基于Drp1-Ser637磷酸化探讨补阴牵正方对PD小鼠脑线粒体的影响

Influence of Buyin Qianzheng Fang on brain mitochondria in mice with Parkinson’s disease: a study based on Drp1-Ser637 phosphorylation

目的通过帕金森病(PD)小鼠脑动力相关蛋白1(Drp1)和其磷酸化位点丝氨酸637(Ser637)表达的变化及对脑线粒体的影响探讨补阴牵正方防治PD的可能机制。方法选用C57BL/6小鼠72只,随机分为正常组、模型组、补阴牵正方组、美多芭组,每组18只。除正常组外,其余3组小鼠腹腔注射1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP)建立PD模型,补阴牵正方组灌胃补阴牵正方水煎剂,每天灌胃2次,连续28 d,美多芭组腹腔注射美多芭,隔天1次,共28 d。爬杆法、抓握法观察小鼠行为学;免疫荧光组化法观察中脑黑质酪氨酸羟化酶(TH)阳性神经元数目;荧光素酶法测定脑线粒体三磷酸腺苷(ATP);JC-1法检测脑线粒体膜电位;蛋白印迹法(Western blot)检测脑蛋白Drp1及其磷酸化蛋白Drp1-Ser637表达。结果相比正常组,模型组小鼠爬杆时间延长(P<0.01),抓握力度减弱(P<0.01),黑质TH阳性神经元数目减少(P<0.01),脑线粒体膜电位及ATP水平降低(P<0.01),脑Drp1蛋白表达增加(P<0.01),Drp1-Ser637磷酸化蛋白表达降低(P<0.01)。与模型组相比,补阴牵正方组小鼠在第14天爬杆时间缩短、握力明显增加(P<0.05),黑质TH数目明显增加(P<0.05),Drp1蛋白表达减少(P<0.01),Drp1-Ser637磷酸化蛋白表达增加(P<0.05),脑线粒体膜电位及ATP水平显著升高(P<0.01);而美多芭组除在第14天行为学较模型组有明显改善外(P<0.05),其余指标均无统计学差异;补阴牵正方组与美多芭组比较其黑质TH数目、ATP水平及膜电位均有差异(P<0.05,P<0.01),Drp1-Ser637磷酸化蛋白表达也显著增加(P<0.01)。结论补阴牵正方可能通过下调维持线粒体动态平衡的分裂蛋白Drp1、上调Drp1-Ser637磷酸化位点的表达改善PD小鼠脑线粒体功能,从而保护中脑黑质多巴胺神经元达到防治PD的作用。

Objective To investigate the possible mechanism of Buyin Qianzheng Fang(BYQZF,Yin-tonifying Healthy qi-leading Formula)in protection and treatment of Parkinson’s disease(PD)through studying the changes and their influence on mitochondria of dynamic-related protein 1(Drp1)and its phosphorylation site serine 637(Drp1-Ser637)in PD mice.Methods C57 BL/6 mice(n=72)were randomly divided into normal group,model group,BYQZF group and madopar group(each n=18).The PD model was established through intraperitoneal injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)in model group,BYQZF group and madopar group.The BYQZF group was orally given decoction of BYQZF twice a day for 28 d,and madopar group was given intraperitoneal injection of madopar once every other day for 28 d.The behaviors of mice were observed by using climbing and gripping methods.The number of positive neurons of tyrosine hydroxylase(TH)in midbrain substantia nigra was observed by using immunofluorescence histochemistry technique.The level of brain mitochondrial adenosine triphosphate(ATP)was detected by using luciferase assay.The brain mitochondrial membrane potential(MMP)was detected by using JC-1 method.The expressions of Drp1 and Drp1-Ser637 were detected by using Western blotting assay.Results Compared with normal group,the climbing time was longer(P<0.01),gripping strength was weaker(P<0.01),number of TH positive neurons decreased(P<0.01),levels of brain MMP and ATP decreased(P<0.01),Drp1 expression increased(P<0.01),and Drp1-Ser637 expression decreased(P<0.01)in model group.Compared with model group,the climbing time was shorter on the 14th d(P<0.05),gripping strength was higher(P<0.05),number of TH positive neurons increased(P<0.01),Drp1 expression decreased(P<0.01),Drp1-Ser637 expression increased(P<0.05),and levels of brain MMP and ATP increased significantly(P<0.01)in BYQZF group.In madopar group only the behaviors of mice were significantly improved on the 14th d(P<0.05),and other indexes had no statistical difference compared with model group.Compared with madopar group,the number of TH positive neurons and levels of ATP and brain MMP had difference(P<0.05,P<0.01),and Drp1-Ser637 expression increased significantly(P<0.01)in BYQZF group.Conclusion Buyin Qianzheng Fang can improve the brain mitochondrial function to protect dopamine neurons in midbrain substantia nigra and prevent and treat PD through down-regulating Drp1 expression and up-regulating Drp1-Ser637 expression in PD mice.