活血益气方药改善心梗气虚血瘀大鼠模型心肌损伤和CX43的研究

【作者】 吴爱明；

【导师】 王硕仁；

【作者基本信息】 北京中医药大学 ， 中西医结合临床， 2010， 硕士

【摘要】 心肌梗死是危害人类健康的主要疾病之一,心肌细胞在缺血缺氧条件下会引起心脏整体结构的改变、心功能的恶化和恶性心律失常发生。心肌的缝隙连接蛋白43(Cx43)与心肌梗死后心律失常的发生关系密切。Cx43所介导的心肌细胞间的缝隙连接通讯是维持心脏电活动协调和稳定的重要结构基础。本研究着眼于威胁人类健康的重大疾病心肌梗死,以心梗气虚血瘀病证结合大鼠模型为研究对象,从Cx43角度探讨了心肌梗死后缝隙连接的分子病理变化及活血益气相关方药的治疗作用。目的：1.建立冠脉结扎致心肌梗死大鼠模型,并对该模型的心脏结构变化和中医证候进行评价。2.研究该病证模型大鼠心肌组织细胞间缝隙连接的分子病理变化。3.观察活血益气相关方药对模型大鼠左心室梗死边缘区Cx43表达的影响。方法：1.用冠脉结扎法建立大鼠心肌梗死模型,用超声心动图观察心脏结构的变化并进行疾病诊断；同时通过仔细观察,紧密联系中医基础知识,有目的、有意识地积极联想和分析,从疾病的外在表现发现和挖掘出具有中医意义的证候要素,对大鼠模型进行中医四诊信息采集,并结合中西医结合临床虚证和血瘀证辨证标准,并将问诊内容替代以具有同等意义的定量指标测试,对该模型进行中医证候评价。2.对该模型大鼠进行Agilent大鼠全基因组表达谱芯片检测,采用GO和Pathway等生物信息学分析方法对差异表达基因进行初步分类,并重点对其中的心肌细胞间缝隙连接的相关基因改变进行具体分析和检索,同时采用实时荧光定量PCR和免疫组化方法验证心室肌细胞间通讯的主要结构组分Cx43的变化。3.通过HE染色和Cx43免疫组化染色,观察活血益气中药丹参片合参芍片和西药倍他乐克治疗该模型大鼠4周后心脏及左心室梗死边缘区Cx43表达的变化.4.采用免疫组化染色方法观察活血祛瘀抗纤维化专方扶正化瘀胶囊和西药卡托普利片治疗该模型大鼠8周后,对左心室梗死边缘区Cx43分布和表达的影响。结果：1.模型大鼠与假手术组比较,术后8周超声心动图检查模型大鼠心脏功能显著受损,左室射血分数(LVEF)、左室短轴缩短率(LVFS)均明显降低(P<0.01),室间隔舒张末期厚度(IVSTd)、左室后壁舒张末期厚度(LVPWTd)、左室后壁收缩末期厚度(LVPWTs)均明显变薄(P<0.01),左室舒张末期内径(LVDd)、左室收缩末期内径(LVDs)明显增大(P<0.01)。天平实测的模型组全心重量／体重明显高于假手术组(P<0.05)。心电图检查可见模型大鼠仍有sT段抬高或病理性Q波存在。模型大鼠的生物学表观在一定程度上反映出了大鼠心肌梗死后神气衰微、体弱正虚、病情较重、血行不畅的特点,符合中医的阴证、虚证和瘀证的特征,并且心率和呼吸频率模型组比假手术显著增快,游泳力竭时间显著缩短。说明模型大鼠的心梗疾病诊断和气虚血瘀的中医证候属性确定。2.对模型和假手术大鼠进行Agilent大鼠全基因组表达谱芯片检测,共筛选出两倍以上变化的差异基因1501条,其中模型组比假手术组上调基因有944条,下调基因有557条。基因功能的GO分析和细胞通路的Pathway分析结果揭示了心梗大鼠病证模型已经涉及了三大物质代谢、能量代谢和细胞结构、分子转导、细胞间通讯等“细胞社会活动”的分子病理过程。我们对研究靶标——心肌细胞间缝隙连接通讯的具体分析显示缝隙连接蛋白发生了改变,并且其正调节因素蛋白激酶1(Casein kinase 1, CK1)出现了下调,负调节因素蛋白激酶C (Protein kinase C, PKC)呈现了上调的趋势。实时荧光定量PCR和免疫组化验证表明模型大鼠心室肌Cx43的mRNA及其编码的蛋白出现了显著的下调。3.丹参片合参芍片及西药倍他乐克对心肌梗死大鼠模型治疗4周后,HE染色可见模型组组心肌细胞胞浆着色不均,核固缩,心肌细胞断裂,结构破坏,排列严重紊乱,大量炎性细胞浸润。丹参片合参芍片治疗组和倍他乐克治疗组的病理改变较模型组略轻。心肌Cx43免疫组化染色显示模型组左心室梗死边缘区的Cx43表达明显减弱甚至消失,界线不清,呈点状甚至片状,分布弥散紊乱,在闰盘、心肌细胞边缘以及心肌细胞胞浆内均可见表达。丹参片合参芍片治疗组和倍他乐克治疗组左心室梗死边缘区Cx43表达减弱,部分阳性表达界线清晰、成线状排列,部分分布在闰盘,部分弥散至心肌细胞边缘以及心肌细胞内。图像分析结果表明丹参片合参芍片治疗组的平均光密度值(AOD)和积分光密度的对数值[log (IOD)]显著高于模型组。4.扶正化瘀胶囊和卡托普利对心肌梗死大鼠模型治疗8周后,在左室梗死边缘区的Cx43的弥散和紊乱情况有部分改善。图像分析结果显示,与假手术组相比,模型组、扶正化瘀胶囊组和卡托普利组的平均光密度值、阳性表达面积和积分光密度显著降低。与模型组比较,扶正化瘀胶囊组和卡托普利组的Cx43的平均光密度值和积分光密度值显著增加,其中卡托普利组的Cx43的阳性表达面积也显著增加。同时左心室梗死区面积扶正化瘀胶囊组和卡托普利组均显著低于模型组。左心室梗死百分比(左心室梗死区面积占左心室总面积的百分比)扶正化瘀胶囊组和卡托普利组也都显著低于模型组。结论：1.冠脉结扎致心肌梗死模型大鼠术后8周时心脏结构的病理变化符合离心性重构的特点和心肌梗死的诊断,其心功能的减损程度已经达到心衰的标准；通过模拟中西医结合临床虚证和血瘀证辨证标准,并将问诊内容替代以具有同等意义的定量指标测试,对该模型进行中医证候评价证明其中医证候特点符合心气虚证和血瘀证的诊断。通过对大鼠模型进行仔细观察,并紧密联系中医基础知识,有目的、有意识地积极联想和分析,就可以从疾病固有的外在生物学表观中发现和挖掘出具有中医意义的证候要素。2.心梗大鼠病证模型的基因改变涉及了三大物质代谢、能量代谢和细胞结构、分子转导、细胞间通讯等众多“细胞社会活动”的分子病理过程。其中心肌Cx43的mRNA表达和其编码蛋白的变化以及Cx43的上游调节因素CKl和PKC的分子病理改变,是心肌梗死后心律失常的重要分子病理基础之一。3.活血益气药丹参片合参芍片及西药倍他乐克具有部分减轻左心室梗死边缘区心肌细胞损伤和炎性细胞浸润程度的作用。并且丹参片合参芍片还可改善心肌梗死后Cx43弥散分布的程度,增加Cx43的平均光密度值和积分光密度值,而倍他乐克对Cx43的作用不明显。采用活血益气法治疗心肌梗死,对于稳定由Cx43介导的心肌细胞间的电偶联有益。4.卡托普利和扶正化瘀胶囊可以改善心肌梗死后Cx43分布的弥散程度,并能够增加左心室梗死边缘区Cx43表达的平均光密度值和积分光密度值,其中卡托普利还可以增加Cx43的阳性表达面积,卡托普利和扶正化瘀胶囊均可以部分改善心肌梗死后心肌Cx43的重构和缩小心肌梗死面积。从抗纤维化角度治疗心肌梗死对改善心肌Cx43的重构有益。更多还原

【Abstract】 Myocardial infarction is one of the major heart diseases to harm public health. Myocardial cells in ischemic and hypoxic conditions cause changes in the overall structure of the heart, deterioration of cardiac function and malignant arrhythmia. The relationship of myocardial connexin 43 (Cx43) and the occurrence of arrhythmia after myocardial infarction is close. The Cx43-mediated gap junctions communication between myocardial cells are the essential structural basis to maintain coordination and stability of cardiac electrical activity. In this study, we focus on the major diseases myocardial infarction that threaten human health, and we take the disease and syndrome model rats with myocardial infarction and the deficiency of Qi and blood stasis syndrome as the object And we study the change in the molecular pathology and the effect after the treatment of promoting blood circulation and supplementing Qi from the perspective of Cx43 gap junctions after myocardial infarction.Objective:1. To establish myocardial infarction rat model by coronary artery ligation, and to evaluate the structural changes in the heart and syndrome of TCM of the model.2. To study the characteristic of molecular pathology changes in the myocardial gap junction intercellular communication of the disease and syndrome model rats.3. To observe the Cx43 expression on left ventricular infarct border zone after the treatment of promoting blood circulation and supplementing Qi.Methods:1. Myocardial infarction rat model were established by coronary artery ligation, then the structural changes of the heart were observed and the diagnosis were given by echocardiography. At the same time the syndrome with significant elements of Chinese medicine from the external manifestations of the disease could be found and collected through careful observation and close contact with basic knowledge of Chinese medicine and the purposeful positive association and analysis. And the syndrome of TCM of the model according to the related criteria of Chinese Integrative Medicine were evaluated through replacing the content of Chinese interrogation with the quantitative indicators which have the same significance of TCM.2. Through the Agilent rat whole genome microarray detection, the differentially expressed genes were classified simply by using GO and Pathway bioinformatics analysis methods. And a specific analysis and retrieval that we focus on were given in the genetic changes of gap junctions between myocardial cells. At the same time the changes of Cx43 which is the main components of myocardial cell communication were verified by Real-time fluorescence quantitative PCR and immunohistochemistry.3. The changes of heart and the expression of Cx43 were observed in the left ventricular infarct border zone, after the 4 week treatment with the metoprolol tablets or with Danshen tablets add up to the Shenshao tablets which have the effect of promoting blood circulation and supplementing Qi, through immunohistochemistry and HE staining.4. The distribution and Expression of Cx43 were studied in the left ventricular infarct border zone by immunohistochemistry, to evaluate the effect of the 8 week treatment with the captopril tablets or with the Fuzhenghuayu capsules which is the special anti-fibrosis drug with the function of promoting blood circulation and dispelling Stasis.Results:1. Echocardiogram parameters include left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), interventricular septum end-diastole thickness(IVSTd), left ventricular posterior wall end-diastole thickness (LVPWTd) and left ventricular posterior wall end-systolic thickness (LVPWTs) reduced (P<0.01), left ventricular end-diastolic dimension (LVDd) and left ventricular end-systolic dimension (LVDs) obviously increased(P<0.01). And whole heart weight/body weight ratio increased(P<0.05). Electrocardiogram shows ST segment elevation or pathologic Q waves in model rats. Heart rate and Breath frequency were accelerated Exhausting swimming period lessen. Above index changes appeared at 8 weeks after operation in model group, compared with that in control group. And the external biological manifestations of the model rats such as weakness, heavy sickness, lack of energy and blood stasis,which could reflects to the TCM feature of the yin syndrome, the deficiency syndrome and the blood stasis syndrome. All of above are strong evidences which could help us to make the myocardial infarction diagnosis and the TCM syndrome diagnosis of the deficiency of Qi and the blood stasis.2. The 1,501 different genes were detected through the Agilent rat whole genome microarray experiments, the model group compare with the sham group, of which there are 944 Up-regulated genes and 557 down-regulated genes. GO analysis and pathway analysis show that the gene changes of this model have been involved in the "social activities of cells" molecular pathological processes, such as three major metabolism, energy metabolism, cell structure, molecular transduction and cell communication. And we focus on and give a specific analysis and retrieval in the genetic changes of gap junctions between myocardial cells. The result show that the connexin have changed, and the positive regulator Casein kinase 1 reduced, and the negative regulator Protein kinase C increased. At the same time the changes of Cx43 gene and protein showed a downward trend according to Real-time fluorescence quantitative PCR and immunohistochemistry.3. After the 4 week treatment with the metoprolol tablets or with Danshen Tablet add up to the Shenshao Tablet, some changes could be found that involve in: HE staining show that uneven coloration, nuclear condensation, myocardial rupture, structural damage, arranged in a serious disorder, and inflammatory cell infiltration in model group, but these pathological changes could be ameliorated in Danshen plus Shenshao tablets group and metoprolol group; The immunohistochemical staining show that Cx43 expression of model group in the left ventricular infarct border zone is significantly decreased or even disappeared, and unclear boundaries, spotty or even flakes and dispersed distribution in the intercalated disk, the edge of myocardial cells and intracellular, but these pathological changes could be ameliorated in Danshen plus Shenshao tablets group and metoprolol group; Image analysis show that the average optical density (AOD) and the logarithm value of the integral optical density [log (IOD)] in Danshen plus Shenshao tablets group and metoprolol group are significantly higher than the model group.4. After the 8 week treatment with the captopril tablets or with the Fuzhenghuayu capsules, the diffusion and confusion of Cx43 in the left ventricular infarct border zone have some improvement. Image analysis show that model group, Fuzhenghuayu capsules group and captopril group all reduced significantly compared with model group in the average optical density value, the positive area and integral optical density. When compared with model group, Fuzhenghuayu capsules group and captopril group increased significantly in the average optical density and integrated optical density, and the positive expression of Cx43 area also increased significantly in captopril group. At the same time, both Fuzhenghuayu capsules group and captopril group significantly reduced compare with model group in data those are the left ventricular infarct area and the percentage of left ventricular infarction (left ventricular infarct area with a percentage of total left ventricular area).Conclusion:1. The pathological manifestations of the coronary artery ligation model rats are eccentric remodeling and myocardial infarction at 8 Weeks after operation. The cardiac functional impairment has reached the standard of heart failure; The TCM syndrome of this model is deficiency of Heart QI and blood stasis, according to the related criteria of Chinese Integrative Medicine, and replacing the content of Chinese interrogation with the quantitative indicators which have the same significance of TCM. We can find and collect the syndrome with significant elements of Chinese medicine from the external manifestations of the disease through careful observation and close contact with basic knowledge of Chinese medicine and the purposeful positive association and analysis.2. The gene changes of this model have been involved in the "social activities of cells" molecular pathological processes, such as three major metabolism, energy metabolism, cell structure, molecular transduction, cell communication and so on. Among all of above any change of the mRNA and protein of Cx43, and its upstream regulator CK1 and PKC was one of the important molecular pathology mechanisms of arrhythmias after myocardial infarction.3. The metoprolol tablets and Danshen tablets plus the Shenshao tablets which have the effect of promoting blood circulation and supplementing Qi can reduce the degree of myocardial cell injury and inflammatory cell invasion to some extent. Danshen tablets plus the Shenshao tablets can ameliorate the diffusion and confusion of Cx43 in the left ventricular infarct border zone, and can increase the average optical density and the logarithm value of the integral optical density of Cx43, but metoprolol tablets have no significant effect on it. The treatment of promoting blood circulation and supplementing Qi can be benefit to the stability of the Cx43-mediated electrical coupling between myocardial cells.4. The captopril tablets and the Fuzhenghuayu capsules can ameliorate the diffusion and confusion of Cx43 in the left ventricular infarct border zone, and can increase the average optical density and the integral optical density of Cx43.Beside above all the captopril tablets also could increase the positive expression of Cx43 area. The two drugs can improve the remodeling of Cx43 after myocardial infarction to some extent From the perspective of anti-fibrosis treatment of myocardial infarction can be benefit to ameliorate the remodeling of Cx43 and can reduce myocardial infarction area.更多还原