芪苈强心胶囊对心力衰竭微血管损伤、心室重构及代谢重构的影响
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摘要
目的:本研究以脉络学说营卫理论为指导，采用胸主动脉缩窄术建立压力超负荷大鼠模型，通过观察心衰时心脏微血管结构与功能损伤、心肌凋亡和纤维化、心肌线粒体为核心的糖脂能量代谢异常，初步揭示微血管病变在心衰发病中的重要作用及其对心室重构、代谢重构的影响与芪苈强心胶囊干预作用；采用灌注衰竭大鼠心脏模型，进一步观察芪苈强心胶囊对能量代谢AMPK/PPARα信号通路的干预作用和对线粒体相关蛋白的影响。通过上述整体与离体实验研究，探讨微血管损伤、心室重构、代谢重构在心力衰竭发病中的影响及芪苈强心胶囊干预作用，为揭示脉络学说营卫“由络以通、交会生化”理论的科学内涵提供实验依据。
方法:
本研究分为以下四部分内容：
1心力衰竭大鼠心肌微血管损伤及芪苈强心胶囊干预作用研究
以SD大鼠为研究对象，采用胸主动脉缩窄术建立压力超负荷致心力衰竭模型，分为正常对照组（Control）、假手术组（Sham）、模型组（Model）、卡托普利组（Captopril）、芪苈强心低、中、高三个剂量组（QLQX-L、QLQX-M、QLQX-H），共7组，每组15只，灌胃给药1次/日，连续6周，正常对照组、假手术和模型组给予同体积羧甲基纤维素钠（CMC-Na）溶剂，给药量均为10ml/kg体重。末次给药后禁食12h，各组大鼠行颈动脉插管观察血流动力学变化；放免法检测N端脑钠肽前体（NT-proBNP）、血管紧张素Ⅱ（AngⅡ）、醛固酮（ALD）含量；ELISA法检测去甲肾上腺素（NE）、内皮素-1（ET-1）、血管性血友病因子（vWF）水平。透射电镜观察各组大鼠心肌组织微血管内皮细胞超微结构变化；运用免疫荧光技术观察芪苈强心对压力超负荷致心力衰竭大鼠心肌微血管密度的影响；采用Real-time PCR检测心肌中与炎症相关的细胞粘附因子ICAM-1、VCAM-1mRNA表达，以及反映血管内皮功能的eNOS mRNA表达量。
2心力衰竭大鼠心室重构及芪苈强心胶囊干预作用研究
实验分组、造模方法同第一部分。心肌组织一般形态学及超微结构变化：HE染色观察心肌组织形态学改变；透射电镜观察超微结构变化。
心肌胶原纤维增生情况：记录心重指数；Masson三色染色观察心肌胶原纤维增生情况；碱水解法检测心肌组织羟脯氨酸水平；Western blot检测心肌组织胶原Ⅰ、Ⅲ（COLⅠ、COLⅢ）表达量。
心肌细胞凋亡情况：采用流式细胞技术及TUNEL原位细胞凋亡检测心肌细胞凋亡率；Real-time PCR检测心肌营养素-1（CT-1）、细胞色素C（CytC）mRNA表达量。
3心力衰竭大鼠代谢重构及芪苈强心胶囊干预作用研究
实验分组、造模方法同第一部分。检测血清乳酸（LA）、乳酸脱氢酶（LDH）、游离脂肪酸（FFA）、尿酸（UA）含量；高效液相色谱法测定ATP、ADP、AMP含量，计算心肌组织总腺苷池及能荷值；运用流式细胞技术检测心肌细胞线粒体膜电位（MMP）；溶解氧电极检测线粒体氧化呼吸活性（RCR）；Real-time PCR检测CPT-Ⅰ、GLUT4、PGC-1α基因相对表达量；Western blot检测p-AMPK、AMPK、PPARα、PGC-1α蛋白表达水平。
4芪苈强心胶囊改善离体衰竭心脏能量代谢的机制研究
本部分研究采用离体Langendorff心脏灌流装置，低钙K-H液灌注制作离体心力衰竭模型，以去乙酰毛花苷注射液为阳性对照药，芪苈强心设立0.025、0.05、0.1、0.2、0.4g/L5个浓度组（分别为QLQX-0.025、QLQX-0.05、QLQX-0.1、QLQX-0.2、QLQX-0.4），另设AMPK阻断剂组（Compound C）和PPARα阻断剂（MK-886）观测芪苈强心对离体衰竭心脏的左室内压、左室负荷变化及冠脉流量的影响；检测各组总腺苷酸池及能荷值；Real-time PCR检测α-MHC、β-MHC、Mfn2、Drp1mRNA表达；Western blot检测离体心脏AMPK、PPARα、p-PI-3K、p-Akt蛋白表达水平。
结果:
1心力衰竭大鼠心肌微血管损伤及芪苈强心胶囊干预作用研究
各组大鼠血流动力学测定结果：与假手术组比较，模型组SBP、LVSP、LVEDP显著升高，±dp/dtmax明显降低（P<0.05）；与模型组比较，各给药组均降低SBP、LVSP、LVEDP水平，升高±dp/dtmax水平（P<0.01），尤以QLQX-H组效果显著。
AngⅡ、ALD、NE、NT-proBNP含量变化：与假手术比较，模型组大鼠上述指标含量均显著增高（P<0.01）；与模型组比较，QLQX-M，QLQX-H组降低NE和NT-proBNP水平（P<0.05，P<0.01）；与模型组比较，QLQX-M、QLQX-H组AngⅡ、ALD水平明显降低（P<0.01）。
心肌组织微血管超微结构显示：模型组毛细血管周围明显水肿，基膜不整、管腔不规则，未见明显线粒体结构，紧密连接模糊，吞饮小泡数目减少。各给药组不同程度改善毛细血管内皮结构，表现在线粒体融合、空泡化减轻，吞饮小泡数目相对增多，紧密连接较明显。
各组心肌组织微血管密度：与假手术组比较，模型组CD34阳性计数和微血管相对密度均显著降低（P<0.01），各组DAPI计数无统计学差异。与模型组CD34阳性数比较，QLQX各剂量组明显增加CD34阳性数（P<0.05，P<0.01）；与模型组比较，各给药组心肌组织微血管相对密度均显著增加（P<0.01）。
血清ET-1、vWF水平：与假手术组比较，模型组ET-1、vWF含量明显增高（P<0.01）；与模型组比较，给药组ET-1含量均显著降低（P<0.01）；与模型组比较，芪苈强心各剂量组显著降低降低vWF含量（P<0.05，P<0.01）。
炎症因子ICAM-1、VCAM-1以及eNOS mRNA表达：与假手术组比较，模型组心肌组织ICAM-1、VCAM-1表达量均显著升高（P<0.01），而eNOS mRNA表达量明显降低（P<0.01）。与模型组比较，QLQX-H组降低ICAM-1表达（P<0.01）；QLQX-M、QLQX-H组均显著降低VCAM-1mRNA表达量（P<0.01）；芪苈强心各剂量组不同程度上调eNOSmRNA表达（P<0.05，P<0.01），以QLQX-H组效果显著。
2心力衰竭大鼠心室重构及芪苈强心胶囊干预作用研究
2.1心肌组织形态学及超微结构观察结果：HE染色结果观察：
与假手术组比较，模型组心肌纤维肥大，局部可见断裂、萎缩变性的肌纤维。给药干预后大体情况基本接近正常。透射电镜观察结果显示模型组大鼠心肌纤维排列紊乱，Z线可见断裂、消失，心肌间质及核周明显水肿，糖原减少；线粒体形态不一、膜结构不清、部分膜融合，嵴紊乱、融合或消失，可见线粒体空化现象。各给药组不同程度减轻心衰大鼠心肌组织上述超微结构改变。
2.2各组大鼠心重指数、胶原纤维、羟脯氨酸含量及心肌COLⅠ、COL Ⅲ
蛋白表达水平的变化：各组大鼠心重指数结果：与正常组、假手术组比较，模型组心重指数明显增加（P<0.01）；与模型组比较，各给药组可明显降低心重指数
（P<0.05，P<0.01），尤以QLQX-H组降低效果显著。心肌组织胶原纤维的变化：Masson三色染色显示，正常组、假手术组心肌纤维束排列紧密有序，未见明显胶原纤维增生；模型组局部可见大面积胶原增生，并蔓延于心肌纤维束间，呈网状分布。各给药组可不同程
度抑制心肌及血管周围胶原纤维的增生。各组心肌组织羟脯氨酸含量的变化：与假手术组比较，模型组羟脯氨酸含量显著增加（P<0.01）；与模型组比较，各给药组明显降低羟脯氨酸
含量（P<0.05，P<0.01），尤以QLQX-H组降低效果最明显。各组大鼠心肌COLⅠ、COLⅢ蛋白表达变化：与假手术组比较，模型组COLⅠ、COLⅢ蛋白显著上调（P<0.01），与模型组比较，QLQX-H组和卡托普利组COLⅠ的表达水平显著下调（P<0.05，P<0.01）；与模型组比较，QLQX-M和QLQX-H组显著降低COLⅢ蛋白表达（P<0.01）。
2.3各组大鼠心肌细胞凋亡的变化：TUNNEL结果显示，模型组心肌组织凋亡细胞明显增多，给予药物治疗后凋亡细胞减少；流式细胞技术结果显示与假手术组比较，模型组心肌细胞凋亡明显增加（P<0.01），与模型组比较，各给药组心肌细胞凋亡
率显著降低，均具有显著统计学意义（P<0.01）。各组心肌组织CT-1、CytC mRNA表达水平的变化：与假手术比较，模型组CT-1，CytC mRNA表达水平显著上调（P<0.01），与模型组比较，各给药组CT-1，CytC mRNA表达水平明显降低（P<0.05，P<0.01）。
3心力衰竭大鼠代谢重构及芪苈强心胶囊干预作用研究各组大鼠血清LA、LDH、FFA、UA含量的变化：与模型组比较，QLQX-L、QLQX-H组大鼠血清LA含量显著降低（P<0.05，P<0.01）。芪苈强心各剂量组均显著降低LDH、FFA、UA水平（P<0.05，P<0.01），其中QLQX-H组降低FFA、LA含量显著优于卡托普利组（P<0.05，P<0.01）。
各组大鼠心肌组织总腺苷酸池及能荷值变化：与假手术组比较，模型组总腺苷酸与能荷值均显著降低（P<0.01）。与模型组比较，药物干预组
腺苷酸池和能荷值均显著增加（P<0.05，P<0.01）各组大鼠心肌组织线粒体MMP与RCR变化：与假手术组比较，模型组心肌线粒体MMP与RCR明显下降（P<0.01）。与模型组比较，各给药组心肌线粒体MMP均升高（P<0.05，P<0.01）；各给药组均可改善
线粒体呼吸功能、提高RCR（P<0.05，P<0.01）。各组CPT-Ⅰ、GLUT4、PGC-1α mRNA表达的变化：与假手术组比较，模型组上述指标mRNA表达量均显著降低（P<0.01）；与模型组比较，QLQX-H组和卡托普利组明显上调CPT-Ⅰ、GLUT4和PGC-1α mRNA
表达（P<0.05，P<0.01）。各组大鼠心肌组织中p-AMPK、AMPK、PPARα、PGC-1α蛋白表达变化：与假手术组比较，模型组p-AMPK蛋白表达无显著变化（P>0.05）；与模型组比较，各给药组显著增加p-AMPK表达水平（P<0.05，P<0.01）。各组AMPK蛋白表达水平未见显著差异（P>0.05）。与假手术组比较，模型组PPARα和PGC-1α蛋白表达量明显降低（P<0.01）；与模型组比较，QLQX-M、QLQX-H组及卡托普利组二者蛋白表达显著上调（P<0.01）。
4芪苈强心胶囊改善离体衰竭心脏能量代谢的机制研究各组左心室压力及心脏负荷的变化：采用低钙K-H液灌注复制心衰模型后，各组左心室压力明显降低，同时心脏负荷增加；与模型组比较，给予芪苈强心干预后，芪苈强心各剂量组左心室压力均有升高，且随着浓度的增加左心室压力升高幅度也增加，其中QLQX-0.2、QLQX-0.4浓度组左心室压力显著升高（P<0.01），有一定的量效关系。给予药物干预后，
各浓度组左心室负荷也有不同程度的降低；各组给药前后自身比较：与低钙灌注致心衰状态时比较，给予药物治疗后芪苈强心各浓度组随着剂量的增加左心室压力升高幅度也增加（P<0.01），QLQX-0.1、QLQX-0.2、QLQX-0.4组和阳性对照药组显著
降低左室负荷幅度（P<0.05，P<0.01）。给予阻断剂后左室压力及负荷变化：与给药时压力比较，加入两组阻断剂后压力均显著降低（P<0.01），心脏负荷均有所提高，其中加入阻断剂MK-886后心脏负荷升高明显（P<0.05）。
各组离体灌注心脏冠脉流出量变化：与正常K-H液灌注组比较，模型组冠脉流量明显降低（P<0.01）；与模型组比较，QLQX-0.05和QLQX-0.1组、阳性对照组冠脉流量显著增加（P<0.05，P<0.01）。与QLQX-0.2组
比较，两阻断剂组的冠脉流量均降低，无统计学差异。各组大鼠心肌组织中总腺苷酸池及能荷值的变化：与正常K-H液灌注组比较，模型组总腺苷酸含量降低，尚无统计学意义，模型组心肌组织能荷值显著降低（P<0.01）；与模型组比较，QLQX-0.05和QLQX-0.4组显著增加总腺苷酸池含量（P<0.05）；与模型组比较，QLQX-0.1、QLQX-0.2、 QLQX-0.4组及阳性对照组显著增加心肌组织能荷值
（P<0.01）。心肌组织α-MHC、β-MHC、Drp1、Mfn2mRNA的表达：与正常K-H液灌注组比较，模型组心肌组织α-MHC、Mfn2mRNA显著下调（P<0.01），而β-MHC、Drp1mRNA表达明显增高（P<0.01）。与模型组比较，QLQX-0.1、QLQX-0.2和QLQX-0.4组增加α-MHC mRNA表达（P<0.01）；与QLQX-0.2组比较，两组阻断剂均降低α-MHC mRNA表达。与模型组比较，QLQX-0.2和QLQX-0.4组显著降低β-MHC表达量（P<0.01），与QLQX-0.2组比较，两组阻断剂增加β-MHC mRNA表达。与模型组比较，QLQX-0.4组显著降低Drp1mRNA表达量（P<0.05），QLQX-0.1、QLQX-0.2
和QLQX-0.4组显著增加Mfn2表达量（P<0.05，P<0.01）。离体心脏p-AMPK、PPARα、p-PI-3K、p-Akt蛋白表达：与正常组K-H液灌注组比较，模型组p-AMPK、PPARα、p-PI-3K、p-Akt蛋白表达显著下调（P<0.01）；与模型组比较，各给药组显著上调p-AMPK蛋白表达（P<0.01）；与QLQX-0.4组比较，Compound C组p-AMPK表达明显下调（P<0.05）。与模型组比较，QLQX-0.2、QLQX-0.4组和阳性药组显著上调PPARα蛋白表达（P<0.01）；与QLQX-0.2组比较，两阻断剂组p-AMPK、PPARα均明显下调（P<0.05）。与模型组比较，各给药组p-PI-3K、p-Akt蛋白表达水平明显上调（P<0.05，P<0.01）；与QLQX-0.2组比较，两阻断剂组p-PI-3K、p-Akt蛋白表达量未见显著变化（P>0.05）；
与阳性药组比较，QLQX-0.4剂量组显著增加p-Akt的蛋白表达（P<0.05）。
结论:1首次以脉络学说营卫理论为指导，探讨微血管损伤在心力衰竭发病中的重要作用，脉络学说认为微血管损伤是心力衰竭重要的发病因素之一，血液动力学和神经体液调节异常共同参与，导致心肌组织结构功能损伤引发心室重构和代谢重构的复杂病理过程。通过实验研究探讨了在心衰时微血管损伤、能量代谢异常和心室重构的病理改变以及三者间的内在联系，验证了脉络学说营卫“由络以通、交会生化”理论科学价值，为从微血管病变切入探讨心力衰竭防治研究提供了新的思路。
2通过整体动物实验，初步揭示了芪苈强心胶囊对心衰微血管损伤、心室重构、能量代谢的干预作用。采用胸主动脉缩窄术建立压力超负荷大鼠模型，证实芪苈强心胶囊通过调节血管活性物质、抑制炎症损伤，促进心肌微血管新生和结构保护、改善血流动力，抑制神经体液过度激活等多重作用，从而有效改善心功能。芪苈强心胶囊抑制心肌成纤维细胞增生，同时减少心肌细胞凋亡，其机制可能与CT-1及CytC介导的线粒体凋亡途径有关。芪苈强心胶囊芪苈强心胶囊通过p-AMPK/PPARα通路调节糖脂代谢途径，改善慢性心衰大鼠能量代谢；通过上调PGC-1α的表达保护心力衰竭大鼠心肌组织线粒体功能与氧化呼吸活性；同时降低循环血中LA、FFA以及UA浓度，减少对内皮损伤作用。
3通过离体实验研究，进一步揭示芪苈强心胶囊改善心肌能量代谢的作用机制与p-AMPK/PPARα信号通路有关；证实芪苈强心胶囊通过上调Mfn2、下调Drp1mRNA保护心肌线粒体，同时芪苈强心胶囊下调Drp1mRNA的表达可抑制CytC介导的心肌细胞凋亡途径，说明能量代谢之线粒体与衰竭心脏的结构重构具有相关性。
Objective: Under the guidance of the Vessel-Collateral Theory, theThoracic Aorta Constriction (TAC) was applied to develop the chronic heartfailure model of rat. The major influence of microvessel injury and effects ofQiliqingxin capsule (QLQX) were disclosed through observing the damage ofmicrovessel structure and function and apoptosis and fibrosis of myocardialtissue and energy metabolism dysfunction of myocardial mitochondria in theanimal model; The Langendorff was adopted to determine the effects ofQLQX on the energy metabolism and mitochondria related protein and signalpathway p-AMPK/PPARαin heart failure. From above experiments, the roleof microvessel and ventricular remodeling and metabolism remodeling toheart failure was explored. These data would give scientific supports to theYing-nutrient and Wei-defense “unblocking collaterals with subsequentconvergence and substance-qi transformation” in the Vessel-CollateralTheory.
Methods:
The study includes four sections:
1The effects and mechanism of QLQX on myocardial microangiopathy inheart failure rats
TAC was used to develop heart failure rats induced by overload pressure.Four weeks later, the survival rats were randomly divided into seven groups(n=15): Control group, Captopril group and high, medium, low-dose QLQXgroups (QLQX-H, QLQX-M, QLQX-L), Model group, Sham operation group(Sham). The rats were administered with QLQX by gavage at the dose of10ml/kg once a day for6weeks, then the same volume of solvent to Sham,Control and Model group. After the last administration and fasted12h, every rat was detected the changes of hemodynamic through carotid arteryintubation. The serum of rats was collected to detect the content of pro-Nterminal brain natriuretic peptide (N-proBNP), angiotensinII (AngII), andaldosterone (ALD) by radioimmunoassay. The ELISA was adopted to detectnorepinephrine (NE), ET-1and vWF. The ultrastructure of microvesselendothelial cells in myocardial tissue was observed by transmission electronmicroscopy. The immunofluorescence technique was used to observe theeffects of QLQX on microvessel density of hypertrophic myocardial tissue inpressure-overload rats. The mRNA expression of ICAM-1VCAM-1andeNOS were detected by Real-time PCR. The content of serum was dected toreflect the endothelial function. The histological staining was adopted toobserve the changes of myocardial cells, cardiac fibroblasts, microvessel andthe intervention effect of QLQX on pathogenesis of heart failure.
2The pathological mechanism of ventricular remodeling in heart failure ratsand the intervention of QLQX
It was the same with part1that experimental animals modeling, grouping,administration and derived method. The HE staining and transmission electronmicroscope were used to observe the ultra-structural changes in myocardium.
The changes of myocardial collagen fiber hyperplasia were determinedwith Masson trachoma staining. The contents of myocardial hydroxyprolinewere detected by the method of alkaline hydrolysis. Western blot was used todetect the protein expression of COLⅠand COLⅢ in myocardial tissue.
The myocardial cell apoptosis rate was detected by flow cytometry andTUNEL. Real-time quantitative PCR was used to analyze the mRNAexpression of nutrition-1(CT-1), cytochrome C (CytC) in myocardial tissue.
3The effects and mechanism of QLQX on metabolic remodeling in heartfailure rats.
It was the same with part1that experimental animals modeling, grouping,administration and derived method. The serum was adopted to detect thecontent of lactic acid (LA), lactate dehydrogenase (LDH), free fatty acid (FFA)and uric acid (UA). The content of ATP, ADP and AMP was determined with high performance liquid chromatography to calculate the total adenosine pooland energy charge in myocardial tissue. Myocardial cell mitochondriamembrane potential (MMP) was detected by flow cytometry. Dissolvedoxygen electrode was used to detect mitochondrial oxidative activity (RCR).Real-time PCR was used to analyze the expression of CPT-Ⅰ, GLUT4andPGC-1α mRNA.Western blot was used to detect p-AMPK, AMPK, PPARαand PGC-1α protein expression level.4The effect and mechanism of QLQX on energy metabolism in the perfusionfailure heart in vitro
The Langendorff perfusion apparatus was adopted to observe the functionof isolated hearts. Low calcium K-H solution induced isolated-heart failure.QLQX was set five concentration (0.025,0.05,0.1,0.2,0.4g/L),andLanatoside as positive control, to observe the effects of QLQX on isolatedcardiac left ventricle pressure, left ventricular load changes and coronary flowand detect the total adenylate pool and energy charge.The mRNA expressionof α-MHC,β-MHC, Mfn2, Drp1was detected by Real-time PCR. Western blotwas used to analyze the protein expression of AMPK, PPARα, p-Akt andp-PI-3K in myocardial tissue.
Results:
1The effects and mechanism of QLQX on myocardial microangiopathy inheart failure rats
Results of hemodynamics in rats: Compared with the sham group, SBP,LVSP, LVEDP in Model group rats were increased and±dp/dtmax decreasedsignificantly (P<0.01). Compared with the Model group, QLQX decreasedSBP, LVSP, LVEDP levels, and increased±dp/dtmax level, estatisticallysignificant changes in QLQX-H and Captopril group (P<0.01). Comparedwith the sham group, no significant difference was detectd in AngⅡ, ALD,NE, NT-proBNP. Compared with the Sham group, these indexes weresignificantly increased in the Model group (P<0.01).Compared with the Modelgroup, the level of Ang II, ALD in QLQX-M, QLQX-H and Captopril groupdecreased significantly (P<0.01). Compared with the Model group, the treatment group decreased NE and NT-proBNP levels, especially QLQX-M(P<0.05), QLQX-H and Captopril group (P<0.01).
The ultrastructure of myocardial micro-vascular: In Model group therewas obvious edema around the capillary, a little in basement membrane, andlumen was irregular, no obvious mitochondrial structure, closely connecttionwas fuzzy, pinocytosis vesicles decreased. Capillary endothelial structure wasbetter in treatment groups, for example, mitochondrial fusion andvacuolization decreased, pinocytosis vesicles relatively increased and tightjunction recoverd obviously.
The microvessel density of cardiac muscle tissue: Compared with shamgroup, the CD34positive count and microvascular relatively density decreasedsignificantly in Model group (P<0.01). The statistical difference of DAPI wasnot showed. Compared with the positive count in Model group, the CD34positive count increased in QLQX-L group (P<0.05), QLQX-M and QLQX-H(P<0.01); compared with the Model group, the relative microvascular densityof cardiac muscle tissue increased in QLQX group, the QLQX-M andQLQX-H group showed statistically difference (P<0.01).
Serum ET-1, vWF levels: Compared with sham group, the ET-1and vWFcontents in Model group increased obviously (P<0.05). Compared with Modelgroup, the ET-1contents in QLQX group decreased significantly (P<0.01).Compared with Model group, the vWF contents decreased significantly inQLQX-M, QLQX-H, Captopril group (P<0.01) and QLQX-L group (P<0.05).
The expression of inflammatory cytokines ICAM-1, VCAM-1and eNOSMrna: Compared with sham group, the ICAM-1and VCAM-1expression ofcardiac tissue of rats in Model group increased significantly (P<0.01).Compared with sham group, the eNOS mRNA expression in Model groupdecreased (P<0.01); compared with Model group, the QLQX group couldpromote the expression of eNOS mRNA at different extents (P<0.05, P<0.01),the QLQX-H group showed remarkable effects (P<0.01).
2Effects of QLQX on ventricular remodeling in pressure-overload heartfailure rats
2.1Myocardial tissue morphological morphology: Compared with the shamgroup, there showed myocardial fiber hypertrophy, partly fracture visible,atrophy of muscle fibers, and diameter in Model group. QLQX inhibitedcardiac hypertrophy, reduced muscle fiber fracture, atrophy and interstitialfibrosis on different degree. Especially QLQX-H group, there was onlypartially myocardial fiber atrophy and myocardial fiber occasionallydegeneration.
Compared with the Sham group, the ultrastructure of cardiac fibersdisplaied disorderly-arranged in Model group. Other changes including Z linesvisible fracture or disappear, myocardial interstitial and obvious edema aroundnuclear, and glycogen decreased. Mitochondrial morphology varied.Membrane structure is not clear, partly membrane fused, and the cristadisordered, fused or disappeared. Treatment groups significantly relieved themyocardial cell stomata edema and mitochondrial injuried, increased theglycogen amount; glycogen particles relatively increased and Z lines werearranged in order.
2.2The effects of QLQX on Collagen fiber hyperplasia of myocardial tissue:
Compared with the control and sham group, HW/BW index of Model ratsincreased obviously (P<0.01).Compared with Model group, the treatmentgroups may lower HW/BW index (P<0.05, P<0.01), especially the index ofQLQX-H rats decreased significantly.The Masson trichromatic staining ofmyocardial tissue showed that myocardial fibers arranged closely, and noobvious hyperplasia of collagen fiber in Control group and the shamgroup.There were mass collagen hyperplasia and spread to the myocardialfiber bundles in Model group. QLQX could inhibit the collagen fiberproliferation and reduce collagen fibrosis size in myocardial tissue and bloodvessels.
Compared with the sham group, the content of hydroxyproline increasedsignificantly in Model group(P<0.01).Compared with the Model group, thehydroxyproline content in treatment group decreased at different degree, inwhich the QLQX-L group, captopril group (P<0.05), QLQX-M and QLQX-H group decreased significantly (P<0.01).
Compared with sham group, the content of COL I, COL III in Modelgroup increased significantly(P<0.01).Compared with Model group, captopriland QLQX-H decreased the expression of COLⅠ(P<0.01,P<0.05). Comparedwith Model group, there was a significant statistical difference between thecaptopril group, QLQX-M and QLQX-H groups in COL Ⅲ protein,significantly in QLQX-H group.
2.3Myocardial cell apoptosis:
The results with TUNNEL analysis showed that the amout of apoptoticcells of myocardial tissue in Model group significantly increased andapoptosis cells decreased after treatment. The results with Flow cytometryanalysis showed that the apoptotic myocardial cells significantly increased(P<0.01) in Model group.Compared with sham group, QLQX reducedapoptotic myocardial cells in pressure-overload heart failure rats statisticallysignificantly (P<0.01).
Compared with sham group, the mRNA expression of CT-1and CytCsignificantly increased in Model group (P<0.01). Compared with the Modelgroup, QLQX reduced CT-1, CytC expression (P<0.01), Captopril groupdecreased CT-1, CytC mRNA expression (P<0.05, P<0.01).
3The effects of QLQX on the metabolic remodeling of rats suffered fromcardiac failure
Compared with Model group, the QLQX-L and QLQX-H group coulddecrease the blood serum LA content of pressure overload rats significantly(P<0.05). The QLQX decreased LDH and UA level greatly (P<0.01).Compared with Model group, FFA content in QLQX groups decreased atdifferent concentrations with statistical difference. QLQX-H group showedobvious effect (P<0.01), which was better than captopril group (P<0.05).
Compared with sham group, the total adenosine and energy charge valuedecreased significantly (P<0.01). Compared with Model group, treatmentgroup increased the adenosine pool and energy value greatly, in which theQLQX-L increased the total adenosine pool content and energy value (P<0.05, P<0.01).
Compared with sham group, the membrane potential of mitochondria ofcardiac muscle decreased in the Model group (P<0.01). Compared with Modelgroup, the MMP of mitochondria of cardiac muscle increased in treatmentgroup, and QLQX-M and QLQX-H group showed significant increase(P<0.01). Treatment groups could improve the respiratory function ofmitochondria and enhance the RCR (P<0.05, P<0.01).
Compared with sham group, the mRNA expression of CPT-Ⅰand GLUT
4and PGC-1α in Model group decreased significantly (P<0.01). Comparedwith Model group, the mRNA expression of above index in QLQX-H groupshowed significant difference (P<0.05, P<0.01).
Compared with sham group, the protein expression of p-AMPK in Modelgroup showed no statisticly difference (P>0.05). Compared with Model group,the expression level of p-AMPK in treatment group increased significantly, inwhich the QLQX-M and QLQX-H group increased obviously (P<0.01). Thetotal amount of AMPK in each group didn’t show significant difference(P>0.05). Compared with sham group, the protein expression level of PPARαand PGC-1α in Model group decreased obviously (P<0.01). Compared withModel group, the expression of the two protein in QLQX-M and QLQX-Hgroup increased significantly (P<0.01).
4The effects and mechanism of QLQX on energy metabolism in the perfusionfailure heart in vitro
Only being perfused with K-H liquid, the left ventricular pressure in eachgroup didn’t show significant difference (P>0.05). When mimicing the cardiacfailure with low calcium K-H liquid perfusion, the left ventricular pressure ineach group decreased obviously and the cardiac loading increased at meanwhile. Compared with Model group, the left ventricular pressure in all QLQXgroups at different concentration increased accompanying withconcentration increasing, in which the left ventricular pressure of QLQX-0.2,QLQX-0.4group increased significantly (P<0.01), and this showed certaindose-effect relationship. After being adding QLQX, the left ventricular loading in each concentration group showed decrease in different degree. After beingadding two blockers, the pressure decreased significantly (P<0.01), and thecardiac load increased at some extent, and the cardiac load after addingblocker MK-886showed significant increase (P<0.05).
Compared with normal K-H perfusion, the coronary flow in Model groupdecreased obviously (P<0.01). Compared with Model group, theQLQX-0.05,QLQX-0.1,QLQX-0.2and QLQX-0.4group increased thecoronary flow obviously (P<0.05, P<0.01).Compared with QLQX-0.2, thecoronary flow with two blockers decreased, but there was no statisticlydifference between these two blocker groups.
Compared with control group, the energy charge value of cardiac muscletissue in Model group decreased (P<0.01).Compared with Model group, thepositive medication, QLQX-0.1, QLQX-0.2and QLQX-0.4showed (P<0.01)statistically significance.
Compared with Model group, the mRNA expression level of Drp1inQLQX-0.4group decreased (P<0.05). Compared with QLQX-0.2group, theexpression with blockers increased, and but showed no significant difference.Compared with Model group, the positive control medication, QLQX-0.2and0.4group increased the expression of Mfn2obviously (P<0.01), andQLQX-0.1showed statistically difference (P<0.05). The positive controlmedication, QLQX-0.1, QLQX-0.2and QLQX-0.4group increased theα-MHC mRNA (P<0.01). Compared with QLQX-0.2group, two groups withblocker decreased the expression of α-MHC mRNA. Compared with Modelgroup, the positive control medication, QLQX-0.2and QLQX-0.4groupdecreased the expression of β-MHC greatly (P<0.01). Compared withQLQX-0.2group, two groups with blocker increased the mRNA expression ofβ-MHC.
Compared with normal K-H perfusion group, the expression level ofp-PI-3K and p-Akt in Model group decreased (P<0.01).Compared with Modelgroup, the treatment increased the expression level of p-PI-3K and p-Aktprotein (P<0.05, P<0.01). Compared with QLQX-0.2, the two blockers up-regulated the protein expression of p-Akt, but this didn’t show statisticdifference (P>0.05).Compared with QLQX-0.2group, the expression ofp-PI-3K with two blockers didn’t show statistic difference (P>0.05).Compared with positive medication, the QLQX-0.4increased the proteinexpression of p-Akt (P<0.05).
Conclusion:
1It is the first time that Ying and Wei theory in Vessels-CollateralTheory was used as guidance to discuss the important role of microvasculardamage in heart failure. Heart failure is multi-dimensional spatio-temporalcomplex pathological processes including ventricular remodeling and energymetabolism disorder, caused by abnormal hemodynamic and neurohumoraltrigger. Through experiment, microvascular injury, abnormal energymetabolism, pathological changes of ventricular remodeling in case of heartfailure and the internal relation between them are discussed. The theory ofscientific value of the Ying-nutrient and Wei-defense “unblocking collateralswith subsequent convergence and substance-qi transformation” of theVessel-Collateral Theory was verified, and it offers new thinking for theprevention and treatment of cardiac failure.
2The effects and mechanism of QLQX on microvascular provention,ventricular remodeling, and energy metabolism were elaborated through theanimal experiment in vivo. TAC was applied to develop the chronic heartfailure model of rat and demonstrate that QLQX could improving the functionof failing heart by adjusting the vasoactive substances, inhibittinginflammatory injury, promoting myocardial angiogenesis and structure,improving hemodynamics, and inhibitting neurohumoral excessivelyactivation. QLQX inhibited cardiac fibroblast proliferation. At the same time,QLQX capsule reduced myocardial apoptosis, the mechanism may be relatedto the mitochondrial apoptotic pathway mediated by CT-1and CytC. QLQXcapsule regulates metabolic pathways of glycolipid through p-AMPK/PPARαpath, improves energy metabolism of rats with chronic heart failure, increasesthe expression of PGC-1α, promotes mitochondrial biosynthesis, protects mitochondrial function and oxidative respiration activity of myocardial tissueof rats with heart failure, reduces the concentration of LA, FFA and UA incirculating blood and reduces endothelial injury.3It was demonstrated the protective effects of QLQX on the structure andfunction of myocardial mitochondrial through the up-regulation of Mfn2mRNA expression and down-regulation expression of Drp1mRNA, andQLQX inhibit myocardial mitochondrial apoptosis pathway mediated by CytCthrough down-regulating Drp1mRNA expression. This illustrates thecorrelation between mitochondria of energy metabolism and structuralremodeling of the failing heart.
方法:
本研究分为以下四部分内容：
1心力衰竭大鼠心肌微血管损伤及芪苈强心胶囊干预作用研究
以SD大鼠为研究对象，采用胸主动脉缩窄术建立压力超负荷致心力衰竭模型，分为正常对照组（Control）、假手术组（Sham）、模型组（Model）、卡托普利组（Captopril）、芪苈强心低、中、高三个剂量组（QLQX-L、QLQX-M、QLQX-H），共7组，每组15只，灌胃给药1次/日，连续6周，正常对照组、假手术和模型组给予同体积羧甲基纤维素钠（CMC-Na）溶剂，给药量均为10ml/kg体重。末次给药后禁食12h，各组大鼠行颈动脉插管观察血流动力学变化；放免法检测N端脑钠肽前体（NT-proBNP）、血管紧张素Ⅱ（AngⅡ）、醛固酮（ALD）含量；ELISA法检测去甲肾上腺素（NE）、内皮素-1（ET-1）、血管性血友病因子（vWF）水平。透射电镜观察各组大鼠心肌组织微血管内皮细胞超微结构变化；运用免疫荧光技术观察芪苈强心对压力超负荷致心力衰竭大鼠心肌微血管密度的影响；采用Real-time PCR检测心肌中与炎症相关的细胞粘附因子ICAM-1、VCAM-1mRNA表达，以及反映血管内皮功能的eNOS mRNA表达量。
2心力衰竭大鼠心室重构及芪苈强心胶囊干预作用研究
实验分组、造模方法同第一部分。心肌组织一般形态学及超微结构变化：HE染色观察心肌组织形态学改变；透射电镜观察超微结构变化。
心肌胶原纤维增生情况：记录心重指数；Masson三色染色观察心肌胶原纤维增生情况；碱水解法检测心肌组织羟脯氨酸水平；Western blot检测心肌组织胶原Ⅰ、Ⅲ（COLⅠ、COLⅢ）表达量。
心肌细胞凋亡情况：采用流式细胞技术及TUNEL原位细胞凋亡检测心肌细胞凋亡率；Real-time PCR检测心肌营养素-1（CT-1）、细胞色素C（CytC）mRNA表达量。
3心力衰竭大鼠代谢重构及芪苈强心胶囊干预作用研究
实验分组、造模方法同第一部分。检测血清乳酸（LA）、乳酸脱氢酶（LDH）、游离脂肪酸（FFA）、尿酸（UA）含量；高效液相色谱法测定ATP、ADP、AMP含量，计算心肌组织总腺苷池及能荷值；运用流式细胞技术检测心肌细胞线粒体膜电位（MMP）；溶解氧电极检测线粒体氧化呼吸活性（RCR）；Real-time PCR检测CPT-Ⅰ、GLUT4、PGC-1α基因相对表达量；Western blot检测p-AMPK、AMPK、PPARα、PGC-1α蛋白表达水平。
4芪苈强心胶囊改善离体衰竭心脏能量代谢的机制研究
本部分研究采用离体Langendorff心脏灌流装置，低钙K-H液灌注制作离体心力衰竭模型，以去乙酰毛花苷注射液为阳性对照药，芪苈强心设立0.025、0.05、0.1、0.2、0.4g/L5个浓度组（分别为QLQX-0.025、QLQX-0.05、QLQX-0.1、QLQX-0.2、QLQX-0.4），另设AMPK阻断剂组（Compound C）和PPARα阻断剂（MK-886）观测芪苈强心对离体衰竭心脏的左室内压、左室负荷变化及冠脉流量的影响；检测各组总腺苷酸池及能荷值；Real-time PCR检测α-MHC、β-MHC、Mfn2、Drp1mRNA表达；Western blot检测离体心脏AMPK、PPARα、p-PI-3K、p-Akt蛋白表达水平。
结果:
1心力衰竭大鼠心肌微血管损伤及芪苈强心胶囊干预作用研究
各组大鼠血流动力学测定结果：与假手术组比较，模型组SBP、LVSP、LVEDP显著升高，±dp/dtmax明显降低（P<0.05）；与模型组比较，各给药组均降低SBP、LVSP、LVEDP水平，升高±dp/dtmax水平（P<0.01），尤以QLQX-H组效果显著。
AngⅡ、ALD、NE、NT-proBNP含量变化：与假手术比较，模型组大鼠上述指标含量均显著增高（P<0.01）；与模型组比较，QLQX-M，QLQX-H组降低NE和NT-proBNP水平（P<0.05，P<0.01）；与模型组比较，QLQX-M、QLQX-H组AngⅡ、ALD水平明显降低（P<0.01）。
心肌组织微血管超微结构显示：模型组毛细血管周围明显水肿，基膜不整、管腔不规则，未见明显线粒体结构，紧密连接模糊，吞饮小泡数目减少。各给药组不同程度改善毛细血管内皮结构，表现在线粒体融合、空泡化减轻，吞饮小泡数目相对增多，紧密连接较明显。
各组心肌组织微血管密度：与假手术组比较，模型组CD34阳性计数和微血管相对密度均显著降低（P<0.01），各组DAPI计数无统计学差异。与模型组CD34阳性数比较，QLQX各剂量组明显增加CD34阳性数（P<0.05，P<0.01）；与模型组比较，各给药组心肌组织微血管相对密度均显著增加（P<0.01）。
血清ET-1、vWF水平：与假手术组比较，模型组ET-1、vWF含量明显增高（P<0.01）；与模型组比较，给药组ET-1含量均显著降低（P<0.01）；与模型组比较，芪苈强心各剂量组显著降低降低vWF含量（P<0.05，P<0.01）。
炎症因子ICAM-1、VCAM-1以及eNOS mRNA表达：与假手术组比较，模型组心肌组织ICAM-1、VCAM-1表达量均显著升高（P<0.01），而eNOS mRNA表达量明显降低（P<0.01）。与模型组比较，QLQX-H组降低ICAM-1表达（P<0.01）；QLQX-M、QLQX-H组均显著降低VCAM-1mRNA表达量（P<0.01）；芪苈强心各剂量组不同程度上调eNOSmRNA表达（P<0.05，P<0.01），以QLQX-H组效果显著。
2心力衰竭大鼠心室重构及芪苈强心胶囊干预作用研究
2.1心肌组织形态学及超微结构观察结果：HE染色结果观察：
与假手术组比较，模型组心肌纤维肥大，局部可见断裂、萎缩变性的肌纤维。给药干预后大体情况基本接近正常。透射电镜观察结果显示模型组大鼠心肌纤维排列紊乱，Z线可见断裂、消失，心肌间质及核周明显水肿，糖原减少；线粒体形态不一、膜结构不清、部分膜融合，嵴紊乱、融合或消失，可见线粒体空化现象。各给药组不同程度减轻心衰大鼠心肌组织上述超微结构改变。
2.2各组大鼠心重指数、胶原纤维、羟脯氨酸含量及心肌COLⅠ、COL Ⅲ
蛋白表达水平的变化：各组大鼠心重指数结果：与正常组、假手术组比较，模型组心重指数明显增加（P<0.01）；与模型组比较，各给药组可明显降低心重指数
（P<0.05，P<0.01），尤以QLQX-H组降低效果显著。心肌组织胶原纤维的变化：Masson三色染色显示，正常组、假手术组心肌纤维束排列紧密有序，未见明显胶原纤维增生；模型组局部可见大面积胶原增生，并蔓延于心肌纤维束间，呈网状分布。各给药组可不同程
度抑制心肌及血管周围胶原纤维的增生。各组心肌组织羟脯氨酸含量的变化：与假手术组比较，模型组羟脯氨酸含量显著增加（P<0.01）；与模型组比较，各给药组明显降低羟脯氨酸
含量（P<0.05，P<0.01），尤以QLQX-H组降低效果最明显。各组大鼠心肌COLⅠ、COLⅢ蛋白表达变化：与假手术组比较，模型组COLⅠ、COLⅢ蛋白显著上调（P<0.01），与模型组比较，QLQX-H组和卡托普利组COLⅠ的表达水平显著下调（P<0.05，P<0.01）；与模型组比较，QLQX-M和QLQX-H组显著降低COLⅢ蛋白表达（P<0.01）。
2.3各组大鼠心肌细胞凋亡的变化：TUNNEL结果显示，模型组心肌组织凋亡细胞明显增多，给予药物治疗后凋亡细胞减少；流式细胞技术结果显示与假手术组比较，模型组心肌细胞凋亡明显增加（P<0.01），与模型组比较，各给药组心肌细胞凋亡
率显著降低，均具有显著统计学意义（P<0.01）。各组心肌组织CT-1、CytC mRNA表达水平的变化：与假手术比较，模型组CT-1，CytC mRNA表达水平显著上调（P<0.01），与模型组比较，各给药组CT-1，CytC mRNA表达水平明显降低（P<0.05，P<0.01）。
3心力衰竭大鼠代谢重构及芪苈强心胶囊干预作用研究各组大鼠血清LA、LDH、FFA、UA含量的变化：与模型组比较，QLQX-L、QLQX-H组大鼠血清LA含量显著降低（P<0.05，P<0.01）。芪苈强心各剂量组均显著降低LDH、FFA、UA水平（P<0.05，P<0.01），其中QLQX-H组降低FFA、LA含量显著优于卡托普利组（P<0.05，P<0.01）。
各组大鼠心肌组织总腺苷酸池及能荷值变化：与假手术组比较，模型组总腺苷酸与能荷值均显著降低（P<0.01）。与模型组比较，药物干预组
腺苷酸池和能荷值均显著增加（P<0.05，P<0.01）各组大鼠心肌组织线粒体MMP与RCR变化：与假手术组比较，模型组心肌线粒体MMP与RCR明显下降（P<0.01）。与模型组比较，各给药组心肌线粒体MMP均升高（P<0.05，P<0.01）；各给药组均可改善
线粒体呼吸功能、提高RCR（P<0.05，P<0.01）。各组CPT-Ⅰ、GLUT4、PGC-1α mRNA表达的变化：与假手术组比较，模型组上述指标mRNA表达量均显著降低（P<0.01）；与模型组比较，QLQX-H组和卡托普利组明显上调CPT-Ⅰ、GLUT4和PGC-1α mRNA
表达（P<0.05，P<0.01）。各组大鼠心肌组织中p-AMPK、AMPK、PPARα、PGC-1α蛋白表达变化：与假手术组比较，模型组p-AMPK蛋白表达无显著变化（P>0.05）；与模型组比较，各给药组显著增加p-AMPK表达水平（P<0.05，P<0.01）。各组AMPK蛋白表达水平未见显著差异（P>0.05）。与假手术组比较，模型组PPARα和PGC-1α蛋白表达量明显降低（P<0.01）；与模型组比较，QLQX-M、QLQX-H组及卡托普利组二者蛋白表达显著上调（P<0.01）。
4芪苈强心胶囊改善离体衰竭心脏能量代谢的机制研究各组左心室压力及心脏负荷的变化：采用低钙K-H液灌注复制心衰模型后，各组左心室压力明显降低，同时心脏负荷增加；与模型组比较，给予芪苈强心干预后，芪苈强心各剂量组左心室压力均有升高，且随着浓度的增加左心室压力升高幅度也增加，其中QLQX-0.2、QLQX-0.4浓度组左心室压力显著升高（P<0.01），有一定的量效关系。给予药物干预后，
各浓度组左心室负荷也有不同程度的降低；各组给药前后自身比较：与低钙灌注致心衰状态时比较，给予药物治疗后芪苈强心各浓度组随着剂量的增加左心室压力升高幅度也增加（P<0.01），QLQX-0.1、QLQX-0.2、QLQX-0.4组和阳性对照药组显著
降低左室负荷幅度（P<0.05，P<0.01）。给予阻断剂后左室压力及负荷变化：与给药时压力比较，加入两组阻断剂后压力均显著降低（P<0.01），心脏负荷均有所提高，其中加入阻断剂MK-886后心脏负荷升高明显（P<0.05）。
各组离体灌注心脏冠脉流出量变化：与正常K-H液灌注组比较，模型组冠脉流量明显降低（P<0.01）；与模型组比较，QLQX-0.05和QLQX-0.1组、阳性对照组冠脉流量显著增加（P<0.05，P<0.01）。与QLQX-0.2组
比较，两阻断剂组的冠脉流量均降低，无统计学差异。各组大鼠心肌组织中总腺苷酸池及能荷值的变化：与正常K-H液灌注组比较，模型组总腺苷酸含量降低，尚无统计学意义，模型组心肌组织能荷值显著降低（P<0.01）；与模型组比较，QLQX-0.05和QLQX-0.4组显著增加总腺苷酸池含量（P<0.05）；与模型组比较，QLQX-0.1、QLQX-0.2、 QLQX-0.4组及阳性对照组显著增加心肌组织能荷值
（P<0.01）。心肌组织α-MHC、β-MHC、Drp1、Mfn2mRNA的表达：与正常K-H液灌注组比较，模型组心肌组织α-MHC、Mfn2mRNA显著下调（P<0.01），而β-MHC、Drp1mRNA表达明显增高（P<0.01）。与模型组比较，QLQX-0.1、QLQX-0.2和QLQX-0.4组增加α-MHC mRNA表达（P<0.01）；与QLQX-0.2组比较，两组阻断剂均降低α-MHC mRNA表达。与模型组比较，QLQX-0.2和QLQX-0.4组显著降低β-MHC表达量（P<0.01），与QLQX-0.2组比较，两组阻断剂增加β-MHC mRNA表达。与模型组比较，QLQX-0.4组显著降低Drp1mRNA表达量（P<0.05），QLQX-0.1、QLQX-0.2
和QLQX-0.4组显著增加Mfn2表达量（P<0.05，P<0.01）。离体心脏p-AMPK、PPARα、p-PI-3K、p-Akt蛋白表达：与正常组K-H液灌注组比较，模型组p-AMPK、PPARα、p-PI-3K、p-Akt蛋白表达显著下调（P<0.01）；与模型组比较，各给药组显著上调p-AMPK蛋白表达（P<0.01）；与QLQX-0.4组比较，Compound C组p-AMPK表达明显下调（P<0.05）。与模型组比较，QLQX-0.2、QLQX-0.4组和阳性药组显著上调PPARα蛋白表达（P<0.01）；与QLQX-0.2组比较，两阻断剂组p-AMPK、PPARα均明显下调（P<0.05）。与模型组比较，各给药组p-PI-3K、p-Akt蛋白表达水平明显上调（P<0.05，P<0.01）；与QLQX-0.2组比较，两阻断剂组p-PI-3K、p-Akt蛋白表达量未见显著变化（P>0.05）；
与阳性药组比较，QLQX-0.4剂量组显著增加p-Akt的蛋白表达（P<0.05）。
结论:1首次以脉络学说营卫理论为指导，探讨微血管损伤在心力衰竭发病中的重要作用，脉络学说认为微血管损伤是心力衰竭重要的发病因素之一，血液动力学和神经体液调节异常共同参与，导致心肌组织结构功能损伤引发心室重构和代谢重构的复杂病理过程。通过实验研究探讨了在心衰时微血管损伤、能量代谢异常和心室重构的病理改变以及三者间的内在联系，验证了脉络学说营卫“由络以通、交会生化”理论科学价值，为从微血管病变切入探讨心力衰竭防治研究提供了新的思路。
2通过整体动物实验，初步揭示了芪苈强心胶囊对心衰微血管损伤、心室重构、能量代谢的干预作用。采用胸主动脉缩窄术建立压力超负荷大鼠模型，证实芪苈强心胶囊通过调节血管活性物质、抑制炎症损伤，促进心肌微血管新生和结构保护、改善血流动力，抑制神经体液过度激活等多重作用，从而有效改善心功能。芪苈强心胶囊抑制心肌成纤维细胞增生，同时减少心肌细胞凋亡，其机制可能与CT-1及CytC介导的线粒体凋亡途径有关。芪苈强心胶囊芪苈强心胶囊通过p-AMPK/PPARα通路调节糖脂代谢途径，改善慢性心衰大鼠能量代谢；通过上调PGC-1α的表达保护心力衰竭大鼠心肌组织线粒体功能与氧化呼吸活性；同时降低循环血中LA、FFA以及UA浓度，减少对内皮损伤作用。
3通过离体实验研究，进一步揭示芪苈强心胶囊改善心肌能量代谢的作用机制与p-AMPK/PPARα信号通路有关；证实芪苈强心胶囊通过上调Mfn2、下调Drp1mRNA保护心肌线粒体，同时芪苈强心胶囊下调Drp1mRNA的表达可抑制CytC介导的心肌细胞凋亡途径，说明能量代谢之线粒体与衰竭心脏的结构重构具有相关性。
Objective: Under the guidance of the Vessel-Collateral Theory, theThoracic Aorta Constriction (TAC) was applied to develop the chronic heartfailure model of rat. The major influence of microvessel injury and effects ofQiliqingxin capsule (QLQX) were disclosed through observing the damage ofmicrovessel structure and function and apoptosis and fibrosis of myocardialtissue and energy metabolism dysfunction of myocardial mitochondria in theanimal model; The Langendorff was adopted to determine the effects ofQLQX on the energy metabolism and mitochondria related protein and signalpathway p-AMPK/PPARαin heart failure. From above experiments, the roleof microvessel and ventricular remodeling and metabolism remodeling toheart failure was explored. These data would give scientific supports to theYing-nutrient and Wei-defense “unblocking collaterals with subsequentconvergence and substance-qi transformation” in the Vessel-CollateralTheory.
Methods:
The study includes four sections:
1The effects and mechanism of QLQX on myocardial microangiopathy inheart failure rats
TAC was used to develop heart failure rats induced by overload pressure.Four weeks later, the survival rats were randomly divided into seven groups(n=15): Control group, Captopril group and high, medium, low-dose QLQXgroups (QLQX-H, QLQX-M, QLQX-L), Model group, Sham operation group(Sham). The rats were administered with QLQX by gavage at the dose of10ml/kg once a day for6weeks, then the same volume of solvent to Sham,Control and Model group. After the last administration and fasted12h, every rat was detected the changes of hemodynamic through carotid arteryintubation. The serum of rats was collected to detect the content of pro-Nterminal brain natriuretic peptide (N-proBNP), angiotensinII (AngII), andaldosterone (ALD) by radioimmunoassay. The ELISA was adopted to detectnorepinephrine (NE), ET-1and vWF. The ultrastructure of microvesselendothelial cells in myocardial tissue was observed by transmission electronmicroscopy. The immunofluorescence technique was used to observe theeffects of QLQX on microvessel density of hypertrophic myocardial tissue inpressure-overload rats. The mRNA expression of ICAM-1VCAM-1andeNOS were detected by Real-time PCR. The content of serum was dected toreflect the endothelial function. The histological staining was adopted toobserve the changes of myocardial cells, cardiac fibroblasts, microvessel andthe intervention effect of QLQX on pathogenesis of heart failure.
2The pathological mechanism of ventricular remodeling in heart failure ratsand the intervention of QLQX
It was the same with part1that experimental animals modeling, grouping,administration and derived method. The HE staining and transmission electronmicroscope were used to observe the ultra-structural changes in myocardium.
The changes of myocardial collagen fiber hyperplasia were determinedwith Masson trachoma staining. The contents of myocardial hydroxyprolinewere detected by the method of alkaline hydrolysis. Western blot was used todetect the protein expression of COLⅠand COLⅢ in myocardial tissue.
The myocardial cell apoptosis rate was detected by flow cytometry andTUNEL. Real-time quantitative PCR was used to analyze the mRNAexpression of nutrition-1(CT-1), cytochrome C (CytC) in myocardial tissue.
3The effects and mechanism of QLQX on metabolic remodeling in heartfailure rats.
It was the same with part1that experimental animals modeling, grouping,administration and derived method. The serum was adopted to detect thecontent of lactic acid (LA), lactate dehydrogenase (LDH), free fatty acid (FFA)and uric acid (UA). The content of ATP, ADP and AMP was determined with high performance liquid chromatography to calculate the total adenosine pooland energy charge in myocardial tissue. Myocardial cell mitochondriamembrane potential (MMP) was detected by flow cytometry. Dissolvedoxygen electrode was used to detect mitochondrial oxidative activity (RCR).Real-time PCR was used to analyze the expression of CPT-Ⅰ, GLUT4andPGC-1α mRNA.Western blot was used to detect p-AMPK, AMPK, PPARαand PGC-1α protein expression level.4The effect and mechanism of QLQX on energy metabolism in the perfusionfailure heart in vitro
The Langendorff perfusion apparatus was adopted to observe the functionof isolated hearts. Low calcium K-H solution induced isolated-heart failure.QLQX was set five concentration (0.025,0.05,0.1,0.2,0.4g/L),andLanatoside as positive control, to observe the effects of QLQX on isolatedcardiac left ventricle pressure, left ventricular load changes and coronary flowand detect the total adenylate pool and energy charge.The mRNA expressionof α-MHC,β-MHC, Mfn2, Drp1was detected by Real-time PCR. Western blotwas used to analyze the protein expression of AMPK, PPARα, p-Akt andp-PI-3K in myocardial tissue.
Results:
1The effects and mechanism of QLQX on myocardial microangiopathy inheart failure rats
Results of hemodynamics in rats: Compared with the sham group, SBP,LVSP, LVEDP in Model group rats were increased and±dp/dtmax decreasedsignificantly (P<0.01). Compared with the Model group, QLQX decreasedSBP, LVSP, LVEDP levels, and increased±dp/dtmax level, estatisticallysignificant changes in QLQX-H and Captopril group (P<0.01). Comparedwith the sham group, no significant difference was detectd in AngⅡ, ALD,NE, NT-proBNP. Compared with the Sham group, these indexes weresignificantly increased in the Model group (P<0.01).Compared with the Modelgroup, the level of Ang II, ALD in QLQX-M, QLQX-H and Captopril groupdecreased significantly (P<0.01). Compared with the Model group, the treatment group decreased NE and NT-proBNP levels, especially QLQX-M(P<0.05), QLQX-H and Captopril group (P<0.01).
The ultrastructure of myocardial micro-vascular: In Model group therewas obvious edema around the capillary, a little in basement membrane, andlumen was irregular, no obvious mitochondrial structure, closely connecttionwas fuzzy, pinocytosis vesicles decreased. Capillary endothelial structure wasbetter in treatment groups, for example, mitochondrial fusion andvacuolization decreased, pinocytosis vesicles relatively increased and tightjunction recoverd obviously.
The microvessel density of cardiac muscle tissue: Compared with shamgroup, the CD34positive count and microvascular relatively density decreasedsignificantly in Model group (P<0.01). The statistical difference of DAPI wasnot showed. Compared with the positive count in Model group, the CD34positive count increased in QLQX-L group (P<0.05), QLQX-M and QLQX-H(P<0.01); compared with the Model group, the relative microvascular densityof cardiac muscle tissue increased in QLQX group, the QLQX-M andQLQX-H group showed statistically difference (P<0.01).
Serum ET-1, vWF levels: Compared with sham group, the ET-1and vWFcontents in Model group increased obviously (P<0.05). Compared with Modelgroup, the ET-1contents in QLQX group decreased significantly (P<0.01).Compared with Model group, the vWF contents decreased significantly inQLQX-M, QLQX-H, Captopril group (P<0.01) and QLQX-L group (P<0.05).
The expression of inflammatory cytokines ICAM-1, VCAM-1and eNOSMrna: Compared with sham group, the ICAM-1and VCAM-1expression ofcardiac tissue of rats in Model group increased significantly (P<0.01).Compared with sham group, the eNOS mRNA expression in Model groupdecreased (P<0.01); compared with Model group, the QLQX group couldpromote the expression of eNOS mRNA at different extents (P<0.05, P<0.01),the QLQX-H group showed remarkable effects (P<0.01).
2Effects of QLQX on ventricular remodeling in pressure-overload heartfailure rats
2.1Myocardial tissue morphological morphology: Compared with the shamgroup, there showed myocardial fiber hypertrophy, partly fracture visible,atrophy of muscle fibers, and diameter in Model group. QLQX inhibitedcardiac hypertrophy, reduced muscle fiber fracture, atrophy and interstitialfibrosis on different degree. Especially QLQX-H group, there was onlypartially myocardial fiber atrophy and myocardial fiber occasionallydegeneration.
Compared with the Sham group, the ultrastructure of cardiac fibersdisplaied disorderly-arranged in Model group. Other changes including Z linesvisible fracture or disappear, myocardial interstitial and obvious edema aroundnuclear, and glycogen decreased. Mitochondrial morphology varied.Membrane structure is not clear, partly membrane fused, and the cristadisordered, fused or disappeared. Treatment groups significantly relieved themyocardial cell stomata edema and mitochondrial injuried, increased theglycogen amount; glycogen particles relatively increased and Z lines werearranged in order.
2.2The effects of QLQX on Collagen fiber hyperplasia of myocardial tissue:
Compared with the control and sham group, HW/BW index of Model ratsincreased obviously (P<0.01).Compared with Model group, the treatmentgroups may lower HW/BW index (P<0.05, P<0.01), especially the index ofQLQX-H rats decreased significantly.The Masson trichromatic staining ofmyocardial tissue showed that myocardial fibers arranged closely, and noobvious hyperplasia of collagen fiber in Control group and the shamgroup.There were mass collagen hyperplasia and spread to the myocardialfiber bundles in Model group. QLQX could inhibit the collagen fiberproliferation and reduce collagen fibrosis size in myocardial tissue and bloodvessels.
Compared with the sham group, the content of hydroxyproline increasedsignificantly in Model group(P<0.01).Compared with the Model group, thehydroxyproline content in treatment group decreased at different degree, inwhich the QLQX-L group, captopril group (P<0.05), QLQX-M and QLQX-H group decreased significantly (P<0.01).
Compared with sham group, the content of COL I, COL III in Modelgroup increased significantly(P<0.01).Compared with Model group, captopriland QLQX-H decreased the expression of COLⅠ(P<0.01,P<0.05). Comparedwith Model group, there was a significant statistical difference between thecaptopril group, QLQX-M and QLQX-H groups in COL Ⅲ protein,significantly in QLQX-H group.
2.3Myocardial cell apoptosis:
The results with TUNNEL analysis showed that the amout of apoptoticcells of myocardial tissue in Model group significantly increased andapoptosis cells decreased after treatment. The results with Flow cytometryanalysis showed that the apoptotic myocardial cells significantly increased(P<0.01) in Model group.Compared with sham group, QLQX reducedapoptotic myocardial cells in pressure-overload heart failure rats statisticallysignificantly (P<0.01).
Compared with sham group, the mRNA expression of CT-1and CytCsignificantly increased in Model group (P<0.01). Compared with the Modelgroup, QLQX reduced CT-1, CytC expression (P<0.01), Captopril groupdecreased CT-1, CytC mRNA expression (P<0.05, P<0.01).
3The effects of QLQX on the metabolic remodeling of rats suffered fromcardiac failure
Compared with Model group, the QLQX-L and QLQX-H group coulddecrease the blood serum LA content of pressure overload rats significantly(P<0.05). The QLQX decreased LDH and UA level greatly (P<0.01).Compared with Model group, FFA content in QLQX groups decreased atdifferent concentrations with statistical difference. QLQX-H group showedobvious effect (P<0.01), which was better than captopril group (P<0.05).
Compared with sham group, the total adenosine and energy charge valuedecreased significantly (P<0.01). Compared with Model group, treatmentgroup increased the adenosine pool and energy value greatly, in which theQLQX-L increased the total adenosine pool content and energy value (P<0.05, P<0.01).
Compared with sham group, the membrane potential of mitochondria ofcardiac muscle decreased in the Model group (P<0.01). Compared with Modelgroup, the MMP of mitochondria of cardiac muscle increased in treatmentgroup, and QLQX-M and QLQX-H group showed significant increase(P<0.01). Treatment groups could improve the respiratory function ofmitochondria and enhance the RCR (P<0.05, P<0.01).
Compared with sham group, the mRNA expression of CPT-Ⅰand GLUT
4and PGC-1α in Model group decreased significantly (P<0.01). Comparedwith Model group, the mRNA expression of above index in QLQX-H groupshowed significant difference (P<0.05, P<0.01).
Compared with sham group, the protein expression of p-AMPK in Modelgroup showed no statisticly difference (P>0.05). Compared with Model group,the expression level of p-AMPK in treatment group increased significantly, inwhich the QLQX-M and QLQX-H group increased obviously (P<0.01). Thetotal amount of AMPK in each group didn’t show significant difference(P>0.05). Compared with sham group, the protein expression level of PPARαand PGC-1α in Model group decreased obviously (P<0.01). Compared withModel group, the expression of the two protein in QLQX-M and QLQX-Hgroup increased significantly (P<0.01).
4The effects and mechanism of QLQX on energy metabolism in the perfusionfailure heart in vitro
Only being perfused with K-H liquid, the left ventricular pressure in eachgroup didn’t show significant difference (P>0.05). When mimicing the cardiacfailure with low calcium K-H liquid perfusion, the left ventricular pressure ineach group decreased obviously and the cardiac loading increased at meanwhile. Compared with Model group, the left ventricular pressure in all QLQXgroups at different concentration increased accompanying withconcentration increasing, in which the left ventricular pressure of QLQX-0.2,QLQX-0.4group increased significantly (P<0.01), and this showed certaindose-effect relationship. After being adding QLQX, the left ventricular loading in each concentration group showed decrease in different degree. After beingadding two blockers, the pressure decreased significantly (P<0.01), and thecardiac load increased at some extent, and the cardiac load after addingblocker MK-886showed significant increase (P<0.05).
Compared with normal K-H perfusion, the coronary flow in Model groupdecreased obviously (P<0.01). Compared with Model group, theQLQX-0.05,QLQX-0.1,QLQX-0.2and QLQX-0.4group increased thecoronary flow obviously (P<0.05, P<0.01).Compared with QLQX-0.2, thecoronary flow with two blockers decreased, but there was no statisticlydifference between these two blocker groups.
Compared with control group, the energy charge value of cardiac muscletissue in Model group decreased (P<0.01).Compared with Model group, thepositive medication, QLQX-0.1, QLQX-0.2and QLQX-0.4showed (P<0.01)statistically significance.
Compared with Model group, the mRNA expression level of Drp1inQLQX-0.4group decreased (P<0.05). Compared with QLQX-0.2group, theexpression with blockers increased, and but showed no significant difference.Compared with Model group, the positive control medication, QLQX-0.2and0.4group increased the expression of Mfn2obviously (P<0.01), andQLQX-0.1showed statistically difference (P<0.05). The positive controlmedication, QLQX-0.1, QLQX-0.2and QLQX-0.4group increased theα-MHC mRNA (P<0.01). Compared with QLQX-0.2group, two groups withblocker decreased the expression of α-MHC mRNA. Compared with Modelgroup, the positive control medication, QLQX-0.2and QLQX-0.4groupdecreased the expression of β-MHC greatly (P<0.01). Compared withQLQX-0.2group, two groups with blocker increased the mRNA expression ofβ-MHC.
Compared with normal K-H perfusion group, the expression level ofp-PI-3K and p-Akt in Model group decreased (P<0.01).Compared with Modelgroup, the treatment increased the expression level of p-PI-3K and p-Aktprotein (P<0.05, P<0.01). Compared with QLQX-0.2, the two blockers up-regulated the protein expression of p-Akt, but this didn’t show statisticdifference (P>0.05).Compared with QLQX-0.2group, the expression ofp-PI-3K with two blockers didn’t show statistic difference (P>0.05).Compared with positive medication, the QLQX-0.4increased the proteinexpression of p-Akt (P<0.05).
Conclusion:
1It is the first time that Ying and Wei theory in Vessels-CollateralTheory was used as guidance to discuss the important role of microvasculardamage in heart failure. Heart failure is multi-dimensional spatio-temporalcomplex pathological processes including ventricular remodeling and energymetabolism disorder, caused by abnormal hemodynamic and neurohumoraltrigger. Through experiment, microvascular injury, abnormal energymetabolism, pathological changes of ventricular remodeling in case of heartfailure and the internal relation between them are discussed. The theory ofscientific value of the Ying-nutrient and Wei-defense “unblocking collateralswith subsequent convergence and substance-qi transformation” of theVessel-Collateral Theory was verified, and it offers new thinking for theprevention and treatment of cardiac failure.
2The effects and mechanism of QLQX on microvascular provention,ventricular remodeling, and energy metabolism were elaborated through theanimal experiment in vivo. TAC was applied to develop the chronic heartfailure model of rat and demonstrate that QLQX could improving the functionof failing heart by adjusting the vasoactive substances, inhibittinginflammatory injury, promoting myocardial angiogenesis and structure,improving hemodynamics, and inhibitting neurohumoral excessivelyactivation. QLQX inhibited cardiac fibroblast proliferation. At the same time,QLQX capsule reduced myocardial apoptosis, the mechanism may be relatedto the mitochondrial apoptotic pathway mediated by CT-1and CytC. QLQXcapsule regulates metabolic pathways of glycolipid through p-AMPK/PPARαpath, improves energy metabolism of rats with chronic heart failure, increasesthe expression of PGC-1α, promotes mitochondrial biosynthesis, protects mitochondrial function and oxidative respiration activity of myocardial tissueof rats with heart failure, reduces the concentration of LA, FFA and UA incirculating blood and reduces endothelial injury.3It was demonstrated the protective effects of QLQX on the structure andfunction of myocardial mitochondrial through the up-regulation of Mfn2mRNA expression and down-regulation expression of Drp1mRNA, andQLQX inhibit myocardial mitochondrial apoptosis pathway mediated by CytCthrough down-regulating Drp1mRNA expression. This illustrates thecorrelation between mitochondria of energy metabolism and structuralremodeling of the failing heart.
引文
1Rosamond W, Flegal K, Friday G, et al. Heart disease and strokestatistics--2007update: a report from the American Heart AssociationStatistics Committee and Stroke Statistics Subcommittee. Circulation,2007,115(5): e69~e171
2中华医学会心血管病学分会,中华心血管病杂志编辑委员会.慢性心力衰竭诊断治疗指南.中华心血管病杂志,2007,35(12):1076~1095
3杨军,蒲世军,蒲国俭,等.慢性心力衰竭注册治疗的临床研究.当代医学,2010,16(10):4~6
4黄海.川芎嗪干预慢性充血性心力衰竭心室重构的实验研究.福建中医学院,2005:88
5董现锋,洪华山.心血管病时冠状动脉微血管重构研究进展.中国动脉硬化杂志,2004,12(1):116~118,120
6Sestito A,Lanza G A, Di Monaco A, et al. Relation between cardiovascularrisk factors and coronary microvascular dysfunction in cardiac syndromeX. J Cardiovasc Med (Hagerstown),2011,12(5):322~327
7Camici P G, Crea F. Coronary microvascular dysfunction. N Engl J Med,2007,356(8):830~840
8den Uil C A,Klijn E, Lagrand W K, et al. The microcirculation in healthand critical disease. Prog Cardiovasc Dis,2008,51(2):161~170
9Neubauer S. The failing heart--an engine out of fuel. N Engl J Med,2007,356(11):1140~1151
10Van Bilsen M, Smeets P J, Gilde A J, et al. Metabolic remodelling of thefailing heart: the cardiac burn-out syndrome? Cardiovasc Res,2004,61(2):218~226
11李佳蓓,黄岚,祝善俊.心力衰竭时心肌能量代谢的研究进展.心血管康复医学杂志,2009,18(1):85~88
12吴以岭,袁国强,贾振华,等.脉络学说的学术地位及其应用价值.中医杂志,2012,(01):3~7
13张建,华琦.心力衰竭的诊断与治疗.北京:人民卫生出版社,2006
14Feng J, Li S S, Liang Q S. Effects of Tanshinone II A on the myocardialapoptosis and the miR-133levels in rats with heart failure. ZhongguoZhong Xi Yi Jie He Za Zhi,2012,32(7):930~933
15孙莉,李树青.高血压致左室肥厚的发生机制及其治疗进展.中国心血管病研究杂志,2007,5(4):315~317
16王玮,方志成,黄从新.心血管疾病与心肌微血管病变关系研究现状,2006,27(4):463~466
17Hu P, Zhang D, Swenson L, et al. Minimally invasive aortic banding inmice: effects of altered cardiomyocyte insulin signaling during pressureoverload. Am J Physiol Heart Circ Physiol,2003,285(3): H1261~H1269
18孙桂芳,刘凤岐.慢性心力衰竭大鼠心肌毛细血管密度及血管内皮生长因子变化.中国组织化学与细胞化学杂志,2005,14(3):339~343
19吴以岭.脉络论.北京:中国科学技术出版社,2010:1301
20Spencer C G,Martin S C, Felmeden D C, et al. Relationship ofhomocysteine to markers of platelet and endothelial activation in "highrisk" hypertensives: a substudy of the Anglo-Scandinavian CardiacOutcomes Trial. Int J Cardiol,2004,94(2-3):293~300
21赵晓民.血压波动性增高致微循环异常和左心室肥厚及其机理的实验研究.山东大学,2008:178
22孙桂芳,刘凤岐,修春红.冠脉微循环障碍参与慢性心力衰竭大鼠心肌重构.中国地方病学杂志,2005,(5):500~502
23鹿欣伦,胡成云,张雨梅.心力衰竭机制的研究进展.山西医药杂志,2011,40(8):766~767
24Spinarova L, Vitovec J. Neurohumoral changes in chronic heart failure.Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub,2007,151(2):201~207
25袁国强,贾振华,杨海涛,等.过度安逸所致神经—内分泌—免疫网络失衡—血管内皮功能障碍发生的可能机制.络病学基础与临床研究,2010,(6):72~77
26农一兵,苏敬泽,温志浩,等.从补益心气与改善心肌能量代谢的关系探讨慢性心力衰竭中西医结合研究的思路.中医杂志,2007,(11):1035~1036
27Yunzeng Zou, Li Lin, Yong Ye, et al. Qiliqiangxin Inhibits theDevelopment of CardiacHypertrophy, Remodeling, and DysfunctionDuring4Weeks of Pressure Overload in Mice. J Cardiovasc Pharmacol,2012,59(3):268~280
28Hong Xiao, You Song, Ya Li, et al. Qiliqiangxin regulates the balancebetween tumor necrosis factor-α and interleukin-10and improves cardiacfunction in rats with myocardial infarction. Cellular Immunology.2009,260:51~55
29Wei Liu, Junjun Chen, Tengfei Xu, et al. Qiliqiangxin Improves CardiacFunction in Spontaneously Hypertensive Rats through the Inhibition ofCardiac Chymase. American Journal of Hypertension,2012,25:250~260
30Yidong Wei, Xiaoyu Liu, Lei Hou, et al. Qiliqiangxin Affects L Type Ca2+Current in the Normal and Hypertrophied Rat Heart. Evidence-BasedComplementary and Alternative Medicine.2012,131830. Doi:10.1155/2012/131830. Epub2012Mar26
31王晓艳.健心汤对心力衰竭的疗效观察及抗心肌重塑机制研究.中南大学,2009
32袁劲松. N-末端脑利钠肽原在心力衰竭中的临床应用进展.国际检验医学杂志,2010,31(12):1417~1419
33汪芳,李卫,黄洁,等.血浆N末端原脑利钠肽水平对慢性心力衰竭患者长期预后的预测价值.中华心血管病杂志,2006,34(1):28~32
34袁慧.关注BNP与NT-proBNP的临床应用.中华检验医学杂志,2012,35(10):870~873
35牛连英,张丽娜,赵晓静.芪苈强心胶囊对老年慢性收缩性心力衰竭疗效及血清BNP水平的影响.疑难病杂志,2012,11(10):741~743
36姚华丽.转录因子激活蛋白-1在血管重构发生中的作用.国际心血管病杂志,2010,37(4):203~206
37Meyer B, Mortl D, Strecker K, et al. Flow-mediated vasodilation predictsoutcome in patients with chronic heart failure: comparison with B-typenatriuretic peptide. J Am Coll Cardiol,2005,46(6):1011~1018
38Schulz R, Rassaf T, Massion P B, et al. Recent advances in theunderstanding of the role of nitric oxide in cardiovascular homeostasis.Pharmacol Ther,2005,108(3):225~256
39唐可京. ICAM-1和VCAM-1的结构与表达调控.国外医学(分子生物学分册),2002,(3):173~177
40刘莉,邹国良,索传涛.利心Ⅰ号对CHF大鼠血流动力学、心肌细胞ICAM-1基因蛋白表达的影响.心脏杂志,2007,19(2):166~169
41杜荣增,汤永庆,邱建平,等. β受体阻断剂对慢性心力衰竭患者血浆vWF及TNF-α的影响.临床心血管病杂志,2011,27(3):232~233
42张三印,沈映君,陈士林,等.葛根素对心肌梗死大鼠心肌HIF-1、eNOS、VEGF基因表达及血清NO浓度的影响.中药药理与临床,2007,23(5):54~57
43张赛丹,全勇,周宏研,等.慢性心力衰竭与内皮功能异常的相关性研究.医学临床研究,2006,23(10):1545~1548
44Kempf T, Wollert K C. Nitric oxide and the enigma of cardiachypertrophy. Bioessays,2004,26(6):608~615
1任崇雷,高长青,李力兵.细胞心肌成形术对梗死后心室重构的影响.中华外科杂志,2007,45(22):1578~1580
2董志峰.系统性免疫调节药物对压力超负荷性大鼠心室重构和心功能干预的实验研究.扬州大学,2006:4
3Le Bousse-Kerdiles M C,Martyre M C, Samson M. Cellular and molecularmechanisms underlying bone marrow and liver fibrosis: a review. EurCytokine Netw,2008,19(2):69~80
4王健,刘全.银丹心脑通软胶囊对大鼠急性心肌梗死后心肌组织TGF-β1表达的影响.中西医结合心脑血管病杂志,2009,7(1):46~47
5Celik A,Sahin S, Koc F, et al. Cardiotrophin-1plasma levels are increasedin patients with diastolic heart failure. Med Sci Monit,2012,18(1):R25~R31
6Gonzalez A, Lopez B, Ravassa S, et al. Cardiotrophin-1in hypertensiveheart disease. Endocrine,2012,42(1):9~17
7Lopez-Andres N, Rousseau A, Akhtar R, et al. Cardiotrophin1is involvedin cardiac, vascular, and renal fibrosis and dysfunction. Hypertension,2012,60(2):563~573
8宋慧文,王琳.苦参素对实验性兔心房颤动左房纤维化细胞间黏附分子－1表达的影响.实用医学杂志,2009,25(12):1920~1922
9李琮辉,窦献蕊.原发性高血压黏附分子及胶原代谢变化的初步探讨.中国循环杂志,2002,17(5):346~349
10李为民,孙宁玲,刘巍,等.缬沙坦对自发性高血压大鼠心肌细胞凋亡的影响.中华医学杂志(英文版),2002,115(3):364~366
11He-Nan Z, Yan W, Miao-Na J, et al. Relation of Cardiotrophin-1(CT-1)and cardiac transcription factor GATA4expression in rat's cardiacmyocytes hypertrophy and apoptosis. Pathol Res Pract,2009,205(9):615~625
12简立国,李恩,孙利强,等. CT-1mRNA在心肌梗死大鼠心肌细胞凋亡中的表达及罗格列酮对其的影响.中外医疗,2009,28(24):18~20
13姚震,陈淑芬,张云波,等.促心肌素C-末端肽对再灌注损伤大鼠心肌细胞凋亡的影响.中国热带医学,2011,11(5):617~619
14Ruggieri M,Avolio C, Scacco S, et al. Glatiramer acetate inducespro-apoptotic mechanisms involving Bcl-2, Bax and Cyt-c in peripherallymphocytes from multiple sclerosis patients. J Neurol,2006,253(2):231~236
1Sambandam N,Lopaschuk G D, Brownsey R W, et al. Energy metabolismin the hypertrophied heart. Heart Fail Rev,2002,7(2):161~173
2van Bilsen M,Smeets P J, Gilde A J, et al. Metabolic remodelling of thefailing heart: the cardiac burn-out syndrome? Cardiovasc Res,2004,61(2):218~226
3李佳蓓,黄岚,祝善俊.心力衰竭时心肌能量代谢的研究进展.心血管康复医学杂志,2009,18(1):85~88
4吴以岭.脉络论.北京:中国科学技术出版社,2010:1301~1302
5王军,白玲,李晶,等.心力衰竭大鼠心肌线粒体蛋白质组学研究.中国科学C辑：生命科学,2009,39(11):1019~1027
6魏聪.基于代谢组学的络气虚滞/郁滞证候病生理基础及通络干预研究.河北医科大学,2011:180
7王红英.卡托普利、心复康口服液对心衰大鼠心肌细胞凋亡和线粒体膜电位的影响.河北医科大学,2008:84
8Ashrafian H,Frenneaux M P, Opie L H. Metabolic mechanisms in heartfailure. Circulation,2007,116(4):434~448
9刘瑛琪,李天德,楮晓雯,等.利用荧光探针JC-1检测心肌细胞线粒体膜电位的改变.解放军医学杂志,2002,27(8):716~718
10Azevedo P S,Minicucci M F, Santos P P, et al. Energy metabolism incardiac remodeling and heart failure. Cardiol Rev,2013,21(3):135~140
11祝善俊,王江.能量代谢疗法能否作为治疗心力衰竭的新靶点?岭南心血管病杂志,2010,16(2):97~98
12祝善俊.代谢重构与慢性心力衰竭.中华老年心脑血管病杂志,2007,(6):361~363
13谌煜.高尿酸血症与心血管疾病的研究进展.疑难病杂志,2012,11(2):147~148
14李鑫德,崔凌凌,任伟,等.高尿酸血症与心血管疾病关系的研究进展.中华内分泌代谢杂志,2011,27(7):614~617
15何青.高尿酸血症与心血管疾病.中国心血管杂志,2010,15(3):169
16Stanley W C, Recchia F A, Lopaschuk G D. Myocardial substratemetabolism in the normal and failing heart. Physiol Rev,2005,85(3):1093~1129
17Essop M F, Opie L H. Metabolic therapy for heart failure. Eur Heart J,2004,25(20):1765~1768
18Aroor A R,Mandavia C H, Sowers J R. Insulin resistance and heart failure:molecular mechanisms. Heart Fail Clin,2012,8(4):609~617
19Taylor M, Wallhaus T R, Degrado T R, et al. An evaluation of myocardialfatty acid and glucose uptake using PET with[18F]fluoro-6-thia-heptadecanoic acid and [18F]FDG in Patients withCongestive Heart Failure. J Nucl Med,2001,42(1):55~62
20蒙荣森. PPARα介导的AMPK激活对心肌肥厚及能量代谢的调节作用.中山大学博士后学位论文,2009:17~40
21Jeninga E H, Schoonjans K, Auwerx J. Reversible acetylation of PGC-1:connecting energy sensors and effectors to guarantee metabolic flexibility.Oncogene,2010,29(33):4617~4624
22Uguccioni G, Hood D A. The importance of PGC-1alpha in contractileactivity-induced mitochondrial adaptations. Am J Physiol EndocrinolMetab,2011,300(2):E361~E371
23Lira V A, Brown D L, Lira A K, et al. Nitric oxide and AMPKcooperatively regulate PGC-1in skeletal muscle cells. J Physiol,2010,588(Pt18):3551~3566
24Yunzhou Dong, Miao Zhang, Shuangxi Wang. Activation ofAMP-Activated Protein Kinase Inhibits Oxidized LDL-TriggeredEndoplasmic Reticulum Stress in Vivo. Diabetes,2010,59(6):1386~1396
25Tran Thi Hien, Won Keun Oh, Phi Hung Nguyen, et al. Nectandrin BActivates Endothelial Nitric-Oxide Synthase Phosphorylation inEndothelial Cells: Role of the AMP-Activated Protein Kinase/EstrogenReceptor α/Phosphatidylinositol3-kinase/Akt Pathway. MolecularPharmacology,2011,80(6):1166~1178
1梁伟涛,Vitali R.离体心脏Langendorff灌流模型稳定性的探讨.国际心血管病杂志,2011,38(1):44~47
2Zhu M,Feng J, Lucchinetti E, et al. Ischemic postconditioning protectsremodeled myocardium via the PI3K-PKB/Akt reperfusion injury salvagekinase pathway. Cardiovasc Res,2006,72(1):152~162
3蔡辉,郭郡浩,商玮,等.压力负荷增加大鼠模型心肌α-MHC和β-MHCmRNA的表达.医学研究生学报,2009,22(10):1032~1035
4Kong Y, Tannous P, Lu G, et al. Suppression of class I and II histonedeacetylases blunts pressure-overload cardiac hypertrophy. Circulation,2006,113(22):2579~2588
5Meng RS, Pei ZH,Yin R,et al. Adenosine monophosphate-activatedprotein kinase inhibits cardiac hypertrophy through reactivatingperoxisome proliferator-activated receptor-alpha signaling pathway. Eur Jpharmacol,2009,620(1-3):63~70
6郝希纯,王东明. Drp1蛋白调节线粒体分裂机制及其在疾病中的作用.广东医学,2011,32(8):1066~1069
7Detmer S A, Chan D C. Functions and dysfunctions of mitochondrialdynamics. Nat Rev Mol Cell Biol,2007,8(11):870~879
8Lee Y J,Jeong S Y, Karbowski M, et al. Roles of the mammalianmitochondrial fission and fusion mediators Fis1, Drp1, and Opa1inapoptosis. Mol Biol Cell,2004,15(11):5001~5011
9Shen T, Zheng M, Cao C, et al. Mitofusin-2is a major determinant ofoxidative stress-mediated heart muscle cell apoptosis. J Biol Chem,2007,282(32):23354~23361
10施冰,冬兰,尹秋生,等.大鼠心肌梗死后心肌组织中线粒体融合蛋白2基因和线粒体分裂蛋白mRNA表达水平的变化.中国临床保健杂志,2012,15(4):392~394
11邓琴琴. EPO通过激活PI3K/Akt途径并上调HSP70的表达对低温下的心衰大鼠起保护作用.南昌大学,2010
1World health organization. Cardiovascular diseases prevention and control.http://www.afro.who.int/en/clusters-a-programmes/dpc/non-communicable-diseases-managementndm/programme-components/cardiovascular-diseases.html.
2顾东风,黄广勇,何江,等.中国心力衰竭流行病学调查及其患病率.中华心血管病杂志,2003,31(1):3~5
3张建,华琦.心力衰竭的诊断与治疗.北京:人民卫生出版社,2006
4姜红,葛均波.心力衰竭药物治疗的现状和前景.中国临床医学,2005,12(3):363~367
5Neubauer S. The failing heart-an engine out of fuel. N Engl J Med,2007,356(11):1140~1151
6Van Bilsen M, Smeets P J, Gilde A J, et al. Metabolic remodelling of thefailing heart: the cardiac burn-out syndrome? Cardiovasc Res,2004,61(2):218~226
7李佳蓓,黄岚,祝善俊.心力衰竭时心肌能量代谢的研究进展.心血管康复医学杂志,2009,18(1):85~88
8祝善俊.代谢重构与慢性心力衰竭.中华老年心脑血管病杂志,2007,9(6):361~363
9Maslov MY, Chacko VP, Stuber M.et al. Altered high-energy phosphatemetabolism predicts contractile dysfunction and subsequent ventricularremodeling in pressure-overload hypertrophy mice. Am J Physiol HeartCirc Physiol,2007,292: H387~391
10能量缺乏是心力衰竭的原因.中华高血压杂志.2008,16(3):273~275
11祝善俊,徐成斌.心力衰竭基础与临床.北京:人民军医出版社,2001:308~520
12祝善俊.代谢重构与慢性心力衰竭.中华老年心脑血管病杂志,2007,9(6):361~363
13Stanley W C, Recchia F A, Lopaschuk G D. Myocardial substratemetabolism in the normal and failing heart. Physiol Rev,2005,85(3):1093~1129
14Essop M F, Opie L H. Metabolic therapy for heart failure. Eur Heart J,2004,25(20):1765~1768
15Aroor A R,Mandavia C H, Sowers J R. Insulin resistance and heart failure:molecular mechanisms. Heart Fail Clin,2012,8(4):609~617
16Taylor M, Wallhaus T R, Degrado T R, et al. An evaluation of myocardialfatty acid and glucose uptake using PET with[18F]fluoro-6-thia-heptadecanoic acid and [18F]FDG in Patients withCongestive Heart Failure. J Nucl Med,2001,42(1):55~62
17Rosenblatt-Velin N, Montessuit C, Papageorgiou I, et al. Postinfarctionheart failure in rats is associated with upregulation of GLUT-1anddownregulation of genes of fatty acid metabolism. Cardiovasc Res,2001,52:407~416
18Kajstura J, Cigola E,Malhotra A, et al. Angiotensin II induces apoptosisof adult ventricular myocytes in Vitro. J Mol Cell Cardiol,1997,29(3):859~870
19何茹.解偶联蛋白家族成员UCP2.北京教育学院学报(自然科学版).2009,4(2):19~23
20Ide T, Tsutsui H, Hayashidani S, et al. Mitochondrial DNA damage anddysfunction associated with oxidative stress in failing hearts aftermyocardial infarction. Circ Res,2001,88:529~535
21Reznick RM, Shulman GI. The role of AMP-activated protein kinase inmitochondrial biogenesis. J Physiol,2006,574:33~39
22李良刚,陈槐卿. CaMK和AMPK信号通路能共调收缩信号诱导的骨骼肌细胞GLUT4基因转录.生物化学与生物物理进展,2009,4:471~479
23Deldicque L, Theisen D, FrancauxM. Regulation of mTOR by amino acidsand resistance exercise in skeletalmuscle. Eur J ApplPhysiol,2005,94(1-2):1~10
24Kanda H, Nohara R, Hasegawa K, et al. A nuclear complex containingPPARalpha/RXRalpha is markedly downregulated in the hypertrophied ratleft ventricular myocardium with normal systolic function. Heart Vessels,2000,15:191~196
25Garnier A, Fortin D, Delomenie C, et al. Depressed mitochondrialtranscription factors and oxidative capacity in rat failing cardiac andskeletal muscles. Physiol,2003,551:491~501
26MF Essop, LH Opie. Metabolic therapy for heart failure. Eur Heart J,2004,25:1814~1821
27P Di Napoli, AA Taccardi, A Barsotti. Long term cardioprotective actionof trimetazidine and potential effect on the inflammatory process inpatients with ischaemic dilated cardiomyopathy. Heart,2005,91:161~165
28Beadle RM,Williams LK,Abozguia K,et al. Metabolic manipulation inchronic heart failure:study protocol for a randomized controlled trial.Trials,2011,12:140
29Kato T, Niizuma S, Inuzuka Y, et al. Analysis of metabolic remodeling incompensated left ventricular hypertrophy and heart failure. Circ Heart Fail,2010,3(3):420~430
30Stanley WC, Chandler MP. Energy metabolism in the normal and failingheart: potential for therapeutic interventions. Heart Fail Rev,2002,7:115~30
31张治宇,殷仁富,李振东,等.雷卡对肾血管性高血压大鼠心肌重塑和脂肪酸代谢的影响.同济大学学报(医学版),2006,27(4):32~35
32汪凡,秦明照."代谢类"抗心肌缺血药物的研究进展.中国医药导刊,2006,8(2):124~126
33宋熔,祝善俊.心肌代谢重构的研究进展.中华高血压杂志,2008,16(3):202~204
34Kristiansen SB. Cardioprotection against ischermia reperfusion injury byL-glutamate and KATP channel openers. Dan Med Bull,2005,52(4):261
1杨淑艳,张雷,郑中华,等.心力衰竭的病理生理机制及药物治疗进展.吉林医药学院学报,2006,27(3):168~171
2吴以岭,主编.脉络论.北京:中国科学技术出版社,2010
3贾美君,蒋梅先,何峥.心力衰竭时动脉功能变化与络病现代病理机制.络病学基础与临床研究(2):397~401
4吴以岭,主编.络病学.北京:中国科学技术出版社,2004:283~285
5王宏涛,唐思文,徐登峰,等.芪苈强心胶囊多目标优化后组合物对实验性心衰大鼠心肌细胞间连接的影响.时珍国医国药,2011,22(10):2375~2377
6唐思文,徐登峰,王玲玲.芪苈强心胶囊多目标优化组合物对实验性心衰大鼠心功能的保护作用.中药新药与临床药理,2011,22(5):532~535
7刘奕训,余河水,康利平.芪苈强心胶囊活性部位中的组成成分研究.中草药,2010,41(7):1060~1065
8康利平,赵阳,余河水,等.采用UPLC-Q-TOF/MSE鉴别芪苈强心胶囊有效部位中的化学成分.药学学报,2011,46(10):1231~1236
9刘建勋,马晓斌,王杨慧.芪苈强心胶囊对实验性心力衰竭犬心脏功能的影响.疑难病杂志,2007,6(3):141~143
10李娅,宋优,程翔,廖玉华.芪苈强心胶囊对大鼠心肌梗死后心肌重构及心功能的影响.中国分子心脏病学杂志,2007,7(4):201~204
11邬真力,许顶立,林昇,等.芪苈强心胶囊对慢性心力衰竭大鼠心脏功能及血浆血管加压素的影响.疑难病杂志,2011,10(2):120~122
12许顶立,芪苈强心对慢性心力衰竭大鼠肾脏AQP-2及心脏功能的影响.中国医学论坛报,2009.12.10
13Xiao H, Song Y, Li Y, et al. Qiliqiangxin regulates the balance betweentumor necrosis factor-α and interleukin-10and improves cardiac functionin rats with myocardial infarction. Cellular immunology,2009,260(1):51~55
14徐涛,郭丽峰,陈立锋,等.芪苈强心胶囊对慢性心力衰竭大鼠PPARα表达的影响.河北医药,2010,32(22):3120~3122
15孙鑫,许静静,陈伟.芪苈强心胶囊对压力超负荷致慢性心衰大鼠的作用及机理研究.中国中医基础医学杂志,2010,16(7):560~562
16林锐波.芪苈强心胶囊对心梗后心衰大鼠心功能及凋亡蛋白caspase-3表达的影响.汕头大学,2010
17徐涛,郭丽峰,陈立锋,等.芪苈强心胶囊对慢性心力衰竭大鼠心肌细胞凋亡的影响.中药新药与临床药理,2010,21(4):366~369
18Briant E B,Richard D O, Barry J C. Anthracycline cardiotoxicity intransgenic mice overexpressing SR Ca2+-ATPase. Biochemical andBiophysical Research Communications,2003,303:504~507
19李佳彧,杨萍.芪苈强心胶囊对心梗后心力衰竭大鼠periostin蛋白表达干预作用的研究.中国实验诊断学,2009,13(2):170~172
20李佳彧,李佳睿,马丕勇,等.通络药物对心梗后心衰大鼠非梗死区心肌纤维化的影响.现代生物医学进展,2011,11(18):3431~3434
21李佳彧,杨萍.通络药物对心肌梗死后心衰大鼠瘢痕区纤维化的影响.中国老年学杂志,2009,29(21):15~17
22杨正央,邓永军.芪苈强心胶囊治疗收缩性心力衰竭的观察.中外医疗,2010,5:113
23吴以岭,谷春华,徐贵成,等.芪苈强心胶囊治疗慢性心力衰竭随机双盲、多中心临床研究.疑难病杂志,2007,6(5):263~266
24刘春香,毛静远,王贤良,等.芪苈强心胶囊治疗慢性心力衰竭的系统评价.中成药,2010,32(4):539~564
25刘军,刘庚,刘江涛,等.芪苈强心胶囊辅治扩张型心肌病心功能不全的疗效观察.疑难病杂志,2009,8(3):159~160
26刘新明,宋一平,顾明峰.芪苈强心胶囊辅治老年难治性心力衰竭疗效观察.临床合理用药,2010,3(18):56~57
27陈丽萍,孟宪文,崔兆文,等.呋塞米持续泵入联合多巴胺及芪苈强心胶囊治疗心肾综合征并发利尿剂抵抗25例临床观察.河北中医,2011,33(5):723~724
28马芳放,路凤月,肖维刚,等.芪苈强心胶囊辅治对慢性心力衰竭患者肱动脉血管内皮舒张功能的影响.疑难病杂志,2008,7(9):543~544
29侯湘岭.芪苈强心胶囊联合西药治疗急性心肌梗死后心力衰竭合并低血压状态46例临床观察.河北中医,2010,32(10):1540~1541
30宋云方.芪苈强心胶囊与辅酶Q10胶囊联合治疗扩张型心肌病60例.中国医药指南,2009,14:81~82
31闫松改.芪苈强心胶囊与螺内酯联合治疗慢性心力衰竭疗效观察.临床医学,2009,29(11):30~31
2中华医学会心血管病学分会,中华心血管病杂志编辑委员会.慢性心力衰竭诊断治疗指南.中华心血管病杂志,2007,35(12):1076~1095
3杨军,蒲世军,蒲国俭,等.慢性心力衰竭注册治疗的临床研究.当代医学,2010,16(10):4~6
4黄海.川芎嗪干预慢性充血性心力衰竭心室重构的实验研究.福建中医学院,2005:88
5董现锋,洪华山.心血管病时冠状动脉微血管重构研究进展.中国动脉硬化杂志,2004,12(1):116~118,120
6Sestito A,Lanza G A, Di Monaco A, et al. Relation between cardiovascularrisk factors and coronary microvascular dysfunction in cardiac syndromeX. J Cardiovasc Med (Hagerstown),2011,12(5):322~327
7Camici P G, Crea F. Coronary microvascular dysfunction. N Engl J Med,2007,356(8):830~840
8den Uil C A,Klijn E, Lagrand W K, et al. The microcirculation in healthand critical disease. Prog Cardiovasc Dis,2008,51(2):161~170
9Neubauer S. The failing heart--an engine out of fuel. N Engl J Med,2007,356(11):1140~1151
10Van Bilsen M, Smeets P J, Gilde A J, et al. Metabolic remodelling of thefailing heart: the cardiac burn-out syndrome? Cardiovasc Res,2004,61(2):218~226
11李佳蓓,黄岚,祝善俊.心力衰竭时心肌能量代谢的研究进展.心血管康复医学杂志,2009,18(1):85~88
12吴以岭,袁国强,贾振华,等.脉络学说的学术地位及其应用价值.中医杂志,2012,(01):3~7
13张建,华琦.心力衰竭的诊断与治疗.北京:人民卫生出版社,2006
14Feng J, Li S S, Liang Q S. Effects of Tanshinone II A on the myocardialapoptosis and the miR-133levels in rats with heart failure. ZhongguoZhong Xi Yi Jie He Za Zhi,2012,32(7):930~933
15孙莉,李树青.高血压致左室肥厚的发生机制及其治疗进展.中国心血管病研究杂志,2007,5(4):315~317
16王玮,方志成,黄从新.心血管疾病与心肌微血管病变关系研究现状,2006,27(4):463~466
17Hu P, Zhang D, Swenson L, et al. Minimally invasive aortic banding inmice: effects of altered cardiomyocyte insulin signaling during pressureoverload. Am J Physiol Heart Circ Physiol,2003,285(3): H1261~H1269
18孙桂芳,刘凤岐.慢性心力衰竭大鼠心肌毛细血管密度及血管内皮生长因子变化.中国组织化学与细胞化学杂志,2005,14(3):339~343
19吴以岭.脉络论.北京:中国科学技术出版社,2010:1301
20Spencer C G,Martin S C, Felmeden D C, et al. Relationship ofhomocysteine to markers of platelet and endothelial activation in "highrisk" hypertensives: a substudy of the Anglo-Scandinavian CardiacOutcomes Trial. Int J Cardiol,2004,94(2-3):293~300
21赵晓民.血压波动性增高致微循环异常和左心室肥厚及其机理的实验研究.山东大学,2008:178
22孙桂芳,刘凤岐,修春红.冠脉微循环障碍参与慢性心力衰竭大鼠心肌重构.中国地方病学杂志,2005,(5):500~502
23鹿欣伦,胡成云,张雨梅.心力衰竭机制的研究进展.山西医药杂志,2011,40(8):766~767
24Spinarova L, Vitovec J. Neurohumoral changes in chronic heart failure.Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub,2007,151(2):201~207
25袁国强,贾振华,杨海涛,等.过度安逸所致神经—内分泌—免疫网络失衡—血管内皮功能障碍发生的可能机制.络病学基础与临床研究,2010,(6):72~77
26农一兵,苏敬泽,温志浩,等.从补益心气与改善心肌能量代谢的关系探讨慢性心力衰竭中西医结合研究的思路.中医杂志,2007,(11):1035~1036
27Yunzeng Zou, Li Lin, Yong Ye, et al. Qiliqiangxin Inhibits theDevelopment of CardiacHypertrophy, Remodeling, and DysfunctionDuring4Weeks of Pressure Overload in Mice. J Cardiovasc Pharmacol,2012,59(3):268~280
28Hong Xiao, You Song, Ya Li, et al. Qiliqiangxin regulates the balancebetween tumor necrosis factor-α and interleukin-10and improves cardiacfunction in rats with myocardial infarction. Cellular Immunology.2009,260:51~55
29Wei Liu, Junjun Chen, Tengfei Xu, et al. Qiliqiangxin Improves CardiacFunction in Spontaneously Hypertensive Rats through the Inhibition ofCardiac Chymase. American Journal of Hypertension,2012,25:250~260
30Yidong Wei, Xiaoyu Liu, Lei Hou, et al. Qiliqiangxin Affects L Type Ca2+Current in the Normal and Hypertrophied Rat Heart. Evidence-BasedComplementary and Alternative Medicine.2012,131830. Doi:10.1155/2012/131830. Epub2012Mar26
31王晓艳.健心汤对心力衰竭的疗效观察及抗心肌重塑机制研究.中南大学,2009
32袁劲松. N-末端脑利钠肽原在心力衰竭中的临床应用进展.国际检验医学杂志,2010,31(12):1417~1419
33汪芳,李卫,黄洁,等.血浆N末端原脑利钠肽水平对慢性心力衰竭患者长期预后的预测价值.中华心血管病杂志,2006,34(1):28~32
34袁慧.关注BNP与NT-proBNP的临床应用.中华检验医学杂志,2012,35(10):870~873
35牛连英,张丽娜,赵晓静.芪苈强心胶囊对老年慢性收缩性心力衰竭疗效及血清BNP水平的影响.疑难病杂志,2012,11(10):741~743
36姚华丽.转录因子激活蛋白-1在血管重构发生中的作用.国际心血管病杂志,2010,37(4):203~206
37Meyer B, Mortl D, Strecker K, et al. Flow-mediated vasodilation predictsoutcome in patients with chronic heart failure: comparison with B-typenatriuretic peptide. J Am Coll Cardiol,2005,46(6):1011~1018
38Schulz R, Rassaf T, Massion P B, et al. Recent advances in theunderstanding of the role of nitric oxide in cardiovascular homeostasis.Pharmacol Ther,2005,108(3):225~256
39唐可京. ICAM-1和VCAM-1的结构与表达调控.国外医学(分子生物学分册),2002,(3):173~177
40刘莉,邹国良,索传涛.利心Ⅰ号对CHF大鼠血流动力学、心肌细胞ICAM-1基因蛋白表达的影响.心脏杂志,2007,19(2):166~169
41杜荣增,汤永庆,邱建平,等. β受体阻断剂对慢性心力衰竭患者血浆vWF及TNF-α的影响.临床心血管病杂志,2011,27(3):232~233
42张三印,沈映君,陈士林,等.葛根素对心肌梗死大鼠心肌HIF-1、eNOS、VEGF基因表达及血清NO浓度的影响.中药药理与临床,2007,23(5):54~57
43张赛丹,全勇,周宏研,等.慢性心力衰竭与内皮功能异常的相关性研究.医学临床研究,2006,23(10):1545~1548
44Kempf T, Wollert K C. Nitric oxide and the enigma of cardiachypertrophy. Bioessays,2004,26(6):608~615
1任崇雷,高长青,李力兵.细胞心肌成形术对梗死后心室重构的影响.中华外科杂志,2007,45(22):1578~1580
2董志峰.系统性免疫调节药物对压力超负荷性大鼠心室重构和心功能干预的实验研究.扬州大学,2006:4
3Le Bousse-Kerdiles M C,Martyre M C, Samson M. Cellular and molecularmechanisms underlying bone marrow and liver fibrosis: a review. EurCytokine Netw,2008,19(2):69~80
4王健,刘全.银丹心脑通软胶囊对大鼠急性心肌梗死后心肌组织TGF-β1表达的影响.中西医结合心脑血管病杂志,2009,7(1):46~47
5Celik A,Sahin S, Koc F, et al. Cardiotrophin-1plasma levels are increasedin patients with diastolic heart failure. Med Sci Monit,2012,18(1):R25~R31
6Gonzalez A, Lopez B, Ravassa S, et al. Cardiotrophin-1in hypertensiveheart disease. Endocrine,2012,42(1):9~17
7Lopez-Andres N, Rousseau A, Akhtar R, et al. Cardiotrophin1is involvedin cardiac, vascular, and renal fibrosis and dysfunction. Hypertension,2012,60(2):563~573
8宋慧文,王琳.苦参素对实验性兔心房颤动左房纤维化细胞间黏附分子－1表达的影响.实用医学杂志,2009,25(12):1920~1922
9李琮辉,窦献蕊.原发性高血压黏附分子及胶原代谢变化的初步探讨.中国循环杂志,2002,17(5):346~349
10李为民,孙宁玲,刘巍,等.缬沙坦对自发性高血压大鼠心肌细胞凋亡的影响.中华医学杂志(英文版),2002,115(3):364~366
11He-Nan Z, Yan W, Miao-Na J, et al. Relation of Cardiotrophin-1(CT-1)and cardiac transcription factor GATA4expression in rat's cardiacmyocytes hypertrophy and apoptosis. Pathol Res Pract,2009,205(9):615~625
12简立国,李恩,孙利强,等. CT-1mRNA在心肌梗死大鼠心肌细胞凋亡中的表达及罗格列酮对其的影响.中外医疗,2009,28(24):18~20
13姚震,陈淑芬,张云波,等.促心肌素C-末端肽对再灌注损伤大鼠心肌细胞凋亡的影响.中国热带医学,2011,11(5):617~619
14Ruggieri M,Avolio C, Scacco S, et al. Glatiramer acetate inducespro-apoptotic mechanisms involving Bcl-2, Bax and Cyt-c in peripherallymphocytes from multiple sclerosis patients. J Neurol,2006,253(2):231~236
1Sambandam N,Lopaschuk G D, Brownsey R W, et al. Energy metabolismin the hypertrophied heart. Heart Fail Rev,2002,7(2):161~173
2van Bilsen M,Smeets P J, Gilde A J, et al. Metabolic remodelling of thefailing heart: the cardiac burn-out syndrome? Cardiovasc Res,2004,61(2):218~226
3李佳蓓,黄岚,祝善俊.心力衰竭时心肌能量代谢的研究进展.心血管康复医学杂志,2009,18(1):85~88
4吴以岭.脉络论.北京:中国科学技术出版社,2010:1301~1302
5王军,白玲,李晶,等.心力衰竭大鼠心肌线粒体蛋白质组学研究.中国科学C辑：生命科学,2009,39(11):1019~1027
6魏聪.基于代谢组学的络气虚滞/郁滞证候病生理基础及通络干预研究.河北医科大学,2011:180
7王红英.卡托普利、心复康口服液对心衰大鼠心肌细胞凋亡和线粒体膜电位的影响.河北医科大学,2008:84
8Ashrafian H,Frenneaux M P, Opie L H. Metabolic mechanisms in heartfailure. Circulation,2007,116(4):434~448
9刘瑛琪,李天德,楮晓雯,等.利用荧光探针JC-1检测心肌细胞线粒体膜电位的改变.解放军医学杂志,2002,27(8):716~718
10Azevedo P S,Minicucci M F, Santos P P, et al. Energy metabolism incardiac remodeling and heart failure. Cardiol Rev,2013,21(3):135~140
11祝善俊,王江.能量代谢疗法能否作为治疗心力衰竭的新靶点?岭南心血管病杂志,2010,16(2):97~98
12祝善俊.代谢重构与慢性心力衰竭.中华老年心脑血管病杂志,2007,(6):361~363
13谌煜.高尿酸血症与心血管疾病的研究进展.疑难病杂志,2012,11(2):147~148
14李鑫德,崔凌凌,任伟,等.高尿酸血症与心血管疾病关系的研究进展.中华内分泌代谢杂志,2011,27(7):614~617
15何青.高尿酸血症与心血管疾病.中国心血管杂志,2010,15(3):169
16Stanley W C, Recchia F A, Lopaschuk G D. Myocardial substratemetabolism in the normal and failing heart. Physiol Rev,2005,85(3):1093~1129
17Essop M F, Opie L H. Metabolic therapy for heart failure. Eur Heart J,2004,25(20):1765~1768
18Aroor A R,Mandavia C H, Sowers J R. Insulin resistance and heart failure:molecular mechanisms. Heart Fail Clin,2012,8(4):609~617
19Taylor M, Wallhaus T R, Degrado T R, et al. An evaluation of myocardialfatty acid and glucose uptake using PET with[18F]fluoro-6-thia-heptadecanoic acid and [18F]FDG in Patients withCongestive Heart Failure. J Nucl Med,2001,42(1):55~62
20蒙荣森. PPARα介导的AMPK激活对心肌肥厚及能量代谢的调节作用.中山大学博士后学位论文,2009:17~40
21Jeninga E H, Schoonjans K, Auwerx J. Reversible acetylation of PGC-1:connecting energy sensors and effectors to guarantee metabolic flexibility.Oncogene,2010,29(33):4617~4624
22Uguccioni G, Hood D A. The importance of PGC-1alpha in contractileactivity-induced mitochondrial adaptations. Am J Physiol EndocrinolMetab,2011,300(2):E361~E371
23Lira V A, Brown D L, Lira A K, et al. Nitric oxide and AMPKcooperatively regulate PGC-1in skeletal muscle cells. J Physiol,2010,588(Pt18):3551~3566
24Yunzhou Dong, Miao Zhang, Shuangxi Wang. Activation ofAMP-Activated Protein Kinase Inhibits Oxidized LDL-TriggeredEndoplasmic Reticulum Stress in Vivo. Diabetes,2010,59(6):1386~1396
25Tran Thi Hien, Won Keun Oh, Phi Hung Nguyen, et al. Nectandrin BActivates Endothelial Nitric-Oxide Synthase Phosphorylation inEndothelial Cells: Role of the AMP-Activated Protein Kinase/EstrogenReceptor α/Phosphatidylinositol3-kinase/Akt Pathway. MolecularPharmacology,2011,80(6):1166~1178
1梁伟涛,Vitali R.离体心脏Langendorff灌流模型稳定性的探讨.国际心血管病杂志,2011,38(1):44~47
2Zhu M,Feng J, Lucchinetti E, et al. Ischemic postconditioning protectsremodeled myocardium via the PI3K-PKB/Akt reperfusion injury salvagekinase pathway. Cardiovasc Res,2006,72(1):152~162
3蔡辉,郭郡浩,商玮,等.压力负荷增加大鼠模型心肌α-MHC和β-MHCmRNA的表达.医学研究生学报,2009,22(10):1032~1035
4Kong Y, Tannous P, Lu G, et al. Suppression of class I and II histonedeacetylases blunts pressure-overload cardiac hypertrophy. Circulation,2006,113(22):2579~2588
5Meng RS, Pei ZH,Yin R,et al. Adenosine monophosphate-activatedprotein kinase inhibits cardiac hypertrophy through reactivatingperoxisome proliferator-activated receptor-alpha signaling pathway. Eur Jpharmacol,2009,620(1-3):63~70
6郝希纯,王东明. Drp1蛋白调节线粒体分裂机制及其在疾病中的作用.广东医学,2011,32(8):1066~1069
7Detmer S A, Chan D C. Functions and dysfunctions of mitochondrialdynamics. Nat Rev Mol Cell Biol,2007,8(11):870~879
8Lee Y J,Jeong S Y, Karbowski M, et al. Roles of the mammalianmitochondrial fission and fusion mediators Fis1, Drp1, and Opa1inapoptosis. Mol Biol Cell,2004,15(11):5001~5011
9Shen T, Zheng M, Cao C, et al. Mitofusin-2is a major determinant ofoxidative stress-mediated heart muscle cell apoptosis. J Biol Chem,2007,282(32):23354~23361
10施冰,冬兰,尹秋生,等.大鼠心肌梗死后心肌组织中线粒体融合蛋白2基因和线粒体分裂蛋白mRNA表达水平的变化.中国临床保健杂志,2012,15(4):392~394
11邓琴琴. EPO通过激活PI3K/Akt途径并上调HSP70的表达对低温下的心衰大鼠起保护作用.南昌大学,2010
1World health organization. Cardiovascular diseases prevention and control.http://www.afro.who.int/en/clusters-a-programmes/dpc/non-communicable-diseases-managementndm/programme-components/cardiovascular-diseases.html.
2顾东风,黄广勇,何江,等.中国心力衰竭流行病学调查及其患病率.中华心血管病杂志,2003,31(1):3~5
3张建,华琦.心力衰竭的诊断与治疗.北京:人民卫生出版社,2006
4姜红,葛均波.心力衰竭药物治疗的现状和前景.中国临床医学,2005,12(3):363~367
5Neubauer S. The failing heart-an engine out of fuel. N Engl J Med,2007,356(11):1140~1151
6Van Bilsen M, Smeets P J, Gilde A J, et al. Metabolic remodelling of thefailing heart: the cardiac burn-out syndrome? Cardiovasc Res,2004,61(2):218~226
7李佳蓓,黄岚,祝善俊.心力衰竭时心肌能量代谢的研究进展.心血管康复医学杂志,2009,18(1):85~88
8祝善俊.代谢重构与慢性心力衰竭.中华老年心脑血管病杂志,2007,9(6):361~363
9Maslov MY, Chacko VP, Stuber M.et al. Altered high-energy phosphatemetabolism predicts contractile dysfunction and subsequent ventricularremodeling in pressure-overload hypertrophy mice. Am J Physiol HeartCirc Physiol,2007,292: H387~391
10能量缺乏是心力衰竭的原因.中华高血压杂志.2008,16(3):273~275
11祝善俊,徐成斌.心力衰竭基础与临床.北京:人民军医出版社,2001:308~520
12祝善俊.代谢重构与慢性心力衰竭.中华老年心脑血管病杂志,2007,9(6):361~363
13Stanley W C, Recchia F A, Lopaschuk G D. Myocardial substratemetabolism in the normal and failing heart. Physiol Rev,2005,85(3):1093~1129
14Essop M F, Opie L H. Metabolic therapy for heart failure. Eur Heart J,2004,25(20):1765~1768
15Aroor A R,Mandavia C H, Sowers J R. Insulin resistance and heart failure:molecular mechanisms. Heart Fail Clin,2012,8(4):609~617
16Taylor M, Wallhaus T R, Degrado T R, et al. An evaluation of myocardialfatty acid and glucose uptake using PET with[18F]fluoro-6-thia-heptadecanoic acid and [18F]FDG in Patients withCongestive Heart Failure. J Nucl Med,2001,42(1):55~62
17Rosenblatt-Velin N, Montessuit C, Papageorgiou I, et al. Postinfarctionheart failure in rats is associated with upregulation of GLUT-1anddownregulation of genes of fatty acid metabolism. Cardiovasc Res,2001,52:407~416
18Kajstura J, Cigola E,Malhotra A, et al. Angiotensin II induces apoptosisof adult ventricular myocytes in Vitro. J Mol Cell Cardiol,1997,29(3):859~870
19何茹.解偶联蛋白家族成员UCP2.北京教育学院学报(自然科学版).2009,4(2):19~23
20Ide T, Tsutsui H, Hayashidani S, et al. Mitochondrial DNA damage anddysfunction associated with oxidative stress in failing hearts aftermyocardial infarction. Circ Res,2001,88:529~535
21Reznick RM, Shulman GI. The role of AMP-activated protein kinase inmitochondrial biogenesis. J Physiol,2006,574:33~39
22李良刚,陈槐卿. CaMK和AMPK信号通路能共调收缩信号诱导的骨骼肌细胞GLUT4基因转录.生物化学与生物物理进展,2009,4:471~479
23Deldicque L, Theisen D, FrancauxM. Regulation of mTOR by amino acidsand resistance exercise in skeletalmuscle. Eur J ApplPhysiol,2005,94(1-2):1~10
24Kanda H, Nohara R, Hasegawa K, et al. A nuclear complex containingPPARalpha/RXRalpha is markedly downregulated in the hypertrophied ratleft ventricular myocardium with normal systolic function. Heart Vessels,2000,15:191~196
25Garnier A, Fortin D, Delomenie C, et al. Depressed mitochondrialtranscription factors and oxidative capacity in rat failing cardiac andskeletal muscles. Physiol,2003,551:491~501
26MF Essop, LH Opie. Metabolic therapy for heart failure. Eur Heart J,2004,25:1814~1821
27P Di Napoli, AA Taccardi, A Barsotti. Long term cardioprotective actionof trimetazidine and potential effect on the inflammatory process inpatients with ischaemic dilated cardiomyopathy. Heart,2005,91:161~165
28Beadle RM,Williams LK,Abozguia K,et al. Metabolic manipulation inchronic heart failure:study protocol for a randomized controlled trial.Trials,2011,12:140
29Kato T, Niizuma S, Inuzuka Y, et al. Analysis of metabolic remodeling incompensated left ventricular hypertrophy and heart failure. Circ Heart Fail,2010,3(3):420~430
30Stanley WC, Chandler MP. Energy metabolism in the normal and failingheart: potential for therapeutic interventions. Heart Fail Rev,2002,7:115~30
31张治宇,殷仁富,李振东,等.雷卡对肾血管性高血压大鼠心肌重塑和脂肪酸代谢的影响.同济大学学报(医学版),2006,27(4):32~35
32汪凡,秦明照."代谢类"抗心肌缺血药物的研究进展.中国医药导刊,2006,8(2):124~126
33宋熔,祝善俊.心肌代谢重构的研究进展.中华高血压杂志,2008,16(3):202~204
34Kristiansen SB. Cardioprotection against ischermia reperfusion injury byL-glutamate and KATP channel openers. Dan Med Bull,2005,52(4):261
1杨淑艳,张雷,郑中华,等.心力衰竭的病理生理机制及药物治疗进展.吉林医药学院学报,2006,27(3):168~171
2吴以岭,主编.脉络论.北京:中国科学技术出版社,2010
3贾美君,蒋梅先,何峥.心力衰竭时动脉功能变化与络病现代病理机制.络病学基础与临床研究(2):397~401
4吴以岭,主编.络病学.北京:中国科学技术出版社,2004:283~285
5王宏涛,唐思文,徐登峰,等.芪苈强心胶囊多目标优化后组合物对实验性心衰大鼠心肌细胞间连接的影响.时珍国医国药,2011,22(10):2375~2377
6唐思文,徐登峰,王玲玲.芪苈强心胶囊多目标优化组合物对实验性心衰大鼠心功能的保护作用.中药新药与临床药理,2011,22(5):532~535
7刘奕训,余河水,康利平.芪苈强心胶囊活性部位中的组成成分研究.中草药,2010,41(7):1060~1065
8康利平,赵阳,余河水,等.采用UPLC-Q-TOF/MSE鉴别芪苈强心胶囊有效部位中的化学成分.药学学报,2011,46(10):1231~1236
9刘建勋,马晓斌,王杨慧.芪苈强心胶囊对实验性心力衰竭犬心脏功能的影响.疑难病杂志,2007,6(3):141~143
10李娅,宋优,程翔,廖玉华.芪苈强心胶囊对大鼠心肌梗死后心肌重构及心功能的影响.中国分子心脏病学杂志,2007,7(4):201~204
11邬真力,许顶立,林昇,等.芪苈强心胶囊对慢性心力衰竭大鼠心脏功能及血浆血管加压素的影响.疑难病杂志,2011,10(2):120~122
12许顶立,芪苈强心对慢性心力衰竭大鼠肾脏AQP-2及心脏功能的影响.中国医学论坛报,2009.12.10
13Xiao H, Song Y, Li Y, et al. Qiliqiangxin regulates the balance betweentumor necrosis factor-α and interleukin-10and improves cardiac functionin rats with myocardial infarction. Cellular immunology,2009,260(1):51~55
14徐涛,郭丽峰,陈立锋,等.芪苈强心胶囊对慢性心力衰竭大鼠PPARα表达的影响.河北医药,2010,32(22):3120~3122
15孙鑫,许静静,陈伟.芪苈强心胶囊对压力超负荷致慢性心衰大鼠的作用及机理研究.中国中医基础医学杂志,2010,16(7):560~562
16林锐波.芪苈强心胶囊对心梗后心衰大鼠心功能及凋亡蛋白caspase-3表达的影响.汕头大学,2010
17徐涛,郭丽峰,陈立锋,等.芪苈强心胶囊对慢性心力衰竭大鼠心肌细胞凋亡的影响.中药新药与临床药理,2010,21(4):366~369
18Briant E B,Richard D O, Barry J C. Anthracycline cardiotoxicity intransgenic mice overexpressing SR Ca2+-ATPase. Biochemical andBiophysical Research Communications,2003,303:504~507
19李佳彧,杨萍.芪苈强心胶囊对心梗后心力衰竭大鼠periostin蛋白表达干预作用的研究.中国实验诊断学,2009,13(2):170~172
20李佳彧,李佳睿,马丕勇,等.通络药物对心梗后心衰大鼠非梗死区心肌纤维化的影响.现代生物医学进展,2011,11(18):3431~3434
21李佳彧,杨萍.通络药物对心肌梗死后心衰大鼠瘢痕区纤维化的影响.中国老年学杂志,2009,29(21):15~17
22杨正央,邓永军.芪苈强心胶囊治疗收缩性心力衰竭的观察.中外医疗,2010,5:113
23吴以岭,谷春华,徐贵成,等.芪苈强心胶囊治疗慢性心力衰竭随机双盲、多中心临床研究.疑难病杂志,2007,6(5):263~266
24刘春香,毛静远,王贤良,等.芪苈强心胶囊治疗慢性心力衰竭的系统评价.中成药,2010,32(4):539~564
25刘军,刘庚,刘江涛,等.芪苈强心胶囊辅治扩张型心肌病心功能不全的疗效观察.疑难病杂志,2009,8(3):159~160
26刘新明,宋一平,顾明峰.芪苈强心胶囊辅治老年难治性心力衰竭疗效观察.临床合理用药,2010,3(18):56~57
27陈丽萍,孟宪文,崔兆文,等.呋塞米持续泵入联合多巴胺及芪苈强心胶囊治疗心肾综合征并发利尿剂抵抗25例临床观察.河北中医,2011,33(5):723~724
28马芳放,路凤月,肖维刚,等.芪苈强心胶囊辅治对慢性心力衰竭患者肱动脉血管内皮舒张功能的影响.疑难病杂志,2008,7(9):543~544
29侯湘岭.芪苈强心胶囊联合西药治疗急性心肌梗死后心力衰竭合并低血压状态46例临床观察.河北中医,2010,32(10):1540~1541
30宋云方.芪苈强心胶囊与辅酶Q10胶囊联合治疗扩张型心肌病60例.中国医药指南,2009,14:81~82
31闫松改.芪苈强心胶囊与螺内酯联合治疗慢性心力衰竭疗效观察.临床医学,2009,29(11):30~31