芍药苷激活自噬对帕金森病小鼠运动能力及多巴胺能神经元的影响

Effect of paeoniflorin on motor behavior and dopamine neurons of mice with Parkinson's disease by activating autophagy

目的探讨芍药苷激活自噬对PD小鼠运动能力及多巴胺能神经元的影响。方法将40只C57BL/6小鼠按随机数字表法分为正常组、模型组、芍药苷组和芍药苷+3-甲基腺嘌呤(3-MA)组,每组10只。后3组小鼠连续7 d腹腔注射1-甲基-4苯基-1,2,3,6-四氢吡啶(MPTP)[30 mg/(kg·d)]制备成亚急性PD模型,期间芍药苷组和芍药苷+3-MA组小鼠分别腹腔注射芍药苷[30 mg/(kg·d)]和芍药苷[30 mg/(kg·d)]+3-MA[2 mg/(kg·d)]。第8天时采用爬杆实验和悬挂实验评估各组小鼠的运动能力,之后取中脑黑质,采用TUNEL染色检测神经细胞凋亡情况,采用免疫组化染色检测酪氨酸羟化酶(TH)阳性细胞数及α-突触核蛋白(α-Syn)、溶酶体相关膜蛋白2A(LAMP2A)、微管相关蛋白1轻链3-Ⅱ(LC3-Ⅱ)表达,采用Western blotting实验检测LAMP2A、LC3-Ⅱ蛋白水平。结果与模型组比较,芍药苷组小鼠的爬杆时间明显缩短,悬挂实验评分明显升高,凋亡神经细胞数量明显减少,TH阳性细胞数及LAMP2A、LC3-Ⅱ表达明显升高,α-Syn表达明显减少,LAMP2A、LC3-Ⅱ蛋白水平明显升高,差异均有统计学意义(P<0.05)。与芍药苷组比较,芍药苷+3-MA组小鼠的爬杆时间明显延长,悬挂实验评分明显降低,凋亡神经细胞数量明显增多,TH阳性细胞数及LAMP2A、LC3-Ⅱ表达明显减少,α-Syn表达明显升高,LAMP2A、LC3-Ⅱ蛋白水平明显降低,差异均有统计学意义(P<0.05)。结论芍药苷通过诱导自噬清除α-Syn,保护多巴胺能神经元,从而改善PD小鼠的运动障碍。

Objective To observe the effect of paeoniflorin on motor behavior and dopamine neurons of mice with Parkinson's disease(PD)by activating autophagy.Methods Forty C57BL/6 mice were randomly divided into normal control group,model group,paeoniflorin group and paeoniflorin+3-methyl adenine(3-MA)group(n=10).The later 3 groups were intraperitoneally injected with 1-methyl-4-phenyl-1,2,3,6-tetmhydropyridine(MPTP)at a dosage of 30 mg/(kg·d)for a consecutive 7 d to establish subacute PD models.Meanwhile,mice in the paeoniflorin group and paeoniflorin+3-MA group were intraperitoneally injected with paeoniflorin(30 mg/[kg·d])or paeoniflorin(30 mg/[kg·d])+3-MA(2 mg/[kg·d]),respectively.Eight d after modeling,pole test and traction test were used to evaluate the locomotor ability of mice in each group.Then,the substantia nigra of the midbrain was taken;TUNEL was used to detect the nerve cell apoptosis;immunohistochemical staining was used to detect the number of tyrosine hydroxylase(TH)positive cells and the expressions ofα-synuclein(α-Syn),lysosomal associated membrane protein 2A(LAMP2A)and microtubule-associated protein 1 light chain 3-II(LC3-II);Western blotting was used to detect the LAMP2A and LC3-II protein levels.Results As compared with model group,paeoniflorin group had significantly shortened rod climbing time,significantly higher scores in traction test,significantly decreased number of apoptotic nerve cells,significantly increased number of TH-positive cells and expressions of LAMP2A and LC3-II,significantly decreasedα-Syn expression,and significantly increased protein levels of LAMP2A and LC3-II(P<0.05).The paeoniflorin+3-MA group had significantly prolonged rod climbing time,significantly lower scores in traction test,significantly increased number of apoptotic nerve cells,significantly decreased number of TH-positive cells and expressions of LAMP2A and LC3-II,significantly increased expression ofα-Syn,and significantly decreased protein levels of LAMP2A and LC3-II as compared with the paeoniflorin group(P<0.05).Conclusion Paeoniflorin can improve dyskinesia in PD mice by inducing autophagy to removeα-Syn and protect dopaminergic neurons.