摘要
【目的】观察益气活血法对缺血性脑中风急性期大鼠脑神经元的保护作用及机制。【方法】将40只SPF级SD大鼠随机分为假手术组、模型组、尼莫地平组、益气活血组,每组10只。除假手术组外,其余各组大鼠采用线栓法制备急性期缺血性脑中风模型。尼莫地平组大鼠给予腹腔注射0.5 mg/kg尼莫地平注射液,益气活血组大鼠给予腹腔注射1.8 mL/kg黄芪注射液和7.2 mg/kg川芎嗪注射液,假手术组和模型组大鼠给予腹腔注射等体积生理盐水,每天1次,连续4 d。治疗前和治疗后12、24、48、72、96 h评估Longa神经功能评分。给药结束后,苏木素-伊红(HE)染色法观察脑神经元损伤情况;脱氧核苷酸末端转移酶(TdT)介导的d UTP缺口末端标记(TUNEL)染色法检测脑神经元凋亡情况;实时定量聚合酶链反应(RT-qPCR)法检测脑组织半胱氨酸蛋白酶3(Caspase-3)、B淋巴细胞瘤-2基因(Bcl-2)、Bcl-2相关X蛋白(Bax)mRNA表达水平;酶联免疫吸附分析(ELISA)检测脑组织白细胞介素6(IL-6)、肿瘤坏死因子α(TNF-α)、丙二醛(MDA)含量和谷胱甘肽过氧化物酶(GSH-Px)、超氧化物歧化酶(SOD)活性;蛋白免疫印迹(Western Blot)法检测脑组织Wnt/β-连环蛋白(β-catenin)信号通路相关蛋白的表达水平。【结果】与假手术组比较,模型组大鼠神经功能缺损评分升高,脑组织神经元凋亡率升高,脑组织IL-6、TNF-α、MDA水平升高,GSH-Px、SOD活性降低,Caspase-3、Bax mRNA表达水平升高,Bcl-2 mRNA表达水平降低、Wnt3a、β-catenin蛋白表达水平降低,糖原合成酶激酶3β(GSK-3β)磷酸化水平升高(均P<0.05),脑组织神经元可见明显损伤。与模型组比较,益气活血组大鼠神经功能缺损评分降低,脑组织神经元凋亡率降低,脑组织IL-6、TNF-α、MDA水平降低,GSHPx、SOD活性升高,Caspase-3、Bax m RNA表达水平降低,Bcl-2 mRNA表达水平升高,Wnt3a、β-catenin蛋白表达水平升高,GSK-3β磷酸化水平降低(均P<0.05),脑组织神经元损伤得到改善。【结论】益气活血法对缺血性脑中风急性期大鼠脑神经元的保护作用机制可能与调控Wnt/β-catenin信号通路有关。
Objective To observe the protective effect and its mechanism of Yiqi Huoxue(invigorating qi and activating blood)therapy on cerebral neurons at the acute stage of ischemic stroke in rats.Methods Forty SPFgrade SD rats were randomly divided into sham-operation group,model group,Nimodipine group and Yiqi Huoxue group,with 10 rats in each group.Except for the sham-operation group,the ischemic stroke model was prepared by the thread-occlusion method in all the other groups.Rats in the Nimodipine group were given an intraperitoneal injection of 0.5 mg/kg of Nimodipine,rats in the Yiqi Huoxue group were given an intraperitoneal injection of 1.8 mL/kg of Astragalus and 7.2 mg/kg of Ligustrazine,and rats in the sham-operation and model groups were given an equal volume of normal saline intraperitoneally,once a day for 4 continuous days.The Longa neurological function scores were assessed before treatment and 12,24,48,72 and 96 hours after treatment.At the end of administration,cerebral neuronal damage was observed by hematoxylin-eosin(HE)staining;cerebral neuronal apoptosis was detected by deoxynucleotide terminal transferase(TdT)-mediated dUTP nick end labeling(TUNEL)staining;mRNA expression levels of cysteine protease 3(Caspase-3),B lymphocytoma-2 gene(Bcl-2)and Bcl-2-associated X protein(Bax)in brain tissues were detected by real time quatitative polymerase chain reaction(RT-qPCR);contents of interleukin 6(IL-6),tumour necrosis factorα(TNF-α)and malondialdehyde(MDA),and activities of glutathione peroxidase(GSH-Px)and superoxide dismutase(SOD)in brain tissues were detected by enzyme-linked immunosorbent assay(ELISA);Western Blot method was used to detect the expression of Wnt/β-catenin signaling pathway-related proteins in brain tissues.Results Compared with the sham-operation group,the neurological deficit score of the model group was increased,the neuronal apoptosis rate of brain tissues was increased,the levels of IL-6,TNF-αand MDA in brain tissues were increased,the activities of GSH-Px and SOD were decreased,and the mRNA expression levels of Caspase-3 and Bax were increased,and Bcl-2mRNA expression level was decreased.The protein expression levels of Wnt3a andβ-catenin were decreased,and the phosphorylation level of GSK-3βwas increased(all P<0.05),and obvious neuron injury in brain tissues was seen.Compared with the model group,the neurological deficit score of Yiqi Huoxue group was decreased,the rate of neuronal apoptosis in brain tissues was decreased,the levels of IL-6,TNF-αand MDA were decreased,the activities of GSH-Px and SOD were increased,and the mRNA expression levels of Caspase-3 and Bax were decreased,and Bcl-2 mRNA expression level was increased;the protein expression levels of Wnt3a andβ-catenin were increased,and the phosphorylation level of GSK-3βwas decreased(all P<0.05),and the neuron injury in brain tissues was improved.Conclusion The mechanism of the protective effect of Yiqi Huoxue therapy on cerebral neurons at the acute stage of ischemic stroke in rats may be related to the regulation and controll of Wnt/β-catenin signaling pathway.
作者
江利敏
桑锋
刘向哲
嵇朋
郭向东
JIANG Li-Min;SANG Feng;LIU Xiang-Zhe;JI Peng;GUO Xiang-Dong(Physical Examination Center,The First Affiliated Hospital of Henan University of Chinese Medicine,Zhengzhou 450003 Henan,China;Key Laboratory of The First Affiliated Hospital of Henan University of Chinese Medicine,Zhengzhou 450003 Henan,China;Ward One of Encephalopathy Department,The First Affiliated Hospital of Henan University of Chinese Medicine,Zhengzhou 450003 Henan,China;Ward Three of Neurology Department,The Third People’s Hospital of Zhengzhou(Cancer Hospital of Henan University),Zhengzhou 450003 Henan,China;Dept.of Otorhinolaryngology,The First Affiliated Hospital of Henan University of Chinese Medicine,Zhengzhou 450003 Henan,China)
出处
《广州中医药大学学报》
CAS
2022年第6期1358-1365,共8页
Journal of Guangzhou University of Traditional Chinese Medicine
基金
河南省中医药科学研究专项课题(编号:2017ZY2021)
作者简介
江利敏(1981-),女,硕士研究生,副主任医师,E-mail:17123207455@163.com;通信作者:郭向东,男,硕士研究生,副主任医师,E-mail:lip56321@163.com
