山柰酚对脑缺血/再灌注损伤的体内外保护作用机制研究
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篇名: 山柰酚对脑缺血/再灌注损伤的体内外保护作用机制研究
TITLE: Study on protective mechanism of kaempferol against cerebral ischemia/reperfusion injury in vitro and in vivo
摘要: 目的 探讨山柰酚(KAE)对脑缺血/再灌注(CI/R)损伤的体内外保护作用机制。方法首先利用网络药理学方法初步筛选KAE治疗CI/R损伤的潜在作用靶点。然后采用AutoDockVina软件将KAE分别与排名前10位的核心靶点进行分子对接,以结合亲和力作为评价标准,进一步明确KAE治疗CI/R损伤的可能作用机制。最后通过体内外实验验证以上结果,即构建氧糖剥夺/再灌注(OGD/R)HT22细胞损伤模型和大脑中动脉阻塞(MCAO)大鼠模型,采用CCK-8法检测细胞活性,分别对大鼠及其脑组织进行神经功能评分和TTC染色,采用Westernblot法检测HT22细胞和大鼠脑组织中蛋白激酶B(Akt)、Src蛋白的磷酸化水平。结果网络药理学筛选结果显示,KAE治疗CI/R损伤与前列腺素内过氧化物合成酶2、基质金属蛋白酶9、JUN、Akt1、肿瘤坏死因子、胱天蛋白酶3、丝裂原激活蛋白激酶8、细胞间黏附分子1、血管细胞黏附分子1、Src蛋白10个核心靶点密切相关。分子对接结果显示,KAE与Akt1、Src蛋白结合稳定。体内外实验结果表明,KAE可显著提高OGD/R损伤HT22的细胞存活率(P<0.05),显著降低MCAO模型大鼠的神经功能评分(P<0.05),显著减小大鼠脑梗死体积(P<0.05),显著升高HT22细胞和大鼠脑组织中Akt、Src蛋白的磷酸化水平(P<0.05),且呈剂量依赖趋势。结论KAE可能通过调控Akt、Src蛋白的磷酸化水平来发挥脑保护作用,从而治疗CI/R损伤。
ABSTRACT: OBJECTIVE To explore the protective mechanism of kaempferol (KAE)on cerebral ischemia/reperfusion (CI/R) injury in vitro and in vivo . METHODS Firstly,the potential targets of KAE in the treatment of CI/R injury were preliminarily screened by network pharmacology. AutoDock Vina software was used to conduct molecular docking between KAE and the top 10 core targets ,and the binding affinity was used as the evaluation standard to further clarify the possible mechanism of KAE in treating CI/R injury. Finally ,the above results were verified by in vivo and in vitro experiments. The oxygen glucose deprivation/ reperfusion(OGD/R)HT22 cell injury model and the middle cerebral artery occlusion (MCAO)rat model were constructed. The cell activity was detected by CCK- 8 method. The neural function score and TTC staining were performed on the rats and their brain tissues. The phosphorylation levels of protein kinase B (Akt)and Src in HT 22 cells and brain tissue of rats were detected by Western blot. RESULTS The results of network pharmacology screening showed that KAE in the treatment of CI/R injury was closely related to 10 core targets including prostaglandin-endoperoxide synthase 2,matrix metalloproteinase 9,JUN,Akt1,tumor necrosis factor ,caspase-3,mitogen activated protein kinase 8,intercellular cell adhesion molecule 1,vascular cell adhesion molecule 1 and Src. The results of molecular docking showed that KAE was stably bound with Akt 1 and Src . The results of in vitro and in vivo experiments showed that KAE could significantly improve the survival rate of OGD/R-injuried HT 22 cells (P<0.05), significantly reduced the neurological function score of MCAO model rats (P<0.05),significantly reduces the volume of cerebral infarction in rats (P<0.05),and significantly increased the phosphorylation levels of Akt and Src in HT 22 cells and brain tissue of rats(P<0.05),which showed a dose dependent trend. CONCLUSIONS KAE may play a neuroprotective role by regulating the phosphorylation expression of Akt and Src ,thus treating CI/R injury.
期刊: 2022年第33卷第17期
作者: 徐芳琴,郭超,王婧雯
AUTHORS: XU Fangqin ,GUO Chao,WANG Jingwen
关键字: 山柰酚;脑缺血/再灌注损伤;网络药理学;分子对接;保护作用;HT22细胞;大鼠
KEYWORDS: kaempferol; cerebral ischemia/reperfusion
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