人參皂苷Rgl對(duì)大鼠心肌缺血再灌注損傷改善作用及機(jī)制探討
發(fā)布時(shí)間：2018-08-24 11:12
【摘要】：背景：缺血再灌注(ischemia/repe rfusion, I/R)是急性冠脈綜合征患者介入或溶栓治療后出現(xiàn)再損傷的主要病理基礎(chǔ),臨床上缺少有效的藥物干預(yù)。能量代謝障礙為起始環(huán)節(jié),及其誘導(dǎo)的炎癥介質(zhì)和凋亡反應(yīng)在I/R損傷中扮演重要角色。心肌缺血缺氧期,ATP合成酶δ亞基ATP 5D低表達(dá),導(dǎo)致了心肌ATP生成不足。另一方面,心肌泵血耗氧,又致ATP降解增多。ATP減少是缺血期心肌收縮蛋白損傷的主要因素之一。I/R引起的能量代謝異常和過量產(chǎn)生的過氧化物破壞了心肌肌絲骨架蛋白。過量產(chǎn)生的活性氧自由基(ROS)一方面可以直接造成細(xì)胞結(jié)構(gòu)破壞和功能受損；另一方面,又可以激活核因子κB (nuclear factor κB, NF-κB),通過NF-κB啟動(dòng)炎性因子和粘附因子的表達(dá),促進(jìn)白細(xì)胞與血管內(nèi)皮細(xì)胞的相互反應(yīng)和游出,加重血管和心肌損傷,進(jìn)一步誘導(dǎo)了細(xì)胞的凋亡。Rho激(Rho-kinase,ROCK)酶信號(hào)通路和磷脂酰肌醇3激酶/蛋白激酶B (PI3K/Akt)信號(hào)通路均是參與細(xì)胞凋亡的重要的信號(hào)通路。PI3K及其下游分子所介導(dǎo)的抗凋亡作用已經(jīng)成為藥物研究領(lǐng)域的焦點(diǎn)。人參皂苷Rgl (ginsenoside Rgl, Rg1)是人參中分離出的單體皂苷之一,具有廣泛的生物學(xué)活性。大量的研究表明人參皂苷對(duì)心血管系統(tǒng)、神經(jīng)系統(tǒng)、免疫系統(tǒng)都有影響,可以抑制細(xì)胞凋亡、擴(kuò)張血管、改善心功能、抗衰老、美容等作用。但目前尚無(wú)體內(nèi)試驗(yàn)的證據(jù),從能量代謝、抑制炎癥和細(xì)胞凋亡的角度,探討人參皂苷Rg1對(duì)大鼠心肌缺血再灌注損傷的改善作用及機(jī)制。目的：本研究通過評(píng)價(jià)Rg1對(duì)I/R的大鼠心肌梗死、心臟表面血流量、心功能、心肌能量代謝、凋亡、炎癥及相關(guān)調(diào)控蛋白的改善作用,觀察Rg1對(duì)心臟缺血再灌注損傷的干預(yù)作用,并通過大鼠心肌NF-κB, Rho激酶和PI3K/Akt信號(hào)通路,探討Rg1的作用機(jī)理。方法：取體重為240-260 g的雄性Spragu-Dawley (SD)大鼠144只,隨機(jī)分為4組,分別為假手術(shù)組(Sham);本底組(Rgl+Sham);模型組(I/R)；預(yù)給藥組(Rgl+I/R)。麻醉下開胸,結(jié)扎冠狀動(dòng)脈左前降支30分鐘,造成缺血,解除結(jié)扎形成再灌注。在缺血前30分鐘,經(jīng)由左側(cè)股靜脈連續(xù)泵入Rgl (1 mg/kg,5 mg/kg,10 mg/kg)至再灌注90分鐘。于缺血前、缺血30分、再灌注30分、再灌注60分、再灌注90分,用激光掃描多普勒血流量?jī)x測(cè)定心臟表面血流量,用心內(nèi)導(dǎo)管法測(cè)定心功能。在再灌注90分,取心肌組織,用2,3,5-氯化三苯基四氮唑和伊文斯氏藍(lán)法染心肌缺血和梗死面積；用蘇木素-伊紅法染心肌形態(tài)結(jié)構(gòu)；用鬼筆環(huán)肽染色法觀察心肌纖維F-actin;用凋亡染色標(biāo)記核斷裂的心肌細(xì)胞；用透射電鏡觀察心肌纖維和線粒體的超微結(jié)構(gòu)。在再灌注90分,從心肌組織中提取RNA,用實(shí)時(shí)定量聚合酶鏈?zhǔn)椒磻?yīng)檢測(cè)ATP合成酶δ亞基(ATP-5D) mRNA的水平；取心肌組織中的蛋白,用酶標(biāo)法檢測(cè)大鼠心肌三磷酸腺苷(adenosinetriphosphate, ATP)、二磷酸腺苷(adenosine diphosphate, ADP)、磷酸腺苷(adenosine monophosphate, AMP)、丙二醛(malondialde hyde, MDA)的含量；取大鼠血清用酶標(biāo)法檢測(cè)肌鈣蛋白I (Cardiac Troponin I, cTnI)的含量；用蛋白免疫印跡法檢測(cè)大鼠心肌組織中B淋巴細(xì)胞瘤-2 (B-cell lymphoma-2, Bcl-2)、Bcl-2相關(guān)X蛋白(Bcl-2 associated x protein, Bax)、活化型半胱天冬酶-3 (cleaved caspase-3)、肌鈣蛋白I (Cardiac Troponin I, cTnI)、Rho激酶(Rho-kinase,ROCK)及其底物肌球蛋白磷酸酶(MYPT-1)磷酸化、磷脂酰肌醇3激酶(Phosphatidyl Inositol 3-kinase,PI3K)P及其磷酸化I3K、 蛋白激酶B(Akt)及其磷酸化Akt、ATP合成酶6亞基(ATP-5D)、磷酸化肌球蛋白輕鏈(phosphorylated myosin light chain,p-MLC)、細(xì)胞漿中和細(xì)胞核中核因子κB (nuclear factor κB, NF-kB)的表達(dá)；用免疫組織化學(xué)染色法檢測(cè)心肌組織中髓過氧化物酶(myeloperoxidase,MPO)、細(xì)胞間黏附分子(Intercellular adhesion molecule,ICAM-1)、中性粒細(xì)胞黏附分子(CD18)的表達(dá)。結(jié)果：在缺血前30 min,連續(xù)性靜脈滴注Rg1至再灌注結(jié)束,可以減輕大鼠心肌梗死面積。5mg/kg的Rg1可以顯著性地：1.減輕I/R引起的大鼠心肌形態(tài)學(xué)改變,包括心肌纖維斷裂、線粒體腫大；2.抑制I/R組大鼠心肌中cTnI水平的降低,血清中的cTnI水平的升高。3.改善I/R引起的大鼠心功能異常；4.抑制I/R引起的大鼠心臟表面血流量的降低；5.抑制I/R引起的大鼠心肌細(xì)胞凋亡,抑制Bcl-2的降低,抑制Bax和Cleaved-caspase3的增加；6.進(jìn)一步升高P-PI3K/PI3K以及P-Akt/Akt的比值,發(fā)揮抗凋亡作用；7.抑制I/R導(dǎo)致的ROCK的激活以及其底物MYPT-1的磷酸化水平；8.抑制I/R引起的大鼠心肌細(xì)胞漿中NF-κB P65的水平降低,細(xì)胞核中NF-κB P65的表達(dá)升高,9.抑制I/R引起的大鼠心肌ADP/ATP、AMP/ATP比值的升高,MDA含量的增加,抑制I/R引起的心肌ATP-5D mRNA水平和蛋白的表達(dá)降低、MLC磷酸化水平的升高。10.抑制I/R引起的MPO、ICAM-1、CD18表達(dá)的增強(qiáng)結(jié)論：本研究證實(shí)了Rgl可以通過抑制I/R引起的大鼠心肌能量代謝異常和氧化應(yīng)激損傷,改善了I/R大鼠的心肌纖維損傷和心肌細(xì)胞凋亡,改善I/R大鼠的心肌結(jié)構(gòu)、心功能和心臟血流量,減輕心臟缺血再灌注損傷。Rg1的作用機(jī)制可能與抑制I/R導(dǎo)致的ROCK的激活及其底物MYPT-1的磷酸化水平,抑制I/R引起的NF-κB核轉(zhuǎn)位及其介導(dǎo)的炎癥反應(yīng),升高P-PI3K/PI3K以及P-Akt/Akt的比值有關(guān)。
[Abstract]:BACKGROUND: Ischemia/repe rfusion (I/R) is the main pathological basis of repe rfusion in patients with acute coronary syndrome (ACS) after interventional or thrombolytic therapy. There is no effective drug intervention in clinic. Energy metabolism disorder is the initial link, and its induced inflammatory mediators and apoptosis play an important role in I/R injury. ATP synthase delta subunit ATP 5D is low expressed during hypoxia, resulting in insufficient ATP production in the myocardium. On the other hand, myocardial pump oxygen consumption leads to increased ATP degradation. ATP reduction is one of the main factors of myocardial contractile protein damage during ischemia. Abnormal energy metabolism and excessive production of peroxides caused by I/R destroy myocardial fibrous skeleton protein. Active oxygen radicals (ROS) can directly damage cell structure and function; on the other hand, ROS can activate nuclear factor kappa B (NF-kappa B) and activate the expression of inflammatory factors and adhesion factors through NF-kappa B, promote the interaction and outflow of leukocytes and vascular endothelial cells, aggravate vascular and vascular functions. Rho-kinase (ROCK) signaling pathway and phosphatidylinositol 3 kinase/protein kinase B (PI3K/Akt) signaling pathway are important signaling pathways involved in apoptosis. The anti-apoptosis effect mediated by PI3K and its downstream molecules has become the focus of drug research. L(ginsenoside Rgl,Rg1) is one of the monomer saponins isolated from Panax ginseng and has a wide range of biological activities.A large number of studies have shown that ginsenoside has effects on cardiovascular system,nervous system and immune system,can inhibit cell apoptosis,dilate blood vessels,improve heart function,anti-aging and beauty. Objective: To evaluate the effects of ginsenoside Rg1 on myocardial ischemia-reperfusion injury in I/R rats by evaluating the effects of ginsenoside Rg1 on myocardial infarction, cardiac surface blood flow, cardiac function, myocardial energy metabolism, apoptosis, inflammation and related regulatory proteins. Methods: 144 male Spragu-Dawley (SD) rats weighing 240-260 g were randomly divided into four groups: sham group (Sham), background group (Rgl+Sham), model group (I/R). Before ischemia, Rgl (1 mg/kg, 5 mg/kg, 10 mg/kg) was continuously pumped through the left femoral vein to reperfusion for 90 minutes. Before ischemia, 30 minutes of ischemia, 30 minutes of reperfusion, 60 minutes of reperfusion and 90 minutes of reperfusion were used. Cardiac surface blood flow was measured by laser scanning Doppler flowmeter and cardiac function was measured by intracardiac catheterization. Myocardial ischemia and infarct size were stained with 2,3,5-triphenyltetrazolium chloride and Evans blue at 90 minutes after reperfusion. Fiber F-actin, stained with apoptosis, and observed the ultrastructure of myocardial fibers and mitochondria by transmission electron microscopy. RNA was extracted from myocardial tissue at 90 minutes after reperfusion and the level of ATP synthase delta subunit (ATP-5D) mRNA was detected by real-time quantitative polymerase chain reaction. The contents of adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP) and malondialde Hyde (MDA) in myocardium of rats were measured, and the contents of cardiac troponin I (cTnI) in serum of rats were detected by enzyme labeling and Western blotting. B-cell lymphoma-2 (Bcl-2), Bcl-2 associated X protein (Bax), cleaved caspase-3, cardiac troponin I (cTnI), Rho-kinase (Rho-kinase, ROCK) and its substrate myosin phosphatase (MYPT-1) phosphorylation, phosphorylation of phosphorylation were detected in rat myocardium. Phosphatidyl Inositol 3-kinase (PI3K) P and its phosphorylated I3K, protein kinase B (Akt) and its phosphorylated Akt, ATP-5D, phosphorylated myosin light chain (p-MLC), nuclear factor kappa B (NF-kB) in cytoplasm and nucleus were expressed in immune tissues. The expression of myeloperoxidase (MPO), intercellular adhesion molecule-1 (ICAM-1) and neutrophil adhesion molecule (CD18) in myocardial tissue was detected by chemical staining. Results: 30 minutes before ischemia, continuous intravenous infusion of Rg 1 to the end of reperfusion could reduce the infarct size of myocardium in rats. G1 can significantly reduce the myocardial morphological changes induced by I/R, including myocardial fiber breakage and mitochondrial enlargement; 2. inhibit the decrease of cTnI level in myocardium and the increase of cTnI level in serum of rats in I/R group; 3. improve the cardiac dysfunction induced by I/R; 4. inhibit the decrease of cardiac surface blood flow induced by I/R; 5. Inhibition of I/R-induced cardiomyocyte apoptosis, inhibition of Bcl-2 reduction, inhibition of Bax and Cleaved-caspase 3 increase; 6. Further increase of P-PI3K/PI3K and P-Akt/Akt ratio to play an anti-apoptotic role; 7. Inhibition of I/R-induced ROCK activation and its substrate MYPT-1 phosphorylation level; 8. Inhibition of I/R-induced cardiomyocyte plasma phosphorylation in rats Inhibition of I/R-induced elevation of ADP/ATP, AMP/ATP ratio, increase of MDA content, inhibition of I/R-induced decrease of ATP-5D mRNA and protein expression, and increase of MLC phosphorylation level. Inhibition of I/R-induced elevation of MPO, ICAM-1 and CD18 expression CONCLUSION: Rgl can ameliorate myocardial fibrous injury and myocardial apoptosis, improve myocardial structure, cardiac function and blood flow of I/R rats, and alleviate myocardial ischemia-reperfusion injury by inhibiting I/R-induced abnormalities of myocardial energy metabolism and oxidative stress. The activation of ROCK and the phosphorylation of its substrate MYPT-1, the inhibition of NF-kappa B Translocation Induced by I/R and its mediated inflammatory response, and the increase of P-PI3K/PI3K and P-Akt/Akt ratio are related.
【學(xué)位授予單位】：北京中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R541.4
[Abstract]:BACKGROUND: Ischemia/repe rfusion (I/R) is the main pathological basis of repe rfusion in patients with acute coronary syndrome (ACS) after interventional or thrombolytic therapy. There is no effective drug intervention in clinic. Energy metabolism disorder is the initial link, and its induced inflammatory mediators and apoptosis play an important role in I/R injury. ATP synthase delta subunit ATP 5D is low expressed during hypoxia, resulting in insufficient ATP production in the myocardium. On the other hand, myocardial pump oxygen consumption leads to increased ATP degradation. ATP reduction is one of the main factors of myocardial contractile protein damage during ischemia. Abnormal energy metabolism and excessive production of peroxides caused by I/R destroy myocardial fibrous skeleton protein. Active oxygen radicals (ROS) can directly damage cell structure and function; on the other hand, ROS can activate nuclear factor kappa B (NF-kappa B) and activate the expression of inflammatory factors and adhesion factors through NF-kappa B, promote the interaction and outflow of leukocytes and vascular endothelial cells, aggravate vascular and vascular functions. Rho-kinase (ROCK) signaling pathway and phosphatidylinositol 3 kinase/protein kinase B (PI3K/Akt) signaling pathway are important signaling pathways involved in apoptosis. The anti-apoptosis effect mediated by PI3K and its downstream molecules has become the focus of drug research. L(ginsenoside Rgl,Rg1) is one of the monomer saponins isolated from Panax ginseng and has a wide range of biological activities.A large number of studies have shown that ginsenoside has effects on cardiovascular system,nervous system and immune system,can inhibit cell apoptosis,dilate blood vessels,improve heart function,anti-aging and beauty. Objective: To evaluate the effects of ginsenoside Rg1 on myocardial ischemia-reperfusion injury in I/R rats by evaluating the effects of ginsenoside Rg1 on myocardial infarction, cardiac surface blood flow, cardiac function, myocardial energy metabolism, apoptosis, inflammation and related regulatory proteins. Methods: 144 male Spragu-Dawley (SD) rats weighing 240-260 g were randomly divided into four groups: sham group (Sham), background group (Rgl+Sham), model group (I/R). Before ischemia, Rgl (1 mg/kg, 5 mg/kg, 10 mg/kg) was continuously pumped through the left femoral vein to reperfusion for 90 minutes. Before ischemia, 30 minutes of ischemia, 30 minutes of reperfusion, 60 minutes of reperfusion and 90 minutes of reperfusion were used. Cardiac surface blood flow was measured by laser scanning Doppler flowmeter and cardiac function was measured by intracardiac catheterization. Myocardial ischemia and infarct size were stained with 2,3,5-triphenyltetrazolium chloride and Evans blue at 90 minutes after reperfusion. Fiber F-actin, stained with apoptosis, and observed the ultrastructure of myocardial fibers and mitochondria by transmission electron microscopy. RNA was extracted from myocardial tissue at 90 minutes after reperfusion and the level of ATP synthase delta subunit (ATP-5D) mRNA was detected by real-time quantitative polymerase chain reaction. The contents of adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP) and malondialde Hyde (MDA) in myocardium of rats were measured, and the contents of cardiac troponin I (cTnI) in serum of rats were detected by enzyme labeling and Western blotting. B-cell lymphoma-2 (Bcl-2), Bcl-2 associated X protein (Bax), cleaved caspase-3, cardiac troponin I (cTnI), Rho-kinase (Rho-kinase, ROCK) and its substrate myosin phosphatase (MYPT-1) phosphorylation, phosphorylation of phosphorylation were detected in rat myocardium. Phosphatidyl Inositol 3-kinase (PI3K) P and its phosphorylated I3K, protein kinase B (Akt) and its phosphorylated Akt, ATP-5D, phosphorylated myosin light chain (p-MLC), nuclear factor kappa B (NF-kB) in cytoplasm and nucleus were expressed in immune tissues. The expression of myeloperoxidase (MPO), intercellular adhesion molecule-1 (ICAM-1) and neutrophil adhesion molecule (CD18) in myocardial tissue was detected by chemical staining. Results: 30 minutes before ischemia, continuous intravenous infusion of Rg 1 to the end of reperfusion could reduce the infarct size of myocardium in rats. G1 can significantly reduce the myocardial morphological changes induced by I/R, including myocardial fiber breakage and mitochondrial enlargement; 2. inhibit the decrease of cTnI level in myocardium and the increase of cTnI level in serum of rats in I/R group; 3. improve the cardiac dysfunction induced by I/R; 4. inhibit the decrease of cardiac surface blood flow induced by I/R; 5. Inhibition of I/R-induced cardiomyocyte apoptosis, inhibition of Bcl-2 reduction, inhibition of Bax and Cleaved-caspase 3 increase; 6. Further increase of P-PI3K/PI3K and P-Akt/Akt ratio to play an anti-apoptotic role; 7. Inhibition of I/R-induced ROCK activation and its substrate MYPT-1 phosphorylation level; 8. Inhibition of I/R-induced cardiomyocyte plasma phosphorylation in rats Inhibition of I/R-induced elevation of ADP/ATP, AMP/ATP ratio, increase of MDA content, inhibition of I/R-induced decrease of ATP-5D mRNA and protein expression, and increase of MLC phosphorylation level. Inhibition of I/R-induced elevation of MPO, ICAM-1 and CD18 expression CONCLUSION: Rgl can ameliorate myocardial fibrous injury and myocardial apoptosis, improve myocardial structure, cardiac function and blood flow of I/R rats, and alleviate myocardial ischemia-reperfusion injury by inhibiting I/R-induced abnormalities of myocardial energy metabolism and oxidative stress. The activation of ROCK and the phosphorylation of its substrate MYPT-1, the inhibition of NF-kappa B Translocation Induced by I/R and its mediated inflammatory response, and the increase of P-PI3K/PI3K and P-Akt/Akt ratio are related.
【學(xué)位授予單位】：北京中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R541.4
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