自噬在MPTP慢性帕金森病小鼠模型中的作用及松果菊苷的干预研究

发布时间：2018-07-04 22:50

  本文选题：帕金森 + MPTP　； 参考：《南京中医药大学》2017年硕士论文

【摘要】：目的:通过1-甲基-4-苯基-1,2,3,6-四氢吡啶(1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride,MPTP)慢 性帕金 森小鼠 模型,观察松果菊苷(Echinacoside,ECH)能否通过自噬途径缓解模型小鼠多巴胺(Dopamine,DA)神经元的损伤,并探讨松果菊苷通过自噬途径保护DA能神经元的作用机制及松果菊苷发挥自噬作用可能的信号通路,为温肾养肝方在帕金森病的临床运用提供理论和科学依据。方法:选用10周龄C57BL/6J雄性小鼠,体重为25～30g,固定时间给予松果菊苷(Echinacoside,ECH)(30mg/kg)/司来吉兰(Selegiline,SEL)(1mg/kg)灌胃处理,1h后给予皮下注射MPTP0.1ml/10g,每周两次,持续四周,建立MPTP慢性帕金森小鼠模型。通过行为学观察(爬杆实验、步态分析)评价小鼠运动迟缓和平衡协调能力及步态异常。运用酪氨酸轻化酶(tyrosine hydroxylase,TH)免疫组化方法,并结合体视学计数,分析模型小鼠黑质部(Substantia nigra,SN)DA能神经元的损伤状况。应用蛋白免疫印迹法,检测自噬相关蛋白mTOR(Mammalian target of rapamycin)的蛋白表达水平,Beclin-1的蛋白表达水平,P62的蛋白表达水平,LC3-Ⅱ(Chain3-Ⅱ)的蛋白表达水平,α-突触核蛋白(α-synuclein,a-SN)的蛋白表达水平,及PI3K(PhosphatidylInositol3-kinase)、AKT(Protein kinase B)、Atg13(autophagy-related gene 13)磷酸化的蛋白表达水平。结果:行为学分析提示松果菊苷能够减少模型小鼠爬杆总时间(杆上停留时间及爬杆时间,P0.05),但步态分析未提供统计学差异(P0.05)。免疫组化提示松果菊苷能减少MPTP慢性帕金森小鼠DA的损伤(P0.01)。蛋白免疫印迹法提示松果菊苷能逆转MPTP慢性模型小鼠中脑Beclin-1表达下调(P0.05),下调MPTP慢性模型小鼠中脑P62的表达(P0.05),促进MPTP慢性模型小鼠中脑LC3-Ⅱ表达增加(P0.05),下调MPTP慢性模型小鼠中脑α-synuclein的表达(P0.05),但PI3K、AKT、mTOR、Atg13磷酸化的蛋白表达水平未提供统计学差异(P0.05)。结论:松果菊苷能够通过自噬途径发挥对MPTP诱导的慢性PD模型小鼠中脑DA神经元的保护作用。
[Abstract]:Objective: to observe whether Echinacoside ECH can attenuate the injury of dopamine DA neurons in mouse model by using 1-Methyl-4-phenyl-6-tetrahydropyridine hydrochloridede MPTP (1-Methyl-4-phenyl-1-tetrahydropyridine hydrochloridede-MPTP) model in Parkinsen mice model by means of 1-methyl-4-phenyl-6-tetrahydropyridine hydrochloridedeform-MPTP (1-Methyl-4-phenyl-1-tetrahydropyridine) -1-methyl-4-phenyl-6-tetrahydropyridine hydrochloridedede-MPTP (MPTP) model. To explore the mechanism of pyrethrin protecting DA neurons through autophagy and the possible signal pathway of autophagy, which provides a theoretical and scientific basis for the clinical application of Wenshen Yanggan recipe in Parkinson's disease. Methods: 10-week-old C57BL / 6J male mice, weighing 25g / 30g, were treated with 30mg/kg / 1mg/kg for 1 h, then subcutaneously injected MPTP 0.1ml / 10g, twice a week for four weeks, to establish the model of chronic Parkinson's disease in mice. The behavioral observation (pole climbing test, gait analysis) was used to evaluate the motor retardation, balance and coordination ability and gait abnormality in mice. The damage of DA neurons in substantia nigra SN of model mice was analyzed by using tyrosine hydroxylase th immunohistochemical method and stereological counting. Western blot was used to detect the protein expression level of autophagy associated protein mTOR and Beclin-1 protein. The protein expression level of P62 and 伪 -synucleinina-SN were determined by Western blotting, and the protein expression of LC3- 鈪，

本文编号：2097782