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,组织间质水肿减轻,变性坏死的神经细胞明显减少,脑组织细胞形态结构明显恢复。结论:益气通脉方可激活PI3K/AKT信号通路,调控下游凋亡相关因子Bax、Bcl-2的表达,从而发挥抗细胞凋亡及保护MCAO/R后受损脑组织的作用。
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 reduced(P<0.05).The ratio of p-PI3K/PI3K,p-AKT/AKT was significantly increased(P<0.01),and the relative expression levels of Bcl-2 protein and genes were significantly increased(P<0.01).HE staining showed that the number of nerve cells in the brain tissue of the model group rats was significantly reduced,the nuclei were pyknotic or disappeared,the cells were significantly ruptured,cavities and inflammatory cell infiltration were observed,some neurons were necrotic and degenerated,arranged irregularly,interstitial edema was observed,and the hierarchy was unclear.The number of brain tissue cells in the Yiqi Tongmai Formula group of rats significantly increased,the morphology of nuclear shrinkage improved,the number of cavities and inflammatory factors decreased,interstitial edema decreased,and the number of degenerated and necrotic nerve cells significantly decreased.The morphology and structure of brain tissue cells were significantly restored.Conclusion:Yiqi Tongmai Formula can activate the PI3K/AKT signaling pathway,regulate the expression of downstream apoptosis related factors Bax and Bcl-2,and thus exert anti apoptotic and protective effects on damaged brain tissue after MCAO/R.
作者
葛文静
陈芳芳
庞伯通
南淞华
秦阳
崔应麟
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)
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)。
