黄芪治疗慢性心衰临床研究的系统评价及黄芪对能量转运影响的研究

【作者】 温志浩；

【导师】 林谦；

【作者基本信息】 北京中医药大学 ， 中西医结合临床， 2009， 博士

【摘要】 慢性心力衰竭(chronic heart failure,CHF)是各种心血管系统疾病发展的终末阶段。随着人口的老龄化,CHF患病率不断升高,已经成为世界性的公共卫生问题。CHF防治指南的广泛应用,使其防治水平得到了很大提高,但病死率和致残率仍很高。很多科学家都在致力探寻更加有效的治疗方法,而针对心肌能量代谢方面的研究已经成为了其中的热点之一,不过目前尚缺乏公认有效的针对心肌能量代谢的特异性药物。运用中药益气法治疗CHF的理念与改善衰竭心脏能量代谢有共通之处,近年来进行了很多有关的临床和实验研究,取得了较肯定的疗效。但这些研究尚未得到系统的评价,在一定程度上制约了其推广和应用。黄芪注射液是益气药的代表药——黄芪的提取物,目前在临床上被广泛用于治疗CHF。本文选择有关该药治疗CHF的临床研究文献进行系统评价和Meta分析,并通过细胞和分子生物学实验探讨其干预作用机制与心肌细胞能量转运的关系,以期为益气法治疗CHF提供循证和实验依据,同时也为寻找改善心肌能量代谢的药物做出有益的探索。一、黄芪注射液治疗CHF临床研究的系统评价和Meta分析方法:(1)以“(题目:黄芪注射液)and(题目:心功能不全or心衰or心力衰竭)—限定:人类”为检索式检索中国生物医学文献数据库,不限制年限;(2)由2位研究者独立进行文献筛选,意见不统一者通过讨论达到一致;(3)获取文献的相关研究特征资料,并加以评价;(4)对合格的随机对照试验(randomized blind controlled trial,RCT),进行Meta分析。结果:(1)共检索到246篇文献;(2)经过两次筛选,剔除不符合纳入标准的文献,最终共117篇文献入选评价;(3)入选文献的研究设计普遍存在缺陷,有8篇文献的Jadad评分为1分,2篇为2分,没有文献得分在2分以上,将这10篇文献,即10个RCT纳入Meta分析,总样本量为711例。(4)Meta分析结果显示,加用黄芪注射液,可以更有效的提高左室射血分数(left ventricularejection fraction,LVEF)(P＜0.001)、短轴缩短率(fraction shortening,FS)(P＜0.00001)、心脏指数(cardiac index,CI)(P＜0.00001)、心输出量(cardiac output,CO)(P＜0.00001)和临床总有效率(P＜0.001);对采用单平面改良的Simpson法测量的两个RCT的结果合并后结果提示加用黄芪注射液对LVEF的影响无统计学意义(P=0.67);加用黄芪注射液与加用硝酸甘油对比,死亡率和心律失常发生率的差异无统计学意义(P值均＞0.05);加用黄芪注射液对左室舒张末期容积(P=0.17)、左室收缩末期容积(P=0.21)和二尖瓣E/A(P=0.75)的影响均无统计学意义。结论:上述分析结果提示在常规治疗基础上加用黄芪注射液可更好地改善CHF患者的心功能。不过根据目前的Meta分析尚不能得出最终结果,真正评价黄芪注射液治疗CHF的疗效还需要大样本、多中心的严格设计的高质量RCT。黄芪注射液治疗CHF的临床研究的方法学质量还有待进一步提高。以下几个方面还有待提高:严格确定研究对象;加强辨证论治的应用;探索合适的阳性对照药或安慰剂和可行的盲法执行方案;选择恰当的疗效评价指标,建立完善的疗效评价体系;完整的随访和不良反应报告。二、黄芪注射液对肥大心肌细胞能量转运的影响方法:体外原代培养乳鼠心肌细胞,运用血管紧张素Ⅱ(angiotensin,AngⅡ)刺激心肌细胞,建立肥大心肌细胞模型。实验分为4组:空白组、模型组、络沙坦组和黄芪注射液组。在24h和48h两个时点,用共聚焦显微镜观察心肌细胞线粒体膜电位(mitochondrial membrane potential,MMP)及黄芪注射液的干预作用;采用生化法和高速电泳法观察肥大心肌细胞中总肌酸激酶活性和同功酶组成的变化和黄芪注射液的干预作用;运用RT-PCR法观察肥大心肌细胞肌酸激酶(creatine kinase,CK)同功酶和腺苷酸转位酶(adenine nucleotide translocator,ANT)异构体mRNA表达的影响和黄芪注射液的干预作用。结果:(1)24h,模型组MMP较空白组显著下降(P＜0.01),各用药组较空白组上升,黄芪注射液组与空白组差异有统计学意义(P＜0.01);48h,模型组MMP较前进一步下降,与其余各组的差异均有统计学意义(P＜0.01),各用药组MMP则趋于正常:(2)ANT的异构体ANT1在肥大心肌细胞中的mRNA表达呈下降趋势,在48h下降明显(P＜0.01),而黄芪注射液有维持其水平的作用。ANT2 mRNA表达水平在肥大心肌细胞未发生明显变化,黄芪注射液在24h有提高其表达水平的作用(P＜0.01),48h各组间的差异无统计学意义;(3)24h,模型组和各用药组的CK-M亚基mRNA表达水平出现明显下降(P值均＜0.05),CK-MM所占比例也出现明显下降(P＜0.01),同时CK-B亚基mRNA表达水平和CK-BB所占比例均明显上升(P＜0.01),此时各组间CK-MB所占比例和总CK活性均无显著差异;48h,模型组CK-M亚基的mRNA表达继续下降(P＜0.01),各用药组的表达水平回升至正常水平,模型组和络沙坦组的CK-MM所占比例均继续下降(P值均＜0.01),而黄芪注射液组则回升至正常水平,模型组心肌细胞CK-B亚基mRNA表达继续上升(P＜0.01),其CK-BB所占比例仍偏高(P＜0.01),而各用药组均出现回落,黄芪注射液组恢复至正常水平,络沙坦组CK-MB比例较空白组显著升高(P＜0.05),模型组的CK活性出现了明显的下降(P＜0.01),而各用药组则维持在正常水平。结论:肥大心肌细胞的MMP较正常心肌细胞明显下降,CK同功酶出现胚胎化改变,到衰竭期则出现ANT1的mRNA表达和CK的总活性降低,说明能量代谢尤其是能量物质转运出现异常。在络沙坦或黄芪注射液的干预下这些变化有所改善。结合上述临床研究的系统评价和实验部分研究,我们推断,黄芪注射液提高CHF患者心功能的机制,有可能与稳定能量物质转运酶系和MMP,从而改善心肌细胞舒缩功能有关。更多还原

【Abstract】 Chronic heart failure(CHF) is the end-stage of all sort of heart disease.As the population is aging,morbidity rate of CHF is getting higher and higher,which becomes a international problem of public health.The guidelines for CHF improve the level of treating CHF,but the mortality and morbidity is still high.The unsatisfactory therapy effect makes people search for better method for treating CHF, the energy metabolism of cardiomyocyte is one of the hot spots,though there is not any generally acknowledged medicine which is against this target.The principle of Traditional Chinese medicine,which treat CHF by replenishing Qi,has something in common with improving the of energy metabolism of failed heart.In resent decades,a lot of clinical and experimental trials have been carried out, which showed us the satisfactory results.But these results have not been systematically evaluated,the generalization and utilization of which is limited up to a certain extent.Radix astragali injection is the extractive of Radix astragali which is the deputy medicine of Qi-tonics.This injection is being generally used for treating CHF.In this thesis,a systematic review of clinical studies on treating CHF with Radix astragali injection will be performed,and some cellular and molecular biological experiments will be executed to investigate the relationship of mechanism of treating with Radix astragali injection and energy transportation of cardiomyocyte.We are aimed to find some evidence-base clinical and experimental evidence for treating CHF by replenishing Qi,and do some significant exploration in searching for the medicine which is able to improving energy metabolism of cardiomyocyte.1 Radix astragali injection for CHF:a systematic review and meta analysisMethods:(1)Search items were:(Title:Radix astragali injection) AND(Title: cardiac insufficiency or heart failure or cardiac failure),the search was applied to the CBM database(the publication date was not limited);(2)Literature bolting was performed by 2 reviewers independently,different opinions would be solved through negotiation;(3)The relation data of literatures were extracted and evaluated;(4)A meta analysis was performed based on qualified randomized blind controlled trials(RCTs).Results:(1) 246 literatures were found out;(2) Through twice bolting,there were 117 literatures met the evaluate procedure.(3) There were many defects in the enrolled literatures.There were 8 literatures’ Jadad scores were 1,2 literatures’ Jadad scores were 2,and there were not any literature’s Jadad score was higher than 2. These 10 literatures contained 10 RCTs which were enrolled into Meta analysis (711.cases).(4) The result of analysis showed that compared with routine therapy, additional of use of Radix astragali injection could raise left ventricular ejection fraction(LVEF)(P＜0.001),cardiac output(CO)(P＜0.00001),cardiac index(CI)(P＜0.00001) and effective rate(P＜0.001) significantly,but not significantly compared with additional of use of nitroglycerine;Two RCTs which used Simpson method enrolled into Meta analysis,and the result showed that compared with routine therapy, additional use of Radix astragali injection couldn’t raised LVEF significantly(P=0.67); Compared with additional use of nitroglycerine,additional use of Radix astragali injection couldn’t lower the mortality and incidence of arrhythmia(P＞0.05);There was no significant difference between routine therapy and additional use of Radix astragali injection in left ventricular end diastolic or systolic volume and E/A(P=0.17, 0.21,0.75).Conclusion:Additional use of Radix astragali injection could improve the function of heart more effectively.However,According to this Meta analysis,we can’t get final answer.We need high qualitative multicenter clinical trial of which the sample is large to evaluate the therapy effective for treating CHF with Radix astragali injection.The clinical trials which treat CHF with Radix astragali injection should improve their technology quality.The following aspects should be improved,such as strict criterion for enrolling,treatment based on syndrome differentiation,searching for suitable positive control or placebo and the way for executing blind method, choosing proper index for evaluating therapy effective,establishing consummate system for evaluating therapy effective,complete follow-up and side-effect reports.2 The effect of Radix astragali injection on energy metabolism of hypertrophic cardiomyocyteMethods:To establish the model of hypertrophic cardiomyocyte,we stimulated the cardiomyocytes of neonatal rat which were primarily cultured in vitro with angiotensin(AngⅡ).The cardiomyocytes were divided into 4 groups:control group, model group,Losartan group and Radix astragali injection group.At 24h and 48h,We investigated the effects of AngⅡ,Losartan and Radix astragali injection,on mitochondrial membrane potential(MMP) of cardiomyocytes by confocal microscopy, on the activity of total creatine kinase(CK) by means of biochemistry method and on CK isoenzymes which were separated using Rapid Electrophoresis system,and on B-CK,M-CK,mi-CK,iso-adenine nucleotide translocator(ANT) mRNA gene expression by using RT-PCR.Results:(1) At 24h,MMP of model group was obviously reduced compared with control(P＜0.01),Radix astragali injection and Losartan could raise MMP,MMP of the cardiomyocytes stimulated by Radix astragali injection was higher than control group significantly(P＜0.01);at 48h,MMP of model group was continually reduced significantly(P＜0.01),the difference among other groups was not significantly.(2) ANT 1 mRNA gene expression of hypertrophic cardiomyocytes were lower,at 48h the difference between model group and control Was significant(P＜0.01),and Radix astragali injection could remain it at the normal level.ANT 2 mRNA gene expressions of hypertrophic cardiomyocytes remained the same with the control.At 24h,Radix astragali injection could raise the level of ANT 2 mRNA gene expression significantly(P＜0.01) and at the 48h not significantly.(3) At 24h,the levels of CK-M mRNA gene expressions(P＜0.05) and the content of CK-MM(P＜0.01) of hypertrophic cardiomyocytes were reduced obviously,at the same time the levels of CK-B mRNA gene expressions and the content of CK-BB were raised significantly(P＜0.01).There were no difference in the content of CK-MB and total CK activity among these groups.At 48h,the level of CK-M mRNA gene expressions of model group was continually reduced,while the levels of the groups treated with Radix astragali injection and Losartan were normalized.The content of CK-MM of model group and L group were raised significantly(P＜0.01),while the content of the group treated with Radix astragali injection remained normal.The level of CK-B mRNA gene expressions and the content of CK-BB of model group were still high(P＜0.01), and there was no difference in the content of CK-BB among other groups.The content of CK-MB of L group was higher than control group obviously(P＜0.05).Total CK activity of model group was depressed significantly(P＜0.01),and Radix astragali injection and Losartan could prevent it from being reduced.Conclusion:The MMP of hypertrophic cardiomyocytes were obviously reduced compared with control,while the activated fetal program made the content of CK change.The level of ANT 1 mRNA gene expressions and CK activity of failed cardiomyocytes decreased.These changes showed that dysfunction of energy metabolism especially energy transportation emerged.Losartan and Radix astragali injection could improved this dysfunction.According to research above,we infer that the mechanism of improving heart function with Radix astragali injection may be concern with stabilizing the enzymes used for transporting energy substances and MMP so as to improving diastolic and systolic function.更多还原