延胡索提取物治疗冠心病室性心律失常的机理研究

【作者】 张萍；

【导师】 陈可冀； 徐凤芹； 马晓昌；

【作者基本信息】 北京中医药大学 ， 中西医结合临床， 2012， 博士

【摘要】 1.研究目的1.1系统评价分析中医药治疗冠心病室性早搏的安全性及有效性,为中医药治疗冠心病室性早搏提供循证医学证据。1.2通过对“冠心病临床科研一体化技术平台”数据库的挖掘分析,研究冠心病室性心律失常的证治规律,探索治疗冠心病室性心律失常的遣方用药特点。1.3从蛋白组学、酶学、免疫组化形态学方面探索延胡索提取物干预大鼠急性心肌梗死后室性心律失常的作用机制,为延胡索提取物治疗冠心病室性心律失常提供实验依据,为延胡索提取物新药的开发奠定基础。2.方法2.1系统评价方法计算机检索PubMed.中国生物医学文献数据库(web版)、中文生物医学期刊数据库(web版)CMCC,VIP中文科技期刊数据库(Web版),CNKI中国期刊全文数据库(Web版),万方数据库,检索至2011年2月各资料库所有可得文献。采用Cochrane协作网提供的Revman5.1.4(Review Manager5.1.4)软件分析中医药治疗冠心病室性早搏的疗效,根据改良Jadad评分对研究的质量进行评价。2.2数据挖掘方法利用SQL Server 2000工具对人口学资料、一般临床特点、证候、治法及方药数据进行转换、加载,利用Business Objects软件进行冠心病室性心律失常临床需求的多维主题查询及Web多维分析；利用Oracle 10g实现冠心病室性心律失常证候分布、遣方用药规律的挖掘分析。利用冠心病临床科研一体化技术平台对室性心律失常的中药配伍规律,药物与证候之间的关系进行复杂网络分析。2.3延胡索提取物治疗大鼠急性心肌梗死后室性心律失常的实验研究方法建立大鼠急性心肌梗死模型,将wistar大鼠随机分为正常组、假手术组、模型组、美托洛尔组、参松养心胶囊组、延胡索提取物高、中、低剂量组。N-BT染色测量心肌梗死面积,ELASA法测定Na+-K+-ATPase和Ca2+-ATPase活性,免疫蛋白印迹法测定缝隙连接蛋白43(Connexin 43,CX43)及其磷酸化CX43 (Phospyo-CX43, P-CX43)的蛋白含量,及免疫组化法观察CX43及P-CX43的表达位置。从以上几个方面来探讨延胡索提取物对大鼠急性心肌梗死后室性心律失常的作用机制。3.结果3.1系统评价结果--中医药治疗冠心病室性早搏的Meta分析与空白对照或西药对照相比,中医药可有效治疗冠心病室性早搏,减少冠心病室性早搏次数,差异有统计学意义(p<0.05)。Jadad评分表明研究质量大部分属于低质量。3.2数据挖掘结果入选了386例冠心病室性心律失常患者,男性201例,女性185例,平均年龄70.06±10.33岁。合并病及疾病亚型主要是高血压、心功能不全、心绞痛、陈旧心肌梗死、糖尿病。冠心病室性心律失常的主要证候为血瘀、气虚、痰浊。患者首次使用的中药主要是茯苓、丹参、当归、半夏、麦冬、甘草、红花、川芎、桃仁、陈皮,首次使用的中药主要为补气药、活血调经药、理气药。最常使用的抗心律失常药为美托洛尔、胺碘酮与慢心律。室性心律失常患者预后在冠心病心律失常患者中较差、无效与死亡率明显高于其他类型心律失常患者。冠心病室性心律失常药物-证候复杂网络挖掘分析显示,常见证候为气虚、血瘀、痰浊,与证候对应的药物主要是丹参、茯苓、当归、麦冬、赤芍、红花、川芎、甘草、半夏、桃仁、陈皮、五味子。3.3延胡索提取物治疗大鼠急性心肌梗死后室性心律失常的实验研究结果3.3.1心梗面积在梗死区面积与梗死区面积／心室总面积上,延胡索提取物小、中剂量组及美托洛尔、参松养心胶囊治疗组均有缩小急性心肌梗死面积的作用(P<0.05)。3.3.2 Na+-K+-ATPase与Ca2+-ATPase活性结果与正常组比较,模型组的Na+-K+-ATPase与Ca2+-ATPase活性明显降低(P<0.01)。延胡索碱提取物组与其他药物治疗组均可以明显提高大鼠急性心肌梗死后降低的Na+-K+-ATPase与Ca2+-ATPase活性(P<0.01)。3.3.3 CX43与P-CX43免疫蛋白印迹结果与正常组比较,模型组的CX43及P-CX43蛋白含量明显减少(P<0.05)。延胡索提取物小、中剂量组及美托洛尔、参松养心胶囊组均能提高急性心肌梗死后降低的CX43与P-CX43的蛋白含量(P<0.05)。其中延胡索提取物中剂量组与美托洛尔组疗效相当。3.3.4各组大鼠心脏大体病理形态学结果HE染色结果显示,延胡索提取物治疗组及其他药物治疗组,肌纤维损伤程度减轻,大部分肌纤维完整,排列整齐,仅见局灶性纤维组织增生,淋巴细胞浸润,而模型组心肌纤维排列紊乱,部分胞浆溶解,纤维组织增生明显,伴玻璃样变,可见肉芽组织形成及坏死组织。3.3.5 CX43与P-CX43免疫组化结果正常组CX43与P-CX43主要分布在细胞端端连接处,即闰盘区域。而模型组,细胞结构被破坏,CX43与P-CX43表达明显降低,且出现侧边化现象明显。延胡索提取物治疗组与美托洛尔组、参松养心胶囊组,CX43与P-CX43表达较模型组明显增加,细胞结构损伤较模型组小,亦有侧边化现象。4.结论4.1系统评价提示中医药对冠心病室性早搏有一定的疗效及安全性,但因纳入研究质量较低,需更多高质量研究进一步验证。4.2冠心病室性心律失常的主要证候为血瘀、气虚、痰浊,常用中药为丹参、茯苓、当归、麦冬、赤芍、红花、川芎、甘草、半夏、桃仁、陈皮、五味子,与证候相应的中药主要为补气药、活血调经药、理气药等。4.3延胡索提取物能减少大鼠急性心肌梗死面积,改善心肌缺血损伤,提高梗死后心肌的Na+-K+-ATPase与Ca2+-ATPase活性和梗死后减少的CX43与P-CX43的蛋白含量,减少CX43与P-CX43侧边化现象,从而降低室性心律失常的发生。更多还原

【Abstract】 1.1 To systematically review the effect and safety of Chinese medical treatment of coronary heart disease (CHD) patients with ventricular premature complexes (VPC), so as to provide evidence for Chinese medicine treating CHD patients with VPC.1.2 Using the data mining technology to dig the data base "CHD platform of integrated clinical and scientific research", to investigate diagnose and treatment regularity of Chinese medicine treating CHD patients with VPC, and to explore the prescription and medication features.1.3 From the aspects of proteomics, enzymology and immunohistochemical morphology, to explore the mechanism of Yanhusuo extract (YE) on rat model of ventricular arrhythmia (VA) after acute myocardial infarction (AMI), so as to provide experimental evidences for YE preventing ventricular arrhythmia from CHD, especially from AMI, and settle the foundation to explore the new medicine of YE.2. Methods2.1 Methods of systemic reviewDatabases of PubMed, CBM, CMCC, VIP, CNKI and WanFang were searched for all available data till February 2011. RevMan5.1.4 software was used to conduct meta-analysis. Jadad score was used to assess the quality of the included trials.2.2 Methods of data miningThe data change-over and data loading for demography information general clinical characteristic、syndromes、therapeutic methods and Chinese medicine prescription were managed by SQL Server 2000 software. Multi-subject inquest and web analysis for the request of clinical need of VA in CHD were managed by Business Objects software. The syndromes distribution and TCM prescription of VA in CHD were dug and analyzed by Oracle 10g software. The Chinese medicine compatibility regularity and the relationship between Chinese medicine and syndromes were explored by complex networks analysis.2.3 Methods of experimental research for YE treating VA after AMI in ratsTo establish rat model of AMI, Wistar rats were randomly divided into normal group, sham group, model group, metoprolol group, Shensong Yangxin Capsule (SYC) group and low dosage、median dosagee and high dosage of YE groups. The infarction area was measured by N-BT staining. The Na+-K+-ATPase and Ca2+-ATPase activities were measured by ELASA method. Western Blot method was used to measure the protein levels of connexin43 (CX43) and phosphor-CX43 (P-CX43), and immnohistochemical method was used to observe the expression location of CX43 and P-CX43. From above all aspects to explore the mechanism of YE on rat VA model after AMI.3. Results3.1 Results of systematic review---the effect of Chinese medicine on CHD patients with VPC, a meta-analysis.Compared with blank control group or western medicine group, Chinese medicine might treat CHD patients with VPC effectively, and also might reduce the VPC frequency (P<0.05). According to Jadad score, the quality of most of the trials was low.3.2 Results of data mining analysis386 CHD patients with VA aged 70.06±10.33 years in average were enrolled, including 201 males and 185 females. The subtypes and combined diseases were hypertension, heart failure, angina pectoris, old myocardial infarction,and diabetes mellitus. The main syndromes of CHD patients with VA were blood stasis, qi deficiency and phlegm-turbid. The first TCM prescription after admission mainly contained Fuling, Danshen, Danggui, Banxia, Maidong, Gancao, Honghua, Chuanxiong, Taoren, and Chenpi. The main functions of these Chinese medicines above were tonifying qi, activating blood and regulating qi. The main anti-arrhythmia medicines were metoprolol, amiodarone and mexiletine. The prognosis of CHD patients with VA was worse than the other types of CHD patients with arrhythmia, the ineffective ratio and mortality were much higher. The complex netmork analysis of medicine-syndrome showed that the main syndromes of CHD patients were qi deficiency, blood stasis and phlegm-turbid. The corresponded medicines for treating the main syndromes were Danshen, Fuling, Danggui, Maidong, Chishao, Honghua, Chuanxiong, Gancao, Banxia, Taoren, Chenpi and Wuweizi.3.3 Results of experimental research into YE treating VA after AMI in rats3.3.1 Result of myocardial infarction areaCompared with the model group, low dosage and median dosage group of Yanhusuo Extract, Metoprolol group and Shensong Yangxin Capsule group could reduce the myocardial infarction area and lower the ratio of myocardial infarction area/ventricle area (P<0.05).3.3.2 Result of Na+-K+-ATPase and Ca2+-ATPase activitiesCompared with the normal group, both the Na+-K+-ATPase and Ca2+-ATPase activities in model group were lowered obviously (P<0.01). The low、median、high dosage of YE and other medicine groups could improve the level of Na+-K+-ATPase and Ca2+-ATPase activities, compared with model group (P<0.01).3.3.3 Result of protein level of CX43 and P-CX43 using Western Blot method.Compared with normal group, the protein levels of CX43 and P-CX43 in model group were obviously lowered (P<0.05). Except the high dosage of YE group, the other medical groups could improve the reduced protein levels of CX43 and P-CX43 after AMI (P<0.05). In addition, the effect of median dosage of YE group was as good as Metoprolol group.3.3.4 Result of pathomorphology in general of the rat hearts in all the groupsHematoxylin and Eosin (HE) staining showed that the low、median、high dosage of YE and other medicine groups could relieve the muscle fiber injury. Most of muscle fibers in the rat hearts of these groups were intact and line up in order. Only local fibroplasias and lymphocytes infiltrating were seen. Nevertheless, in the model group, cardiac muscle fibers lined up disorderly. Plasmolysis, fibroplasias, glassy degeneration, granulation tissue and necrotic tissue were seen.3.3.5 Result of immunohistochemical CX43 and P-CX43In the normal group, CX43 and P-CX43 were mainly located at end-to-end apposition, also known as the myocardial cell intercalated disc region. In the model group, cellular structure was destroyed; CX43 and P-CX43 expression decreased obviously, and dislocated from end-to-end to side-to-side connections, also called lateralization. Compared with model group, the expression of CX43 and P-CX43 in YE groups, Metoprolol group and Shensong Yangxin Capsule group increased obviously, and just some of the cellular structures suffered from injury. However, some cardiocytes appeared lateralization.4. Conclusion4.1 The result of systematic review suggested that Chinese medicine could treat CHD patients with VPC effectively and safely. However, much more high quality researches are still needed to back up this result. 4.2 Blood stasis, qi deficiency and phlegm-turbid were the main syndrome elements of CHD patients with VA. The most of Chinese medicine used were Danshen, Fuling, Danggui, Chishao, Honghua, Chuanxiong, Gancao, Banxia, Taoren, Chenpi, and Wuweizi. Corresponded with the main syndrome, the functions of these Chinese medicines were tonifying qi, activating blood and regulating qi.4.3 Yanhuosuo Extract could reduce the acute myocardial infarction area on rat AMI model, enhance the protective effect of ischemia injury, increase the activities of Na+-K+-ATPase and Ca2+-ATPase and the reduced protein level of CX43 and P-CX43 after AMI, and reduce the lateralization of CX43 and P-CX43, and accordingly reduce the genesis of ventricular arrhythmia. The mechanism of YE treating ventricular arrhythmia might be related to above all.更多还原