牛膝和白芍配伍对帕金森病肝阳上亢证小鼠的协同神经保护作用及机制研究

Synergistic neuroprotective effect of compatibility of Achyranthis bidentatae radix and Paeoniae radix alba on Parkinson disease mice with the syndrome of hyperactivity of liver yang and related mechanism

目的探讨牛膝和白芍配伍对帕金森病(PD)肝阳上亢证小鼠的协同神经保护作用及可能的作用机制。方法C57BL/6小鼠随机分为对照组、模型组、镇肝熄风汤组、牛膝组、白芍组、牛膝和白芍配伍组,采用附子汤灌胃联合腹腔注射MPTP法制备PD肝阳上亢证小鼠模型。通过激惹实验、旷场实验及转棒实验观察小鼠行为学改变;免疫组化方法测定TH阳性表达水平;硫代巴比妥酸法、羟胺法、微量酶标法分别检测脑组织氧化应激指标谷胱甘肽(GSH)、超氧化物歧化酶(SOD)、丙二醛(MDA)水平;蛋白免疫印迹法检测脑组织氧化应激相关蛋白核因子NF-E2相关因子2(Nrf2)、血红素氧合酶1(HO-1)的表达。结果与模型组相比,牛膝组、白芍组、牛膝和白芍配伍组均可明显改善小鼠激惹程度,增加旷场活动总距离,延长转棒掉落潜伏期,增加TH阳性表达率;牛膝组及配伍组可明显提高小鼠脑组织SOD及GSH水平,降低MDA含量,激活Nrf2核转位并上调HO-1蛋白的表达,对氧化应激有一定程度的改善,而白芍组对改善氧化应激作用不明显;与单独用药组相比,配伍组治疗效果最为显著。结论牛膝和白芍配伍可能通过牛膝激活Nrf2/HO-1信号通路抑制氧化应激,对PD肝阳上亢证小鼠发挥协同增效的神经保护作用。

Objective To determine the synergistic neuroprotective effect of the compatibility of Achyranthis bidentatae radix(ABR)and Paeoniae radix alba(PRA)on Parkinson disease(PD)mice with the syndrome of hyperactivity of liver yang and its possible mechanism.Methods C57BL/6 mice were randomly divided into 6 groups:a control group,a model group,a Zhengan Xifeng decoction group,an ABR group,a PRA group and an ABR+PRA group.The mice were given Fuzi decoction intragastrically and MPTP intraperitioneally to establish the PD mice models with the syndrome of hyperactivity of liver yang.The behavioral changes of the mice were determined with irritable test,open field experiment and rotating-stick experiment.Then immunohistochemical method was used to detect the positive expression level of tyrosine hydroxylase(TH)in the substantia nigra.Thiobarbituric acid,hydroxylamine and microenzyme labeling methods were used respectively to detect the oxidative stress indicators glutathione(GSH),superoxide dismutase(SOD)and malondialdehyda(MDA)in the brain tissue.Western blot was used to detect the expression of oxidative stress-related proteins nuclear factor NF-E2 ralated factor 2(Nrf2)and heme oxygenase 1(HO-1).Results As compared with the model group,the irritable degree of mice was reduced,the total distance in the open field experiment was increased,the drop latency was prolonged and the positive expression rate of TH was improved in the ABR group,the PRA group and the ABR+PRA group.In the ABR group and ABR+PRA group,the levels of SOD and GSH were enhanced,the content of MDA were reduced,the nuclear transfer of Nrf2 were activated and expression of HO-1 were up-regulated,and oxidative stress were improved to a certain extent,but the PRA group had no obvious effect.Compared with the single medication group,the ABR+PRA group showed the most remarkable effect of treatment.Conclusion Combination of ABR and PRA exerts synergistic neuroprotective effects in PD mice with the syndrome of hyperactivity of liver yang,whose mechanism may be related to the activation of Nrf2/HO-1 signaling pathway by ABR to inhibit the oxidative stress.