黄芪总提物及黄芪有效成分治疗心衰的心肌力能学机制研究
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摘要
背景
心力衰竭(Heart Failure, HF)是一种由于各种器质性或功能性心脏疾病使心室或射血能力受损的综合症,其病死率约50%,已成为21世纪最重要的心血管病症。在过去三十年,急性心梗的病死率已经有所下降,但心衰的伴发率却持续升高,已严重影响了人类健康。对于心衰的了解,早在2000年前就被希腊Hippocrates所描述,但对其治疗,虽然取得了一定的进展,却一直不理想,因此,如何治疗心衰,阻止心衰的病程发展一直是医学研究的热点问题。
HF的发病是由多种因素决定的,早在1939年,Herrmann和Decherd发现慢性心衰心肌中肌酸含量明显降低,因而提出在慢性心衰时存在心肌能量的耗竭。目前越来越多的临床研究和实验研究均显示,衰竭的心脏都存在着心肌能量的耗竭,因而能量代谢在心衰进展中起着非常重要的作用。事实上,心肌能量代谢障碍是心衰的重要标志之一。研究显示,心肌能量缺乏、代谢酶类过表达以及基因的异常能够引起左心室重塑,是心衰进展中必不可少的因素。在胎儿时期,葡萄糖作为代谢底物的主要供能者,游离脂肪酸氧化率很低。提供ATP的量很少,心脏中的糖酵解是能量的主要来源,并且糖酵解酶类具有更高的活性。出生后游离脂肪酸的氧化率增长了10倍,同时葡萄糖的氧化率下降,而心衰晚期游离脂肪酸氧化减少葡萄糖氧化增加,这恰好和一些研究观察到的结果相反。如果新生儿罹患心肌肥大,脂肪酸氧化的关键酶类活性及表达将保持“胎儿状态”。许多参与脂肪酸氧化的关键酶类在从胎儿代谢状态转化到成人状态时都发生了改变,表明对应于慢性心肌应力(变化)而发生的不是一个简单的胎儿代谢调节的转变。心衰状态下代谢重塑的影响不但与脂肪酸供给的代谢底物的转变有关,而且还与代谢中的关键酶类的“胎儿化”有关。
可见,心衰实质上是由于能量不足造成基因表达异常而引起的一种超负荷性心肌病。对心衰心肌中高能磷酸化合物的检测也证实,与能量代谢障碍相伴的心室重构是心衰的主要病理生理学机制。能量代谢障碍贯穿于心肌从代偿性肥大到最终衰竭的全过程中,是心衰发生、发展和恶化的重要因素。鉴于心衰的发展与心肌机械收缩力学以及心肌力能学障碍和紊乱等众多因素有关,有学者提出可通过调整心肌能量代谢的方法来治疗心衰和提高心肌工作效率。纠正代谢失衡的疗法可能为心衰及心肌缺血等与力能学过程紊乱有关的疾病的治疗开辟出一条新的治疗途径。到目前为止,一些直接改善心肌能量代谢的药物已被证实能够提高心衰患者的心功能。因此,研究改善能量代谢的措施,对于防治心衰的发生发展具有重要意义,甚至Stephan等认为未来心衰治疗的一个目标就是改善心肌的能量代谢。
运用中药益气法治疗心衰的理念与改善衰竭心脏能量代谢有共通之处。从黄芪中所提取出的黄芪甲苷,黄芪多糖,黄芪总提物都对心功能改善均具有很好的作用,目前在临床上被广泛用于治疗心脏疾患。研究表明,黄芪甲苷主要是通过其抗氧化作用而保护心脏功能。虽然有研究展示黄芪等补气中药及/或有效成分也有调节其他组织器官脂质和糖代谢的功能,但对于其是否通过直接参与调解心肌细胞能量底物的选择、利用及产能而发挥其改善心脏功能尚无人涉足。
故研究从心肌机械收缩力学以及心肌力能学(能量代谢)的角度,通过比较黄芪总提物及黄芪有效成分治疗后的一系列变化,包括心肌收缩做功、心肌对能量底物如游离脂肪酸、葡萄糖、丙酮酸和乳酸等的选择及利用、心肌氧耗、心肌能量(底物)转运体、心肌能量代谢的关键酶类等的改变,系统探讨黄芪总提物及黄芪有效成分对阿霉素所致心衰的保护机制。从而为补气中药治疗心衰的机制从一个新的视角上提供更新的科学依据和研究示范。这种纠正能量代谢失衡的方法可能为中医药治疗心衰及心肌缺血等与力能学过程紊乱有关的疾病开辟出一条新的治疗途径。
目的
采用阿霉素致实验性心衰大鼠模型为对象,通过电生理技术,分子生物学技术,HPLC及试剂盒检测技术,从心肌收缩机械力学和力能学角度,观察包括心肌能量底物的选择、氧耗、中间产物的转运、能量代谢的关键酶类、能量的储存和利用等,系统探讨黄芪总提物及黄芪有效成分(黄芪皂苷/黄芪多糖等)保护实验性心衰心肌功能损伤的科学机制。
方法
本研究将SD大鼠分为7组,包括正常(Control)组、HF组、地高辛(Digoxigein,DG)、黄芪甲苷(Astragaloside, ASIV)组、黄芪多糖(Astragalus Polysaccharide, APS)组、黄芪总皂苷(Total Saponins of Astragalus, AST)组、黄芪总提物(Astragalus Extract Mixture, AEM)组,除HF组18只大鼠外,其余各组均12只,采用阿霉素(或称盐酸多柔比星,Adriamycin, ADR)制造心衰模型,分别给予生理盐水(正常组)及治疗药物(地高辛及黄芪单体,总提物),从能量代谢的角度观察其对HF的治疗作用及机制。实验主要包括以下疗效确定和作用机制研究两个部分。
1疗效确定
采用langendroff灌流系统,观察比较正常组、模型组及治疗组大鼠一般状态、生存率、心重/体重等基本状态及心脏机械力学指标,包括左心室收缩压(Left Ventricular Systolic Pressure, LVSP)、左心室舒张终末压(Left Ventricular End-diastolic Pressure, LVEDP)、心率(Heart Rate, HR)、左心室内压最大上升和下降速率(±dp/dtmax)等的变化,以确定黄芪总提物及黄芪有效成分对改善实验性心衰心脏功能的作用效果。
2机制研究
21黄芪总提物及黄芪有效成分改善心衰心脏功能的力能学基础
于前部分疗效确定工作的同时,分别检测比较各组大鼠心脏灌流前后的灌流液中能量底物包括葡萄糖、游离脂肪酸(Free Fatty Acid, FFA)和丙酮酸的摄取利用、氧分压以及乳酸生成量等的改变情况,并与心肌收缩功能相联系,从力能学角度进一步探讨。
2.2黄芪总提物及黄芪有效成分改善心衰心肌力能学的机制研究
在上述工作的基础上,进一步采用分子生物学、HPLC检测等技术手段,对心肌能量底物利用、能量的产生、转运及储存等相关的关键酶类、转运体以及能量储存量等进行检测并比较,主要包括如下内容：
2.2.1探讨黄芪总提物及黄芪有效成分是否通过影响能量代谢底物转运体而改善心脏功能的作用
分别检测包括游离脂肪酸转运过程中所涉及到的脂肪酸转运体(Fatty Acid Translocase, FAT)、转运体膜结合脂肪酸蛋白(Plasma Membrane-associated Fatty Acid Binding Protein, FABPpm)及位于线粒体膜上的肉毒碱棕榈酰转移酶(Carnitine Palmitoyltransferase, CPT)以及电子传递链过程中的脱偶联蛋白(Mitochondrial Uncoupling Protein, UCP)的基因表达。
2.2.2阐明黄芪总提物及黄芪有效成分是否通过影响能量代谢酶类而改善心脏功能的作用
检测包括糖酵解过程的关键酶6磷酸果糖激酶(Phosphofructokinase, PFK),丙酮酸激酶(Pyruvate Kinase, PK),脂肪酸β氧化过程中的关键酶中链辅酶A脱氢酶(Medium-chain acyl-CoA Dehydrogenase, MACD)、长链辅酶A脱氢酶(Long-chain acyl-CoA Dehydrogenase, LCAD)以及磷酸肌酸和肌酸转化过程中的酶一肌酸激酶(Creatine Kinase, CK)基因表达的变化。
2.2.3阐明黄芪总提物及黄芪有效成分是否通过影响能量利用及其储存而发挥作用
分别检测和计算能量池——磷酸肌酸(Phosphocreatine, PCr)、直接供能物质ATP及产生能量的底物ADP的变化。
结果
1疗效确定
机械力学指标的检测结果表明：黄芪总提物及黄芪有效成分均可明显改善大鼠一般状态,使HF症状减轻；除APS组外,均显著提高ADR致HF大鼠的存活率(P<0.01或P<0.05,)；除AEM组外,均能增加心脏/体重比值(P<0,01或P<0,05)；均能显著提高LVSP(P<0.01)及+dp/dtmax(除APS组P<0.05外,均P<0.01)；亦能显著提高心脏做功指标一心率脉压乘积(Rate-Pressure Product, RPP),与HF组比差异显著(P<0.01)；其中AEM组还可以增加HR(P<0.01),同时缩短T-dp/dtmax(O.Ol),表明其心脏收缩力学改善作用更为明显,且明显强于阳性对照药物DG(P<0.01或P<0.05)。有效成分ASIV和AST作用大致相仿,而APS组亦能显著提高LVSP及+dp/dt max,对HF具有一定的治疗作用。
2机制研究
2.1黄芪总提物及黄芪有效成分改善心衰心脏功能的力能学基础
本实验结果显示,阿霉素所致实验性心衰后心肌对能量代谢利用发生紊乱并产生障碍,心肌由利用游离脂肪酸转向利用葡萄糖为主,这与心肌机械收缩力学障碍相伴随。黄芪总提物及黄芪有效成分均可明显提高氧气的摄取和利用(P<0.05或P<0.01)；提高游离脂肪酸及丙酮酸的摄取和利用(P<0.01或P<005)；降低对葡萄糖的摄取和利用,提高做功与葡萄糖摄取的比值(P<0.01)；除APS外,均能提高做功与摄取丙酮酸之比(P<0.01)；显著减少乳酸堆积现象(P<0.01或P<0.05)；使得心肌做功与摄取葡萄糖比上FFA之比亦显著升高(P<001)；表明黄芪总提物及黄芪有效成分能在一定程度上缓解HF心肌能量代谢紊乱现象,其中ASIV组对游离脂肪酸的摄取,AEM组对葡萄糖的摄取改善作用更为明显(P<0.01)。
2.2黄芪总提物及黄芪有效成分改善心衰心肌力能学的机制
2.2.1黄芪总提物及黄芪有效成分对心衰心肌能量代谢酶类的影响
实验结果表明,心衰后心肌PFK含量显著升高,大约为Control组的1.3倍(P<0.01),但各治疗组与HF组比较均无统计学差异；各组大鼠心肌中PK的含量变化并不明显(P>0.05),提示该酶可能没有参与介导黄芪总提物及黄芪有效成分保护HF心肌的作用；HF组线粒体脂肪酸β氧化的关键酶类的含量都普遍降低,其中MCAD的表达约下降到Control组50%。ASIV及AEM组能够提高HF心肌中MCAD含量(P<0.01)；ASIV及AEM组同样显示出能够提高LACD基因表达的作用(P<0.01),使之趋近于Control组的水平,而APS组此作用不明显；HF组大鼠心肌中CKMB的含量显著升高(P<0.01),APS及AEM能够显著降低其含量(P<0.05)。
2.2.2黄芪总提物及黄芪有效成分对心衰心肌能量代谢底物转运体的影响
本实验结果表明,HF组中UCP表达升高,升幅约为Control组的50%；治疗组中APS及AEM组能显著降低UCP的含量(P<0.01),减少能量在传递过程中的损失；HF组CPT-I的含量显著下降,为正常组的0.32倍(P<0.01),而黄芪总提物及黄芪各有效成分均能显著增加其表达(P<0.01),尤其是ASIV组及AEM组比阳性对照药物DG组效果还要好(P<0.01),提示黄芪总提物及其有效成分可通过影响CPT的表达而起到保护心肌损伤的作用；HF组FAT-CD36表达显著下降,而治疗组中ASIV组及AEM组则可显著增加FAT-CD36含量(P<0.01),使之达到与Control组相仿的水平,而DG组及APS组与HF组差别不大,未见明显的统计学差异；对于FABP蛋白,HF后其含量显著降低(P<0.01),但黄芪总提物及黄芪各有效成分治疗组与HF组比较无统计学差异(P<0.05)。
223黄芪总提物及黄芪有效成分对心衰心肌能量利用及其储存的影响
实验结果表明,虽然ATP的含量在各组间变化不显著(P>0.05),但HF组与Control组比较,PCr的含量明显下降(P<0.01),这一结果提示HF虽然尚未严重影响ATP含量,但已经产生了从线粒体ATP向能量池PCr储存能力障碍。治疗组ASIV及AEM组能够显著增加HF状态下PCr含量(P<001),使其趋近于Control组,改善能量储存不足的现象；HF组大鼠心肌中ADP含量与正常组比较,亦见明显下降(P<0.01),AST则能够增加ADP含量(P<0.05),而其他治疗组如DG、 APS及AEM组等虽然也可小幅度提高ADP含量,但与HF组比较未见明显差异(P<0.05),因此不能肯定上述治疗组可以通过调整ADP含量来发挥其治疗和保护作用。
结论
1.黄芪总提物及黄芪有效成分均可明显改善ADR所致HF大鼠一般状态,增加心脏／体重比,可明显提高其存活率,改善心肌收缩机械力,进而改善其左心室做功状况。
2.HF后存在心肌能量底物利用的紊乱,出现游离脂肪酸供能代谢比例下降和葡萄糖代谢功能比例增加的“胚胎化”现象,上述心肌供能代谢的紊乱可能是HF心肌机械收缩功能受损的基础。
3黄芪总提物及黄芪有效成分均在很大程度上逆转ADR致HF过程中的各种能量代谢紊乱和底物利用障碍,改善HF心肌力能学,从而保护心衰受损的心肌,而无多余负性的血流动力学效应。
4.黄芪总提物及黄芪有效成分通过减少能量代谢底物转运体中UCP、增加CPT-I及FAT-CD36的表达,从而起到对ADR致HF的保护作用。
5.增加能量代谢酶类中MCAD、 LACD及减少CKMB的基因表达可能是黄芪总提物及黄芪有效成分纠正ADR所致HF时的心肌力能学紊乱的机制之一,由此而进一步改善其受损的心肌机械收缩力学性能。
6.提高线粒体ATP向能量池PCr的储存能力及调节ADP含量可能也参与介导了黄芪总提物及黄芪有效成分纠正ADR所致HF时的心肌力能学紊乱,从而进一步改善其受损的心肌机械收缩力学性能。
总之,黄芪总提物及黄芪有效成分可以通过调节阿霉素所致的实验性心衰心肌能量代谢过程中的关键酶类、能量代谢相关转运体以及能量产生/储存相关因素,从而纠正心衰时心肌能量底物选择和利用等力能学过程的紊乱,并最终改善心衰心肌的机械收缩力学性能。
Background
Heart failure (HF) refers to a serious phase of many cardiac diseases caused by various factors and is the final consequence of most cardiovascular diseases. HF is the leading cause of death and disability in the industrialized world. The mortality rate of HF is about50%, and although there has been a reduction in mortality from acute myocardial infarction over the last30years, there has been a concomitant rise in morbidity of HF. The syndrome of HF was described by Hippocrates over two millennia ago but therapeutic efficacy, even at present, is so poor that its death rate is always on high level. Thereby, finding out the new ways to enhance the therapeutic efficacy is very significant for the treatment of HF.
The incidence of HF is determined by a variety of factors, while a growing number of clinical and experimental studies have shown that there is a depletion of myocardial energy in HF. Myocardial energy metabolism plays an important role in the development of heart failure. In fact, one of the hallmarks of HF is lack of energy and obstruction of energy metabolism is one of the main indicators of HF. Modern research showed that in the progression of HF there are several crucial causes including energy deficiency, over-activity of metabolic enzymes and abnormal gene expression, which jointly induce the remodeling of left ventricle. In the fetal and newborn heart, glucose is the primary energy substrate for energy production, while fatty acid oxidation as a way to produce energy (ATP) in myocardium is at a low level, providing only a small proportion of overall ATP production. The heart is very reliant on glycolysis as a source of energy during this period due to the higher activities of the enzymes in the glycolytic pathway. However, after birth, there is a dramatic10-fold increase in fatty acid oxidation, which is accompanied by a parallel decrease in glycolytic rates. This is opposite to the switch towards reduced fatty acid oxidation and increased glucose oxidation observed in some forms of severe HF. Interestingly, if the newborn heart is subjected to a volume-overload hypertrophy, the expression and activity of key enzymes controlling fatty acid oxidation remain in the "fetal state". A number of key enzymes involved in fatty acid oxidation are altered in the transitions from fetal to adult metabolism, illustrating that there is not a simple switch to fetal metabolic regulation in response to chronic cardiac stress. Presumably, impact of the metabolic remodelling in failing heart is associated with not only the substrate metabolism switch about FFA supply but also the key enzyme convert to "fetal state".
Therefore, HF is actually an overloaded cardiomyopathy induced by the abnormal gene expression following the energy deficiency. It was confirmed by the study with measuring high energy phosphates in myocardium of the patient with HF that the ventricular remodeling accompanied with the disorder of energy metabolism is pathophysiologically the fundamental mechanism. Occurring in the whole course of HF development throughout from compensatory myocardial hypertrophy to terminal cardiac failure, disorder of energy metabolism plays an important role in the progression of HF. Based on the fact that both impairment of cardiac mechanical contractility and disorder of energy metabolism are the dominant manifestations of HF, it was proposed by the scientists that modulating myocardial energy metabolism may enhance the cardiac work efficiency so as to treat HF effectively. More and more studies showed that modulating myocardial substrate utilization can significantly improve LV function and mechanical efficiency of failed heart., suggesting that the metabolic modulation as a new therapeutic way is very significant for the treatment of HF and even other heart diseases like myocardial ischemia which is known to be also related to the disorder of energy metabolism.
Reinforcing qi to treat heart failure differentiated as the type of deficiency of cardiac qi in Chinese medicine is conceptually similar with modulating the disorder of energy metabolism. Astragalus membranaceus is a Chinese herbal medicine with function of supplementing qi. It was reported that Astragalus membranaceus can improve cardiac function significantly and has been widely using in the treatment of various heart diseases. Astragalus membranaceus contains various active components with cardiac effect, including astragalosides, polysaccharides and flavones.
As one of the primary active components of Astragulas, Astragaloside IV has been found to produce potent cardioprotective effects. Both in vivo and in vitro studies have provided evidences indicating that an antioxidant effect may be one of the underlying mechanisms by which astragaloside IV protects myocardium. Although there were previous studies involving the metabolic modulation in other organ or tissues by Astragalus membranaceus or its components, no any one of them was emphasized on exploring whether Astragalus membranaceus or its components can directly modulate the course of myocardial energetics such as the utilization of metabolic substrates in energy production so as to improve the function of failed heart.
With aim at systemically exploring the energetic mechanisms underlying the improvement of cardiac mechanical function produced by the extract and effective components of AEM, the present study was conducted to compare the various mechanic and energetic alterations before and after administrating the extract and the effective components of AEM. We compared and analyzed the changes in the utilization of energetic substrates by myocardium, partial pressure of oxygen (PO2), myocardial energetic enzymes and transporters for transportation of energy or energetic substrates in myocardium. It was anticipated that the evidence shown in present study could provide a scientific explanation from a new angle of view on the mechanisms underlying the improvement of cardiac mechanical contractility produced by qi-invigorating herbal medicine. Modulating the disorder of myocardial energetic metabolism may potentially be an effective new way to screen Chinese herbal medicine effective in the treatment of various diseases characterized with the disorder of energetic metabolism such as heart failure and myocardial ischemia.
Objective
As aforementioned, the present study was aimed at systemically exploring the energetic mechanisms underlying the improvement of cardiac mechanical function produced by the extract and the effective components of AEM, the present study was conducted to compare the various mechanic and energetic alterations before and after administrating the extract and the effective components of AEM. The study contains two parts of experiments. The actions of the extract and the effective components of AEM on the cardiac mechanic contractility in the rats with experimental heart failure were confirmed firstly with evaluating system of cardiac contractile function; based on the above confirmation of the effects produced by AEM, the second part of the study was conducted to address the energetic mechanism underlying the cardiac mechanic effect of AEM. The evidence provided by present study is prospected to be helpful to create a new way (with focus on modulating energetic metabolism) for screening more Chinese herbs which are effective in treating the heart diseases characterized with disorder of energetic metabolism.
Methods
Ninety male SD rats were randomized into7groups with18rats in HF group and12rats in each of other groups, including Control group, Digoxigein (DG) group, Astragaloside (ASIV) group, Astragalus Polysaccharide (APS) group, Total Saponins of Astragalus (AST) group and Astragalus Extract Mixture (AEM) group. The animals in all groups except Control group were treated with intraperitoneal injection of adriamycin once two days, while the animals in all groups except Control and HF groups were treated by intragastric administration of astragalus extract mixture and active components in the therapeutic groups at the same time. At the last day of treatment, the hearts were then isolated and perfused with modified Krebs buffer. Thereafter, two parts of experiments including effect confirming and mechanism analyzing ones were conducted.
1. In the first part of the experiments cardiovascular hemodynamic indices and the changes of substrate energy metabolism were measured to evaluate the actions of AEM and the effective components on the cardiac mechanic contractility in the rats with the experimental HF induced by ADR.
Animal survival rate (SR) was evaluated and the indices of cardiac mechanic contractility including left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), heart rate (HR), T-dp/dtmax and Rate-Pressure Product (RPP) were recorded by a transducer connecting with a cardiac catheter inserted into left ventricle.
2. In the second part of the experiments, the energetic mechanisms underlying the effects of AEM and the effective components in improvement of cardiac mechanic contractility of failed heart were explored. The changes in the utilization of energetic substrates by myocardium, partial pressure of oxygen (PO2), myocardial energetic enzymes and transporters for transportation of energy or substrates were detected.
2.1. Fundamental energetic mechanisms underlying the cardiac mechanic effects of AEM and the effective components of AS were addressed as follows:
By using of the techniques of biochemistry and HPLC etc., FFA, glucose, pyruvic acid and PO2in the perfusion buffer before and after perfusion and the lactic acid in perfusion buffer after perfusion were measured so as to explore the mechanisms mentioned above.
2.2. Further mechanisms underlying energetic alterations responsible for the production of cardiac mechanic effects of AEM and the effective components of AS were explored in the following experiments:
2.2.1. Effects of AEM and the effective components of AS on the transporters of the myocardial energetic metabolism in the failed heart induced by ADR
Also, the contents of the transporters for the transportation of energy and energetic substrates, including FAT, FABPpm, CPT and UCP protein were measured by Western Blotting technique to determine which one(s) is (are) involved in the mediation of the cardiac mechanic and energetic effects of AEM and the effective components of AS.
2.2.2. Effects of AEM and the effective components of AS on the key enzymes of the myocardial energetic metabolism in the failed heart induced by ADR
In this part of experiment, the contents or gene expression of the energetic metabolism-related key enzyme(s) in myocardium including PFK, PK, MACD, LCAD and CK were measured to determine which enzyme(s) is(are) involved in the mediation of the cardiac mechanic and energetic effects of AEM and the effective components of AS.
2.2.3. Effects of AEM and the effective components of AS on the myocardial energy storage in the failed heart induced by ADR
Furthermore, The content of ATP, ADP and PCr were also determined by HPLC to explore if the state of energy production and storage contribute to the mediation of the cardiac mechanic and energetic effects of AEM and the effective components of AS.
Results
1. Confirmation of the improvement of cardiac mechanic contractility produced by AEM and the effective components of AS in the rats with ADR-induced HF.
The results showed that the general physical condition, the ratio of heart weight over body weight and the survival rate were declined in HF group as compared with normal control group (P<0.05or P<0.01). An amelioration of the general physical condition was observed in all treating groups (P<0.01or P<0.05). The ratio of heart of weight over body weight was elevated in all treating groups except AEM group (P<0.05or P<0.01). In the HF group all the hemodynamic indices were impaired significantly as compared with normal control group (P<0.05or P<0.01); As compared with HF group, LVSP and+dp/dtmax were significantly enhanced in all treating groups (P<0.05or P<0.01). In addition, Rate-Pressure Product (RPP) was also improved significantly by AEM and the components of AS in comparison with that in HF group (P<0.01). Comparatively, the strongest cardiac mechanic effect was observed in AEM group among all of treating groups including DG group. Further experiment showed that AEM can even enhance HR and shorten T-dp/dtmax in the rats with HF. The results also showed that with similar cardiac mechanic effect of AST, APS significantly enhanced LVSP and+dp/dt max.
2. Exploration of the mechanisms underlying the effects of AEM and the effective components in the improvement of cardiac mechanic contractility of failed heart
2.1. Fundamental energetic mechanisms underlying the cardiac mechanic effects of AEM and the effective components of AS
The results showed that there was a metabolic disorder exhibited as a sharp switch away from fatty acid towards carbohydrate oxidation in HF group in comparison with normal control group. The switch of substrate uptake in HF group was evidenced by a sharp rise in glucose uptake and a fall in fatty acid uptake that coincided with contractile dysfunction. PO2was greatly enhanced by AEM and the effective components of AS(P <0.01or P<0.05as compared with HF group); An Obvious increase in the uptake of free fatty acid and pyruvic acid was found in all of treating groups(P<0.01or P<0.05); Likewise, as compared with HF group, the glucose uptake and the ratio of cardiac work (indicated as RPP) over glucose uptake were significantly reduced by AEM and the effective components of AS(P<0.01); The ratio of cardiac work over pyruvic acid uptake was raised in all treating groups except APS group(P<0.01as compared with HF group); In all treating groups, both the concentration of Lactic acid and the ratio of the uptake of glucose over the uptake of free fatty acid were reduced significantly(P <0.01or P<0.05). The data above suggests that the disorder in energetic metabolism in rats with ADR-induced HFwas significantly attenuated by AEM and the effective components of AS. Comparatively, the effect of AEM on modulation of glucose uptake and that of ASIV on regulation of FFA uptake were comparatively stronger than other effective components of AS respectively.
2.2. Further mechanisms underlying energetic alterations responsible for the production of cardiac mechanic effects of AEM and the effective components of AS
2.2.1. Effects of AEM and the effective components of AS on the transporters of the myocardial energetic metabolism in the failed heart induced by ADR
The results showed that the expression of UCP in HF group was elevated to1.5folds of that in control group; while the HF-induced enhancement of UCP expression was significantly diminished by APS and AEM respectively (P<0.01as compared with HF group).
In HF group CPT-I, a key transferase for energy production and transportation, was substantially reduced to a very low level as only32%of that in control group(P<0.01). As compared with HF group, the content of CPT-I was significantly enhanced by AEM and the effective components of AS (P<0.01). The CPT-I-enhancing effect in ASIV and AEM groups was even obviously stronger than that in DG group P<0.01), suggesting CPT-I is involved in the mediation of both cardiac mechanic and energetic effects produced by AEM and the components of AS. The content of FAT-CD36was reduced in HF group (P<0.01as compared with control group) and the HF-reduced FAT-CD36was significantly enhanced in ASIV and AEM group to the control level, while in DG and APS group the FAT-CD36content was not significantly different from that of HF group (P>0.05). The protein of FABP, measured also by Western Blotting technique, was diminished markedly in HF group (P<0.01as compared with control group). However, no statistical difference was shown in terms of FABE content between HF group and each of all treating groups including AEM, ASIV, APS and AST groups respectively (P>0.05).
2.2.2. Effects of AEM and the effective components of AS on the key enzymes of the myocardial energetic metabolism in the failed heart induced by ADR
The content of PFK in HF group increased to about1.3folds of that in normal control group (P<0.01). However, PFK in all the treating groups was not significantly different from that in HF group. There is no significant difference in content of PK among all groups. The results above suggest that PFK and PK are not involved in the mediation of both the cardiac mechanic and energetic effects produced by AEM and the effective components of AS.
As another important enzyme in the beta-oxidation of free fatty acid, MCAD was reduced in HF group, to about50%of that in control group. In ASIV and AEM groups both protein content of MCAD and the gene expression of LACD were enhanced respectively as compared with HF group (P<0.01). No significant effect on LACD was observed in APS group. Further results showed that CKMB content was enhanced obviously in HF group (P<0.01as compared with Control group). The HF-induced increment of CKMB was significantly attenuated by APS and AEM respectively (P<0.05).
2.2.3. Effects of AEM and the effective components of AS on the myocardial energy storage in the failed heart induced by ADR
The results achieved in present study showed that there was no significant difference in terms of ATP content among all groups. However, PCr content in HF group was significantly lower than that in control group (P<0.01), indicating that although ATP content wasn't influenced by HF, the capability of energy pool for energy storage which is manifested as a transition from ATP to PCr was impaired in the failed heart. PCr content in ASIV and AEM groups were recovered almost to the level of control group (P<0.01as compared with HF group). Myocardial ADP content in HF group was markedly reduced as compared with control group (P<0.01). The HF-reduced ADP content was significantly enhanced by AST (P<0.05), but was only mildly elevated by DG, APS and AEM without statistical significance as compared with HF group (P>0.05).
Conclusion
1. Under the condition of the ADR-induced experimental HF a bad general physical condition, reduced ratio of heart weight over body weight and impaired cardiac mechanic contractility are observed. AEM and the effective components of AS significantly improve the general physical condition, increase the ratio of heat/body weight and survival rate and enhance the cardiac mechanic contractility in the rats with ADR-induced HF;
2. A significant disorder of myocardial energetic metabolism is obvious in the ADR-induced heart failure. The disorder is actually an abnormal alteration in the uptake and use of energetic substrates including FFA, glucose, pyruvic acid, lactic acid and even oxygen. In the ADR-induced HF a so-called "fetal state" is shown as that myocardial uptake of FFA as the substrate for energy production is reduced while glucose uptake is concomitantly enhanced. The disorder of energetics may be responsible for the impairment cardiac mechanics as the energetic disorder is concomitant with mechanic impairment in myocardium.
3. The energetic disorder in the uptake of substrates is significantly ameliorated by AEM and the effective components of AS so as to improve the HF-impaired cardiac mechanic contractility.
4. AEM and the effective components of AS significantly attenuate the content of UCP, enhance protein expression of CPT-I and FAT-CD36, which may contribute to the improvement of the HF-induced energetic disorder.
5. Another mechanism underlying the improvement of the HF-induced energetic disorder is that AEM and the effective components of AS significantly modulate the contents of the key enzymes of energetic metabolism, including increase in MCAD and LACD and decrease in the gene expression of CKMB.
6. Elevating the PCr level by AEM and the components of AS means increase in the energy storage, which is also one of the mechanisms underlying the cardiac mechanic and energetic effects produced by AEM and the effective components of AS in the ADR-induced HF.
In summary, AEM and the effective components of AS significantly enhance the cardiac mechanic contractility in ADR-induced heart failure via modulating the myocardial key enzymes of energetic metabolism, transporters of energy and substrates and the energy storage so as to correct the energetic disorder including in particular the abnormal uptake of energetic substrates by myocardium.
心力衰竭(Heart Failure, HF)是一种由于各种器质性或功能性心脏疾病使心室或射血能力受损的综合症,其病死率约50%,已成为21世纪最重要的心血管病症。在过去三十年,急性心梗的病死率已经有所下降,但心衰的伴发率却持续升高,已严重影响了人类健康。对于心衰的了解,早在2000年前就被希腊Hippocrates所描述,但对其治疗,虽然取得了一定的进展,却一直不理想,因此,如何治疗心衰,阻止心衰的病程发展一直是医学研究的热点问题。
HF的发病是由多种因素决定的,早在1939年,Herrmann和Decherd发现慢性心衰心肌中肌酸含量明显降低,因而提出在慢性心衰时存在心肌能量的耗竭。目前越来越多的临床研究和实验研究均显示,衰竭的心脏都存在着心肌能量的耗竭,因而能量代谢在心衰进展中起着非常重要的作用。事实上,心肌能量代谢障碍是心衰的重要标志之一。研究显示,心肌能量缺乏、代谢酶类过表达以及基因的异常能够引起左心室重塑,是心衰进展中必不可少的因素。在胎儿时期,葡萄糖作为代谢底物的主要供能者,游离脂肪酸氧化率很低。提供ATP的量很少,心脏中的糖酵解是能量的主要来源,并且糖酵解酶类具有更高的活性。出生后游离脂肪酸的氧化率增长了10倍,同时葡萄糖的氧化率下降,而心衰晚期游离脂肪酸氧化减少葡萄糖氧化增加,这恰好和一些研究观察到的结果相反。如果新生儿罹患心肌肥大,脂肪酸氧化的关键酶类活性及表达将保持“胎儿状态”。许多参与脂肪酸氧化的关键酶类在从胎儿代谢状态转化到成人状态时都发生了改变,表明对应于慢性心肌应力(变化)而发生的不是一个简单的胎儿代谢调节的转变。心衰状态下代谢重塑的影响不但与脂肪酸供给的代谢底物的转变有关,而且还与代谢中的关键酶类的“胎儿化”有关。
可见,心衰实质上是由于能量不足造成基因表达异常而引起的一种超负荷性心肌病。对心衰心肌中高能磷酸化合物的检测也证实,与能量代谢障碍相伴的心室重构是心衰的主要病理生理学机制。能量代谢障碍贯穿于心肌从代偿性肥大到最终衰竭的全过程中,是心衰发生、发展和恶化的重要因素。鉴于心衰的发展与心肌机械收缩力学以及心肌力能学障碍和紊乱等众多因素有关,有学者提出可通过调整心肌能量代谢的方法来治疗心衰和提高心肌工作效率。纠正代谢失衡的疗法可能为心衰及心肌缺血等与力能学过程紊乱有关的疾病的治疗开辟出一条新的治疗途径。到目前为止,一些直接改善心肌能量代谢的药物已被证实能够提高心衰患者的心功能。因此,研究改善能量代谢的措施,对于防治心衰的发生发展具有重要意义,甚至Stephan等认为未来心衰治疗的一个目标就是改善心肌的能量代谢。
运用中药益气法治疗心衰的理念与改善衰竭心脏能量代谢有共通之处。从黄芪中所提取出的黄芪甲苷,黄芪多糖,黄芪总提物都对心功能改善均具有很好的作用,目前在临床上被广泛用于治疗心脏疾患。研究表明,黄芪甲苷主要是通过其抗氧化作用而保护心脏功能。虽然有研究展示黄芪等补气中药及/或有效成分也有调节其他组织器官脂质和糖代谢的功能,但对于其是否通过直接参与调解心肌细胞能量底物的选择、利用及产能而发挥其改善心脏功能尚无人涉足。
故研究从心肌机械收缩力学以及心肌力能学(能量代谢)的角度,通过比较黄芪总提物及黄芪有效成分治疗后的一系列变化,包括心肌收缩做功、心肌对能量底物如游离脂肪酸、葡萄糖、丙酮酸和乳酸等的选择及利用、心肌氧耗、心肌能量(底物)转运体、心肌能量代谢的关键酶类等的改变,系统探讨黄芪总提物及黄芪有效成分对阿霉素所致心衰的保护机制。从而为补气中药治疗心衰的机制从一个新的视角上提供更新的科学依据和研究示范。这种纠正能量代谢失衡的方法可能为中医药治疗心衰及心肌缺血等与力能学过程紊乱有关的疾病开辟出一条新的治疗途径。
目的
采用阿霉素致实验性心衰大鼠模型为对象,通过电生理技术,分子生物学技术,HPLC及试剂盒检测技术,从心肌收缩机械力学和力能学角度,观察包括心肌能量底物的选择、氧耗、中间产物的转运、能量代谢的关键酶类、能量的储存和利用等,系统探讨黄芪总提物及黄芪有效成分(黄芪皂苷/黄芪多糖等)保护实验性心衰心肌功能损伤的科学机制。
方法
本研究将SD大鼠分为7组,包括正常(Control)组、HF组、地高辛(Digoxigein,DG)、黄芪甲苷(Astragaloside, ASIV)组、黄芪多糖(Astragalus Polysaccharide, APS)组、黄芪总皂苷(Total Saponins of Astragalus, AST)组、黄芪总提物(Astragalus Extract Mixture, AEM)组,除HF组18只大鼠外,其余各组均12只,采用阿霉素(或称盐酸多柔比星,Adriamycin, ADR)制造心衰模型,分别给予生理盐水(正常组)及治疗药物(地高辛及黄芪单体,总提物),从能量代谢的角度观察其对HF的治疗作用及机制。实验主要包括以下疗效确定和作用机制研究两个部分。
1疗效确定
采用langendroff灌流系统,观察比较正常组、模型组及治疗组大鼠一般状态、生存率、心重/体重等基本状态及心脏机械力学指标,包括左心室收缩压(Left Ventricular Systolic Pressure, LVSP)、左心室舒张终末压(Left Ventricular End-diastolic Pressure, LVEDP)、心率(Heart Rate, HR)、左心室内压最大上升和下降速率(±dp/dtmax)等的变化,以确定黄芪总提物及黄芪有效成分对改善实验性心衰心脏功能的作用效果。
2机制研究
21黄芪总提物及黄芪有效成分改善心衰心脏功能的力能学基础
于前部分疗效确定工作的同时,分别检测比较各组大鼠心脏灌流前后的灌流液中能量底物包括葡萄糖、游离脂肪酸(Free Fatty Acid, FFA)和丙酮酸的摄取利用、氧分压以及乳酸生成量等的改变情况,并与心肌收缩功能相联系,从力能学角度进一步探讨。
2.2黄芪总提物及黄芪有效成分改善心衰心肌力能学的机制研究
在上述工作的基础上,进一步采用分子生物学、HPLC检测等技术手段,对心肌能量底物利用、能量的产生、转运及储存等相关的关键酶类、转运体以及能量储存量等进行检测并比较,主要包括如下内容：
2.2.1探讨黄芪总提物及黄芪有效成分是否通过影响能量代谢底物转运体而改善心脏功能的作用
分别检测包括游离脂肪酸转运过程中所涉及到的脂肪酸转运体(Fatty Acid Translocase, FAT)、转运体膜结合脂肪酸蛋白(Plasma Membrane-associated Fatty Acid Binding Protein, FABPpm)及位于线粒体膜上的肉毒碱棕榈酰转移酶(Carnitine Palmitoyltransferase, CPT)以及电子传递链过程中的脱偶联蛋白(Mitochondrial Uncoupling Protein, UCP)的基因表达。
2.2.2阐明黄芪总提物及黄芪有效成分是否通过影响能量代谢酶类而改善心脏功能的作用
检测包括糖酵解过程的关键酶6磷酸果糖激酶(Phosphofructokinase, PFK),丙酮酸激酶(Pyruvate Kinase, PK),脂肪酸β氧化过程中的关键酶中链辅酶A脱氢酶(Medium-chain acyl-CoA Dehydrogenase, MACD)、长链辅酶A脱氢酶(Long-chain acyl-CoA Dehydrogenase, LCAD)以及磷酸肌酸和肌酸转化过程中的酶一肌酸激酶(Creatine Kinase, CK)基因表达的变化。
2.2.3阐明黄芪总提物及黄芪有效成分是否通过影响能量利用及其储存而发挥作用
分别检测和计算能量池——磷酸肌酸(Phosphocreatine, PCr)、直接供能物质ATP及产生能量的底物ADP的变化。
结果
1疗效确定
机械力学指标的检测结果表明：黄芪总提物及黄芪有效成分均可明显改善大鼠一般状态,使HF症状减轻；除APS组外,均显著提高ADR致HF大鼠的存活率(P<0.01或P<0.05,)；除AEM组外,均能增加心脏/体重比值(P<0,01或P<0,05)；均能显著提高LVSP(P<0.01)及+dp/dtmax(除APS组P<0.05外,均P<0.01)；亦能显著提高心脏做功指标一心率脉压乘积(Rate-Pressure Product, RPP),与HF组比差异显著(P<0.01)；其中AEM组还可以增加HR(P<0.01),同时缩短T-dp/dtmax(O.Ol),表明其心脏收缩力学改善作用更为明显,且明显强于阳性对照药物DG(P<0.01或P<0.05)。有效成分ASIV和AST作用大致相仿,而APS组亦能显著提高LVSP及+dp/dt max,对HF具有一定的治疗作用。
2机制研究
2.1黄芪总提物及黄芪有效成分改善心衰心脏功能的力能学基础
本实验结果显示,阿霉素所致实验性心衰后心肌对能量代谢利用发生紊乱并产生障碍,心肌由利用游离脂肪酸转向利用葡萄糖为主,这与心肌机械收缩力学障碍相伴随。黄芪总提物及黄芪有效成分均可明显提高氧气的摄取和利用(P<0.05或P<0.01)；提高游离脂肪酸及丙酮酸的摄取和利用(P<0.01或P<005)；降低对葡萄糖的摄取和利用,提高做功与葡萄糖摄取的比值(P<0.01)；除APS外,均能提高做功与摄取丙酮酸之比(P<0.01)；显著减少乳酸堆积现象(P<0.01或P<0.05)；使得心肌做功与摄取葡萄糖比上FFA之比亦显著升高(P<001)；表明黄芪总提物及黄芪有效成分能在一定程度上缓解HF心肌能量代谢紊乱现象,其中ASIV组对游离脂肪酸的摄取,AEM组对葡萄糖的摄取改善作用更为明显(P<0.01)。
2.2黄芪总提物及黄芪有效成分改善心衰心肌力能学的机制
2.2.1黄芪总提物及黄芪有效成分对心衰心肌能量代谢酶类的影响
实验结果表明,心衰后心肌PFK含量显著升高,大约为Control组的1.3倍(P<0.01),但各治疗组与HF组比较均无统计学差异；各组大鼠心肌中PK的含量变化并不明显(P>0.05),提示该酶可能没有参与介导黄芪总提物及黄芪有效成分保护HF心肌的作用；HF组线粒体脂肪酸β氧化的关键酶类的含量都普遍降低,其中MCAD的表达约下降到Control组50%。ASIV及AEM组能够提高HF心肌中MCAD含量(P<0.01)；ASIV及AEM组同样显示出能够提高LACD基因表达的作用(P<0.01),使之趋近于Control组的水平,而APS组此作用不明显；HF组大鼠心肌中CKMB的含量显著升高(P<0.01),APS及AEM能够显著降低其含量(P<0.05)。
2.2.2黄芪总提物及黄芪有效成分对心衰心肌能量代谢底物转运体的影响
本实验结果表明,HF组中UCP表达升高,升幅约为Control组的50%；治疗组中APS及AEM组能显著降低UCP的含量(P<0.01),减少能量在传递过程中的损失；HF组CPT-I的含量显著下降,为正常组的0.32倍(P<0.01),而黄芪总提物及黄芪各有效成分均能显著增加其表达(P<0.01),尤其是ASIV组及AEM组比阳性对照药物DG组效果还要好(P<0.01),提示黄芪总提物及其有效成分可通过影响CPT的表达而起到保护心肌损伤的作用；HF组FAT-CD36表达显著下降,而治疗组中ASIV组及AEM组则可显著增加FAT-CD36含量(P<0.01),使之达到与Control组相仿的水平,而DG组及APS组与HF组差别不大,未见明显的统计学差异；对于FABP蛋白,HF后其含量显著降低(P<0.01),但黄芪总提物及黄芪各有效成分治疗组与HF组比较无统计学差异(P<0.05)。
223黄芪总提物及黄芪有效成分对心衰心肌能量利用及其储存的影响
实验结果表明,虽然ATP的含量在各组间变化不显著(P>0.05),但HF组与Control组比较,PCr的含量明显下降(P<0.01),这一结果提示HF虽然尚未严重影响ATP含量,但已经产生了从线粒体ATP向能量池PCr储存能力障碍。治疗组ASIV及AEM组能够显著增加HF状态下PCr含量(P<001),使其趋近于Control组,改善能量储存不足的现象；HF组大鼠心肌中ADP含量与正常组比较,亦见明显下降(P<0.01),AST则能够增加ADP含量(P<0.05),而其他治疗组如DG、 APS及AEM组等虽然也可小幅度提高ADP含量,但与HF组比较未见明显差异(P<0.05),因此不能肯定上述治疗组可以通过调整ADP含量来发挥其治疗和保护作用。
结论
1.黄芪总提物及黄芪有效成分均可明显改善ADR所致HF大鼠一般状态,增加心脏／体重比,可明显提高其存活率,改善心肌收缩机械力,进而改善其左心室做功状况。
2.HF后存在心肌能量底物利用的紊乱,出现游离脂肪酸供能代谢比例下降和葡萄糖代谢功能比例增加的“胚胎化”现象,上述心肌供能代谢的紊乱可能是HF心肌机械收缩功能受损的基础。
3黄芪总提物及黄芪有效成分均在很大程度上逆转ADR致HF过程中的各种能量代谢紊乱和底物利用障碍,改善HF心肌力能学,从而保护心衰受损的心肌,而无多余负性的血流动力学效应。
4.黄芪总提物及黄芪有效成分通过减少能量代谢底物转运体中UCP、增加CPT-I及FAT-CD36的表达,从而起到对ADR致HF的保护作用。
5.增加能量代谢酶类中MCAD、 LACD及减少CKMB的基因表达可能是黄芪总提物及黄芪有效成分纠正ADR所致HF时的心肌力能学紊乱的机制之一,由此而进一步改善其受损的心肌机械收缩力学性能。
6.提高线粒体ATP向能量池PCr的储存能力及调节ADP含量可能也参与介导了黄芪总提物及黄芪有效成分纠正ADR所致HF时的心肌力能学紊乱,从而进一步改善其受损的心肌机械收缩力学性能。
总之,黄芪总提物及黄芪有效成分可以通过调节阿霉素所致的实验性心衰心肌能量代谢过程中的关键酶类、能量代谢相关转运体以及能量产生/储存相关因素,从而纠正心衰时心肌能量底物选择和利用等力能学过程的紊乱,并最终改善心衰心肌的机械收缩力学性能。
Background
Heart failure (HF) refers to a serious phase of many cardiac diseases caused by various factors and is the final consequence of most cardiovascular diseases. HF is the leading cause of death and disability in the industrialized world. The mortality rate of HF is about50%, and although there has been a reduction in mortality from acute myocardial infarction over the last30years, there has been a concomitant rise in morbidity of HF. The syndrome of HF was described by Hippocrates over two millennia ago but therapeutic efficacy, even at present, is so poor that its death rate is always on high level. Thereby, finding out the new ways to enhance the therapeutic efficacy is very significant for the treatment of HF.
The incidence of HF is determined by a variety of factors, while a growing number of clinical and experimental studies have shown that there is a depletion of myocardial energy in HF. Myocardial energy metabolism plays an important role in the development of heart failure. In fact, one of the hallmarks of HF is lack of energy and obstruction of energy metabolism is one of the main indicators of HF. Modern research showed that in the progression of HF there are several crucial causes including energy deficiency, over-activity of metabolic enzymes and abnormal gene expression, which jointly induce the remodeling of left ventricle. In the fetal and newborn heart, glucose is the primary energy substrate for energy production, while fatty acid oxidation as a way to produce energy (ATP) in myocardium is at a low level, providing only a small proportion of overall ATP production. The heart is very reliant on glycolysis as a source of energy during this period due to the higher activities of the enzymes in the glycolytic pathway. However, after birth, there is a dramatic10-fold increase in fatty acid oxidation, which is accompanied by a parallel decrease in glycolytic rates. This is opposite to the switch towards reduced fatty acid oxidation and increased glucose oxidation observed in some forms of severe HF. Interestingly, if the newborn heart is subjected to a volume-overload hypertrophy, the expression and activity of key enzymes controlling fatty acid oxidation remain in the "fetal state". A number of key enzymes involved in fatty acid oxidation are altered in the transitions from fetal to adult metabolism, illustrating that there is not a simple switch to fetal metabolic regulation in response to chronic cardiac stress. Presumably, impact of the metabolic remodelling in failing heart is associated with not only the substrate metabolism switch about FFA supply but also the key enzyme convert to "fetal state".
Therefore, HF is actually an overloaded cardiomyopathy induced by the abnormal gene expression following the energy deficiency. It was confirmed by the study with measuring high energy phosphates in myocardium of the patient with HF that the ventricular remodeling accompanied with the disorder of energy metabolism is pathophysiologically the fundamental mechanism. Occurring in the whole course of HF development throughout from compensatory myocardial hypertrophy to terminal cardiac failure, disorder of energy metabolism plays an important role in the progression of HF. Based on the fact that both impairment of cardiac mechanical contractility and disorder of energy metabolism are the dominant manifestations of HF, it was proposed by the scientists that modulating myocardial energy metabolism may enhance the cardiac work efficiency so as to treat HF effectively. More and more studies showed that modulating myocardial substrate utilization can significantly improve LV function and mechanical efficiency of failed heart., suggesting that the metabolic modulation as a new therapeutic way is very significant for the treatment of HF and even other heart diseases like myocardial ischemia which is known to be also related to the disorder of energy metabolism.
Reinforcing qi to treat heart failure differentiated as the type of deficiency of cardiac qi in Chinese medicine is conceptually similar with modulating the disorder of energy metabolism. Astragalus membranaceus is a Chinese herbal medicine with function of supplementing qi. It was reported that Astragalus membranaceus can improve cardiac function significantly and has been widely using in the treatment of various heart diseases. Astragalus membranaceus contains various active components with cardiac effect, including astragalosides, polysaccharides and flavones.
As one of the primary active components of Astragulas, Astragaloside IV has been found to produce potent cardioprotective effects. Both in vivo and in vitro studies have provided evidences indicating that an antioxidant effect may be one of the underlying mechanisms by which astragaloside IV protects myocardium. Although there were previous studies involving the metabolic modulation in other organ or tissues by Astragalus membranaceus or its components, no any one of them was emphasized on exploring whether Astragalus membranaceus or its components can directly modulate the course of myocardial energetics such as the utilization of metabolic substrates in energy production so as to improve the function of failed heart.
With aim at systemically exploring the energetic mechanisms underlying the improvement of cardiac mechanical function produced by the extract and effective components of AEM, the present study was conducted to compare the various mechanic and energetic alterations before and after administrating the extract and the effective components of AEM. We compared and analyzed the changes in the utilization of energetic substrates by myocardium, partial pressure of oxygen (PO2), myocardial energetic enzymes and transporters for transportation of energy or energetic substrates in myocardium. It was anticipated that the evidence shown in present study could provide a scientific explanation from a new angle of view on the mechanisms underlying the improvement of cardiac mechanical contractility produced by qi-invigorating herbal medicine. Modulating the disorder of myocardial energetic metabolism may potentially be an effective new way to screen Chinese herbal medicine effective in the treatment of various diseases characterized with the disorder of energetic metabolism such as heart failure and myocardial ischemia.
Objective
As aforementioned, the present study was aimed at systemically exploring the energetic mechanisms underlying the improvement of cardiac mechanical function produced by the extract and the effective components of AEM, the present study was conducted to compare the various mechanic and energetic alterations before and after administrating the extract and the effective components of AEM. The study contains two parts of experiments. The actions of the extract and the effective components of AEM on the cardiac mechanic contractility in the rats with experimental heart failure were confirmed firstly with evaluating system of cardiac contractile function; based on the above confirmation of the effects produced by AEM, the second part of the study was conducted to address the energetic mechanism underlying the cardiac mechanic effect of AEM. The evidence provided by present study is prospected to be helpful to create a new way (with focus on modulating energetic metabolism) for screening more Chinese herbs which are effective in treating the heart diseases characterized with disorder of energetic metabolism.
Methods
Ninety male SD rats were randomized into7groups with18rats in HF group and12rats in each of other groups, including Control group, Digoxigein (DG) group, Astragaloside (ASIV) group, Astragalus Polysaccharide (APS) group, Total Saponins of Astragalus (AST) group and Astragalus Extract Mixture (AEM) group. The animals in all groups except Control group were treated with intraperitoneal injection of adriamycin once two days, while the animals in all groups except Control and HF groups were treated by intragastric administration of astragalus extract mixture and active components in the therapeutic groups at the same time. At the last day of treatment, the hearts were then isolated and perfused with modified Krebs buffer. Thereafter, two parts of experiments including effect confirming and mechanism analyzing ones were conducted.
1. In the first part of the experiments cardiovascular hemodynamic indices and the changes of substrate energy metabolism were measured to evaluate the actions of AEM and the effective components on the cardiac mechanic contractility in the rats with the experimental HF induced by ADR.
Animal survival rate (SR) was evaluated and the indices of cardiac mechanic contractility including left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), heart rate (HR), T-dp/dtmax and Rate-Pressure Product (RPP) were recorded by a transducer connecting with a cardiac catheter inserted into left ventricle.
2. In the second part of the experiments, the energetic mechanisms underlying the effects of AEM and the effective components in improvement of cardiac mechanic contractility of failed heart were explored. The changes in the utilization of energetic substrates by myocardium, partial pressure of oxygen (PO2), myocardial energetic enzymes and transporters for transportation of energy or substrates were detected.
2.1. Fundamental energetic mechanisms underlying the cardiac mechanic effects of AEM and the effective components of AS were addressed as follows:
By using of the techniques of biochemistry and HPLC etc., FFA, glucose, pyruvic acid and PO2in the perfusion buffer before and after perfusion and the lactic acid in perfusion buffer after perfusion were measured so as to explore the mechanisms mentioned above.
2.2. Further mechanisms underlying energetic alterations responsible for the production of cardiac mechanic effects of AEM and the effective components of AS were explored in the following experiments:
2.2.1. Effects of AEM and the effective components of AS on the transporters of the myocardial energetic metabolism in the failed heart induced by ADR
Also, the contents of the transporters for the transportation of energy and energetic substrates, including FAT, FABPpm, CPT and UCP protein were measured by Western Blotting technique to determine which one(s) is (are) involved in the mediation of the cardiac mechanic and energetic effects of AEM and the effective components of AS.
2.2.2. Effects of AEM and the effective components of AS on the key enzymes of the myocardial energetic metabolism in the failed heart induced by ADR
In this part of experiment, the contents or gene expression of the energetic metabolism-related key enzyme(s) in myocardium including PFK, PK, MACD, LCAD and CK were measured to determine which enzyme(s) is(are) involved in the mediation of the cardiac mechanic and energetic effects of AEM and the effective components of AS.
2.2.3. Effects of AEM and the effective components of AS on the myocardial energy storage in the failed heart induced by ADR
Furthermore, The content of ATP, ADP and PCr were also determined by HPLC to explore if the state of energy production and storage contribute to the mediation of the cardiac mechanic and energetic effects of AEM and the effective components of AS.
Results
1. Confirmation of the improvement of cardiac mechanic contractility produced by AEM and the effective components of AS in the rats with ADR-induced HF.
The results showed that the general physical condition, the ratio of heart weight over body weight and the survival rate were declined in HF group as compared with normal control group (P<0.05or P<0.01). An amelioration of the general physical condition was observed in all treating groups (P<0.01or P<0.05). The ratio of heart of weight over body weight was elevated in all treating groups except AEM group (P<0.05or P<0.01). In the HF group all the hemodynamic indices were impaired significantly as compared with normal control group (P<0.05or P<0.01); As compared with HF group, LVSP and+dp/dtmax were significantly enhanced in all treating groups (P<0.05or P<0.01). In addition, Rate-Pressure Product (RPP) was also improved significantly by AEM and the components of AS in comparison with that in HF group (P<0.01). Comparatively, the strongest cardiac mechanic effect was observed in AEM group among all of treating groups including DG group. Further experiment showed that AEM can even enhance HR and shorten T-dp/dtmax in the rats with HF. The results also showed that with similar cardiac mechanic effect of AST, APS significantly enhanced LVSP and+dp/dt max.
2. Exploration of the mechanisms underlying the effects of AEM and the effective components in the improvement of cardiac mechanic contractility of failed heart
2.1. Fundamental energetic mechanisms underlying the cardiac mechanic effects of AEM and the effective components of AS
The results showed that there was a metabolic disorder exhibited as a sharp switch away from fatty acid towards carbohydrate oxidation in HF group in comparison with normal control group. The switch of substrate uptake in HF group was evidenced by a sharp rise in glucose uptake and a fall in fatty acid uptake that coincided with contractile dysfunction. PO2was greatly enhanced by AEM and the effective components of AS(P <0.01or P<0.05as compared with HF group); An Obvious increase in the uptake of free fatty acid and pyruvic acid was found in all of treating groups(P<0.01or P<0.05); Likewise, as compared with HF group, the glucose uptake and the ratio of cardiac work (indicated as RPP) over glucose uptake were significantly reduced by AEM and the effective components of AS(P<0.01); The ratio of cardiac work over pyruvic acid uptake was raised in all treating groups except APS group(P<0.01as compared with HF group); In all treating groups, both the concentration of Lactic acid and the ratio of the uptake of glucose over the uptake of free fatty acid were reduced significantly(P <0.01or P<0.05). The data above suggests that the disorder in energetic metabolism in rats with ADR-induced HFwas significantly attenuated by AEM and the effective components of AS. Comparatively, the effect of AEM on modulation of glucose uptake and that of ASIV on regulation of FFA uptake were comparatively stronger than other effective components of AS respectively.
2.2. Further mechanisms underlying energetic alterations responsible for the production of cardiac mechanic effects of AEM and the effective components of AS
2.2.1. Effects of AEM and the effective components of AS on the transporters of the myocardial energetic metabolism in the failed heart induced by ADR
The results showed that the expression of UCP in HF group was elevated to1.5folds of that in control group; while the HF-induced enhancement of UCP expression was significantly diminished by APS and AEM respectively (P<0.01as compared with HF group).
In HF group CPT-I, a key transferase for energy production and transportation, was substantially reduced to a very low level as only32%of that in control group(P<0.01). As compared with HF group, the content of CPT-I was significantly enhanced by AEM and the effective components of AS (P<0.01). The CPT-I-enhancing effect in ASIV and AEM groups was even obviously stronger than that in DG group P<0.01), suggesting CPT-I is involved in the mediation of both cardiac mechanic and energetic effects produced by AEM and the components of AS. The content of FAT-CD36was reduced in HF group (P<0.01as compared with control group) and the HF-reduced FAT-CD36was significantly enhanced in ASIV and AEM group to the control level, while in DG and APS group the FAT-CD36content was not significantly different from that of HF group (P>0.05). The protein of FABP, measured also by Western Blotting technique, was diminished markedly in HF group (P<0.01as compared with control group). However, no statistical difference was shown in terms of FABE content between HF group and each of all treating groups including AEM, ASIV, APS and AST groups respectively (P>0.05).
2.2.2. Effects of AEM and the effective components of AS on the key enzymes of the myocardial energetic metabolism in the failed heart induced by ADR
The content of PFK in HF group increased to about1.3folds of that in normal control group (P<0.01). However, PFK in all the treating groups was not significantly different from that in HF group. There is no significant difference in content of PK among all groups. The results above suggest that PFK and PK are not involved in the mediation of both the cardiac mechanic and energetic effects produced by AEM and the effective components of AS.
As another important enzyme in the beta-oxidation of free fatty acid, MCAD was reduced in HF group, to about50%of that in control group. In ASIV and AEM groups both protein content of MCAD and the gene expression of LACD were enhanced respectively as compared with HF group (P<0.01). No significant effect on LACD was observed in APS group. Further results showed that CKMB content was enhanced obviously in HF group (P<0.01as compared with Control group). The HF-induced increment of CKMB was significantly attenuated by APS and AEM respectively (P<0.05).
2.2.3. Effects of AEM and the effective components of AS on the myocardial energy storage in the failed heart induced by ADR
The results achieved in present study showed that there was no significant difference in terms of ATP content among all groups. However, PCr content in HF group was significantly lower than that in control group (P<0.01), indicating that although ATP content wasn't influenced by HF, the capability of energy pool for energy storage which is manifested as a transition from ATP to PCr was impaired in the failed heart. PCr content in ASIV and AEM groups were recovered almost to the level of control group (P<0.01as compared with HF group). Myocardial ADP content in HF group was markedly reduced as compared with control group (P<0.01). The HF-reduced ADP content was significantly enhanced by AST (P<0.05), but was only mildly elevated by DG, APS and AEM without statistical significance as compared with HF group (P>0.05).
Conclusion
1. Under the condition of the ADR-induced experimental HF a bad general physical condition, reduced ratio of heart weight over body weight and impaired cardiac mechanic contractility are observed. AEM and the effective components of AS significantly improve the general physical condition, increase the ratio of heat/body weight and survival rate and enhance the cardiac mechanic contractility in the rats with ADR-induced HF;
2. A significant disorder of myocardial energetic metabolism is obvious in the ADR-induced heart failure. The disorder is actually an abnormal alteration in the uptake and use of energetic substrates including FFA, glucose, pyruvic acid, lactic acid and even oxygen. In the ADR-induced HF a so-called "fetal state" is shown as that myocardial uptake of FFA as the substrate for energy production is reduced while glucose uptake is concomitantly enhanced. The disorder of energetics may be responsible for the impairment cardiac mechanics as the energetic disorder is concomitant with mechanic impairment in myocardium.
3. The energetic disorder in the uptake of substrates is significantly ameliorated by AEM and the effective components of AS so as to improve the HF-impaired cardiac mechanic contractility.
4. AEM and the effective components of AS significantly attenuate the content of UCP, enhance protein expression of CPT-I and FAT-CD36, which may contribute to the improvement of the HF-induced energetic disorder.
5. Another mechanism underlying the improvement of the HF-induced energetic disorder is that AEM and the effective components of AS significantly modulate the contents of the key enzymes of energetic metabolism, including increase in MCAD and LACD and decrease in the gene expression of CKMB.
6. Elevating the PCr level by AEM and the components of AS means increase in the energy storage, which is also one of the mechanisms underlying the cardiac mechanic and energetic effects produced by AEM and the effective components of AS in the ADR-induced HF.
In summary, AEM and the effective components of AS significantly enhance the cardiac mechanic contractility in ADR-induced heart failure via modulating the myocardial key enzymes of energetic metabolism, transporters of energy and substrates and the energy storage so as to correct the energetic disorder including in particular the abnormal uptake of energetic substrates by myocardium.
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