人参皂苷Rgl对大鼠心肌缺血再灌注损伤改善作用及机制探讨
发布时间：2018-08-24 11:12
【摘要】：背景：缺血再灌注(ischemia/repe rfusion, I/R)是急性冠脉综合征患者介入或溶栓治疗后出现再损伤的主要病理基础,临床上缺少有效的药物干预。能量代谢障碍为起始环节,及其诱导的炎症介质和凋亡反应在I/R损伤中扮演重要角色。心肌缺血缺氧期,ATP合成酶δ亚基ATP 5D低表达,导致了心肌ATP生成不足。另一方面,心肌泵血耗氧,又致ATP降解增多。ATP减少是缺血期心肌收缩蛋白损伤的主要因素之一。I/R引起的能量代谢异常和过量产生的过氧化物破坏了心肌肌丝骨架蛋白。过量产生的活性氧自由基(ROS)一方面可以直接造成细胞结构破坏和功能受损；另一方面,又可以激活核因子κB (nuclear factor κB, NF-κB),通过NF-κB启动炎性因子和粘附因子的表达,促进白细胞与血管内皮细胞的相互反应和游出,加重血管和心肌损伤,进一步诱导了细胞的凋亡。Rho激(Rho-kinase,ROCK)酶信号通路和磷脂酰肌醇3激酶/蛋白激酶B (PI3K/Akt)信号通路均是参与细胞凋亡的重要的信号通路。PI3K及其下游分子所介导的抗凋亡作用已经成为药物研究领域的焦点。人参皂苷Rgl (ginsenoside Rgl, Rg1)是人参中分离出的单体皂苷之一,具有广泛的生物学活性。大量的研究表明人参皂苷对心血管系统、神经系统、免疫系统都有影响,可以抑制细胞凋亡、扩张血管、改善心功能、抗衰老、美容等作用。但目前尚无体内试验的证据,从能量代谢、抑制炎症和细胞凋亡的角度,探讨人参皂苷Rg1对大鼠心肌缺血再灌注损伤的改善作用及机制。目的：本研究通过评价Rg1对I/R的大鼠心肌梗死、心脏表面血流量、心功能、心肌能量代谢、凋亡、炎症及相关调控蛋白的改善作用,观察Rg1对心脏缺血再灌注损伤的干预作用,并通过大鼠心肌NF-κB, Rho激酶和PI3K/Akt信号通路,探讨Rg1的作用机理。方法：取体重为240-260 g的雄性Spragu-Dawley (SD)大鼠144只,随机分为4组,分别为假手术组(Sham);本底组(Rgl+Sham);模型组(I/R)；预给药组(Rgl+I/R)。麻醉下开胸,结扎冠状动脉左前降支30分钟,造成缺血,解除结扎形成再灌注。在缺血前30分钟,经由左侧股静脉连续泵入Rgl (1 mg/kg,5 mg/kg,10 mg/kg)至再灌注90分钟。于缺血前、缺血30分、再灌注30分、再灌注60分、再灌注90分,用激光扫描多普勒血流量仪测定心脏表面血流量,用心内导管法测定心功能。在再灌注90分,取心肌组织,用2,3,5-氯化三苯基四氮唑和伊文斯氏蓝法染心肌缺血和梗死面积；用苏木素-伊红法染心肌形态结构；用鬼笔环肽染色法观察心肌纤维F-actin;用凋亡染色标记核断裂的心肌细胞；用透射电镜观察心肌纤维和线粒体的超微结构。在再灌注90分,从心肌组织中提取RNA,用实时定量聚合酶链式反应检测ATP合成酶δ亚基(ATP-5D) mRNA的水平；取心肌组织中的蛋白,用酶标法检测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；取大鼠血清用酶标法检测肌钙蛋白I (Cardiac Troponin I, cTnI)的含量；用蛋白免疫印迹法检测大鼠心肌组织中B淋巴细胞瘤-2 (B-cell lymphoma-2, Bcl-2)、Bcl-2相关X蛋白(Bcl-2 associated x protein, Bax)、活化型半胱天冬酶-3 (cleaved caspase-3)、肌钙蛋白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亚基(ATP-5D)、磷酸化肌球蛋白轻链(phosphorylated myosin light chain,p-MLC)、细胞浆中和细胞核中核因子κB (nuclear factor κB, NF-kB)的表达；用免疫组织化学染色法检测心肌组织中髓过氧化物酶(myeloperoxidase,MPO)、细胞间黏附分子(Intercellular adhesion molecule,ICAM-1)、中性粒细胞黏附分子(CD18)的表达。结果：在缺血前30 min,连续性静脉滴注Rg1至再灌注结束,可以减轻大鼠心肌梗死面积。5mg/kg的Rg1可以显著性地：1.减轻I/R引起的大鼠心肌形态学改变,包括心肌纤维断裂、线粒体肿大；2.抑制I/R组大鼠心肌中cTnI水平的降低,血清中的cTnI水平的升高。3.改善I/R引起的大鼠心功能异常；4.抑制I/R引起的大鼠心脏表面血流量的降低；5.抑制I/R引起的大鼠心肌细胞凋亡,抑制Bcl-2的降低,抑制Bax和Cleaved-caspase3的增加；6.进一步升高P-PI3K/PI3K以及P-Akt/Akt的比值,发挥抗凋亡作用；7.抑制I/R导致的ROCK的激活以及其底物MYPT-1的磷酸化水平；8.抑制I/R引起的大鼠心肌细胞浆中NF-κB P65的水平降低,细胞核中NF-κB P65的表达升高,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水平和蛋白的表达降低、MLC磷酸化水平的升高。10.抑制I/R引起的MPO、ICAM-1、CD18表达的增强结论：本研究证实了Rgl可以通过抑制I/R引起的大鼠心肌能量代谢异常和氧化应激损伤,改善了I/R大鼠的心肌纤维损伤和心肌细胞凋亡,改善I/R大鼠的心肌结构、心功能和心脏血流量,减轻心脏缺血再灌注损伤。Rg1的作用机制可能与抑制I/R导致的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核转位及其介导的炎症反应,升高P-PI3K/PI3K以及P-Akt/Akt的比值有关。
[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.
【学位授予单位】：北京中医药大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：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.
【学位授予单位】：北京中医药大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R541.4
【相似文献】
相关期刊论文 前10条
1 陈同度,张昌颖;素食大鼠的贫血现象[J];营养学报;1957年04期
2 陈伟强;赵善广;;自制注射用大鼠固定装置[J];上海实验动物科学;1992年04期
3 肖柳英,林培英,冯昭明,张丹;不同周龄的SD大鼠生理、生化及体重的正常值测定[J];中药新药与临床药理;1996年03期
4 李淑云;简易大鼠灌胃器的制作[J];锦州医学院学报;2001年04期
5 杨明智,陈积圣;一种大鼠抓取与固定的新工具介绍[J];上海实验动物科学;2001年03期
6 戴英,陆群;复方H_(505)对Wistar大鼠外周血的血液流变学指标的影响[J];中国血液流变学杂志;2001年01期
7 韦应波,孙喜庆,曹新生,姚永杰,冯岱雅,杨长斌;+Gz暴露时间对大鼠记忆功能和行为的影响[J];航天医学与医学工程;2003年01期
8 吕学军,郭俊生,李敏,周利梅,张永娟;晕船大鼠体内铁含量的变化[J];中国职业医学;2003年04期
9 汤仁仙,王迎伟,王慧,周峰;201A中药合剂对大鼠抗肾小球基底膜肾炎病变的影响[J];徐州医学院学报;2003年06期
10 孙同柱,付小兵,翁立新,梁雪梅,陈伟;介绍一种简易的大鼠保定方法[J];上海实验动物科学;2004年01期
相关会议论文 前10条
1 尹音;孙振宇;胡敏;李冬霞;;持续性高正加速度对大鼠颞颌关节损伤的作用[A];第八届全国颞下颌关节病学及（牙合）学大会论文汇编[C];2011年
2 祝~=骧;iJ梊霞;洃克琴;崔素英;文允摪;，
本文编号：2200642
本文链接：https://www.wllwen.com/yixuelunwen/mazuiyixuelunwen/2200642.html
最近更新
教材专著
