基于Nrf2信号通路研究丁苯酞对高危缺血性卒中的保护作用

Preventive effect of butylphthalide based on Nrf2 signaling pathway on high-risk ischemic stroke

目的:在急性脑梗死小鼠模型中,检测预使用丁苯酞(n-butylphthalide,NBP)是否可通过核因子红系2相关因子2(nuclear factor-erythroid 2-related factor 2,Nrf2)通路减轻缺血性脑损伤。方法:Nrf2-/-纯合子和Nrf2+/+野生型小鼠随机分为3组,分别为对照组(C)、低剂量给药组(20 mg/kg)和高剂量给药组(60 mg/kg),每组6只。将丁苯酞用植物油按30 mg/mL溶解,低剂量给药组以20 mg/kg每天一次灌胃,高剂量给药组以60 mg/kg每天一次灌胃,对照组小鼠给予同等剂量的植物油灌胃,1次/d,持续给药4周。4周末采用电凝法制备小鼠局灶性脑梗死模型(d MCAO)。在脑梗死模型3 d及10 d后,通过Longa评分评估小鼠的脑神经功能。通过氯化三苯基四氮唑(triphenyltetrazolium chloride,TTC)染色检测小鼠的脑梗死体积,并通过Western blot及免疫荧光技术检测小鼠脑组织中Nrf2及H奎尼酮氧化还原酶1(NADPH:quinone oxidoreductase 1,NQO1)、血红素加氧酶(heme oxygenase 1,HO-1)的蛋白表达水平。结果:与Nrf2野生型小鼠比较,Nrf2基因敲除小鼠在dMCAO术后体重下降幅度更大,小鼠改良Longa评分更高,小鼠脑梗体积更大,小鼠脑组织中Nrf2及下游NQO1、HO-1的蛋白表达水平均显著上调。在Nrf2+/+野生型小鼠中丁苯酞高剂量给药组(60+/+)小鼠改良Longa评分低于未给药小鼠(C+/+),脑梗体积减少,高剂量NBP对高危缺血性脑卒中脑损伤具有预防作用;在敲除Nrf2基因的小鼠,高剂量NBP对小鼠脑梗水平及神经功能的保护作用消失,无法诱导下游血红素加氧酶(HO-1)和NAD(P)H奎尼酮氧化还原酶1(NQO-1)的蛋白表达水平的上调。结论:预使用丁苯酞对于高危缺血性脑卒中的脑损伤可能具有一定保护作用,能够保护缺血性脑卒中小鼠的神经运动平衡功能,减少梗死体积。Nrf2的敲除加重了缺血性脑梗死小鼠的脑损伤,大剂量丁苯酞能够上调小鼠脑组织中Nrf2及其下游NQO-1与HO-1蛋白水平的表达,发挥其生理功能。

Objective:To investigate whether pre-administration of butylphthalide can alleviate ischemic brain injury through the nuclear factor erythroid 2-related factor 2(Nrf2)pathway in a mouse model of acute cerebral infarction.Methods:Nrf2-/-homozygous and Nrf2+/+wild-type mice were randomly divided into 3 groups:control group(C),low-dose butylphthalide group(20mg/kg)and high-dose butylphthalide group(60mg/kg),6 mice in each group.Butylphthalide was dissolved in vegetable oil at 30 mg/ml.The low-dose group was given 20 mg/kg body weight by gavage,the high-dose group was given 60 mg/kg body weight by gavage,and the control group was given the same dose of vegetable oil by gavage.Once daily for 4 weeks.The mouse focal cerebral infarction model(dMCAO)was established by electrocoagulation at the end of 4 weeks.After 3 days and 10 days of the cerebral infarction model,the neurological function of the mice was evaluated by Longa score.The cerebral infarction volume of mice was detected by TTC,and the protein expression levels of Nrf2,NQO-1 and HO-1 in mouse brain tissue were detected by Western blot and immunofluorescence techniques.Results:Compared with Nrf2 wild-type mice,Nrf2 knockout mice showed greater weight loss after dMCAO,higher modified Longa score,larger cerebral infarct volume,and Nrf2 and downstream HO-1 and NQO-1 protein expression levels were significantly up-regulated.In Nrf2+/+wild-type mice,the modified Longa score of the high-dose butylphthalide group(60+/+)was lower than that of the non-dose butylphthalide mice(C+/+),the volume of cerebral infarction was reduced,and the high-dose butylbenzene Phthalophthalene(NBP)has a preventive effect on high-risk ischemic stroke brain injury;in mice knocked out of the Nrf2 gene,the protective effect of high-dose NBP on the level of cerebral infarction and neurological function in mice disappeared,and the downstream heme oxygenation could not be induced.Up-regulation of protein expression levels of enzyme(HO-1)and NAD(P)H quinidone oxidoreductase 1(NQO-1).Conclusion:Pre-administration of butylphthalide may have a certain protective effect on the brain injury of high-risk ischemic stroke,which can protect the neuromotor balance function and reduce the infarct size in ischemic stroke mice.Knockout of Nrf2 aggravated the brain injury in mice with ischemic cerebral infarction.High-dose butylphthalide can up-regulate the expression of Nrf2 and its downstream NQO-1 and HO-1 proteins in the brain tissue of mice,exerting its physiological function.