【作者】 尹慧秋；

【导师】 张继东；

【作者基本信息】 山东大学 ， 中西医结合临床， 2009， 博士

【摘要】 第一部分舒脉胶囊对心肌缺血大鼠促血管新生的研究背景目前,缺血性心脏病使全世界数百万人经受着病痛的折磨。改善心肌缺血在缺血性心脏病的防治研究中具有重要意义。治疗性血管新生可促进缺血组织周边侧支循环的建立,心脏冠脉侧支循环的形成和开放能改善心肌缺血、坏死,延缓缺血性心肌病的形成,改善患者的临床症状和预后,已成为心血管研究领域的热点之一。冠心病的中西医结合防治研究也已从单纯改善病变血管供血转向同时促进侧支循环的建立。中医药益气化瘀的治疗法则及其临床有效性,为中西医结合促血管新生的研究提供了理论和临床依据。特异性内皮细胞丝裂原血管内皮生长因子(VEGF)家族在血管新生过程中起关键作用。但是,通过单一促血管生成因子诱导产生的新生血管结构以内皮细胞(EC)为主,没有血管平滑肌细胞(VSMC)和周细胞包被形成完整的动脉中膜结构,因此血管容易出血渗出,亦无法输送血流,最终会导致新生血管退化。而血小板衍生生长因子(PDGF)家族成员尤其是PDGF-BB是作用很强的促动脉生成因子,主要作用于血管壁细胞包括周细胞和VSMC。PDGF-BB与细胞表面的酪氨酸蛋白激酶受体结合,募集周细胞和VSMC包绕新生血管网络形成完整的血管结构从而发挥稳定血管的作用。血管生成(angiogenesis)和动脉生成(arteriogenesis)都有助于心脏的血管新生,但对于改善心肌血流量,动脉生成是最重要的过程。PI3K/Akt信号通路激活在介导生长因子促血管新生的作用已引起重视。PI3K/Akt通过诱导VEGF的表达达到促血管新生的作用。抑制PI3K/Akt信号通路活性可降低VEGF、PDGF受体VEGFR2及PDGFR活性及其作用底物的磷酸化。随着中西医结合的发展,中药在心血管疾病的防治中的作用越来越受到重视。既往研究表明中药单体成分及复方制剂可不同程度的促进VEGF、bFGF等mRNA及蛋白的表达,从而促进血管新生。但中药对信号通路介导促血管新生的分子机制的研究及对促动脉生成因子的研究仍属崭新的研究领域。舒脉胶囊是导师根据多年的临床经验研制而成,是大量应用于临床治疗缺血性心脏病的中药复方制剂。本研究通过对心肌缺血(Myocardial ischemia,MI)大鼠缺血心肌组织学、形态学改变的观察,通过超声心动图分析左室心功能,通过实时定量RT-PCR技术检测缺血心肌VEGF、PDGF-BB mRNA的表达,以及通过Western blot检测缺血心肌磷酸化PI3K/Akt、VEGF、PDGF-BB的蛋白表达的改变,通过免疫组化检测缺血心肌微血管密度,探讨舒脉胶囊在缺血心肌中促血管新生的作用,并探讨其信号调控机制,为其临床防治缺血性心血管疾病提供更为可靠的科学依据。目的1.通过观察舒脉胶囊对实验性大鼠缺血心肌组织学、形态学及心功能改变,及对毛细血管与微动脉的新生的影响,探讨舒脉胶囊促血管新生的疗效。2.观察舒脉胶囊对大鼠缺血心肌磷酸化PI3K、Akt蛋白表达,及VEGF、PDGF-BB的蛋白及mRNA表达影响,探讨舒脉胶囊促进稳定而有功能的血管新生的信号调控机制。方法1.研究对象建立大鼠心肌缺血实验模型Wistar大鼠结扎左冠状动脉前降支建立心肌缺血模型。造模成功的大鼠随机分为舒脉大剂量/LY294002组(SMCH/LY)、舒脉小剂量组(SMCL)、舒脉大剂量组(SMCH)、贝复济组(bFGF)、模型组(MIR);另设假手术组(Sham)。每组24只,2周、4周每个时间段各12只。SMCH组灌胃给予舒脉胶囊1.71g/(kg.d)(相当于临床用量的30倍),SMCL组灌胃给予舒脉胶囊342mg/(kg.d)(相当于临床用量的6倍),SMCH/LY组灌胃给予舒脉胶囊1.71g/(kg.d)(相当于临床用量的30倍),腹腔注射给予PI3K抑制剂LY294002(0.3mg/kg,溶解于双蒸水中,每3天给药一次),bFGF组给予贝复剂(bFGF),于开胸结扎冠脉后立即在结扎处周围予bFGF心肌注射1次(125IU),术后始皮下注射肝素1250U/kg,每日1次,连续5天。MIR组和Sham组分别灌胃给予等量蒸馏水。灌胃给药大鼠,均为每日1次,连续给药2周和4周。末次给药后禁食24 h,不禁水。10%水合氯醛,300mg/kg,腹腔注射麻醉后进行后续实验。2.研究内容(1)心功能检测;(2)Western blot检测缺血心肌VEGF、PDGF-BB、PI3K、p-Akt蛋白表达;(3)实时定量RT-PCR(Real-time Quantitative RT-PCR)检测缺血心肌VEGF、PDGF-BB mRNA表达;(4)检测缺血心肌微血管密度(小静脉:vWF免疫组化染色法,小动脉:αSMA免疫荧光染色法);(5)Masson染色检测缺血心肌胶原含量的变化。结果1.心功能检测治疗第2周后,SMCH、SMCL、bFGF组的左室射血分数(LVEF)较MIR组显著增高(P＜0.05);SMCH与SMCL、bFGF组比较差异无显著性意义(P＞0.05)。SMCH、SMCL、bFGF组左心室收缩末期内径(LVESD)、左心室舒张末期内径(LVEDD)较MIR组显著降低(P＜0.05)。应用SMCH+LY294002(SMCH/LY)较MIR组LVEF、LVESD、LVEDD无显著性差别(P＞0.05)。进一步组间两两比较发现,应用大剂量舒脉胶囊组(SMCH)明显高于SMCH/LY组(P＜0.05)以及Sham组(P＜0.05)、MIR组(P＜0.05)。SMCH组较bFGF组或应用小剂量舒脉胶囊组(SMCL)LVEF、LVESD、LVEDD无显著性差异(P＞0.05)。治疗第4周后,SMCH、SMCL、bFGF组的LVEF较MIR组显著增高(P＜0.05);LVESD、LVEDD较MIR组显著降低(P＜0.05)。但应用SMCH+LY(LY294002)较MIR组LVESD、LVEDD无显著性差异(P＞0.05);但LVEF较MIR组升高。进一步两两比较分析,SMCH组与bFGF组差别无统计学意义,SMCH治疗组较SMCL或SMCH/LY的LVEF、LVEDD显著增高(P＜0.05)。SMCH组较SMCL组LVESD无显著性差别(P＞0.05)。2.Western blot检测分析治疗第2周后,SMCH、SMCL、bFGF组VEGF、PDGF-BB、PI3K、p-Akt蛋白表达较MIR组显著升高(P＜0.05)。SMCH/LY组VEGF、PDGF-BB、PI3K、p-Akt蛋白表达与MIR组差异无显著性意义(P＞0.05)。进一步组间两两比较分析表明,SMCH组与bFGF组或SMCL组之间差别无统计学意义(P＞0.05)。SMCH组较SMCH/LY组VEGF、PDGF-BB、PI3K、p-Akt蛋白表达明显增高。治疗第4周后,SMCH组、SMCL组、bFGF组VEGF、PDGF-BB、PI3K、p-Akt蛋白表达较MIR组显著升高(P＜0.005)。SMCH/LY组VEGF、PDGF-BB、PI3K、p-Akt蛋白表达与MIR组差异无显著性意义(P＞0.05)。进一步组间两两比较分析表明,SMCH组较bFGF组或SMCL组或SMCH/LY组VEGF、PDGF-BB、PI3K、p-Akt蛋白增高。与治疗2周后比较,SMCH组、SMCL组、bFGF组治疗4周后VEGF、PDGF-BB、PI3K、p-Akt蛋白表达降低。3.实时定量RT-PCR(Real-time Quantitative RT-PCR)检测治疗第2周后,SMCH组、SMCL组、bFGF组VEGF、PDGF-BB mRNA表达较MIR组显著升高(P＜0.05)。SMCH/LY组VEGF、PDGF-BB mRNA表达与MIR组差异无显著性意义(P＞0.05)。进一步组间两两比较分析表明,SMCH组与bFGF组或SMCL组之间差别无统计学意义(P＞0.05)。SMCH组较SMCH/LY组VEGF、PDGF-BBmRNA明显增高(P＜0.05)。治疗第4周后,SMCH组、SMCL组、bFGF组VEGF、PDGF-BB mRNA表达较MIR组显著升高(P＜0.05)。SMCH/LY组VEGF、PDGF-BBmRNA表达与MIR组差异无显著性意义(P＞0.05)。进一步组间两两比较分析表明,SMCH组较SMCL组或SMCH/LY组VEGF、PDGF-BB mRNA增高。与治疗2周后比较,SMCH组、SMCL组、bFGF组治疗4周后VEGF、PDGF-BB mRNA表达降低。4.缺血心肌微血管密度(小静脉、小动脉)的测定治疗2周后,SMCH组、SMCL组、bFGF组的毛细血管密度均较MIR组明显增高。SMCH/LY组毛细血管密度与MIR组差异无显著性意义(P＞0.05)。进一步组间两两比较分析表明,SMCH组与bFGF组之间差别无统计学意义。SMCH组(36.93±5.49)较SMCH/LY组毛细血管密度明显增高。SMCH组、SMCL组、bFGF组的小动脉密度均较MIR组(17.76±5.19)明显增高。SMCH/LY组小动脉密度与MIR组差异无显著性意义。组间两两比较分析表明,SMCH组的小动脉密度显著高于SMCL(P＜0.05)或SMCH/LY(P＜0.05)组以及Sham组(9.44±2.68)(P＜0.05)。SMCH组与bFGF组之间差别无统计学意义(P＞0.05)。治疗4周后,SMCH组、SMCL组、bFGF组的毛细血管密度均较MIR组明显增高(P＜0.05)。SMCH/LY组毛细血管密度与MIR组差异无显著性意义。进一步组间两两比较分析表明,SMCH组毛细血管密度较SMCH/LY组、SMCL组、bFGF组明显增高(P＜0.05)。SMCH组、SMCL组、bFGF组的小动脉密度均较MIR组明显增高(P＜0.05)。组间两两比较分析表明,SMCH组的小动脉密度显著高于SMCL(P＜0.05)或SMCH/LY(P＜0.05)组以及Sham组(P＜0.05)。PI3K抑制剂LY294002显著抑制了舒脉胶囊所致的毛细血管和微动脉的新生,说明PI3K信号通路介导舒脉胶囊促血管新生。5.Masson染色检测缺血心肌胶原含量的变化光镜下Masson染色显示,心肌细胞染色呈黄色,间质胶原呈蓝绿色。治疗2周后,MIR组大鼠缺血心肌肌原纤维间隙明显增宽,排列紊乱,心肌细胞肥大,可见局灶性肌丝溶解。心肌梗死区及非梗死区均可见大量胶原纤维沉积。粗大胶原纤维相互连接成网状,排列紊乱,分布不匀;Sham组大鼠心肌组织未见胶原纤维增生,心肌细胞排列整齐,明暗带清晰,形态结构正常。SMCH、SMCL、bFGF组大鼠心肌肌原纤维和肌丝排列有序,形态较为正常,胶原纤维分布比较均匀、纤细,心肌间质纤维化较MIR组明显减少,但并不能完全阻止其发生。进一步两两比较分析,SMCH组较SMCL组和bFGF组心肌间质胶原含量差异无显著性意义。SMCH/LY组心肌间质纤维化面积与SMCH组比较组明显增加。结论1.本研究首次证明舒脉胶囊对心肌缺血(MI)大鼠具有显著的促血管新生疗效,改善MI大鼠心功能。表现为微血管密度(小动脉、小静脉)的增加,心功能指标LVEF的提高,LVESD、LVEDD降低。且舒脉胶囊的疗效具有剂量依赖性。证实了舒脉胶囊通过对缺血心肌促血管新生作用达到了改善心功能的终极目标。2.与MIR组比较,舒脉胶囊单独治疗显著提高了缺血心肌VEGF、PDGF-BB的表达水平,及磷酸化PI3K、Akt蛋白的表达水平。证实舒脉胶囊促毛细血管与微动脉新生的作用是通过促进VEGF、PDGF-BB、及磷酸化PI3K、Akt蛋白的表达实现的。3.与舒脉胶囊单独治疗比较,PI3K抑制剂(LY294002)和舒脉胶囊联合治疗显著抑制PI3K/Akt信号通路的活性,同时明显抑制舒脉胶囊促VEGF、PDGF-BB的高表达,及降低舒脉胶囊促毛细血管与微动脉新生的作用。证实舒脉胶囊促血管新生的分子机制是通过激活PI3K/Akt信号通路介导VEGF、PDGF-BB的表达、促进VEGF、PDGF-BB介导的毛细血管以及稳定的侧支循环的建立。第二部分舒脉胶囊改善心肌缺血大鼠左室重构的研究背景左室重构(left ventricular remodeling,LVR)是心脏功能由代偿向失代偿转变阶段所发生的心脏结构与形态的改变。左室重构不仅使心肌梗死患者左室功能严重受损,并发症增多,而且死亡率亦明显增加,因此抗心室重构已成为当前心血管领域中最为重要的研究内容之一。而心肌纤维化是左室重构发展的重要因素。血浆与心肌中致炎细胞因子与心肌纤维化和左室重构的病情发展密切相关。在AMI急性期存在炎症反应增强,早期即可发生心室重构,抗炎治疗可以减轻心室重构。肿瘤坏死因子-alpha(tumor necrosis factor-alpha,TNF-α)被认为是致炎细胞因子中最为重要的导致基质纤维化的细胞因子。TNF受体(TNFR)基因敲除小鼠与组织基质金属蛋白酶抑制因子-1(tissue inhibitor of metalloproteinase,TIMP-1)的上调相关,并通过TIMP-1抑制胶原降解,促进纤维化的发展。TNF-α与CFs的增殖也有密切联系。因此抑制TNF-α的分泌对改善左室重构至关重要。炎性刺激激活细胞内许多信号转导通路,包括核因子-κB信号通路和三个通过ERK、JNK、p38MAPK调控的信号通路。p38 MAPK被认为是调控炎症的重要信号通路。体内外研究证实在缺血与其它病理条件下,心肌内p38 MAPK被激活。激活的p38 MAPK促进心肌内分泌足够的TNF-α,从而促进心肌纤维化和心室重构。选择性的应用p38MAPK抑制剂SB203580可抑制p38 MAPK活性,降低TNFα的分泌,并且通过逆转MMPs和TIMP-1的表达比例,降低心肌纤维化,改善心室重构。由此可以推断p38 MAPK信号通路介导的TNFα的分泌是促进心肌纤维化和左室重构的潜在的信号调控机制。有些中药已经被证实具有明显的抗炎疗效。但是,很少有中药复方对炎症因子介导的心肌纤维化和左室重构影响及其作用机制的研究。舒脉胶囊是根据中药组方原则合成的复方制剂,在临床已经多年被用于治疗缺血性心脏病。本研究通过观察心肌缺血大鼠缺血心肌组织学、形态学的病理改变及超微结构改变,通过实时定量RT-PCR,免疫组化检测缺血心肌心肌胶原Ⅰ(collagenⅠ)mRNA及蛋白的表达,免疫组化检测缺血心肌α-SMA蛋白的表达,通过Western blot检测缺血心肌p38 MAPK、TNF-α、及TIMP-1的蛋白表达,通过放免法检测血浆TNF-α的水平,并通过超声心动图检测大鼠整体与局部心功能,探讨舒脉胶囊抑制心肌缺血大鼠炎症因子TNFα介导的心肌成纤维细胞增殖、心肌纤维化和左室重构的疗效及信号调控机制。目的1.通过观察舒脉胶囊对实验性大鼠缺血心肌组织学、形态学及超微结构的改变、整体与局部心功能、α-SMA蛋白表达、及collagenⅠmRNA与蛋白表达的影响,探讨舒脉胶囊减少心肌成纤维细胞增殖、降低心肌纤维化、及改善左室重构的疗效。2.观察舒脉胶囊对实验性大鼠缺血心肌磷酸化应激激活细胞丝裂原活化蛋白激酶(p-p38 MAPK)、TNF-α、金属蛋白酶组织抑制因子(TIMP)-1蛋白表达的影响,探讨舒脉胶囊改善左室重构的机制。方法1.研究对象建立大鼠心肌梗死实验模型Wistar大鼠结扎左冠状动脉前降支建立心肌缺血模型。造模方法同第一部分。造模成功的大鼠随机分为舒脉大剂量组(SMCH)、舒脉小剂量组(SMCL)、p38MAPK抑制剂SB203580(SB)、模型组(MIR);另设假手术组(Sham)。每组24只,1、6周每个时间段各12只。2.研究内容(1)超声心动检测;(2)放射免疫法检测血浆TNF-α含量;(3)Western blot检测缺血心肌磷酸化p38 MAPK(p-p38 MAPK)、TNF-α、TIMP-1蛋白表达;(4)实时定量RT-PCR检测缺血心肌collagenⅠmRNA表达;(5)免疫组化染色检测缺血心肌collagenⅠ、α-SMA蛋白表达;(6)HE染色;(7)天狼猩红染色检测胶原含量的变化;(8)透射电镜观测心肌超微结构变化。结果1.左心室整体与局部功能超声心动图分析在治疗后第1、6周时进行了超声检查。左室射血分数(LVEF)是目前临床上评价左心室整体功能最常用的指标。治疗第1周后,SMCH组、SMCL组、SB组的LVEF、BW较MIR组显著增高(P＜0.05),而LVESD、LVEDD、LVW/BW较MIR组显著降低。局部室壁增厚率(WT)可用来反应心肌局部功能。治疗第1周后,SMCH组、SMCL组、SB组的WT较MIR组显著增高(P＜0.05)。进一步组间两两比较发现,应用大剂量舒脉胶囊组(SMCH)较小剂量舒脉胶囊组(SMCL)或SB组WT无显著性差异(P＞0.05)。治疗第6周后,SMCH组、SMCL、SB组的LVEF、BW、WT较MIR组显著增高(P＜0.05),LVESD、LVEDD、HW/BW较MIR组显著降低(P＜0.05)。进一步两两比较分析,SMCH较SMCL组LVEF、WT增高,SMCH组LVESD、LVEDD、HW/BW较SMCL组降低(P＜0.05)。SMCH组较SB组各指标差异差别无统计学意义(P＞0.05)。2.血浆TNF-α水平的测定通过放免法检测SMC的抗炎症的作用。治疗1周后,MIR组、SMCH组、SMCL组血浆TNF-α水平较Sham组明显升高。但是,SMCH组、SMCL组(剂量依赖性)、SB组血浆TNF-α水平较MIR组明显降低(P＜0.05)。治疗6周后SMCH组、SMCL组、SB组血浆TNF-α水平较MIR组降低(P＜0.05),且较1周时血浆TNF-α水平降低。3.Western blot检测分析治疗1周后,MIR组大鼠心肌组织p-p38MAPK、TNF-α、TIMP-1蛋白的表达较Sham组明显增加。SMCH、SMCL、SB组心肌组织p-p38MAPK、TNF-α、TIMP-1蛋白表达较MIR组明显减少。进一步两两比较分析,SMCH组较SMCL组或SB组心肌组织p-p38MAPK、TNF-α、TIMP-1蛋白表达差异无显著性意义(P＞0.05)。治疗6周后,SMCH、SMCL、SB组心肌组织p-p38MAPK、TNFα、TIMP-1蛋白表达较MIR组明显减少(P＜0.05)。进一步两两比较分析,SMCH组较SMCL组心肌组织p-p38MAPK、TNF-α、TIMP-1蛋白表达减少(P＜0.05)。SMCH组较SB组心肌组织p-p38MAPK、TNF-α、TIMP-1蛋白表达差异无显著性意义(P＞0.05)。除外Sham组,各组心肌组织p-p38MAPK、TNF-α、TIMP-1蛋白的表达较1周时明显降低。4.实时定量RT-PCR通过实时定量RT-PCR检测大鼠心肌组织collagenⅠmRNA的表达。治疗1周后,MIR组大鼠心肌组织collagenⅠmRNA的表达较Sham组明显增加(P＜0.05)。SMCH、SMCL、SB组心肌组织collagenⅠmRNA表达较MIR组明显减少(P＜0.05)。进一步两两比较分析,SMCH组较SMCL组或SB组心肌组织collagenⅠmRNA表达差异无显著性意义。治疗6周后,SMCH、SMCL、SB组心肌组织collagenⅠmRNA表达较MIR组明显减少。进一步两两比较分析,SMCH组较SMCL组心肌组织collagenⅠmRNA表达减少。SMCH组较SB组心肌组织collagenⅠmRNA表达差异无显著性意义。5.免疫组化染色分析5.1α-SMA表达α-平滑肌激动蛋白(α-SMA)是检测CFs增殖的可靠指标。通过免疫组化染色法可使阳性指标呈棕色或棕黄色染色。α-SMA主要表达于VSMC与CFs中。结果表明,Sham组大鼠只在心肌VSMC中有表达。而在MIR组,α-SMA广泛表达,并见有密集的阳性表达的CFs核聚集。舒脉胶囊与SB203580均明显降低α-SMA在心肌组织的广泛表达,表明其具有抑制CFs增殖的作用。6周时,除了Sham组,各组α-SMA表达较1周时增加。5.2 CollagenⅠ表达治疗1周后,通过免疫组化检测到MIR组心肌间质中较Sham组明显增多的棕褐色阳性表达。SMCH、SMCL、SB治疗组的Ⅰ型胶原(collagenⅠ)的阳性表达较MIR组显著降低。进一步两两比较分析,SMCH组较SMCL组或SB组心肌间质collagenⅠ的表达无显著性差异。治疗6周后,SMCH、SMCL、SB治疗组的Ⅰ型胶原(collagenⅠ)的阳性表达较MIR组显著降低。进一步两两比较分析,SMCH组较SMCL组心肌间质collagenⅠ的表达减少。SMCH组较SB组心肌间质collagenⅠ的表达无显著性差异。6.HE染色Sham组大鼠心肌细胞细长,细胞分界较清楚,胞核大小均一,染色正常。肌纤维排列紧密整齐,壁内小动脉管壁及管腔正常,管周纤维组织少;MIR组大鼠心肌细胞减少,细胞核大小不甚规则,心肌细胞肥大,分界不清楚。肌纤维排列较紊乱,壁内小动脉管壁厚,管腔狭小,管周纤维组织增多。CFs大量增殖、聚集。SMCH、SMCL、SB组大鼠心肌细胞肥大减轻,肌纤维排列较稀疏,间质纤维化减轻,心内膜下纤维组织减少。各组心肌内有一些散在小血管断面。7.天狼猩红染色胶原含量的变化6周时,各组大鼠心肌纤维化面积均有不同程度的提高。MIR组大鼠心肌存在广泛的心肌纤维化,且心肌纤维化面积明显高于1周组(P＜0.05)。SMC的治疗明显改善了大鼠的心肌纤维化,且呈剂量依赖性,大剂量组优于小剂量组。应用p38MAPK抑制剂(SB组)明显降低心肌纤维化的面积,与SMC治疗组比较差异无显著性(P＞0.05)。与MIR组比较,SMC+LY组大鼠心肌纤维化程度也有降低,但降低水平明显小于舒脉胶囊治疗组与SB组。8.透射电镜观测心肌超微结构变化Sham组大鼠心肌肌原纤维排列整齐、紧密,无断裂,肌丝清晰,细胞核发育良好,线粒体体积大数量多,嵴丰富排列紧密呈Z字型,肌浆网体积大,数量多,胞浆丰富。MIR组大鼠心肌细胞肿胀、肌原纤维排列紊乱,核异形、呈分叶状,核膜已染色质边聚,常染色质呈团块状,Z线消失,核周细胞器呈点状、碎片状等变化;多数线粒体呈多形性,大小不等、嵴断裂溶解呈现絮状、空泡样变。SMCH与SB203580组心肌肌原纤维大都排列整齐,未见肌丝溶解,肌浆网扩张减少;线粒体无明显肿胀;细胞核轻度肿胀。SMCL组与SMC+LY组局部心肌细胞核异型;线粒体形状不规则;核周有点状、碎片状改变。结论1.舒脉胶囊对MI大鼠左室重构有明显的逆转作用,其具体表现为:改善MI大鼠心肌组织形态学指标,降低心肌CFs增殖与ECM胶原含量,即降低心肌α-SMA与collagenⅠ的表达,降低心肌纤维化,改善心肌超微结构的病理改变。2.舒脉胶囊改善左室重构的分子机制为舒脉胶囊抑制p38MAPK信号通路,从而抑制心肌TNFα的蛋白表达。进而下调TIMP-1及αSMA的蛋白表达,抑制CFs的增殖与胶原的沉积。3.舒脉胶囊改善左室整体与局部心功能。表现为左室射血分数(LVEF)的提高,左心室收缩末期内径(LVESD)、左心室舒张末期内径(LVEDD)的降低,左室缺血区室壁厚度分数(WT%)的提高。心功能改善是舒脉胶囊对缺血心肌保护作用的终极指标。更多还原

【Abstract】 Introduction:Ischemic heart disease is one of the leading causes of morbidity and afflicts more than hundreds of millions worldwide.It’s necessary to improve regional myocardial blood flow in prevention and treatment of ischemic heart disease.Theraputic angiogenesis can stimulate the formation of bypass circuit,and improve ischemic injury and has been the hot issue of cardialvascular research.Herbal medicines have been shown to exhibit numerous biological activities;however, compared to synthesized chemical medicines,systematic scientific evidence and proof of efficacy and safety are generally lacking.The underlying mechanisms are largely unclear. Therefore,it is important to do systematic research to assess the actions of herb medicines. Ischemic heart disease is one of the leading causes of morbidity and mortality worldwide. Myocardial ischemia（MI） subsequently contributes to angina,myocardial infarction,heart failure and death.So it is important to improve myocardial perfusion and function after MI.Shu-Mai Capsule（SMC） is one of the most frequently used Chinese prescriptions in traditional Chinese medicine（TCM） clinical practice for the treatment of ischemic heart disease.According to the concepts of traditional Chinese medicine,SMC consists of 7 medicinal compositions.Astragaloside,Salvianolic acid,Sanchinoside,and Ginsenoside Rg1 were identified as the major effective constituents.SMC,a prepared compound of traditional Chinese medicine,has been used as an anti-ischemic agent for years in clinic.In the present study,the changes of the pathology of ischemic myocardium were observed in myocardial ischemic（MI） rats,and the variation of VEGF and PDGF-BB mRNA and protein expression was detected by real-time RT-PCR and Western blot,and the variation of PI3K,p-Akt was detected by Western blot.In order to discover novel agents on stimulating stable collaterogenesis and improving cardiac function,the possible mechanism of SMC on stimulating angiogenesis and arteriogenesis in the ischemic myocardium was investigated,which could provide scientific evidence for the treatment of ischemic heart disease in clinic.Aim1.To investigate the effects of SMC on angiogenesis,arteriogenesis through observing the expression von Willebrand factor（vWF） andα-smooth muscle actin（α-SMA）,and detecting the cardiac function in rats with myocardial ischemia（MI）..2.To investigate the potential mechanism of SMC on stimulating angiogenesis, arteriogenesis in myocardial ischemia rat models through observing the phosphorylation of PI3K/Akt and detecting the expression of vascular endothelial growth factor（VEGF）, platelet derived growth factor（PDGF-BB） in the ischemical myocardium.MethodsAll animal care and experimental protocols complied with the Animal Management Rules of the Ministry of Health of the People’s Republic of China（document No 55,2001） and the guidelines for the Care and Use of Laboratory Animals of Shandong University of Traditional Chinese Medicine,China.Wistar rats weighing 230-290 g were purchased from the Animal Center of Shandong University（Shandong,Jinan）.Rats were housed in temperature-（22±2℃） and humidity-（55±5%） controlled rooms with a 12/12-hr light/dark cycle.Solid rodent chow and tap water were given freely.Animals were allowed to acclimatize for 1 week before entry into the experimental protocol.Then they were intraperitoneally anesthetized by use of 10%chloral hydrate（300 mg/kg） and ventilated with a VIP Bird ventilator（tidal volume,3.0 ml;respiratory rate,60 cycles/min）.The MI model was created by ligation of the left anterior descending coronary artery approximately 3 mm distal from its origin with use of a 6-0 polypropylene suture.Twenty four rats were randomly chosen as the sham-operated group,which underwent a similar surgical procedure but without coronary artery ligation.The rats with MI were randomly divided into five groups of 24 rats,including high- and low-dose SMC（SMCH and SMCL）,high-dose SMC and PI3K inhibitor LY294002（SMCH/LY）,bFGF,and myocardial ischemia rat models（MIR）.We also include the sham operated rats（Sham）.SMCL rats received low doses of SMC（342mg/kg; equal to six fold the clinic dosage）,SMCH rats received high doses（1.71 g mg/kg;equal to 30-fold the clinic dosage）,and MIR rats received an equal volume of distilled water as did Sham rats for 2（n=12 rats/group） and 4（n=12 rats/group） weeks.LY294002 （phosphatidylinositol 3-kinase（PI3K） inhibitor） was dissolved in sterile double H₂O and given at the indicated dose（0.3mg/kg）.The rats received the LY294002 via intraperitoneal administration once every three days for 2 and 4 weeks.After the last administration,all rats were fasted with free access to water for 24 h, and anesthetized with intraperitoneal injection of 10%chloral hydrate（300 mg/kg） which was followed by other experiments.The detection contents listed below:（1） Echocardiographic Analysis;（2） Determination of the levels of VEGF,PDGF-BB,PI3K, p-Akt in the ischemic myocardium with western blot;（3） Analysis of VEGF and PDGF-BB mRNA expression by real-time RT-PCR;（4） Determination of the myocardial microvessel density（MVD）（angiogenesis:expression of vWF;arteriogenesis:expression ofα-SMA）; （5） Determination of the area of fibrosis in the ischemic myocardium by Masson stainning.Statistical AnalysisAll values are expressed as means±SD.Data analysis involved use of SPSS v11.5 （SPSS;Chicago,IL）.Intergroup differences were analyzed by one-way ANOVA followed by the Bonferroni-corrected post-hoc analysis for multiple comparisons.Data with non-normal distribution were analyzed by nonparametric statistics.A P＜0.05 was considered significant.Results1.Echocardiographic AnalysisAt week 2 after treatment,a significant improvement of LVEF was observed in the SMC-treated groups（SMCH and SMCL） and bFGF group as compared with the MIR group（P＜0.05）.There was no significant difference between SMCH and SMCL or bFGF group（P＞0.05）.LVEDD and LVESD did not differ significantly between the SMCH/LY group and MIR group（P＞0.05）,and were higher than those in the Sham group（P＜0.05）. Taken together,the early improvement indicated that SMC stimulation rescued cardiac structure and function after injury.To determine whether SMC treatment has a long-term effect,we performed an additional and separate blinded and randomized study and treated the rats for 4 weeks.SMC therapy increased LVEF and decreased LVEDD in a dose dependent manner as compared to the MIR（P＜0.05）.There was no significant difference between the SMCH and bFGF（P＞0.05）.These findings demonstrate that SMC therapy remarkably stimulates myocardial collaterogenesis,leading to a significant improvement of regional as well as global myocardial contractile functions.2.Western blot analysesVEGF plays important role in angiogenesis because of its ability to stimulate vessel hyperpermeability,endothelial-cell proliferation,migration and capillary formation. PDGF-BB,are important arteriogenic factors for stabilization of the newly formed vasculature.To investigate whether SMC on stimulates stable collaterogenesis through high induction of VEGF and PDGF-BB via PI3K/Akt pathway,we determine the expression phosphorylation of Akt（p-Akt）,PI3K,VEGF,and PDGF-BB in the myocardium.At week 2,p-Akt,PI3K,VEGF,and PDGF-BB protein expression were significantly increased in the SMCH,SMCL,and bFGF as compared with the MIR（P＜0.05）,and with little detectable p-Akt,PI3K,VEGF,and PDGF-BB levels in the Sham rats.Administration of PI3K inhibitor L294002 blocked SMC-induced VEGF and PDGF-BB production,and inhibited the phosphorylation of Akt.At week 4,p-Akt,PI3K, VEGF,and PDGF-BB protein expression were significantly increased in the SMC-treated groups as dose dependence as compared with the MIR（P＜0.05）,and with little detectable p-Akt,PI3K,VEGF,and PDGF-BB levels in the Sham rats.There was no significant difference between the SMCH group and bFGF group（P＞0.05）.Administration of PI3K inhibitor L294002 blocked SMC-induced VEGF and PDGF-BB production,and inhibited the phosphorylation of Akt.These data showed that SMC could promote VEGF and PDGF-BB biosynthesis as dose dependence in the ischemic myocardium via PI3K/Akt signal pathway.3.Comparison of expression of VEGF and PDGF-BB in the myocardium by real time RT-PCR At week 2,the expression of VEGF and PDGF-BB mRNA was increased in the MIR group than that in the Sham group（P＜0.05）.The expression of VEGF and PDGF-BB mRNA was increased significantly in the SMCH group,the SMCL group and the bFGF group as compared with that in the MIR group or SMCH/LY group（all P＜0.05）.There was no significant difference between the SMCH group and the bFGF group or the SMCL group（P＞0.05）.At week 4,the expression of VEGF and PDGF-BB mRNA in the SMCL group was lower than that in the SMCH group（P＜0.05）.There was no significant difference between the SMCH group and the bFGF group（P＞0.05）.The expression of VEGF and PDGF-BB mRNA in the SMCH at week 4 was slightly downregulated as compared with that at week 2.4.Determination of the myocardial capillaries and arterioles densityQuantitative analysis confirmed that capillary density stained for the presence of vWF in the ischemic border zone was significantly higher in the SMCH（36.93±5.49）,SMCL, and bFGF groups than that in the SMCH/LY together treated group（15.13±5.11）,MIR （17.76±5.19） and Sham groups（9.44±2.68） at week 2（P＜0.05）.Capillary density was higher in the SMCH,SMCL,and bFGF at week 4 than that at week 2 but was significantly lower in the MIR and SMCH/LY.Additionally,the sections were stained for the presence ofα-SMA,a marker expressed in both the pericytes and the smooth muscle cells of mature vessels（arterioles）.Of course, vascular smooth muscle cells（VSMCs） are necessary for vascular maturation and arteriogenesis,which is the process by which capillaries acquire a VSMC coat to become arterioles with the ability to regulate blood flow.The formation of mature vessels was thought to be more important than capillary formation in restoring functional blood flow. The mature vessels index was significantly higher in the SMC-treated groups and bFGF group as compared with the SMCH/LY together treated group.Long-term follow-up analysis（week 4） showed that the collateral index continued to increase and remained stable in the SMC-treated groups（dose dependent） and bFGF group. Administration of PI3K inhibitor LY294002 blocked SMC-mediated angiogenesis and arteriogegesis.It suggested that high production of VEGF and PDGF-BB mediated by SMC promotes formation of capillaries and mature vessels via the PI3K/Akt pathway. 5.Myocardial collagen depositionAt week 2,the MIR group showed many newly formed collagen deposits（Red） between myocardial interstices and myocardial disarrangement and cellular swelling as compared with the Sham group.Myocardial collagen deposition was greater in the SMCH, SMCL and bFGF than in the Sham group（P＜0.05）,but was less than that in the MIR group （P＜0.05）.There was no significant difference between the SMCH/LY group and MIR group.The Sham myocardium showed a normal array of myocardial fibers and very little interstitial collagen in myocardium.At week 4,the area of fibrosis in the SMCL was higher than the SMCH and lower than the MIR.There was no significant difference between the SMCH and bFGF.Conclusion1.The findings of the current study illustrate for the first time that treatment with SMC induces significantly increase of capillaries and arterioles in a dose-dependent manner in the ischemic myocardium,which lead to functional improvement such as promoting LVEF,decresing LVESD and LVEDD.To improve the cardiac function is the aim for SMC to protect the ischemic myocardium.2.SMC can stimulate the expression of VEGF and PDGF-BB,and enhance the phosphorylation of PI3K and Akt as compared with the MIR group,which indicated that SMC stimulated angiogenesis,arteriogenesis through upregulating the phosphorylation of PI3K,Akt,and the expression of VEGF and PDGF-BB.3.PI3K inhibition（LY294002） significant inhibits the activity of PI3K/Akt signaling pathway,and also decrease the effect of SMC on stimulating angiogenesis and arteriogenesis.The mechanism is the therapeutic effect of SMC on promoting VEGF and PDGF-BB-mediated angiogenesis,arteriogenesis,and stabilization of myocardial collateral networks through the PI3K/Akt signal pathway. IntroductionPersistent remodeling contributes to functional decompensation and,eventually,heart failure and even death.Cardiac fibrosis is an important factor in the process of LV remodeling after MI.Thus,it is crucial to improve regional myocardial blood flow and attenuate LV myocardial fibrosis in ischemic tissues.Pro-inflammatory cytokines play important roles in the pathogenesis and pathophysiology of ischemic heart diseases.In both patients and experimental animal models,the induction of proinflammatory cytokines is closely associated with myocardial fibrosis and the onset and progression of cardiac remodeling.Among the pro-inflammatory cytokines,tumor necrosis factor-alpha（TNF-α） is considered to be the predominant cytokine to induce interstitial fibrosis.Findings in TNFR null mice suggest that TNF-αnormally account for the up-regulation of tissue inhibitor of matrix metalloproteinase （TIMP）-1 and may promote fibrosis by inhibiting collagen degradation.TNF-αalso is responsible for the proliferation of fibroblasts.Given the key role of TNFαas a mediator of myocardial fibrosis and the fact that fibrosis is a serious problem in left ventricular（LV） remodeling,inhibiting the induction of TNF-αis important on attenuation of myocardial fibrosis.In clinical and animal studies,elevated plasma and cardiac levels of TNF-αwere linked to interstitial fibrosis and ventricular remodeling.Inflammatory stimuli activate many intracellular signaling pathways,including the nuclear factor-κB pathway and three MAPK pathways mediated through extracellular-signal-regulated kinase（ERK）,c-Jun N-terminal kinase（JNK） and p38 MAPK,with p38 MAPK considered a central regulator of inflammation.Treatment with the selective p38 inhibitor（SB203580[SB]）reduced p38 MAPK activity in transgenic hearts,blocked TNF-αsecretion,and attenuated extracellular matrix（ECM） remodeling.Some studies have provided direct in vivo and in vitro evidence that a stress-activated mitogen-activated protein kinase（p38 MAPK） is activated in hearts under ischemia and other pathological conditions.The activation of p38 MAPK is a critical regulator of inflammatory response and is sufficient to induce the release of TNF-αin cardiomyocytes with significant contribution to marked myocardial fibrosis and pathological remodeling. Administration of the selective p38 MAPK inhibitor blocks the secretion of TNF-α,which reduces myocardial fibrosis and attenuates cardiac remodeling by reversing the balance between matrix metalloproteinases（MMPs） activity and tissue inhibitor of metalloproteinase（TIMP）-1 expression.This leads us to propose that p38-mediated TNF-αinduction represents a potential signaling mechanism that contributes to myocardial fibrosis and progressive LV remodeling.Evidence gathered from a systematic review shows that herbal medicines,which seem to be relatively safe and convenient,may offer a much-needed alternative.Some herbal medicines have been proved to be effective on anti-inflammation.However,limited data are available about the efficacy and cost-effectiveness of complex prescriptions of herbal medicine on anti-inflammatory cytokines-induced myocardial fibrosis and LV remodeling. SMC-an extract of traditional Chinese medicine（TCM）,having been used for symptomatic treatment of MI in clinic for years,is a modified herbal drug based on a traditional Chinese hepatotherapeutic formula.The aim of this study was to investigate the efficacy of SMC on anti-inflammatory cytokine TNF-α-induced myocardial fibrosis and LV remodeling in MI rats,as well as the potential mechanism.Aim1.To investigate the effects of Shu-Mai Capsule（SMC） on attenuating left ventricular （LV） remodeling and myocardial fibrosis through detecting the cardiac function and observing the proliferation of CFs（through detecting the expression ofα-SMA ） and the expression collagenⅠin the ischemical myocardium in rats with myocardial ischemia （MI）. 2.To investigate the potential mechanism of SMC on attenuating LV remodeling in myocardial ischemia rat models through observing the changes of the morphology and the ultrastructure,the phosphorylation of a stress-activatedmitogen-activated protein kinase （p-p38 MAPK）,and detecting the expression of tumor necrosis factor-alpha（TNF-α）, tissue inhibitor of metalloproteinase（TIMP）-1 in the ischemical myocardium.MethodsAll animal care and experimental protocols complied with the Animal Management Rules of the Ministry of Health of the People’s Republic of China（document No 55,2001） and the guidelines for the Care and Use of Laboratory Animals of Shandong University of Traditional Chinese Medicine,China.Wistar rats weighing 230-290 g were purchased from the Animal Center of Shandong University（Shandong,Jinan）.Rats were housed in temperature-（22±2℃） and humidity-（55±5%） controlled rooms with a 12/12-hr light/dark cycle.Solid rodent chow and tap water were given freely.Animals were allowed to acclimatize for 1 week before entry into the experimental protocol.Then they were intraperitoneally anesthetized by use of 10%chloral hydrate（300 mg/kg） and ventilated with a VIP Bird ventilator（Bird Products Corp.;Palm Springs,CA）（tidal volume,3.0 ml; respiratory rate,60 cycles/min）.The MI model was created by ligation of the left anterior descending coronary artery approximately 3 mm distal from its origin with use of a 6-0 polypropylene suture.Twenty rats were randomly chosen as the sham-operated group, which underwent a similar surgical procedure but without coronary artery ligation.The rats with MI were randomly divided into four groups of 24 rats,including highand low-dose SMC（SMCH and SMCL）,p38 MAPK inhibitor SB203580（SB）,and myocardial ischemia rat models（MIR）.We also include the sham operated rats（Sham）. SMCL rats received low doses of SMC（342mg/kg;equal to six fold the clinic dosage）, SMCH rats received high doses（1.71 g/kg;equal to 30-fold the clinic dosage）,and MIR rats received an equal volume of distilled water as did Sham rats.SB203580[p38 mitogen-activated protein kinase（p38 MAPK） inhibitor]was dissolved in sterile double H₂O and given at the indicated dose（2 mg/kg body weight）.The rats received the SB203580 via intraperitoneal administration once every three days for 1 and 4 weeks. These five groups were further divided into 2 groups each（n=12 each） for 1- and 6-week treatment,respectively.After the last administration,all rats were fasted with free access to water for 24 h, and anesthetized with intraperitoneal injection of 10%chloral hydrate（300 mg/kg） which was followed by other experiments.The detection contents listed below:（1） Echocardiographic analysis;（2） Determination of the levels of TNF-αby radioimmunoassay（RIA） assay;（3） Determination of the levels of p38 MAPK,TNF-α、TIMP-1 protein with western blot;（4） Analysis of collagenⅠmRNA expression by real-time RT-PCR;（5） Determination of the protein expression of collagenⅠ、α-SMA with immunohistochemistry assay;（6） Determination of the area of fibrosis in the ischemic myocardium;（7） Ultrastructural observation and Transmission Electron Microscope（TCM） study.Statistical AnalysisAll values are expressed as means±SD.Data analysis involved use of SPSS v11.5 （SPSS;Chicago,IL）.Intergroup differences were analyzed by one-way ANOVA followed by the Bonferroni-corrected post-hoc analysis for multiple comparisons.Data with non-normal distribution were analyzed by nonparametric statistics.A P＜0.05 was considered significant.Results1.Eehocardiographie analysisAt week 1 after treatment,a significant improvement of LVEF was observed in the SMC-treated groups（SMCH and SMCL） and SB group as compared with the MIR group. There was no significant difference between SMCH and SMCL or SB group.LVEDD and LVESD were higher in the MIR group than those in the Sham group.Taken together,the early improvement indicated that SMC stimulation rescued cardiac structure and function after injury.To determine whether SMC treatment has a long-term effect,we performed an additional and separate blinded and randomized study and treated the rats for 6 weeks. SMC therapy increased LVEF and decreased LVEDD in a dose-dependent manner as compared to the MIR（P＜0.05）.There was no significant difference between the SMCH and SB.These findings demonstrate that SMC therapy remarkably attenuated left ventricular remodeling,leading to a significant improvement of regional as well as global myocardial contractile functions.2.Inhibition effects of SMC on serum TNF-αlevelAnti-inflammation activity of SMC was investigated by radioimmunoassay.At week 1 and 6 week,the level of TNF-αin the MIR,SMCL,SMCH,and SB group was higher than that in the Sham group（P＜0.05）.However,the serum level of TNF-αin the SMC-treated groups dropped gradually and a significant decrease was found at both 1 and 6 weeks in a dose-dependent manner as compared with the MIR（P＜0.05）.No significant difference was shown between the SMCH and the SB group at both week 1 and 6.3.Western blot analysisThe p38 mitogen-activated protein kinase（MAPK） has been paid much attention, which mediates inflammatory cytokines induction and is associated with cardiac remodeling and contractile dysfunction.In TNFα,acting primarily via TNFR2,promotes fibrosis by stimulating myofibroblast proliferation and reducing collagen degradation by inducing TIMP-1.To investigate whether SMC on attenuating left ventricular remodeling through downregulation of TNFαand TIMP-1 via p38 MAPK pathway,we determine the expression of phosphorylation of p38 MAPK（p-p38）,TNF-α,and TIMP-1 in the myocardium at both week 1 and 6.p-p38 MAPK,TNF-α,and TIMP-1 protein expression were significantly decreased in the SMC-treated rats in a dose dependent manner and SB group as compared with the MIR group,and with little detectable p-p38 MAPK,TNF-α, and TIMP-1 levels in the Sham rats at both week 1 and 6.Administration of SMC significant inhibited the phosphorylation of p-38 MAPK as the p38 MAPK inhibitor SB203580.These data showed that SMC could reduce TNF-αand TIMP-1 biosynthesis in ischemic myocardium via the inhibiting the p38 MAPK signal pathway.4.Analysis of collagenⅠmRNA expression by real-time RT-PCRAt week 1,the expression of collagenⅠwas increased in the MIR group than that in the Sham group（P＜0.05）.The expression of collagenⅠmRNA was decreased significantly in the SMCH group,the SMCL group and the SB group as compared with that in the MIR group（all P＜0.05）.There was no significant difference between the SMCH group and the SB group or the SMCL group（P＞0.05）.At week 6,the level of collagenⅠwas higher in the MIR group than all the other groups.The expression of collagenⅠin the SMCL group was higher in the SMCL than that in the SMCH group.There was no significant difference between the SMCH group and the SB group（all P＞0.05）.The expression of collagenⅠmRNA in the all groups except for the Sham group at week 6 was slightly downregulated as compared with that at week 1.5.Expression ofα-SMA and and CollagenⅠprotein by immunohistochemistryα-SMA was detected within the media of the arteries and arterioles in the normal myocardium of the Sham group.An increased accumulation ofα-SMA-positive cells was also observed in the ischemic myocardium of the MIR group.SMC and SB treatment decreased the accumulation ofα-SMA positive cells in the myocardium,which indicated that number of fibroblasts in the MIR were higher than the SMC-treated groups（in a dose dependent manner）,and SB at both week 1 and 6.No significant difference was shown between the SMCH and SB（p38 MAPK inhibitor SB203580） group.CollagenⅠwas detected widely in extracellular matrix in the MIR group at week 6.However,the expression of collagenⅠwas attenuated in the SMC-treated groups in a dose dependent manner and the SB group.6.Myocardial fibrosisThe MIR group showed many newly formed collagen deposits（Red） between myocardial interstices and myocardial disarrangement and cellular swelling as compared with the Sham group.The Sham myocardium showed a normal array of myocardial fibers and very little interstitial collagen in myocardium.Myocardial collagen deposition was greater in the SMC-treated group and the SB group than in the Sham group（P＜0.05）,but was less than that in the MIR group at both week 1 and 6（P＜0.05）.7.Ultrastruetural observation and transmission electron microscope studyStructural studies using transmission electron microscope（TEM） were performed. Cardiac muscle fibers in the Sham group were abundant,with regular arrays of myofibrils closely arranged within the sarcomere and normal size of the mitochondria with normal numbers in myocardium.In the MIR group,the structure of mitochondria was damaged seriously.There were apparent cellular and tissue swelling.Most myofibrils were either disappeared or disorganized.Mitochondria were swollen obviously and loosely arranged. Mitochondrial membranes were vague or partly ruptured and cristae were obviously loose and dissolved,a lot of vacuoluses were formed.A significant ultrastructural organization change in mitochondria and myofibrils were ameliorated in the SMC-treated groups and the SB group.ConclusionIn summary,the findings of the current study illustrate for the first time that SMC therapy showed benefits in preservation of cardiac structure and function in the MI rat model.The mechanism is that SMC improves cardiac function through attenuating inflammatory-induced LV myocardial fibrosis after MI.It attenuates inflammatory cytokine TNF-α-induced myocardial fibrosis via inhibiting the p38 MAPK pathway and downregulated the expression of TIMP-1 andα-SMA.更多还原