枸杞多糖基于AMPK-mTOR通路对鱼藤酮诱导的帕金森病大鼠神经元保护作用研究

Study on the neuro-protective effect of lycium barbarum polysaccharide on neurons of rats with rotenone-induced Parkinson’s disease via AMPK-mTOR signaling pathway

目的研究枸杞多糖基于腺苷酸活化蛋白激酶(AMPK)-哺乳动物雷帕霉素靶蛋白(mTOR)通路对鱼藤酮诱导的帕金森病大鼠神经元的保护作用。方法将45只雄性SD大鼠随机分为对照组、帕金森病组及枸杞多糖组,每组15只。枸杞多糖组给予枸杞多糖水溶液50 mg/kg灌胃预处理,帕金森病组及对照组给予等量蒸馏水灌胃预处理,均1次/d,持续3 d。预处理结束后,帕金森病组及枸杞多糖组采用颈背部皮下注射鱼藤酮方法建立帕金森病模型,对照组颈背部皮下注射等量溶剂。注射3 h后,枸杞多糖组给予枸杞多糖水溶液50 mg/kg灌胃,对照组及帕金森病组给予等量蒸馏水灌胃,均1次/d,连续28 d。干预结束后,使用透射电镜技术观察各组大鼠黑质溶酶体数目及线粒体损伤情况,采用ELISA法测定各组大鼠纹状体线粒体氧化呼吸酶Ⅰ及AMPK-mTOR通路相关蛋白AMPK、p-AMPK、mTOR、p-mTOR含量。结果透射电镜显示,对照组大鼠线粒体形态正常;帕金森病组大鼠黑质内神经元出现线粒体肿胀变性、嵴消失等不可逆性损伤;与帕金森病组相比,枸杞多糖组大鼠神经元内溶酶体增多,线粒体发生可逆性的轻度水肿。与对照组比较,帕金森病组大鼠纹状体线粒体氧化呼吸酶Ⅰ含量明显升高(P<0.05),p-AMPK/AMPK比值明显降低(P<0.05);与帕金森病组比较,枸杞多糖组大鼠纹状体线粒体氧化呼吸酶Ⅰ含量明显降低(P<0.05),p-AMPK/AMPK比值明显升高(P均<0.05);3组p-mTOR/mTOR比值比较差异均无统计学意义(P均>0.05)。结论枸杞多糖可通过AMPK-mTOR通路减少异常蛋白蓄积,减轻帕金森病大鼠黑质纹状体线粒体损伤,从而产生神经保护作用。

Objective It is to study the neuro-protective effect of lycium barbarium polysaccharide(LBP)on neurons of rats with rotenone-induced Parkinson’s disease via Adenylate-activated protein kinase(AMPK)-mammalian target of rapamycin(mTOR)signaling pathway.Methods Forty-five male SD rats were randomly divided into control group,rotenone group and LBP group,15 rats in each group.The LBP group was pretreated with 50 mg/kg aqueous solution of LBP by gavage,and the Parkinson’s disease group and the control group were pretreated with the same volume of distilled water by gavage,all once a day,continuously pretreatd for 3 days.After the pretreatment,the Parkinson’s disease group and the LBP group were subcutaneously injected with rotenone on the back of the neck to establish Parkinson’s disease models,and the control group was subcutaneously injected with the same amount of solvent on the back of the neck.After 3 hours of injection,the LBP group was given 50 mg/kg aqueous solution of LBP by gavage,and the control group and Parkinson’s disease group were given the same amount of distilled water by gavage,all once a day,continuously treated for 28 days.After the intervention,the number of lysosomes and mitochondrial damage in the substantia nigra of the rats in each group were observed by transmission electron microscopy,and the contents of striatal mitochondrial oxidative respiratory enzyme I and AMPK-mTOR pathway-related proteins AMPK,p-AMPK,mTOR,p-mTOR were detected by ELISA.Results Transmission electron microscopy showed that the mitochondrial morphology of the rats in the control group was normal;the neurons in the substantia nigra of the Parkinson’s disease group had irreversible damage such as mitochondrial swelling,degeneration and cristae disappearance;compared with the Parkinson’s disease group,the lysosomes in neurons of the rats in the LBP group increased,and reversible mild edema occurred in mitochondria.Compared with the control group,the content of mitochondrial oxidative respiratory enzymeⅠin the Parkinson’s disease group was significantly increased(P<0.05),and the ratio of p-AMPK/AMPK was significantly decreased(P<0.05);compared with the Parkinson’s disease group,the content of mitochondrial oxidative respiratory enzymeⅠin the LBP group was significantly decreased(P<0.05),and the ratio of p-AMPK/AMPK was significantly increased(P<0.05);there was no significant difference in the ratio of p-mTOR/mTOR among the three groups(P>0.05).Conclusion LBP can reduce the accumulation of abnormal protein through the AMPK-mTOR pathway,reduce the mitochondrial damage in the nigrostriatal of Parkinson’s disease rats,and thus to play its neuroprotective effects.