【作者】 陈琳琳；

【导师】 李志樑；

【作者基本信息】 南方医科大学 ， 内科学（专业学位）， 2014， 博士

【摘要】 研究背景：心肌梗死(myocardial infarction, MI)是心血管疾病致死及致残的主要原因。我国每年约百万余患者死于急性心肌梗死(Acute myocardial infarction, AMI),该病被视为“人类健康第一杀手”。近年来随着溶栓、心脏介入手术的广泛开展及药物治疗在心肌梗死治疗中的迅猛发展,有效地挽救了心肌梗死患者濒临死亡的心肌,预后得到了极大改善,但仍有许多患者由于各种原因未能及时地进行血运重建,心肌发生了不可逆的死亡,引起心室重构(ventricular remodeling, VR)。心室重构是指心肌梗死后由神经体液调节机制激活、炎症、细胞因子引起心肌细胞及细胞间质形态结构的改变,使心脏结构功能按照一定的模式进行重塑,发生的左心室的进行性扩张和外形改变,包括心室容积、形状、室壁厚度、心肌结构等方面的改变,导致心脏形态结构和血流动力学异常,左室进行性扩大,收缩功能逐渐降低,最终导致心力衰竭(heart failure, HF)和死亡。心室重构与急性心肌梗死早期心脏破裂、室壁瘤形成等严重并发症有关,是影响心肌梗死近、晚期预后的主要原因。心肌梗死的严重程度不仅取决于梗死面积大小,其与炎症细胞的浸润及炎症细胞分泌的细胞外基质金属蛋白酶(MMPs)也密切相关；而梗死心肌的重构也与梗死面积、梗死后炎症反应及梗死心肌的修复密切相关。心肌梗死后的修复过程中存在生理性和病理性免疫炎症反应,生理性的炎症反应促进了自身修复和保护,病理性免疫炎症反应则介导了心室重构和心力衰竭的发生。梗死心肌的修复有赖于炎症介质的释放及白细胞介导的坏死细胞及细胞碎片的清除,免疫反应在整个修复过程中发挥重要的调节作用,其具体机制尚未完全明确。动物实验发现,通过适时的调控免疫细胞、炎症因子的表达,抑制免疫炎症反应,可以在一定程度上改善心肌的重构,减少了心力衰竭的发生。因此,心室重构中免疫调节机制可能成为心室重构相关疾病治疗的新靶点,有效地控制心肌梗死后的炎症反应,可能是延缓、逆转心肌梗死后心室重构及预防心力衰竭的关键所在。姜黄素(Curcumin)是姜黄(Curcuma)中提取的酚类物质,目前己经有研究表明,姜黄素对心肌的缺血性再灌注损伤具有保护作用,且这种作用呈一定的剂量依赖关系；姜黄素还可抑制左室压力超负荷导致的不良心室重构,改善左室功能；在大鼠心肌梗死中,姜黄素改善心功能的作用也被多次证实。但姜黄素心肌保护作用的作用仍不明确。姜黄素的药理作用广泛,其在免疫调控方面的功效也备受关注。研究发现,姜黄素可调节促炎与抗炎细胞因子的表达、抑制4F-κB信号通路活化、清除氧自由基以及抑制细胞凋亡等,有效地降低血液循环中白介素-1(IL-1)、白介素-6(IL-6)、白介素-12(IL-12)、TNF-α水平,同时还可抑制转化细胞生长因子(TGF-β)和白介素-10(IL-10)的升高,这些研究结果提示姜黄素具有双向免疫调控作用,促使机体的炎症反应处于一种低水平的平衡状态,避免组织过度的炎症反应导致的机体损伤。基于对心肌梗死后心室重构中炎症反应过程的理解和姜黄素对炎症性疾病及改善心功能的疗效,我们推断姜黄素可能通过免疫调控抑制心肌梗死后的炎症反应,从而发挥抗心室重构的作用。本研究分为两部分：在第一部分研究中,我们通过结扎小鼠冠状动脉前降支来制备心肌梗死的动物模型,通过观测不同剂量姜黄素对心肌梗死后4周小鼠心脏功能及结构、心肌组织病理学、心肌胶原重构以及心肌细胞凋亡的影响,并设定阳性对照组,以明确姜黄素在改善心肌梗死后心功能方面的确切疗效；第二部分研究,我们通过检测姜黄素对梗死心肌组织炎症反应的影响,旨在阐明姜黄素改善心肌梗死后心功能可能存在的免疫调控机制,为心肌梗死后心室重构的预防和临床救治提供新的思路。第一部分姜黄素对小鼠心肌梗死后心功能及心室重构的影响目的：探讨姜黄素对心肌梗死后小鼠心功能及心室重构的影响。方法：1.小鼠心肌梗死模型的制备与分组选用SPF级雄性C57BL/6小鼠,周龄为8-10周,体重20-25g,结扎左冠状动脉前降支建立心肌梗死模型。开胸结扎前降支,观测结扎部位以下心肌颜色变苍白,心电图显示心肌有缺血表现,即证明造模成功。将心肌梗死模型成功的小鼠随机分为4组：模型对照组,依那普利组,姜黄素低剂量组,姜黄素高剂量组；假手术组为开胸后冠状动脉前降支只穿线,不结扎。本部分研究共5组：Sham、MI组分别给予6%乙醇和6%聚乙醇400混合液1ml/kg.d,腹腔注射；依那普利组(阳性对照组)：依那普利剂量为7.5mg/kg.d,研磨后用蒸馏水溶解后灌胃,并予以6%乙醇和6%聚乙二醇400混合液4ml/kg.d腹腔注射；姜黄素低、高剂量组分别给予姜黄素溶液4ml/kg.d(两组药物浓度分别为12.5mg/ml、25mg/ml),腹腔注射。各组小鼠均正常饮食饮水,连续给药4周。2.超声心动图评价心肌梗死后小鼠心功能于术后4周检测超声心动图,探头频率为30MHz。采集胸骨旁长轴切面、心尖四腔切面B-Mode图像。取胸骨旁左室短轴,2D超声示左室短轴切面,在乳头肌水平应用M型超声记录左心室运动情况。形态学指标包括：左室舒张末期内径(]VEDd)、左室收缩末期内径(LVESd),收缩功能指标包括：射血分数(LVEF)、短轴收缩率(-VFS)。比较各组小鼠心脏形态及功能变化。3.体重、心脏重量/体重比测定及心脏大体形态学观察小鼠测过超声后,称量并记录各组小鼠体重,用1%戊巴比妥钠(50mg/kg)腹腔麻醉后,于左胸触及心尖搏动处用1ml注射器抽取血液1ml左右,加入抗凝管中,静置2h后,离心(2000r/min,15min)后取上层清液,放入-20℃冰箱待检。心脏采血后处死小鼠,迅速开胸,剪下心脏,迅速放入冰生理盐水中,洗尽血液,肉眼观察心脏大体形态,剪除左、右心耳及残余大血管后以滤纸滤干,电子天平称量心脏实际重量(湿重),并计算其与体重的比值。取出心脏称重后直接放入-80℃超低温冰箱中保存备做心肌结缔组织染色。组织染色准备时,将心脏从超低温冰箱中取出后沿着结扎线,并垂直于心脏的长轴将心脏剪成两半,留取心脏结扎线至心尖的那一部分,置于4%的福尔马林溶液中固定保存,用于免疫组织化学研究。4.小鼠心肌结缔组织染色4周后处死小鼠,小鼠心肌组织用HE染色和Masson三色染色法进行组织病理学观察；免疫组化法检测心肌组织中Ⅰ型胶原、Ⅲ型胶原的蛋白表达。5.姜黄素对心肌梗死小鼠梗死边缘区心肌细胞凋亡的影响TUNEL染色检测心肌梗死梗死边缘区心肌细胞凋亡,比较各组小鼠心肌细胞凋亡情况。6.数据分析与统计方法数据采用SPSS13.0统计软件进行分析,所有实验结果以均数±标准差(X±S)表示,多组间比较采用单因素方差分析(one-way ANOVA).方差齐时,组间多重比较则采用LSD法；若方差不齐则采用Welch稳健估计,组间多重比较则采用Dunnett’s T3法。P<0.05被认为差异有统计学意义。结果：1.小鼠心脏超声观察结果结果显示：各组间彩色多普勒超声心动图心功能指标LVEDd、LVESd、LVEF、 LVFS差异具有统计学意义(P<0.01)。与假手术组相比,模型对照组、姜黄素高低剂量组及依那普利组小鼠心功能指标LVEDd、LVESd显著升高(P<0.01),LVEF、LVFS显著降低(P<0.01)。与模型对照组比较,依那普利组及姜黄素高、低剂量组LVEDd、LVESd均显著降低,而LVEF、LVFS显著升高,差异具有统计学意义(P<0.01),说明姜黄素、依那普利均能有效改善心功能,减少左室扩张。与依那普利组相比,姜黄素低剂量组LVFS降低、LVEF有所降低(P<0.01),说明姜黄素低剂量组改善心功能方面不及依那普利组显著；与依那普利组比较,姜黄素高剂量组LVESd有所降低(P<0.01)、FS有所升高(P<0.01),说明姜黄素高剂量组较依那普利在改善心功能方面可能存在更好的疗效。与姜黄素低剂量组比较,姜黄素高剂量组LVEDd、LVESd降低,而LVEF、LVFS明显升高(P<0.01)。以上说明,在改善心梗后小鼠心功能方面,姜黄素高剂量组疗效优于姜黄素低剂量组,与依那普利效果相当。2.小鼠心肌梗死后体重、心脏重量及心脏重量/体重比结果结果显示：与假手术组相比,模型对照组小鼠心脏重量、心脏重量/体重比明显升高,体重明显减轻,差异具有统计学意义(P<0.01),说明冠脉结扎所致的心肌梗死后心室发生重构,心脏重量增加；与心梗模型比较,依那普利组、姜黄素高、低剂量组的心脏重量、心脏重量/体重比降低明显,体重增加,差异具有统计学意义(P<0.01),说明依那普利和姜黄素干预均可以改善心肌梗死后不良心室重构；依那普利组、姜黄素低剂量组、姜黄素高剂量组在心脏重量、心脏重量/体重比无统计学差异(P>0.05),说明依那普利和姜黄素能明显缓解心室肥厚,效果无明显差异。3.小鼠心脏大体形态观察结果显示：与假手术组比较,模型对照组小鼠心脏体积明显增大,左心室室腔明显扩大,心脏已不能维持正常几何形状；与模型对照组比较,姜黄素低、高剂量组心脏基本能维持正常几何形状,体积缩小,梗死区面积缩小,梗死区仍残存心肌组织,弹性及韧性较好；心室腔扩张程度减轻,且与依那普利组差异不明显。4.HE染色结果假手术组心肌着色均匀,横纹排列整齐,细胞界限清晰,排列规整,显示正常心肌细胞形态。模型对照组心肌纤维增粗、变长、排列紊乱、间隙增宽,细胞核大而深染,可见炎症细胞浸润。姜黄素高、低剂量组与依那普利组边缘区见少量心肌细胞肥大增生,有点状纤维增生,非梗死区心肌细胞排列整齐,炎症细胞浸润较模型对照组减轻。其中姜黄素高剂量组与依那普利组其纤维增生程度较姜黄素低剂量组减弱明显,炎性细胞浸润亦减轻。5.小鼠心肌梗死后纤维化面积的变化Masson染色检测心肌梗死后4周小鼠心肌纤维化的面积。结果显示：与假手术组比较,模型对照组小鼠心脏梗死区及梗死边缘区域可见纤维增生、排列紊乱；与模型对照组比较,姜黄素高、低剂量组及依那普利组心肌纤维化的面积减少,差异有统计学意义(P<0.01)；与依那普利组比较,姜黄素低剂量纤维化面积增加,差异有统计学意义(P<0.01),姜黄素高剂量组纤维化面积无统计学差异(P=0.771)。6.免疫组织化学法检测心肌非梗死区Ⅰ型胶原、Ⅲ型胶原的蛋白表达以及Ⅰ/Ⅲ胶原的比值结果结果显示：假手术组未见明显胶原蛋白表达,无基质纤维化表现；与假手术组比较,模型对照组、依那普利组、姜黄素低剂量组、姜黄素高剂量组左室非梗死区纤维化表现明显,可见Ⅰ型、Ⅲ型胶原蛋白表达明显增加,Ⅰ/Ⅲ胶原蛋白的比值显著增加(P<0.01)。与模型对照组比较,依那普利组、姜黄素高低剂量组胶原纤维表达程度有所减轻,Ⅰ型、Ⅲ型胶原蛋白表达明显减少,Ⅰ/Ⅲ胶原蛋白的比值较模型对照组明显减小(P<0.01)。与依那普利组比较,姜黄素高、低剂量组胶原纤维化程度稍重,但胶原蛋白表达量无统计学差异(P>0.05)；姜黄素低剂量组Ⅰ/Ⅲ胶原蛋白的比值有所增加(P<0.01),姜黄素高剂量组Ⅰ/Ⅲ胶原蛋白的比值差异无统计学意义(P=0.138)。与姜黄素低剂量组比较,依那普利组、姜黄素高剂量组纤维化程度无明显差异,但两组Ⅰ/Ⅲ胶原蛋白的比值下降明显(P<0.01)。7.小鼠心肌细胞凋亡的测定结果模型制备及干预后3d,用TUNEL试剂盒检测梗死边缘区心肌细胞凋亡水平。结果显示：与模型对照组比较,姜黄素高、低剂量组及依那普利组心肌细胞凋亡减少,差异有统计学意义(P<0.01)；姜黄素低剂量较依那普利组及姜黄素高剂量组心肌细胞凋亡增多,差异有统计学意义(P<0.01)；姜黄素高剂量组与依那普利组心肌细胞凋亡无统计学差异(P=0.356)。结论：1.采用结扎模型小鼠冠状动脉左前降支,观察局部心肌颜色、活动度及心电图变化,成功制作了AMI小鼠模型,其组织形态学、心功能及胶原蛋白表达检测均表明模型小鼠存在心室重构的病理改变。2.姜黄素可改善心肌梗死后小鼠的心功能,抑制心肌肥厚,减缓心室重塑,与阳性对照药依那普利有相近的实验结果。3.姜黄素可调节不同类型胶原蛋白的表达,减少心肌梗死后纤维化面积。4.姜黄素能减少心肌细胞凋亡,提示这可能是姜黄素改善AMI后心室重构的作用机制之一。5.姜黄素低、高剂量组均可不同程度改善心肌梗死后小鼠心室重构的各项检测指标,且姜黄素高剂量的作用效果优于姜黄素低剂量组,其心脏保护作用有一定的剂量依赖关系。第二部分姜黄素对小鼠心肌梗死后炎症反应的影响及机制研究目的：炎症反应是心肌梗死后心室重构的重要环节,是慢性心力衰竭发展的基本机制。NF-κB信号通路活化可以促进炎症反应及心肌细胞凋亡。有研究报道姜黄素可抑制TLR4、NF-κB信号通路的激活,所以我们推测姜黄素可能通过下调梗死心肌组织中的TLR4的表达,并抑制其下游NF-κB信号通路的活化,从而逆转心肌梗死后心室重构,发挥改善心肌梗死后心功能的作用。本实验研究目的是观察姜黄素对小鼠心脏梗死边缘区树突状细胞的浸润、梗死心肌组织中炎症细胞因子IL-1β、TNF-α、IL-6、IL-10的表达、梗死心肌组织中TLR4的表达及其下游NF-κB炎症信号通道的影响。方法：1.实验分组选用SPF级雄性C57BL/6小鼠,8-10周龄。按开胸后是否结扎前降支,分为假手术组及心肌梗死组。心肌梗死造模成功后,将存活小鼠随机分为MI组、姜黄素组。实验共分为三组：Ⅰ.假手术组(Sham);II.模型对照组(MI)；Ⅲ.姜黄素组(Cur)。Sham、MI组分别给予6%乙醇和6%聚乙二醇400混合液4ml/kg.d腹腔注射；姜黄素组分别给予姜黄素溶液4ml/kg.d(浓度25mg/ml)腹腔注射。各组小鼠正常饮食饮水,连续给药4周。Ⅰ、Ⅱ、Ⅲ三组按模型制备及干预后3天(A)、7天(B)、14天(C)、28天(D)分为4个亚组。2.标本收集心脏采血后处死小鼠,迅速开胸,剪下心脏,冰生理盐水中洗尽血液,肉眼观察心脏大体形态,剪除左、右心耳及残余大血管后以滤纸滤干,以电子天平称取心脏实际重量(湿重),分别计算与体重的比值。各组分别取4只用于qPCR及Western-blot研究。心肌梗死边缘区域,判断梗死区主用通过肉眼观察到心肌变白,梗死边缘区主要为梗死瘢痕外约lmm以内区域。在取材过程中,为了更好的界定梗死边缘区与非梗死的左心室,先将梗死瘢痕外约1.5mm以外的心肌组织去除,剪下梗死边缘区域心肌组织后,其余心肌组织快速地置于液氮中,然后转入-80℃冰箱保存(假手术组样本留取相对应的左室心肌组织)。其余小鼠留取心脏用于免疫组织化学研究。沿着结扎线,并垂直于心脏的长轴将心脏剪成两半,留取心脏结扎线至心尖的那一部分,置于4%的福尔马林溶液中固定保存,用于免疫组织化学研究。3.免疫荧光法检测梗死边缘区心肌组织中树突状细胞(dendritic cells,DCs)的变化选取模型制备及干预后3d、7d、14d、28d组小鼠(每组各5只,假手术组仅取3d组作为对照),留取小鼠梗死及边缘区心肌,石蜡包埋切片后,用免疫荧光法检测梗死边缘区心肌组织中CD11c阳性表达。4.Western Blot法测定小鼠梗死心肌组织中蛋白的表达选取模型制备及干预后7d组小鼠(每组各4只),提取梗死心肌中的总蛋白,通过western blot检测TLR4、iNOS以及NF-κB信号通路蛋白——NF-κB p65、 IκBα和IκB蛋白激酶α/β(IKKα/β)的总蛋白及磷酸化表达水平的表达。5.qPCR检测梗死区心肌组织中TLR4及炎症因子的表达选取模型制备及干预后3d、7d、28d组小鼠(每组各4只,假手术组仅取3d组作为对照),提取梗死心肌中的RNA,通过qPCR检测梗死心肌组织中TLR4mRNA以及炎症细胞因子IL-1β、TNF-α、IL-6、IL-10mRNA的表达。6.数据分析与统计方法数据采用SPSS13.0统计软件进行分析,所有实验结果以均数±标准差(X±S)表示,多组间比较采用单因素方差分析(one-way ANOVA)。方差齐时,组间多重比较则采用LSD法；若方差不齐则采用Welch稳健估计,组间多重比较则采用Dunnett’s T3法。P<0.05被认为差异有统计学意义。结果：1.姜黄素对梗死边缘区心肌组织中树突状细胞浸润的影响采用石蜡切片免疫荧光法检测心梗边缘区表面抗原为CD11c阳性的树突状细胞浸润情况。结果显示：与假手术组(仅取3d组作为对照)比较,模型对照组梗死边缘区心肌组织中CDllc阳性的树突状细胞浸润在心梗3d组明显增多,心梗7d组达到峰值,其后开始逐渐减少,28d后基本恢复正常。与模型对照组比较,在各对应时间点姜黄素亚组中,梗死边缘区心肌组织中的树突状细胞浸润均有所减少(P<0.05)。2.姜黄素对梗死心肌组织中炎症细胞因子的影响qPCR检测了梗死心肌组织中促炎因子IL-1β、TNF-α、IL-6及抑炎因子IL-10的mRNA表达情况。结果显示：与假手术比较(仅取3d组作为对照),模型对照组梗死心肌组织中促炎因子IL-1β、TNF-α、IL-6mRNA表达上调,3d组最高,7d组有所下降,28d基本恢复正常；抑炎因子IL-10在3d组、7d组基本没有变化,28天组表达有所上调(P=0.031)。与模型对照组比较,姜黄素在3d、7d各对应亚组中梗死心肌组织中促炎因子IL-1β、TNF-α、IL-6mRNA表达均有所下调(P<0.05),28d时基本都恢复正常；抑炎因子IL-10在心梗后3d、7d、28d时与模型对照组无明显差异,但较假手术组有所上升(P=0.002)。3.姜黄素对梗死心肌组织中iNOS蛋白的影响Western blot法检测假手术组、模型对照组、姜黄素组7d亚组小鼠梗死心肌组织中iNOS蛋白的表达。结果显示：与假手术组比较,模型对照组小鼠梗死心肌组织中iNOS蛋白水平显著增加(P<0.05),而姜黄素组iNOS蛋白水平也显著增加(P<0.05)；而与模型对照组比较,姜黄素组iNOS表达水平显著降低(P<0.05)。4.姜黄素对梗死心肌组织中TLR4水平的影响Western blot和qPCR分析检测假手术组、模型对照组、姜黄素组7d亚组小鼠梗死心肌组织中TLR4蛋白和mRNA的表达情况。结果显示：与假手术组比较,模型对照组小鼠梗死心肌组织中TLR4mRNA和蛋白水平显著增加(P<0.05),姜黄素组TLR4表达水平明显降低(P<0.05)。5.姜黄素对NF-κB炎症信号通路的影响为了进步一阐明姜黄素抑制NF-κB炎症信号通路的活化,本研究采用western blot法检测了假手术组、模型对照组、姜黄素组7d亚组小鼠梗死心肌中NF-κB p65.IκBα和IκB蛋白激酶α/β(IKKα/β)的总蛋白及磷酸化表达水平。结果显示：与假手术组比较,模型对照组IκBα及IKKα/β的磷酸化水平增加(P<0.05),表明IKK复合物活化使IκBa处于磷酸化水平,从而激活TNF-κBp65,使p-NF-κBp65表达明显增多(P<0.05),而姜黄素组较模型对照组IκBα及IKKα/β的磷酸化水平降低(P<0.05)。表明姜黄素通过抑制IKKα/β活化来抑制IκBα磷酸化,最终抑制NF-κBp65信号通路的激活,发挥了抑制炎症反应的作用。结论：1.心肌梗死急性期早期,炎症反应增强,梗死心肌局部的促炎症细胞因子IL-1p、IL-6、TNF-α表达上调,梗死心肌边缘区树突状细胞浸润增多,而姜黄素可以调控树突状细胞在梗死心肌边缘区的浸润,并下调促炎症因子的表达,抑制梗死心肌局部的炎症反应；急性心梗的炎症反应晚期,抑炎症细胞因子IL-10表达上调,姜黄素可以上调其表达。这些结果提示我们姜黄素在心室重构炎症反应早期,可通过抑制促炎因子的表达来减轻炎症反应,以免过度的炎症反应导致不良的心室重构；炎症反应后期可通过上调抑炎因子的表达,从而发挥炎症清除作用。这可能是姜黄素发挥心肌梗死后心室重构保护作用的免疫调控机制之一。2.iNOS在心肌损伤后触发非特异性免疫反应的过程中发挥着重要作用,有研究证实iNOS的表达与促炎症因子诱导的心肌细胞凋亡及纤维化正相关。姜黄素可抑制心梗后梗死心肌中iNOS蛋白的表达,这可能是其减少心肌细胞凋亡及心肌梗死纤维化面积的机制之一。3.姜黄素可下调梗死心肌中TLR4的表达,并可以通过阻断其下游NF-κB的经典激活途径,其主要是通过抑制KKα的活化来抑制IjBα磷酸化,最终抑制NF-κBp65的激活。NF-κB的经典激活途径主要参与机体的急慢性炎症反应,NF-κB(?)是心肌梗死后炎症反应的关键分子之一。姜黄素可抑制TNF-κB通路的活化,这也可能是姜黄素发挥抑制心肌梗死后炎症反应的作用途径之一。更多还原

【Abstract】 Backgroud:Myocardial infarction(MI) is a major cause of cardiovascular disease to death and disability.In our country, every year about millions of patients died of Acute myocardial infarction (AMI), the disease is regarded as "the first killer" to human health. Over the last30years, advanced coronary care and early reperfusion strategies have dramatically improved survival rates in patients suffering an acute myocardial infarction. However, this impressive success has resulted in a larger pool of patients who, having survived the acute infarction, are at risk of developing heart failure. Despite therapeutic advances, the risk of heart failure following myocardial infarction has remained high; in fact, some studies have suggested an increased incidence of postinfarction heart failure in recent decades that parallels the decreasing acute mortality rates. Development of heart failure following myocardial infarction is closely associated with profound alterations in cardiac geometry, function, and structure, also referred to as "ventricular remodeling."The molecular and cellular changes in the remodeling heart affect both the area of necrosis and the noninfarcted segments of the ventricle and manifest clinically as increased chamber dilation and sphericity, myocardial hypertrophy, and worsened cardiac function. Cardiac remodeling is linked to heart failure progression and is associated with poor prognosis in patients surviving a myocardial infarction. The extent of postinfarction remodeling is dependent on the size of the infarct and on the quality of cardiac repair. Cardiac repair is dependent on a superbly orchestrated inflammatory response that serves to clear the wound from dead cells and matrix debris, but also provides key molecular signals for activation of reparative cells. It is becoming increasingly apparent that timely repression and containment of inflammatory signals are needed to ensure optimal formation of a supportive scar in the infarcted area and to prevent development of adverse remodeling. Because even relatively subtle alterations in myocardial architecture, matrix compo sition, and cellular phenotype profoundly affect chamber geometry and ventricular function, defects, and aberrations in temporal and spatial regulation of the postinfarction inflammatory reaction may have catastrophic consequences. Excessive early inflammation may augment matrix degradation causing cardiac rupture. Prolongation of the inflammatory reaction may impair collagen deposition leading to formation of a scar with reduced tensile strength, thus increasing chamber dilation. Enhanced expression of proinflammatory mediators may activate proapoptotic pathways inducing further loss of cardiomyocytes. Finally defective containment of the inflammatory reaction may lead to extension of the inflammatory infiltrate into the noninfarcted myocardium enhancing fibrosis and worsening diastolic function.Because of the importance of inflammatory and reparative mechanisms in cardiac remodeling, defects in resolution of inflammation may be responsible for remodeling, injury, and heart failure in a large number of patients surviving a myocardial infarction. Emerging concepts on the significance of endogenous inhibitory pathways in cardiac repair may provide insight into the reasons for the failure of previous strategies targeting the inflammatory response in patients with myocardial infarction.Curcumin, the natural yellow pigment extracted from the rhizomes of the plant curcuma longa, has been demonstrated to exhibit a variety of potent beneficial effects, acting as an antioxidant, anti-inflammatory and anti-fibrotic. Curcumin attenuates plasma inflammatory cytokines surge and cardiomyocytic apoptosis following cardiac ischemia/reperfusion by inhibition of NF-kappaB activation. Curcumin can inhibit adverse ventricular remodeling caused by left ventricular pressure overload and improve left ventricular function.Curcumin can also improve cardiac function after myocardial infarction in rats. But the role of myocardial protective effect of curcumin is still not clear.Curcumin can regulate the expression of proinflammatory and anti-inflammatory cytokine, inhibit the NF-kappa B signaling pathway activation, removal of oxygen free radicals and inhibition of apoptosis, effectively reduce the level of interleukin1(IL-1), interleukin6(IL-6), interleukin12(IL-12), TNF alpha. At the same time can also induce up-regulation of transformed cell growth factor (TGF-beta) and interleukin10(IL-10). These results indicate that curcumin have the bidirectional immune regulation function, keeping the body’s inflammatory response in a low level of equilibrium, avoiding tissue damage caused by excessive inflammation.Based on the basis of inflammatory reaction is critical factor in ventricular remodeling after myocardial infarction, and curcumin is efficacious for inflammatory and cardiacvuacular disease, We infer that curcumin may through immuneregulation and inhibiting the inflammatory reaction after myocardial infarction, which play the role of anti-ventricular remodeling.In this study, we attempt to investigate the cardioprotection of curcumin after myocardial infarction. The study was divided into two parts as bellow. Part I was designed to explore the effects of curcumin on cardiac function and ventricular remodeling after myocardial infarction. Part II was to clarify curcumin could improving cardic function after myocardial infarction may be through immune regulation mechanism, and to finding a new thought of prevention for ventricular remodeling. Part I:Effects of curcumin on cardiac function in the mice model of myocardial infarctionObjective:To explore the effects of lycopene on cardiac function and ventricular emodeling after myocardial infarction.Method:Selecting male C57BL/6with SPF level, ligating the left coronary artery a id resulting in the myocardial infarction. Dividing those falling into4groups: Model control group, Curcumin group with high dosage, low dosage, Enalapril group; plus Sham operation group. A total of5groups:Curcumin group with low dosage:50mg/kg·d, intraperitoneal injection; Curcumin group with high d ssage:100mg/kg·d, intraperitoneal injection; Enalapril group:enalapril7.5mg/kg.1,and the same amount of solvent, intraperitoneal injection; Sham operation gro up and Model control groups:the same amount of solvent of curcumin, intra peritoneal injection. Continuous administration for4weeks. Using echocardiogr iphy to checkout cardiac function on4weeks after operation. Then all mice were killed, the heart mass index(HMI) was calculated and the myocardial tiss aes were sampled and then pathologically observed through HE staining techni c and Masson trichrome staining technic. Testing the protein of myocardial tiss ae collagen I, collagen Ⅲ through immunohistochemical methods; observing th e effect of Curcumin on the apoptosis of cardiomyocyte after myocardial infarc tion by TUNEL staining.Statistical analysis:All statistical anlyses were performed with SPSS13.0software. All values are presented as mean±SD. Statistical significance was determined using one-way ANOVA, multiple comparisons using Fisher’s LSD post hoc tests. When the variance of arrhythmia, the use of robust estimation Welch, then the use of T3compared methods. Significance was taken as P<0.05.Result: 1. Results of cardiac ultrasonographyCompared with the sham group, model group mice had significantly increased LVEDd, LVESd values and declined EF, FS values (P<0.01),Curcumin group with high dosage, low dosageg and Enalapril group can had significantly declined LVEDd, LVESd values and increased EF、FS values (P<0.01,)compared with the model group, but there was no significant difference between Curcumin group with high dosage and Enalapril group. These are both better than Curcumin group with low dosage.2. The Result of heart weight/body weight constitutions in experiment miceCompared with the sham group, model control group mice had significantly increased heart weight and HMI significantly(P<0.01); Compared with the model group, heart weight and HMI decreased significantly in curcumin group with high dosage, low dosage and Enalapril group mice, the difference was significant (P<0.01), but there was no significant difference between them(P>0.05).3. Result of HE stainingSham operation group:the pigmentation of myocardium is even and in neat band, the cell boundary is clear, indicating normal myocardial morphology. Model control group:thickening, elongation, disordered arrangement and widened interspace of myocardial fibers, the cell nucleus is big and dark stained indicating the infiltration of inflammatory cells. The junctional zone of curcumin group with high dosage, low dosage and Enalapril shows mild myocardial cell hypertrophy and hyperplasia with spotted fibroplasias, clear and neat myocardial cells display around the non infarct zone, less inflamed cell infiltration compared with the Model control group and curcumin group with high dosage is better than group with high dosage.4. Result of Masson trichrome stainingCompared with MI model group, the myocardial fibrosis area of curcumin group with high dosage, low dosage and enalapril is reduced, the difference was statistically significant (P<0.01); Compared with enalapril group, fibrosis area of curcumin with low dosage increases, the difference was statistically significant (P<0.01), fibrosis area of curcumin with high dosage group is no statistical difference (P=0.771).5. Expression results of Immunohistochemical detection of myocardial collagen type I, III collagen proteinSham operation group:no significant expression of collagen,no stromal fibrosis; Model control group:obvious fibrosis, showing significant expression of collagen; Curcumin group with high dosage, low dosage and Enalapril group:a small amount of collagen fiber, obviously much less than that in Model control group, but the occurrence is still inevitable. The content of myocardium collagen and the ratio of myocardial collagen type I to III collagen protein in model control group were obviously higher than the sham operation group(P<0.01); the content of myocardium collagen and the ratio of myocardial collagen type I to III collagen protein in Curcumin group with high dosage, low dosage and Enalapril group were lower than which in model control group(P<0.01); the ratio of myocardial collagen type I to III collagen protein in Cucumin group with high dosage, low dosage and Enalapril group was no significant (P>0.05). And Curcumin with high dosage is better than Curcumin with low dosage.6. Effect of curcumin on cardiomyocyte apoptosis in myocardial infarction miceAfter3days of MI model operation and drug intervention, TUNEL kit was used to detecting cardiomyocyte apoptosis in border of infarted myocardium. Compared with model control group, curcumin group with high dosage, low dosage and enalapril can reduce cardiomyocyte apoptosis, the difference was statistically significant (P<0.01); Compared with Enalapril group and Curcumin group with low dosage, Curcumin group with low dosage increased cardiomyocyte apoptosis, the difference was statistically significant(P<0.01);the cardiomyocyte apoptosis index of Curcumin group with high dosage and enalapril was no significant (P=0.356).Conclusion:1. The left anterior descending coronary artery is subjected to ligation to establishing model in mice with myocardial infarction. If the AMI model mice was successfully made, the local myocardial would be color and electrocardiogram (ECG) changed. The tissue morphology, cardiac function and collagen expression detection have been showed to detected the pathological changes of the model exists ventricular remodeling in mice. 2. The treatments used in Curcumin can improve cardiac function of the mice, reduce myocardial fibrosis, maintain cardiomyocyte’ normal morphology, inhibit the cardiomyocyte apoptosis in myocardial infarction mice. Curcumin with high dosage same effect as Enalapril.3. Curcumin might be one of Effective medicine for anti-ventricular remodeling after myocardial infarction. Curcumin with low and high dosage both improve cardiac function and ameliorate ventricular remodeling after myocardial infarction in mice, and the effects of curcumin with high dosage is better than the group with low dosage, the protect effective has a certain dosage-response relationship. Part Ⅱ:Effects of curcumin on inflammatory response in the mice model of myocardial infarctionObjective:Inflammatory response is the basic mechanism of the development of chronic heart failure. The activation of NF-kappa B signaling pathway can promote inflammation and cardiomyocyte apoptosis. Studies have reported that curcumin can inhibit the activation of TLR4and the NF-kappa B signaling pathway, so we speculated that curcumin may inhibit the inflammatory response through suppression the expression of TLR4in and the activation of NF-kappa B signaling pathways in infracted myocardium, thereby effectively reversing ventricular remodeling and improving the cardiac function after myocardial infarction. The purpose of this experimental research is to observe influence of curcumin on the infiltration of dendritic cells in the edge area of infracted myocardium and the expression of inflammatory cytokine IL-1β, tumor necrosis factor a, IL-6and IL-10, and the expression of TLR4and the signal pathway downstream of NF-kappa B in infarcted heart.Methods:Selecting male C57BL/6with SPF level, ligating the left coronary artery and resulting in the myocardial infarction. Dividing those falling into2groups:Model control group, Curcumin group; plus Sham operation group. A total of3groups: Curcumin group with high dosage:100mg/kg-d, intraperitoneal injection; Sham operation group and Model control groups:the same amount of solvent of curcumin, intraperitoneal injection. And those groups will be divided into4subgroups:the group of3days,7days,14days and28days after the MI model operation and intervention. Immunofluorescence staining was observed to dendritic cells infiltration. The expression of TLR4and iNOS, and NF-κB pathway activation in ischemic zone surrounding infarted myocardium were detected by western blot analysis. The relative mRNA expression of TLR, the inflammatory cytokine IL-1β, tumor necrosis factor a, IL-6and IL-10were detected by quantitative real-time PCR. Statistical analysis:All statistical anlyses were performed with SPSS13.0software. All values are presented as mean±SD. Statistical significance was determined using one-way ANOVA, multiple comparisons using Fisher’s LSD post hoc tests. When the variance of arrhythmia, the use of robust estimation Welch, then the use of T3compared methods. Significance was taken as P<0.05.Results:1. The influence of curcumin on the infiltration of dendritic cells in the border of infarcted myocardiumImmunofluorescence analysis of paraffin section is used to detect the expression of CD11c+dendritic cells. Results show that:the infiltration of CD11c+dendritic cells into infarcted myocardium3days, and reached its peak7days after MI in MI control group was significantly higher than in sham group(P<0.05), and returned to normal level on day28after MI. The infiltration of dendritic cells could be reduced by curcumin (P<0.05).2. The influence of curcumin on the expression of inflammatory cytokines in infarcted heartReal time PCR was used to test the mRNA expression of proinflammatory factor IL-1beta, tumor necrosis factor a, IL-6and anti-inflammation factor IL-10. Results show that:IL-1β, tumor necrosis factor a, IL-6, which are proinflammatory, increased in3days after MI and gradually deseased thereafter. The expression of those proinflammatory factor in MI control group was significantly higher than in sham group; The relative mRNA expression of proinflammatory factor could be reduced by curcumin compared with MI control group (P<0.05).3. The influence of curcumin on the expression of iNOS in infarcted heartiNOS was upregulated in the ischemic myocardium and attenuated by curcumin campared with mice in MI control group(P<0.05).4. The influence of curcumin on the expression of TLR4and activation of NF-κB signaling pathway in infarcted heartPhosphorylation and degradation of IκBα and phosphorylation of NF-κB p65 were induced with mice after MI but were significantly suppressed in crucumin group. Curcumin attenuate TLR4expression and inhibite NF-κB signaling pathway activation campared with mice in MI control group(P<0.05).Conclusion:Curcumin reduce the infiltration of dendritic cells in border of infracted myocardium and the expression of TLR4, proinflammatory factor, iNOS in infracted myocardium. Curcumin reduce inflammatory response after MI through inhibiting activation of NF-κB signaling pathway, which could be the mechanism of cardioprotective effects of curcumin.更多还原