保护组细胞损伤减弱,活细胞数量增多,细胞活性增强;25、50μmol/L剂量芍药苷保护组显著降低MPP^(+)诱导的PC12细胞LDH释放率(P<0.01)、ROS水平(P<0.01)和Ca^(2+)浓度(P<0.01),显著缓解了MPP^(+)诱导的细胞凋亡(P<0.01);MPTP诱导可显著减少小鼠的自主活动次数(P<0.01)和滚轴时间(P<0.01),经芍药苷预处理后显著增加小鼠自发站立次数(P<0.05)和滚轴时间(P<0.01);25、50μmol/L芍药苷保护组显著增加了细胞内Bcl-2/Bax表达(P<0.01),同时降低了Caspase-3的表达(P<0.01)。结果表明,芍药苷对MPP^(+)和MPTP诱导帕金森病体内、外模型均具有神经保护作用,其作用机制是通过增加细胞内Bcl-2/Bax表达,降低了Caspase-3的表达实现的。

Parkinson's disease(PD)is a common neurodegenerative disease,which has seriously affected the physical health of elderly people.To investigate the anti-PD activity and mechanism of peoniflorin,1-methyl-4-phenylpyridinium(MPP^(+))-induced PC12 Parkinson's cell model was established in this study.Cell activity,release of lactatedehydrogenase(LDH),intracellular Ca^(2+)concentration,levels of reactive oxygen species(ROS)and apoptosis were detected by morphology observation.To evaluate the neuroprotective effect of paeoniflorin,autonomic dysfunction and roller ability were detected,and anti-PD effect of paeoniflorin was verified using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine,MPTP)-induced PD mouse model.On this basis,the expression levels of the proteins related to apoptosis,Bcl-2,Bax and Caspase-3,were detected by Western blot.The results showed that compared with the normal control group,the model group showed a decrease in the number of cells and a significant reduction in the cell body;the cell activity was significantly reduced(P<0.01);MPP^(+)significantly increased the rate of LDH release(P<0.01),the level of ROS(P<0.01),and the concentration of Ca^(2+)of PC12cells,while the rate of apoptosis was significantly increased(P<0.01);compared with the model group,the drug-protected group showed attenuated cell damage,increased number of viable cells,and enhanced cell activity.The level of MPP^(+)-induced LDH release from PC12cells(P<0.01),ROS level(P<0.01),and Ca^(2+)concentration(P<0.01)significantly decreased in 25μmol/L and 50μmol/L paeoniflorin-protected group,and it significantly mitigated MPP^(+)-induced apoptosis(P<0.01);MPTP induction could significantly reduce the number of voluntary activities(P<0.01)and rolling time(P<0.01),and significantly increase the number of spontaneous standing(P<0.05)and rolling time(P<0.01)in mice pretreated with paeoniflorin.Mice in50μmol/L paeoniflorin-protected group significantly increased the expression of intracellular Bcl-2/Bax(P<0.01),and simultaneously decreased the Caspase-3expression(P<0.01).In conclusion,paeoniflorin exerted neuroprotective effects on both in vivo and ex vivo models of MPP^(+)and MPTP-induced PD,and its mechanism of action was realized by increasing the intracellular Bcl-2/Bax expression and decreasing the expression of Caspase-3.