橄榄苦苷对心肌缺血再灌注损伤的保护作用及机制研究

发布时间：2018-05-08 18:36

本文选题：橄榄苦苷 + 心肌　；参考：《兰州大学》2017年博士论文

【摘要】：研究背景:冠心病是世界范围内导致死亡和残疾的主要原因之一,其临床表现为心肌缺血再灌注损伤(MIRI)。但是,到目前为止,其确切的发病机制仍不十分清楚。目前的研究主要集中在氧自由基形成、细胞内钙超载、细胞凋亡、氧及能量利用障碍等方面,尤其认为氧化应激损伤和细胞凋亡与MIRI的发生密切相关。橄榄苦苷是油橄榄叶中的主要活性成分之一,具有强大的抗氧化和清除氧自由基作用。早在2004年Manna等人首次报道橄榄苦苷对由缺血再灌注引起的离体心脏氧化性心肌损伤具有一定的保护作用,但此后很少有学者进行在体实验研究,也很少对其作用机制进行进一步研究。研究目的:评价橄榄苦苷对心肌缺血再灌注损伤的保护作用,并对其机制进行研究。实验方法:建立大鼠心肌缺血再灌注损伤模型和原代培养乳鼠心肌细胞缺血再灌注损伤模型,采用ELISA法检测CK、SOD、LDH、CAT、MDA和GSH-PX的活性;TTC染色法观察梗死心肌面积;HE染色观察心肌损伤;TUNEL法和流式细胞术检测心肌细胞凋亡;荧光探针DCFH-DA检测细胞内的ROS水平;JC-1染色法检测细胞线粒体膜电位;Western blot法检测凋亡相关蛋白Cyt-C,Caspase-3,Caspase-9,Bcl-2/Bax,细胞外信号调节激酶(ERK1/2)和蛋白激酶B(PKB/Akt)的磷酸化表达水平。实验结果:1)与大鼠缺血再灌注模型组相比,橄榄苦苷(100,200,400mg/kg)预处理明显抑制大鼠缺血再灌注诱导的CK、LDH和MDA的升高(P0.01;P0.05;P0.05)和SOD、CAT和GSH-PX的降低(P0.05;P0.05;P0.05)。模型组大鼠心肌梗死比率达18.9%,与空白对照组相比,差异具有统计学意义(P0.001)。与模型组相比,橄榄苦苷预处理明显降低梗死比率(P0.05)和心肌细胞凋亡指数(P0.05)。与模型组相比,橄榄苦苷(400mg/kg)明显升高Bcl-2/Bax比值(P0.01);明显增加p-ERK1/2和p-Akt的表达(P0.05,P0.05)。2)与原代培养乳鼠心肌细胞缺血再灌注损伤模型组比较,橄榄苦苷(100,200,400μg/m L)呈浓度依赖地升高缺血再灌注诱导的原代培养乳鼠心肌细胞存活率(P0.001),降低Hoechst33258荧光染色阳性细胞比率(P0.01)和心肌细胞凋亡率(P0.001)。橄榄苦苷呈浓度依赖地降低CK、LDH和ROS水平(P0.01,P0.01,P0.001),抑制MDA的过度释放(P0.001),升高SOD、CAT和GSH-PX活性(P0.05,P0.05,P0.05)。橄榄苦苷降低绿色/红色荧光强度比值(P0.001)。抑制Bax、Cyt-C、Caspase-3和Caspase-9的高表达(P0.05,P0.05,P0.05,P0.01),增强Bcl-2表达(P0.05),升高Bcl-2/Bax比值(P0.05)。橄榄苦苷(400μg/m L)诱导Akt和ERK磷酸化的高表达(P0.05,P0.05)并升高p-Akt/t-Akt比值(P0.01)和p-ERK/t-ERK比值(P0.05)。加入PI3K/Akt的特异性抑制剂LY294002则能够抑制橄榄苦苷诱导的Akt磷酸化高表达(P0.05),并降低p-Akt/t-Akt比值,加入ERK的特异性抑制剂U0126则能够抑制橄榄苦苷诱导的ERK磷酸化高表达(P0.05),并降低p-ERK/t-ERK比值。结论:橄榄苦苷可有效保护心肌缺血再灌注损伤,其作用机制可能与其抑制氧化应激诱导心肌细胞线粒体凋亡途径和激活RISK途径有关。
[Abstract]:Background: coronary heart disease (CHD) is one of the leading causes of death and disability worldwide. However, so far, its exact pathogenesis is still not very clear. The current studies mainly focus on the formation of oxygen free radicals, intracellular calcium overload, apoptosis, oxygen and energy use disorders, especially the oxidative stress damage and apoptosis are closely related to the occurrence of MIRI. Oligopicrin is one of the main active components in olive leaves and has strong antioxidant and scavenging effects on oxygen free radicals. As early as 2004, Manna et al reported for the first time that olivopicrin has a protective effect on oxidative myocardial injury induced by ischemia reperfusion in vitro, but few scholars have carried out in vivo experimental studies since then. Little further research has been done on the mechanism of its action. Objective: to evaluate the protective effect of olivopicrin on myocardial ischemia reperfusion injury and its mechanism. Methods: the model of myocardial ischemia-reperfusion injury in rats and the model of myocardial ischemia reperfusion injury in primary cultured neonatal rat cardiomyocytes were established. ELISA method was used to detect the activity of GSH-PX and the activity of GSH-PX. The area of infarcted myocardium was observed by HE staining and Tunel and flow cytometry were used to detect the apoptosis of myocardial cells. The level of ROS in cells was detected by fluorescence probe DCFH-DA and the phosphorylation of apoptosis-related protein Cyt Caspase-3, Caspase-3, Caspase-9, extracellular signal regulated kinase ERK1 / 2) and protein kinase (PKB / Akt) were detected by JC-1 staining and Western blot. Results: (1) compared with the model group of ischemia and reperfusion in rats, the preconditioning of olivopicrin 100200 mg / kg significantly inhibited the elevation of MDA and LDH in ischemic reperfusion induced by ischemia and reperfusion in rats (P0.01, P0.05, P0.05), and the decrease of SODCAT and GSH-PX, P0.05P0.05P0.05. The myocardial infarction ratio in the model group was 18.9%, which was significantly higher than that in the blank control group (P 0.001). Compared with the model group, olivopicrin pretreatment significantly decreased the infarct ratio (P 0.05) and myocardial apoptosis index (P 0.05). Compared with the model group, the ratio of Bcl-2/Bax and the expression of p-ERK1/2 and p-Akt were increased significantly (P 0.05, P 0.05, P 0.05, P 0.05, P 0.05, P 0.05, P 0.05, P 0.05, P 0.05, P 0.05, P 0.05, P 0.05, P 0.05, P 0.05, P 0.05, P 0.05, P 0.05, P 0.05, P 0.05, P 0.05), respectively. Oligopicrin 100200 渭 g / mL increased the survival rate of primary cultured neonatal rat cardiomyocytes induced by ischemia-reperfusion in a concentration-dependent manner (P0.001), and decreased the ratio of Hoechst33258 positive cells (P0.01) and cardiomyocyte apoptosis (P0.001). In a concentration-dependent manner, olive glycoside decreased the levels of LDH and ROS, inhibited the excessive release of MDA, and increased the activities of MDA, such as P0.05, P0.05, P0.05, P0.05, P0.05, P0.05, P0.05, P0.05, P0.05, and P0.05, P0.05, P0.05, P0.05, P0.05, and P0.05, respectively. Oligopicrin decreased the ratio of green / red fluorescence intensity (P 0.001). The high expression of Caspase-3 and Caspase-9 was inhibited by BaxtCon, and the expression of Bcl-2 was enhanced by P0.05P0.05P0.05P0.01, and the ratio of Bcl-2/Bax was increased by P0.05. Oligopicrin (400 渭 g / m L) induced the phosphorylation of Akt and ERK (P0.05P0.05) and increased the p-Akt/t-Akt ratio (P0.01) and the p-ERK/t-ERK ratio (P0.05). LY294002, a specific inhibitor of PI3K/Akt, could inhibit the high expression of Akt phosphorylation induced by Oligopicrin and decrease the ratio of p-Akt/t-Akt. U0126, a specific inhibitor of ERK, could inhibit the phosphorylation of ERK induced by Oligopicrin and decrease the ratio of p-ERK/t-ERK. Conclusion: olivopicrin can effectively protect myocardial ischemia-reperfusion injury, and its mechanism may be related to its inhibition of oxidative stress-induced mitochondrial apoptosis and activation of RISK pathway.
【学位授予单位】：兰州大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R541.4

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