附子联合MPTP诱导肝阳上亢型PD小鼠亚急性模型与慢性模型的建立及比较

Establishment and Comparison of Subacute Model and Chronic Model Induced by FU ZI Combined with MPTP in Parkinson′s Disease Mice with Hyperactivity of Liver Yang

目的附子联合1-甲基-4-苯基-1,2,3,6-四氢吡啶(anendons combined 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine,MPTP)诱导C57BL/6小鼠肝阳上亢PD亚急性模型和肝阳上亢PD慢性模型的建立,通过行为学、病理学及分子生物学方法对模型进行评估。方法小鼠随机分为正常组、模型组、肝阳上亢PD亚急性组MPTP[25mg/(kg·d)]注射7天、肝阳上亢PD慢性组[25mg/(kg·d)],每3.5天注射1次,注射5周,在注射MPTP前,各模型组附子煎液灌胃4周。爬杆、转棒实验检测小鼠行为学变化;免疫组化检测小鼠中脑黑质酪氨酸羟化酶(trosine hydroxylase,TH)的表达,免疫印迹检测各组小鼠蛋白含量变化,实时定量PCR检测TH mRNA表达变化。结果与正常组比较,肝阳上亢PD慢性组小鼠行为变化明显,模型组及肝阳上亢PD亚急性组小鼠变化较低,在爬杆实验中,肝阳上亢PD慢性组小鼠爬杆时间明显长于模型组及肝阳上亢PD亚急性组(P<0.05),转棒实验中,肝阳上亢PD慢性组小鼠在棒时间明显短于模型组及肝阳上亢PD亚急性组(P<0.05)。中脑黑质区TH阳性细胞表达含量方面,与正常对照组比较,肝阳上亢PD慢性组TH表达量显著下降(P<0.05),模型组及肝阳上亢PD亚急性组表达量高于肝阳上亢PD慢性组;免疫印迹及实时定量PCR检测结果表明,肝阳上亢PD慢性组小鼠在蛋白及mRNA表达量均为最低(P<0.05)。结论肝阳上亢PD慢性组小鼠与单纯MPTP模型组及肝阳上亢PD亚急性组比较,损伤程度更高,周期更长,更具有与人类PD相似的临床症状,提示该模型有望成为PD发病机制研究潜在的稳定模型。

Objective To establish subacute model and chronic model induced by FU ZI combined with anendons combined 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)in Parkinson′s disease(PD)mice with hyperactivity of liver Yang and the model was evaluated by behavioral,pathological and molecular biological methods.Methods Mice were randomly divided into normal group,PD model group,subacute model of PD with hyperactive liver Yang group[MPTP 25mg/(kg·d)injection for 7days],chronic model of PD with hyperactive liver Yang group[MPTP 25mg/(kg·d)injection once every 3.5 days for 5 weeks],before MPTP injection,all model groups were gavaged with aconiti decoction for 4 weeks.The behavioral changes of mice were detected by pole climbing and pole turning experiments;the expression of trosine hydroxylase(TH)in the substantia nigra of mouse midbrain was detected by immunohistochemistry;the protein content of each group was detected by Western blot;and the mRNA expression of TH by real-time quantitative polymerase chain reaction(RT-qPCR).Results Compared with the normal group,the behavioral changes in the chronic model with hyperactive liver Yang group were obvious,while there were no significant changes in the model group and the subacute model with hyperactive liver Yang group.In the pole climbing experiment,the time of chronic model with hyperactive liver Yang group was significantly longer than that of model group and subacute model with hyperactive liver Yang group(P<0.05);in the rotarod test,the suspension time of chronic model with hyperactive liver Yang chronic group was significantly shorter than that of model group and subacute model with hyperactive liver Yang group(P<0.05);compared with normal group,the expression of TH positive cells in the substantia nigra region of chronic model with hyperactive liver Yang group was significantly decreased(P<0.05),and that in the model group and subacute model with hyperactive liver Yang group were higher than that in the chronic model with hyperactive liver Yang group.The results of Western blot and RT-qPCR showed that the expression levels of protein and mRNA in chronic model with hyperactive liver Yang group were the lowest(P<0.05).Conclusion Compared with MPTP model and subacute model with hyperactive liver Yang,the chronic model with hyperactive liver Yang of mice has higher degree of injury,longer cycle,and more clinical symptoms similar to PD in human.which can be a potential stable model for the pathogenesis of PD in the future.