紫草素对心肌缺血再灌注损伤的保护作用研究

Protective effect of shikonin on myocardial ischemia reperfusion injury

目的:本文旨在探究紫草素(Shi)对心肌缺血再灌注(I/R)损伤小鼠心肌组织病变、细胞凋亡、氧化应激及炎症反应的影响及其机制。方法:随机将小鼠分为5组:对照组(Ctrl);I/R模型组(I/R);紫草素处理组(I/R+Shi)(12.5、25、50 mg/kg)。苏木素-伊红(HE)染色观察组织病变。脱氧核糖核苷酸末端转移酶介导的缺口末端标记(TUNEL)检测细胞凋亡。酶联免疫吸附试验(ELISA)检测白细胞介素(IL)-6、IL-1β和肿瘤坏死因子α(TNF-α)水平。蛋白印迹检测Bax、Bcl-2、caspase-3、caspase-9、PI3K、p-AKT、AKT、p-mTOR和mTOR蛋白水平。结果:I/R组小鼠心肌梗死面积大于Ctrl组(P<0.01)。与I/R组相比,I/R+Shi(12.5、25、50 mg/kg)组小鼠心肌梗死面积下降(P<0.01)。紫草素可减轻心肌缺血再灌注损伤小鼠心肌组织病变。I/R组AST、cTnⅠ、CK-MB和LDH水平高于Ctrl组(P<0.01)。与I/R组相比,I/R+Shi(12.5、25、50 mg/kg)组AST、cTnⅠ、CK-MB和LDH水平下降(P<0.01)。与Ctrl组相比,I/R组细胞凋亡、Bax、caspase-3和caspase-9表达上升,Bcl-2表达降低(P<0.01)。与I/R组相比,I/R+Shi(25、50 mg/kg)组细胞凋亡、Bax、caspase-3和caspase-9表达下降,Bcl-2表达升高(P<0.01)。与Ctrl组相比,I/R组SOD和GSH水平下降,MDA水平上升(P<0.01)。与I/R组相比,I/R+Shi(12.5、25、50 mg/kg)组SOD和GSH水平升高,MDA水平降低(P<0.01)。而且,I/R组IL-6、IL-1β和TNF-α水平高于Ctrl组(P<0.01)。I/R+Shi(12.5、25、50 mg/kg)组IL-6、IL-1β和TNF-α水平低于I/R组(P<0.01)。另外,I/R组PI3K表达及p-AKT/AKT和p-mTOR/mTOR低于Ctrl组(P<0.01)。与I/R组相比,I/R+Shi(25、50 mg/kg)组PI3K表达及p-AKT/AKT和p-mTOR/mTOR上升(P<0.01)。PI3K/AKT信号通路抑制剂LY294002可逆转紫草素对心肌缺血再灌注损伤小鼠PI3K/AKT信号通路相关蛋白表达、心肌损伤标记物、细胞凋亡、氧化应激及炎症反应的影响。结论:紫草素可通过激活PI3K/AKT信号通路减轻心肌缺血再灌注损伤小鼠心肌组织病变、细胞凋亡、氧化应激和炎症反应。

Objective: The study aims to explore the effect of shikonin(Shi)on the pathological changes of tissues,apoptosis,oxidative stress and inflammatory response in myocardial ischemia reperfusion(I/R)injury mice. Methods: Mice were randomly divided into five groups,Ctrl group,I/R group and I/R+Shi(12.5,25,50 mg/kg)group.The pathological changes of tissues were observed by hematoxylin-eosin(HE)staining.The apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick labeling(TUNEL).The levels of interleukin(IL)-6,IL-1β and tumor necrosis factor alpha(TNF-α)were measured by enzyme-linked immunosorbent assay(ELISA).The protein levels of Bax,Bcl-2,caspase-3,caspase-9,PI3K,p-AKT,AKT,p-mTOR and mTOR were detected by Western blot. Results: The size of myocardial infarction in I/R group was larger than Ctrl group( P <0.01).Compared with I/R group,the size of myocardial infarction in I/R+Shi (12.5,25,50 mg/kg) group were decreased P <0.01).The pathological changes of myocardial tissues were alleviated by shikonin.The levels of AST,cTnⅠ,CK-MB and LDH in I/R group were higher than Ctrl group( P <0.01).Compared with I/R group,the levels of AST,cTnⅠ,CK-MB and LDH in I/R+Shi (12.5,25,50 mg/kg) group were reduced ( P <0.01).Compared with Ctrl group,the apoptosis and the expression of Bax,caspase-3 and caspase-9 in I/R group was increased with declined expression of Bcl-2( P <0.01).Compared with I/R group,the apoptosis and the expression of Bax,caspase-3 and caspase-9 in I/R+Shi(25,50 mg/kg)group was decreased with enhancive expression of Bcl-2 ( P <0.01).Compared with Ctrl group,the levels of SOD and GSH in I/R group were reduced with elevated levels of MDA ( P <0.01).Compared with I/R group,the levels of SOD and GSH in I/R+Shi(12.5,25,50 mg/kg)group were enhanced with decreased levels of MDA ( P <0.01).Moreover,the levels of IL-6,IL-1β and TNF-α in I/R group were higher than Ctrl group ( P <0.01).The levels of IL-6,IL-1β and TNF-α in I/R+Shi(12.5,25,50 mg/kg) group were lower than I/R group( P <0.01).In addition,the expression of PI3K and the rate of p-AKT/AKT and p-mTOR/mTOR in I/R group was lower than Ctrl group ( P <0.01).Compared with I/R group,the expression of PI3K and the rate of p-AKT/AKT and p-mTOR/mTOR in I/R+Shi(25,50 mg/kg) group was increased( P <0.01).The effects of shikonin on the expression of PI3K/AKT related proteins,the levels of myocardial injury markers,apoptosis,oxidative stress and inflammation in myocardial ischemia reperfusion injury mice were reversed by PI3K/AKT pathway inhibitor LY294002. Conclusion: Shikonin alleviates the pathological changes of tissues,apoptosis,oxidative stress and inflammatory response in myocardial ischemia reperfusion injury mice via activating the PI3K/AKT signaling pathway.