丹蒌对大鼠急性心肌缺血损伤的保护作用及其机制
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摘要
丹蒌是由瓜蒌皮、薤白、葛根、川芎、丹参、赤芍、泽泻、黄芪、骨碎补、郁金组成的纯植物药制剂,君药瓜蒌皮、薤白取自张仲景《金匮要略》治疗胸痹心痛之经典明方“瓜蒌薤白白酒汤”,秉承中医几千年的痰瘀同治法,对原方进行加减制成的新配方制剂。有临床研究表明丹蒌对心血管系统有明显的药理作用,可减少心绞痛发作次数,改善心电图异常,降脂,改善血液流变学等作用。丹蒌在临床研究中取得良好疗效,但揭示其药理作用和机制的实验研究报道甚为少见。本研究通过观察丹蒌对异丙肾上腺素所致急性心肌缺血大鼠的心电图、血清酶学、病理形态学、心肌坏死面积、凋亡相关蛋白以及基质金属蛋白酶等指标的影响,初步探讨丹蒌对心肌缺血损伤的保护作用及其可能机制。
本实验采用一次性大剂量皮下多点注射异丙肾上腺素(Isoproterenol,ISO)制备大鼠急性心肌缺血动物模型。用Ⅱ导联心电图监测心电图(Extracellularmatrix,ECG)J点变化;HE染色观察心肌组织病理形态学变化;逐区点测量法测定心肌梗死面积(myocardial infarction scope,MIS);腹主动脉取血全自动生化分析仪测定谷草转氨(aspartate transa- minase,AST)、肌酸激酶(creatine kinase,CK)、乳酸脱氢酶(lactate dehydrogenase,LDH)活性;免疫组织化学法测定心肌原癌基因Bcl-2 (B-cell lymphoma/ leulemia-2, Bcl-2)、半胱氨酸天门冬氨酸蛋白酶-3(cysteinyl aspartates pecific proteinase-3,Caspase-3)、基质金属蛋白酶-2(matrix metalloproteinases-2,MMP-2)、基质金属蛋白酶-9(matrix metalloproteinases-9, MMP-9)、基质金属蛋白酶组织抑制剂-1(tissue inhibitors ofmetallo- proteinases,TIMP-1)蛋白表达变化。
实验结果表明:丹蒌能明显对抗心肌缺血大鼠心电图J点的下移,缩小心肌坏死面积,改善心肌缺血引起的心肌组织病理损伤,减少炎性细胞浸润;降低血清CK、LDH和AST活性;升高缺血心肌凋亡相关蛋白Bcl-2的表达、降低Caspase-3表达;并能明显减少缺血损伤心肌MMP-2、MMP-9蛋白表达,对TIMP-1蛋白表达无明显影响。
本实验说明,丹蒌对心肌缺血损伤具有明显保护作用,并能防治缺血损伤后梗死扩展,促进梗死愈合,防止早期心室重塑,其作用机制可能与抑制炎症反应,减少心肌凋亡,抑制心肌胶原降解有关。
Coronary heart disease is myocardial ischemia disease, which due to coronary atherosclerosis, leading to stenosis or obstruction, or together with spasm, thrombosis, resulting in lumen obstruction, causing angina, myocardial infarction, heart failure and a series of serious consequences. As the economic and social changing, cardiovascular disease has spread in developing countries, the incidence rate showed an increasing trend, and age is getting younger, as the first of the world's disease burden and causes of death. Therefore, in-depth study for control strategies is important. Although Western medicine has made rapid progress in myocardial ischemia treatment, a number of synthetic drugs adverse reactions, not suitable for the patient long-term use, but these aspects of Chinese medicine is an advantage. Dan Lou is pure herbal preparation based on classical Chinese medicine side, adhering to the Traditional Chinese Medicine for thousands of years Phlegm and Blood Stasis, multi-center clinical study has shown its unique effect in myocardial ischemia treatment , which can reduce the frequency of angina attack, improve the abnormal ECG, reduce the dosage of available coronary dilatation drugs significantly. However, hardly any animal experimental studies on the protection of Dan Lou were studied on myocardial and its mechanism. This study was designed to understand the protection of Dan Lou on ischemia myocardial and its mechanism, and the efficacy of Dan Lou, as the guide to provide more experimental basis for clinical treatment.
Objective: we observe the effect of different doses of Dan Lou on electrocardiogram in acute myocardial ischemia, myocardial enzymes, area of myocardial necrosis, myocardial pathologic morphology, and observation the impact on Bcl-2, Caspase-3, MMP-2, MMP-9, TIMP-1 protein expression by the establishment of animal models of acute myocardial ischemia caused by is oproterenol.
Methods: 80 healthy male Wistar rats with the weight 200-250g (provided by the Experimental Animal Center of Jilin University), were randomly divided into 4 groups:①Control group: normal saline.②model control group: normal saline.③large dose group: Dan Lou powder 900mg/kg, administered with normal saline preparation of suspension.④small dose group: Dan Lou powder 450mg/kg, administered with normal saline preparation of suspension. 20 rats in each group, the volume of distribution are 10ml/kg, were intragastric once day, last for 14 days and fed a normal diet, no limitation in drinking water. 1 hour after the last administration, the model group, large and small dose group, were given subcutaneous injection of isoproterenol once, 15mg/kg, to prepare acute myocardial ischemia model, the control group injected the same volume normal saline instead.Continuous observation of the ECG changes was given before and 20 minutes after injection. 24h after the injection, 10 rats of each group were selected and anesthesia randomly, abdominal aortic blood was taken, serum CK, LDH and AST activity were measured by automatic biochemical analyzer. Those rats were killed, and cardiac apical 1/3 was cross-sected then made of paraffin. HE staining was used to observe pathological changes in myocardial,myocardial necrosis area measured by district-point measurement and myocardial Bcl-2, Caspase-3 protein expression in each group detected by immunohistochemistry. The remaining rats were sacrificed after intragastric for 7 days, with myocardial cross-section, paraffin sections for HE staining and immunohis- tochemistry as before to observe the pathological changes of heart, measure myocardial necrosis area, then detect the expression of myocardial MMP-2, MMP-9, TIMP-1 protein.
Results:
(1) Electrocardiogram J point in normal rats showed mild elevation, or on the base line. J point down shifted significantly (P<0.01) in model group rats injected with ISO, however, large doses Dan Lou inhibited (P<0.01) J point downshift much more significantly, and small dose Dan Lou inhibited slightly .Dan Lou can antagonize myocardial ischemia.
(2) Dan Lou significantly decreased serum CK, LDH and AST activity (P<0.01), it is suggested that Dan Lou can protect myocardial cells and stabilize cell membrane.
(3) After single 15mg/kg dose injection of isoproterenol, can cause chip spotty myocardial necrosis, area of myocardial necrosis were significantly reduced in 24h after modeling, 7 days after the high dose group (P<0.01), the area of myocardial necrosis in the latter group reduce even more than the the formerone, and the difference was significant (P<0.01).
(4) Myocardial pathological observation showed that, compared with the control group, widespread integration of cardiac disease, focal necrosis of muscle fiber-chip, fracture, myocardial nuclear fragmentation, dissolution, irregular arrangement of myocardial fibers, structural disorder, interstitial edema and the inflammatory cell infiltration within necrosis were found in the model group; the extent of myocardial lesions in the large group significantly reduced compared with model group. 7 days after modeling, myocardial lesions significantly retraction, fibrosis can be seen, only a small amount of inflammatory cell infiltration, lesions tend to heal in the large group, but the model group myocardial lesions have increased compared with 24h after the modeling.
(5) Myocardial Bcl-2 protein expression was definitely high in model, large and small dose group compared with the control group (P<0.01); large group were significantly higher compared with the model group (P<0.01), small group increased slightly, but no significantly difference in comparison with the model group (P>0.05); differences in large and small dose group were also obvious (P<0.05).Myocardial expression of Caspase-3 protein was significantly increase in model, large and small dose group (P<0.01); there was no significant difference among model group, large and small dose group (P>0.05); either between large and small dose group (P>0.05).
(6) 1 week after injection of ISO test showed: myocardial MMP-2, MMP-9 expression was significantly high in model, large and small dose group compared with the control group (P<0.01); the expression in large dose group was significantly low compared with the control group (P<0.01); there was no significant difference between model group and small dose group (P>0.05); however, large difference was seen between low-dose group and high dose group (P<0.05). TIMP-1 expression was significantly increased in model, large and small dose group compared with the control group; the difference was significant (P<0.01); there was no significant difference among model group ,large and small dose group (P> 0.05); either between large and small dose group (P>0.05).
Conclusion:
(1) Dan Lou significantly inhibits J point on ECG in the model of actue ischemia rats;(P<0.01).
(2) Dan Lou can significantly reduce the myocardial necrosis area in rats with acute myocardial ischemia induced by isoproterenol, decreased serum AST, LDH and CK activity, reduce the pathological damage,and has a protective effect on the injury.
(3) Dan Lou can up-regulate the expression of Bcl-2 , down-regulate the expression of Caspase-3,as the same time,Dan Lou may protect ischemia through relieving apoptosis;
(4) MMP-2, MMP-9 and TIMP-1 expression was significantly decreased , which suggested that Dan Lou can inhibite the enlargement of infarction and have a therapeutic effect on ventricular remodeling;
(5) Dan Lou has no impact on the expression of TIMP-1, the inhibition of the activity of MMP may not comlishment by TIMP-1.
本实验采用一次性大剂量皮下多点注射异丙肾上腺素(Isoproterenol,ISO)制备大鼠急性心肌缺血动物模型。用Ⅱ导联心电图监测心电图(Extracellularmatrix,ECG)J点变化;HE染色观察心肌组织病理形态学变化;逐区点测量法测定心肌梗死面积(myocardial infarction scope,MIS);腹主动脉取血全自动生化分析仪测定谷草转氨(aspartate transa- minase,AST)、肌酸激酶(creatine kinase,CK)、乳酸脱氢酶(lactate dehydrogenase,LDH)活性;免疫组织化学法测定心肌原癌基因Bcl-2 (B-cell lymphoma/ leulemia-2, Bcl-2)、半胱氨酸天门冬氨酸蛋白酶-3(cysteinyl aspartates pecific proteinase-3,Caspase-3)、基质金属蛋白酶-2(matrix metalloproteinases-2,MMP-2)、基质金属蛋白酶-9(matrix metalloproteinases-9, MMP-9)、基质金属蛋白酶组织抑制剂-1(tissue inhibitors ofmetallo- proteinases,TIMP-1)蛋白表达变化。
实验结果表明:丹蒌能明显对抗心肌缺血大鼠心电图J点的下移,缩小心肌坏死面积,改善心肌缺血引起的心肌组织病理损伤,减少炎性细胞浸润;降低血清CK、LDH和AST活性;升高缺血心肌凋亡相关蛋白Bcl-2的表达、降低Caspase-3表达;并能明显减少缺血损伤心肌MMP-2、MMP-9蛋白表达,对TIMP-1蛋白表达无明显影响。
本实验说明,丹蒌对心肌缺血损伤具有明显保护作用,并能防治缺血损伤后梗死扩展,促进梗死愈合,防止早期心室重塑,其作用机制可能与抑制炎症反应,减少心肌凋亡,抑制心肌胶原降解有关。
Coronary heart disease is myocardial ischemia disease, which due to coronary atherosclerosis, leading to stenosis or obstruction, or together with spasm, thrombosis, resulting in lumen obstruction, causing angina, myocardial infarction, heart failure and a series of serious consequences. As the economic and social changing, cardiovascular disease has spread in developing countries, the incidence rate showed an increasing trend, and age is getting younger, as the first of the world's disease burden and causes of death. Therefore, in-depth study for control strategies is important. Although Western medicine has made rapid progress in myocardial ischemia treatment, a number of synthetic drugs adverse reactions, not suitable for the patient long-term use, but these aspects of Chinese medicine is an advantage. Dan Lou is pure herbal preparation based on classical Chinese medicine side, adhering to the Traditional Chinese Medicine for thousands of years Phlegm and Blood Stasis, multi-center clinical study has shown its unique effect in myocardial ischemia treatment , which can reduce the frequency of angina attack, improve the abnormal ECG, reduce the dosage of available coronary dilatation drugs significantly. However, hardly any animal experimental studies on the protection of Dan Lou were studied on myocardial and its mechanism. This study was designed to understand the protection of Dan Lou on ischemia myocardial and its mechanism, and the efficacy of Dan Lou, as the guide to provide more experimental basis for clinical treatment.
Objective: we observe the effect of different doses of Dan Lou on electrocardiogram in acute myocardial ischemia, myocardial enzymes, area of myocardial necrosis, myocardial pathologic morphology, and observation the impact on Bcl-2, Caspase-3, MMP-2, MMP-9, TIMP-1 protein expression by the establishment of animal models of acute myocardial ischemia caused by is oproterenol.
Methods: 80 healthy male Wistar rats with the weight 200-250g (provided by the Experimental Animal Center of Jilin University), were randomly divided into 4 groups:①Control group: normal saline.②model control group: normal saline.③large dose group: Dan Lou powder 900mg/kg, administered with normal saline preparation of suspension.④small dose group: Dan Lou powder 450mg/kg, administered with normal saline preparation of suspension. 20 rats in each group, the volume of distribution are 10ml/kg, were intragastric once day, last for 14 days and fed a normal diet, no limitation in drinking water. 1 hour after the last administration, the model group, large and small dose group, were given subcutaneous injection of isoproterenol once, 15mg/kg, to prepare acute myocardial ischemia model, the control group injected the same volume normal saline instead.Continuous observation of the ECG changes was given before and 20 minutes after injection. 24h after the injection, 10 rats of each group were selected and anesthesia randomly, abdominal aortic blood was taken, serum CK, LDH and AST activity were measured by automatic biochemical analyzer. Those rats were killed, and cardiac apical 1/3 was cross-sected then made of paraffin. HE staining was used to observe pathological changes in myocardial,myocardial necrosis area measured by district-point measurement and myocardial Bcl-2, Caspase-3 protein expression in each group detected by immunohistochemistry. The remaining rats were sacrificed after intragastric for 7 days, with myocardial cross-section, paraffin sections for HE staining and immunohis- tochemistry as before to observe the pathological changes of heart, measure myocardial necrosis area, then detect the expression of myocardial MMP-2, MMP-9, TIMP-1 protein.
Results:
(1) Electrocardiogram J point in normal rats showed mild elevation, or on the base line. J point down shifted significantly (P<0.01) in model group rats injected with ISO, however, large doses Dan Lou inhibited (P<0.01) J point downshift much more significantly, and small dose Dan Lou inhibited slightly .Dan Lou can antagonize myocardial ischemia.
(2) Dan Lou significantly decreased serum CK, LDH and AST activity (P<0.01), it is suggested that Dan Lou can protect myocardial cells and stabilize cell membrane.
(3) After single 15mg/kg dose injection of isoproterenol, can cause chip spotty myocardial necrosis, area of myocardial necrosis were significantly reduced in 24h after modeling, 7 days after the high dose group (P<0.01), the area of myocardial necrosis in the latter group reduce even more than the the formerone, and the difference was significant (P<0.01).
(4) Myocardial pathological observation showed that, compared with the control group, widespread integration of cardiac disease, focal necrosis of muscle fiber-chip, fracture, myocardial nuclear fragmentation, dissolution, irregular arrangement of myocardial fibers, structural disorder, interstitial edema and the inflammatory cell infiltration within necrosis were found in the model group; the extent of myocardial lesions in the large group significantly reduced compared with model group. 7 days after modeling, myocardial lesions significantly retraction, fibrosis can be seen, only a small amount of inflammatory cell infiltration, lesions tend to heal in the large group, but the model group myocardial lesions have increased compared with 24h after the modeling.
(5) Myocardial Bcl-2 protein expression was definitely high in model, large and small dose group compared with the control group (P<0.01); large group were significantly higher compared with the model group (P<0.01), small group increased slightly, but no significantly difference in comparison with the model group (P>0.05); differences in large and small dose group were also obvious (P<0.05).Myocardial expression of Caspase-3 protein was significantly increase in model, large and small dose group (P<0.01); there was no significant difference among model group, large and small dose group (P>0.05); either between large and small dose group (P>0.05).
(6) 1 week after injection of ISO test showed: myocardial MMP-2, MMP-9 expression was significantly high in model, large and small dose group compared with the control group (P<0.01); the expression in large dose group was significantly low compared with the control group (P<0.01); there was no significant difference between model group and small dose group (P>0.05); however, large difference was seen between low-dose group and high dose group (P<0.05). TIMP-1 expression was significantly increased in model, large and small dose group compared with the control group; the difference was significant (P<0.01); there was no significant difference among model group ,large and small dose group (P> 0.05); either between large and small dose group (P>0.05).
Conclusion:
(1) Dan Lou significantly inhibits J point on ECG in the model of actue ischemia rats;(P<0.01).
(2) Dan Lou can significantly reduce the myocardial necrosis area in rats with acute myocardial ischemia induced by isoproterenol, decreased serum AST, LDH and CK activity, reduce the pathological damage,and has a protective effect on the injury.
(3) Dan Lou can up-regulate the expression of Bcl-2 , down-regulate the expression of Caspase-3,as the same time,Dan Lou may protect ischemia through relieving apoptosis;
(4) MMP-2, MMP-9 and TIMP-1 expression was significantly decreased , which suggested that Dan Lou can inhibite the enlargement of infarction and have a therapeutic effect on ventricular remodeling;
(5) Dan Lou has no impact on the expression of TIMP-1, the inhibition of the activity of MMP may not comlishment by TIMP-1.
引文
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