穿心莲内酯通过诱导线粒体自噬对实验性帕金森病小鼠改善作用研究

目的探讨穿心莲内酯(AG)对实验性帕金森病小鼠的改善作用及其机制是否与诱导线粒体自噬有关。方法C57BL/6雄性小鼠,10~12周龄,体重22~25g,均购于辽宁成大生物股份有限公司。共将40只小鼠按照实验目的分为5组:对照组、模型组、AG低剂量组、AG中剂量组和AG高剂量组,各8只。除对照组外,模型组、AG低剂量组、AG中剂量组和AG高剂量组均通过腹腔注射MPTP诱导C57BL/6小鼠发生实验性帕金森病。AG低、中、高剂量组依次分别给予2.5、5.0、10.0mg·kg^-1·d^-1的AG灌胃,对照组和模型组给予等量0.9%氯化钠溶液灌胃,每日1次,连续14d。测试各组小鼠的神经行为学功能;实时荧光定量PCR检测各组小鼠脑SNpc区域IL-1β、NLRP3的mRNA表达;蛋白质免疫印迹检测其parkin、LC3-Ⅱ、LC3-Ⅰ、p62的蛋白表达情况;免疫组织化学检测其parkin表达情况。结果与对照组相比,模型组小鼠神经行为学受损,表现为爬杆时间延长和转棒停留时间下降,其脑SNpc区域IL-1β、NLRP3的mR NA表达水平显著升高,parkin、LC3-Ⅱ/Ⅰ表达水平显著降低,p62蛋白表达增加,差异均有统计学意义(P<0.05);与模型组比较,AG中、高剂量组可显著恢复小鼠神经行为学功能,脑SNpc区域IL-1β、NLRP3的mRNA水平显著降低,parkin、LC3-Ⅱ/Ⅰ表达显著增加,p62蛋白的表达显著减少,差异均有统计学意义(P<0.05),并均呈剂量依赖性。结论AG可显著改善实验性帕金森病小鼠神经行为学功能,其机制主要通过诱导脑线粒体自噬并减轻神经炎症实现,且在本研究设置剂量范围内其作用效果呈剂量依赖性。

Andrographolide ameliorates experimental Parkinson's disease in mice via induction of parkin-mediated mitophagy

Objective To investigate the ameliorative effect of andrographolide(AG)on experimental Parkinson's disease mice and whether the mechanism is related to the induction of mitochondrial autophagy.Methods C57BL/6 male mice,10-12 weeks old,weight 22-25 g,were purchased from Liaoning Chengda Biological Co.,Ltd.A total of 40 mice were used for follow-up experiments.The mice were divided into 5 groups according to the purpose of the experiment:control group,model group,AG low-dose group,AG medium-dose group and AG high-dose group,each with 8 animals.In addition to the control group,the model group,the AG low-dose group,the AG medium-dose group and the AG high-dose group were induced experimental Parkinson's disease in C57BL/6 mice by intraperitoneal injection of MPTP.The three AG dose groups were given 2.5,5.0,10.0 mg·kg^-1·d^-1 respectively by gavage.The control group,model group were given the same amount of 0.9%sodium chloride solution by gavage,once a day for 14 consecutive days.The neurobehavioral tests were carried out in each group.The mRNA expressions of IL-1βand NLRP3 in SNPC region were detected by real-time quantitative PCR,the protein expressions of Parkin,LC3-Ⅱ,LC3-Ⅰand p62 were detected by Western blotting,and the expression of Parkin was detected by immunohistochemistry.Results Compared with the control group,the neurobehavior of the model group mice was impaired,the rod climbing time was prolonged and the rotor stay time was decreased,the levels of IL-1βand NLRP3 mRNA expression in the SNpc area of the brain were significantly increased,and the level of parkin,LC3-Ⅱ/Ⅰexpression were significantly decreased,and the level of p62 protein expression was significantly increased,and the differences were statistically significant(P<0.05).Compared with the model group,the neurobehavioral function of the mice in the AG medium and high dose groups recovered significantly,the levels of IL-1βand NLRP3 mRNA in the brain SNpc area were significantly decreased,the level of parkin,LC3-Ⅱ/Ⅰexpression were significantly increased,and the level of p62 protein expression was significantly decreased,the differences were statistically significant(P<0.05),and all were dose-dependent.Conclusion In this study,AG can significantly improve the neurobehavioral function of mice with experimental Parkinson's disease by inducing autophagy of brain mitochondria and reducing neuro-inflammation.And its effect is dose-dependent within the dose range set in this study.