益气通脉方调控PI3K/AKT信号通路改善脑缺血再灌注损伤大鼠神经元凋亡  

Yiqi Tongmai Formula Regulating PI3K/AKT Signaling Pathway to Improve Neuronal Apoptosis in Rats with Cerebral Ischemia-Reperfusion Injury

作　　者：葛文静 陈芳芳 庞伯通 南淞华 秦阳 崔应麟[1,2] GE Wenjing;CHEN Fangfang;PANG Botong;NAN Songhua;QIN Yang;CUI Yinglin(The Second Clinical Medical College,Henan University of Chinese Medicine,Zhengzhou Henan China 450002;Key Laboratory of Chinese Medicine in Henan Province for Prevention and Treatment of Cardiovascular and Cerebrovascular Diseases,Zhengzhou Henan China 450002)

机构地区：[1]河南中医药大学第二临床医学院,河南郑州450002 [2]河南省中医药防治心脑血管病重点实验室,河南郑州450002

出　　处：《中医学报》2024年第1期63-70,共8页Acta Chinese Medicine

基　　金：河南省科技攻关计划项目(GZY-KSJ-2021-016,2023ZY1007);国家中医药管理局科研基金项目(212102311123)。

摘　　要：目的:基于磷脂酰肌醇3-激酶/蛋白激酶B(phosphatidylinositol 3-kinases/protein kinase B,PI3K/AKT)信号通路探讨益气通脉方对大脑中动脉阻塞再灌注(middle cerebral artery occlusion and recirculate,MCAO/R)模型大鼠神经功能损伤及凋亡的作用机制。方法:从68只健康SPF级雄性SD大鼠中选取12只为假手术组,其余大鼠采用改良线栓法制备MCAO/R模型。将36只MACO/R模型成功大鼠分为模型组、益气通脉方组(1.08 g·kg^(-1))和尼莫地平片组(10.8 mg·kg^(-1)),每组12只,给予大鼠进行相应药物干预,灌胃体积10 mL·kg^(-1),连续14 d,每天1次。假手术组和模型组大鼠灌胃给予等体积生理盐水。选用改良神经功能缺损程度评分(modified neurological severity score,mNSS)法评估大鼠的神经功能;HE染色观察各组大鼠脑组织形态改变;TUNEL染色检测细胞凋亡情况;Western blot检测缺血半暗带脑组织PI3K、p-PI3K、AKT、p-AKT、B淋巴细胞瘤-2(B cell lymphoma-2,Bcl-2)、Bcl-2相关的X基因(Bcl2-associated X,Bax)的蛋白表达水平;RT-qPCR法检测大鼠脑组织Bax mRNA、Bcl-2 mRNA表达水平。结果:与假手术组比较,模型组大鼠mNSS评分、神经细胞凋亡率、Bax蛋白及基因的相对表达量显著升高(P<0.01),p-PI3K/PI3K、p-AKT/AKT的比值显著下调(P<0.01),Bcl-2蛋白及基因的相对表达量均显著下调(P<0.01);与模型组比较,益气通脉方组大鼠第3、7、14天mNSS评分明显降低(P<0.05),神经细胞凋亡率、Bax蛋白及基因的相对表达量明显下降(P<0.05),p-PI3K/PI3K、p-AKT/AKT的比值显著升高(P<0.01),Bcl-2蛋白及基因的相对表达量均显著升高(P<0.01)。HE染色显示,模型组大鼠脑组织神经细胞明显减少,细胞核固缩或消失,细胞破裂明显,出现空腔和炎症细胞浸润,部分神经元坏死变性,排列不规整,组织间质水肿,层次不清;益气通脉方组大鼠脑组织细胞数目明显增多,细胞核皱缩形态改善,空腔及炎症因子减少,组织间质水肿�Objective:Based on the signal pathway of phosphatidylinositol 3-kinases/protein kinase B(PI3K/AKT),to explore the mechanism of Yiqi Tongmai Formula on neural function damage and apoptosis in middle cerebral artery occlusion and reperfusion(MCAO/R)model rats.Method:Twelve healthy SPF grade male SD rats were selected as the sham surgery group from 68 individuals,while the remaining rats were used to prepare MCAO/R models using an improved suture method.36 successful MACO/R model rats were divided into model group,Yiqi Tongmai Formula group(1.08 g·kg^(-1))and Nimodipine tablet group(10.8 mg·kg^(-1)),with 12 rats in each group.The rats were given corresponding drug intervention,with a gastric lavage volume of 10 mL·kg^(-1) for 14 consecutive days,once a day.Rats in the sham surgery group and model group were given equal volumes of physiological saline by gavage.Use the Modified Neurological Severity Score(mNSS)method to evaluate the neural function of rats;HE staining was used to observe the morphological changes of brain tissue in each group of rats;TUNEL staining was used to detect cell apoptosis;Western blot was used to detect the protein expression levels of PI3K,p-PI3K,AKT,p-AKT,B cell lymphoma-2(Bcl-2),and Bcl-2 associated X genes(Bcl-2 associated X,Bax)in ischemic penumbra brain tissue;RT qPCR was used to detect the expression levels of Bax mRNA and Bcl-2 mRNA in rat brain tissue.Results:Compared with the sham surgery group,the mNSS score,neuronal apoptosis rate,relative expression levels of Bax protein and genes in the model group rats were significantly increased(P<0.01),while the ratios of p-PI3K/PI3K and p-AKT/AKT were significantly down regulated(P<0.01).The relative expression levels of Bcl-2 protein and genes were significantly down regulated(P<0.01);Compared with the model group,the mNSS score of rats in the Yiqi Tongmai Formula group was significantly reduced(P<0.05)on the 3rd,7th,and 14th day,while the apoptosis rate of neurons,relative expression of Bax protein and genes were significantly reduce

关 键 词：益气通脉方 PI3K/AKT信号通路 神经元细胞凋亡 脑缺血再灌注损伤 

分 类 号：R285.5[医药卫生—中药学]