本文選題：橄欖苦苷 + 心肌�。� 參考：《蘭州大學(xué)》2017年博士論文

【摘要】：研究背景:冠心病是世界范圍內(nèi)導(dǎo)致死亡和殘疾的主要原因之一,其臨床表現(xiàn)為心肌缺血再灌注損傷(MIRI)。但是,到目前為止,其確切的發(fā)病機(jī)制仍不十分清楚。目前的研究主要集中在氧自由基形成、細(xì)胞內(nèi)鈣超載、細(xì)胞凋亡、氧及能量利用障礙等方面,尤其認(rèn)為氧化應(yīng)激損傷和細(xì)胞凋亡與MIRI的發(fā)生密切相關(guān)。橄欖苦苷是油橄欖葉中的主要活性成分之一,具有強(qiáng)大的抗氧化和清除氧自由基作用。早在2004年Manna等人首次報(bào)道橄欖苦苷對由缺血再灌注引起的離體心臟氧化性心肌損傷具有一定的保護(hù)作用,但此后很少有學(xué)者進(jìn)行在體實(shí)驗(yàn)研究,也很少對其作用機(jī)制進(jìn)行進(jìn)一步研究。研究目的:評價(jià)橄欖苦苷對心肌缺血再灌注損傷的保護(hù)作用,并對其機(jī)制進(jìn)行研究。實(shí)驗(yàn)方法:建立大鼠心肌缺血再灌注損傷模型和原代培養(yǎng)乳鼠心肌細(xì)胞缺血再灌注損傷模型,采用ELISA法檢測CK、SOD、LDH、CAT、MDA和GSH-PX的活性;TTC染色法觀察梗死心肌面積;HE染色觀察心肌損傷;TUNEL法和流式細(xì)胞術(shù)檢測心肌細(xì)胞凋亡;熒光探針DCFH-DA檢測細(xì)胞內(nèi)的ROS水平;JC-1染色法檢測細(xì)胞線粒體膜電位;Western blot法檢測凋亡相關(guān)蛋白Cyt-C,Caspase-3,Caspase-9,Bcl-2/Bax,細(xì)胞外信號調(diào)節(jié)激酶(ERK1/2)和蛋白激酶B(PKB/Akt)的磷酸化表達(dá)水平。實(shí)驗(yàn)結(jié)果:1)與大鼠缺血再灌注模型組相比,橄欖苦苷(100,200,400mg/kg)預(yù)處理明顯抑制大鼠缺血再灌注誘導(dǎo)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)。模型組大鼠心肌梗死比率達(dá)18.9%,與空白對照組相比,差異具有統(tǒng)計(jì)學(xué)意義(P0.001)。與模型組相比,橄欖苦苷預(yù)處理明顯降低梗死比率(P0.05)和心肌細(xì)胞凋亡指數(shù)(P0.05)。與模型組相比,橄欖苦苷(400mg/kg)明顯升高Bcl-2/Bax比值(P0.01);明顯增加p-ERK1/2和p-Akt的表達(dá)(P0.05,P0.05)。2)與原代培養(yǎng)乳鼠心肌細(xì)胞缺血再灌注損傷模型組比較,橄欖苦苷(100,200,400μg/m L)呈濃度依賴地升高缺血再灌注誘導(dǎo)的原代培養(yǎng)乳鼠心肌細(xì)胞存活率(P0.001),降低Hoechst33258熒光染色陽性細(xì)胞比率(P0.01)和心肌細(xì)胞凋亡率(P0.001)。橄欖苦苷呈濃度依賴地降低CK、LDH和ROS水平(P0.01,P0.01,P0.001),抑制MDA的過度釋放(P0.001),升高SOD、CAT和GSH-PX活性(P0.05,P0.05,P0.05)。橄欖苦苷降低綠色/紅色熒光強(qiáng)度比值(P0.001)。抑制Bax、Cyt-C、Caspase-3和Caspase-9的高表達(dá)(P0.05,P0.05,P0.05,P0.01),增強(qiáng)Bcl-2表達(dá)(P0.05),升高Bcl-2/Bax比值(P0.05)。橄欖苦苷(400μg/m L)誘導(dǎo)Akt和ERK磷酸化的高表達(dá)(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的特異性抑制劑LY294002則能夠抑制橄欖苦苷誘導(dǎo)的Akt磷酸化高表達(dá)(P0.05),并降低p-Akt/t-Akt比值,加入ERK的特異性抑制劑U0126則能夠抑制橄欖苦苷誘導(dǎo)的ERK磷酸化高表達(dá)(P0.05),并降低p-ERK/t-ERK比值。結(jié)論:橄欖苦苷可有效保護(hù)心肌缺血再灌注損傷,其作用機(jī)制可能與其抑制氧化應(yīng)激誘導(dǎo)心肌細(xì)胞線粒體凋亡途徑和激活RISK途徑有關(guān)。
[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.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R541.4

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