代谢综合征心肌纤维化发病机制及药物干预的研究
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摘要
背景
过去的二十年里,全球代谢综合征(MS)的患病率逐年递增,糖尿病及肥胖人数也随之增加。随着MS带来的心血管病发病率的上升,我们认识到防治这一全球性疾病迫在眉睫。MS是包括糖耐量异常、胰岛素抵抗、腹型肥胖、血脂异常及高血压等心血管疾病危险因素的一组症候群。胰岛素抵抗被认为是该综合征的核心的病理生理学基础。而炎症可能在MS中起重要作用。
临床研究证实MS患者有不同程度的左室舒张功能障碍,表现为心肌松弛性减低和僵硬度增大。心肌纤维化是左室舒张功能障碍发生发展的重要机制,但在MS中的发生机制尚不明确。研究表明炎症是除肾素-血管紧张素-醛固酮(RASS)系统和高血压之外致心肌纤维化的重要因素,许多炎症因子参与了心肌纤维化的形成和发展。而MS是一种慢性炎症状态,血中炎症因子高敏C反应蛋白(hs-CRP)、白介素-6(IL-6)、白介素-18(IL-18)等都有升高。IL-18作为IL-1家族成员之一,可以通过激活JNK信号转导通路参与一些反应。JNK信号转导通路是将信息从细胞表面传到细胞核内的一系列丝氨酸/苏氨酸酶,主要对细胞的生长发育起介导作用。位于JNK信号转导通路下游的核因子AP-1包含c-jun/c-fos蛋白,JNK通过激活AP-1使存在AP-1结合位点的基因表达增强。与心肌纤维化有关的基因,例如Ⅰ型胶原、Ⅲ型胶原、基质金属蛋白酶-2(MMP-2)、MMP-9基因,都存在AP-1的结合位点。因此我们推断IL-18可以通过激活JNK/AP-1信号转导通路加重心肌纤维化参与MS的发生发展,这在国内外均未见研究报道。
本研究在综合分析心肌纤维化研究动态和细胞信号转导途径的基础上,提出“IL-18/JNK/AP-1”信号转导通路在MS心肌纤维化发病中可能起重要作用的假说,利用转染IL-18腺病毒载体技术,深入研究IL-18在MS心肌纤维化发生发展中的作用。
目的
(1)建立果糖喂养Wistar大鼠MS的动物模型;
(2)观察MS大鼠模型心肌组织病理学和超微结构的变化,明确心肌纤维化存在及分布特点;
(3)明确“IL-18/JNK/AP-1”信号转导通路各关键分子在MS大鼠心肌组织的表达;
(4)构建IL-18腺病毒载体并转染MS大鼠,验证“IL-18/JNK/AP-1”信号转导途径在MS心肌纤维化发生发展的作用;
(5)采用超声心动图技术和心导管技术全面评价MS大鼠模型以及转染IL-18腺病毒载体后MS大鼠心脏结构和功能的变化。
方法
雄性Wistar大鼠50只,随机分为两组:正常对照组(n=12只),MS组(n=38只)。正常对照组以标准大鼠饲料喂养,并给予普通饮水:MS组大鼠以标准大鼠饲料喂养,并给予10%高果糖饮水。高果糖喂养32周后,将MS组大鼠随机分为三组:MS组(n=9只),继续给予高果糖饮水;空载体组(n=9只),继续给予高果糖饮水的同时通过尾静脉注射1×10~(10)PFU含GFP腺病毒载体;IL-18载体组(n=13只),继续给予高果糖饮水的同时通过尾静脉注射1×10~(10)PFU IL-18腺病毒载体;正常对照组及MS组给予等体积的生理盐水尾静脉注射。转染腺病毒载体6周后,处死动物,取大鼠心肌组织进行实验。实验过程中,进行以下检测:(1)各组大鼠每两周测量体重及尾动脉血压一次;(2)分别于实验前,高果糖喂养32周后,转染载体后1周、2周、3周、4周、6周抽取各组大鼠颈静脉窦血2ml检测空腹血糖、空腹胰岛素、甘油三酯、总胆固醇、血中IL-18浓度,并计算胰岛素抵抗指数(HOMA);(3)分别于实验前、高果糖喂养32周后、实验末进行超声心动图检查;(4)于实验末记录各组大鼠心电图,以心导管法进行血流动力学测定;(5)心肌组织超微结构和病理学检查;(6)应用Masson染色观察心肌纤维化程度:(7)免疫组织化学检测IL-18、Ⅰ型胶原、Ⅲ型胶原的蛋白表达;(8)实时定量RT-PCR法检测IL-18、Ⅰ型胶原、Ⅲ型胶原的mRNA表达;(9)Western-blot检测JNK和磷酸化JNK蛋白的表达;(10)凝胶电泳迁移分析检测转录因子AP-1活性。
结果
1体重、尾动脉收缩压及生化指标
高果糖喂养32周后,与正常对照组相比,MS组大鼠体重增加(P＜0.01),尾动脉收缩压升高(P＜0.001);血清甘油三酯(P＜0.01)及空腹胰岛素(P＜0.001)升高,HOMA增加(P＜0.001),而总胆固醇及空腹血糖无统计学意义。
转染IL-18腺病毒载体后6周,与MS组和空载体组比较,IL-18载体组血胰岛素水平(P＜0.001)及HOMA(P＜0.05)升高;而体重、尾动脉收缩压、甘油三酯、总胆固醇在IL-18载体组与MS组和空载体组间无统计学意义。
2血清IL-18水平变化
转染前MS组大鼠血IL-18水平较正常对照组明显升高(P＜0.001)。IL-18腺病毒转染后1周,IL-18载体组大鼠血清IL-18水平较空载体组显著升高(P＜0.001),转染后2周达到最高水平,约为空载体组血清IL-18水平的4倍(P＜0.001),于转染后3周开始降低,但仍高于空载体组IL-18水平(P＜0.001),4周时与空载体组IL-18水平相当。
3超声心动图检测
正常对照组12只大鼠,均未见二尖瓣或三尖瓣返流。MS组9只大鼠,轻度二尖瓣返流2只,中度二尖瓣返流1只,重度二尖瓣返流0只;轻度三尖瓣返流2只,中度三尖瓣返流0只,重度三尖瓣返流1只;其中2只二尖瓣、三尖瓣均存在返流;二尖瓣、三尖瓣均无返流5只。空载体组9只大鼠,轻度二尖瓣返流1只,中度二尖瓣返流2只,重度二尖瓣返流1只;轻度三尖瓣返流1只,中度三尖瓣返流0只,重度三尖瓣返流1只;其中2只二尖瓣、三尖瓣均存在返流;二尖瓣、三尖瓣均无返流5只。IL-18载体组13只大鼠,轻度二尖瓣返流3只,中度二尖瓣返流2只,重度二尖瓣返流1只;轻度三尖瓣返流2只,中度三尖瓣返流1只,重度三尖瓣返流1只;其中3只二尖瓣、三尖瓣均存在返流;二尖瓣、三尖瓣均无返流6只。正常对照组大鼠无瓣膜返流发生,MS组及空载体组大鼠瓣膜返流发生率均为44.44%,IL-18载体组大鼠瓣膜返流发生率为53.85%。Fisher检验显示:与对照组相比,MS组大鼠瓣膜返流发生率增加,差异有统计学意义(P＜0.01)。与空载体组相比,IL-18载体组大鼠瓣膜返流发生率增加,差异有统计学意义(P＜0.05)。
与正常对照组相比,MS组大鼠左室舒张末期内径(LVDd)明显扩大(P＜0.01),舒张末期室间隔(IVSd)、左室后壁同步增厚(LVPWd)(P＜0.05),左房内径(LA)明显扩大(P＜0.001)。与正常对照组相比,MS组大鼠E峰、Ea峰明显降低(P＜0.001),等容舒张时间(IVRT)延长(P＜0.01),Tei指数增加(P＜0.001),左室射血分数(EF)、短轴缩短率(FS)、相对室壁厚度(RWth)及E/Ea峰比值无统计学意义。
与MS组和空载体组相比,IL-18载体组大鼠LVDd、IVSd、LVPWd、LA及RWth均无统计学意义。与MS组和空载体组相比,IL-18载体组大鼠E峰、Ea峰降低,但无统计学意义;IVRT(P＜0.01);Tei指数增加(P＜0.01):EF、FS及E/Ea峰比值无统计学意义。
4心电图变化
正常对照组12只大鼠心电图均基本正常。MS组和空载体组各9只大鼠中6只心电图出现QRS波正负交替变化为特征的心电图改变,1只大鼠出现复杂心律失常(两种以上的心电图异常或宽大畸形的QRS波),2只大鼠心电图基本正常。IL-18载体组13只大鼠中10只心电图出现QRS波正负交替变化为特征的心电图改变,2只大鼠出现复杂心律失常,1只大鼠心电图基本正常。正常对照组无心律失常发生,MS组和空载体组心律失常发生率是77.78%,IL-18载体组心律失常发生率是92.31%。Fisher检验显示:与正常对照组相比,MS组大鼠心律失常发生率明显增加,差异有统计学意义(P＜0.001);与MS组和空载体组相比,IL-18载体组大鼠心律失常发生率明显增加,差异有统计学意义(P＜0.01)。
5血液动力学
与正常对照组相比,MS组大鼠左室收缩压(LVSP)、左室舒张末压(LVEDP)升高(P＜0.001);左室压力最大下降速率(-dp/dt)下降(P＜0.01),-dp/dt/LVSP明显下降(P＜0.001);左室松弛时间常数(T)延长(P＜0.001)。心率(HR)及左室压力最大上升速率(+dp/dt)均无统计学意义。
与MS组和空载体组相比,IL-18载体组大鼠LVEDP升高(P＜0.001);-dp/dt、-dp/dt/LVSP下降(P＜0.05);T延长(P＜0.05)。心率及+dp/dt均无统计学意义。
6透射电镜观察超微结构的改变
正常对照组可见心肌细胞排列整齐,质膜连续;粗细肌丝排列规整,肌小节及明暗各带清晰可见;线粒体丰富,大小均一;细胞间质可见成纤维细胞和少量胶原纤维。
MS组可见心肌细胞排列紊乱,质膜部分断裂;粗细肌丝部分溶解丧失;增生的线粒体呈多形性,大小不一;间质充满了增生的胶原纤维,胶原纤维走形紊乱,断裂;成纤维细胞核不规则,胞浆内质网肿胀,线粒体嵴减少。
空载体组可见心肌细胞排列紊乱,质膜部分断裂;粗细肌丝部分溶解丧失;增生的线粒体呈多形性,大小不一;间质充满了增生的胶原纤维,胶原纤维走形紊乱,断裂。
IL-18载体组可见心肌细胞排列紊乱,质膜断裂;肌原纤维呈灶性溶解,肌丝扭曲、断裂,肌节对位不齐;线粒体明显增多,大小不一呈堆积状;间质可见大量的胶原纤维。
7组织病理观察
HE染色显示,正常对照组心肌细胞排列整齐,细胞核大小均一,胞浆染色均匀。MS组及空载体组心肌细胞排列紊乱,细胞内可见肌纤维增粗、变长、有较多分支,细胞核增大、深染蓝色,胞质丰富红染。IL-18载体组心肌细胞肥大,排列紊乱更为严重,细胞内可见肌纤维断裂、排列疏松紊乱,细胞核大小不甚规则,胞浆染色不均匀。
8 Masson染色胶原含量的检测
心肌细胞呈红色,胶原纤维呈绿色。正常对照组胶原组织分布均匀,相邻细胞和血管周围的胶原纤维网完好:MS组和空载体组心肌间和血管周围的胶原组织增多,围绕心肌细胞的胶原纤维网部分断裂;IL-18载体组心肌间和血管周围的胶原组织明显增多,围绕心肌细胞的胶原纤维网断裂、排列紊乱。
与正常对照组相比,MS组左室心肌组织间(P＜0.001)和血管周围(P＜0.01)胶原含量升高。与MS组和空载体组相比,IL-18载体组左室心肌组织间(P＜0.05)和血管周围(P＜0.05)胶原含量升高。
相关性分析显示,MS组大鼠左室心肌间质和血管周围胶原含量与IL-18腺病毒载体转染后第二周血清IL-18浓度呈正相关(CVF:P＜0.01;PCVA:P＜0.05)。
9免疫组织化学检测
IL-18:染色阳性信号为棕色颗粒,定位于心肌细胞胞浆内。正常对照组心肌细胞胞浆内几乎未见IL-18表达;MS组与空载体组心肌细胞内可见分布均匀、稀疏的浅棕色颗粒;IL-18载体组心肌细胞内可见浓密的深棕色颗粒。以各视野累计光密度值(IOD)作为反应IL-18蛋白表达的定量指标结果显示,与正常对照组比较,MS组IOD值升高(P＜0.001);与MS组和空载体组比较,IL-18载体组IOD值升高(P＜0.001)。
Ⅰ型胶原:染色阳性信号为棕色条索状纤维,定位于心肌细胞间质和血管周围。正常对照组心肌细胞间和血管周围可见少数纤细的浅棕色条索状纤维;MS组与空载体组心肌细胞间和血管周围可见较多粗大、排列紊乱的棕色条索状纤维;IL-18载体组心肌细胞间和血管周围可见充满粗大、排列紊乱的深棕色条索状纤维。以各视野IOD值作为反应Ⅰ型胶原蛋蛋白表达的定量指标结果显示,与正常对照组比较,MS组IOD值升高(P＜0.001);与MS组和空载体组比较,IL-18载体组IOD值升高(P＜0.001)。
Ⅲ型胶原:染色阳性信号为棕色条索状纤维,定位于心肌细胞间质和血管周围。正常对照组心肌细胞间和血管周围可见少数纤细的浅棕色条索状纤维;MS组与空载体组心肌细胞间和血管周围可见较多粗大、排列紊乱的棕色条索状纤维;IL-18载体组心肌细胞间和血管周围可见充满粗大、排列紊乱的深棕色条索状纤维。以各视野IOD值作为反应Ⅲ型胶原蛋白表达的定量指标结果显示,与正常对照组比较,MS组IOD值升高(P＜0.05);与MS组和空载体组比较,IL-18载体组IOD值升高(P＜0.05)。
相关性分析显示,MS组大鼠左室Ⅰ、Ⅲ型胶原蛋白表达水平比值与IL-18腺病毒载体转染后第二周血清IL-18浓度呈正相关(P＜0.05);MS组大鼠左室Ⅰ、Ⅱ型胶原蛋白表达水平比值与IL-18蛋白表达水平呈正相关(P＜0.05):MS组大鼠左室IL-18蛋白表达水平与左室心肌间质和血管周围胶原含量呈正相关(P＜0.05)。
10实时定量RT-PCR检测各关键分子mRNA表达
与正常对照组比较,MS组大鼠左室心肌组织IL-18 mRNA表达增加(P＜0.001);MS组大鼠左室心肌组织Ⅰ型胶原(P＜0.001)及Ⅲ型胶原(P＜0.01)mRNA表达增加。
与MS组和空载体组比较,IL-18载体组大鼠左室心肌组织IL-18mRNA表达增加(P＜0.001);IL-18载体组大鼠心肌组织Ⅰ、Ⅲ型胶原mRNA表达增加(P＜0.01)。
相关性分析显示,MS组大鼠左室Ⅰ、Ⅲ型胶原mRNA表达水平比值与IL-18腺病毒载体转染后第二周血清IL-18浓度呈正相关(P＜0.05);MS组大鼠左室Ⅰ、Ⅲ型胶原mRNA表达水平比值与IL-18mRNA表达水平呈正相关(P＜0.05);MS组大鼠左室IL-18mRNA表达水平与左室心肌间质和血管周围胶原含量呈正相关(CVF:P＜0.05:PCVA:P＜0.01)。
11各组大鼠关键分子蛋白质表达水平的比较
各组大鼠左室心肌组织总JNK蛋白水平比较:四组大鼠左室心肌组织JNK总蛋白质表达水平无统计学意义。
各组大鼠左室心肌组织磷酸化JNK蛋白表达水平比较:与正常对照组比较,MS组磷酸化JNK蛋白含量升高(P＜0.001);与MS组和空载体组比较,IL-18载体组磷酸化JNK蛋白含量升高(P＜0.01)。
12 AP-1转录因子活性的比较
以EMSA法检测各组大鼠左室心肌组织AP-1活性,结果显示与正常对照组比较,MS组AP-1活性增强,具体表现为探针与核蛋白结合产生的条带变宽,条带颜色加深;与MS组和空载体组相比,IL-18载体组AP-1活性增强,具体表现为探针与核蛋白结合产生的条带变宽,条带颜色加深。
结论
(1)给予Wistar大鼠10%的高果糖饮水32周后,大鼠出现尾动脉收缩压升高,体重增加,血液中甘油三酯、胰岛素水平升高,反应胰岛素抵抗的指标HOMA增加,提示成功建立了符合人类MS特点的大鼠模型;
(2)超声心动图检测显示,MS大鼠心脏E峰、Ea均降低,IVRT延长,Tei指数增加,心导管检查显示LVSP、LVEDP升高。-dp/dt、-dp/dt/LVSP下降,T延长,提示MS大鼠存在心脏舒张功能减低;
(3)Masson染色证实心肌间质及血管周围胶原含量增加,免疫组织化学染色和实时定量RT-PCR证实Ⅰ型胶原和Ⅲ型胶原蛋白和mRNA表达水平升高,提示MS大鼠存在心肌纤维化,这可能是MS心脏舒张功能的障碍的主要病理学和分子生物学基础;
(4)MS大鼠血液中IL-18浓度升高,免疫组织化学染色和实时定量RT-PCR证实心肌组织中炎症因子IL-18蛋白和mRNA表达水平升高,且与心肌胶原含量及Ⅰ、Ⅲ型胶原蛋白和mRNA表达水平呈显著相关关系,提示IL-18可能是MS心肌纤维化的重要机制之一。MS大鼠心肌组织磷酸化JNK水平升高,转录因子AP-1活性增强,提示在MS大鼠模型中,IL-18可能通过“JNK/AP-1”信号途径导致心肌纤维化;
(5)构建IL-18腺病毒载体并转染MS大鼠,血液中和心肌组织中IL-18水平显著增加,同时伴有心肌纤维化程度加重,出现左室舒张功能进一步减低,心肌组织磷酸化JNK蛋白表达水平和转录因子AP-1活性进一步增强,提示“IL-18/JNK/AP-1”信号转导通路是引起MS心肌纤维化和心脏舒张功能减低的重要分子机制。
背景
代谢综合征(MS)是严重威胁人类健康的疾病。MS包括高血压、腹型肥胖、糖耐量异常、高甘油三脂血症、低高密度脂蛋白血症、胰岛素抵抗等一组心血管危险因素。以世界卫生组织(WHO)、美国国家胆固醇教育计划成人治疗组(NCEP-ATPIII)MS诊断标准及美国国家健康和营养普查III(NHANESIII)结果判断，美国MS的发病率为23．7％，60岁以上者发病率达43．5％。研究表明MS患者发生冠心病及中风的危险性增加3倍，心血管死亡率也显著增加，即使在无基础心血管疾病及糖尿病患者中，MS也显著增加心血管疾病的发生率和死亡率。提示MS潜在的心血管损害可能是其心血管并发症高发的原因。因此，寻找能逆转或延缓MS患者心血管损害的药物对于更好的治疗MS患者至关重要。
二氢吡啶类钙通道阻滞剂(CCB)已被广泛用于治疗高血压、心绞痛等疾病。临床与基础研究提示CCBs在降压的同时还具有一定的靶器官保护作用。CAMELOT研究显示氨氯地平能降低血压正常的冠心病患者心血管事件发生率，血管内超声提示氨氯地平能延缓动脉粥样硬化的进展。INSIGHT研究表明CCBs能降低伴糖尿病的高血压患者总死亡率，不增加新发糖尿病的发生率。对多项大规模临床试验结果进行荟萃分析示CCBs能有效降低伴糖尿病的高血压患者心血管事件死亡率。给予高血压患者CCBs治疗能显著改善患者心脏收缩及舒张功能。动物实验结果显示。长效CCBs能降低血管源性生长因子的表达并能阻止心肌重构。体内培养心肌成纤维细胞发现，CCBs可减少血管紧张素II介导的Ca2+内流，从而阻止血管紧张素II介导的心肌纤维化。因此，有学者建议要重视CCBs在代谢紊乱中的应用。但CCBs的的靶器官保护作用的机制尚不清楚，一些间接和直接的证据提示可能与CCBs的抗炎作用有关。研究发现，氨氯地平能改善肥胖的2型糖尿病患者胰岛素抵抗，降低血TNF"G水平。动物试验证实氨氯地平能够通过持续抑制NO合成而抑制炎症，改善动脉粥样硬化，同时降低MCP-1、TGF-[11等炎症因子，改善氧化应激。国外一些研究证实硝苯地平、尼伐地平、贝尼地平能通过减弱NF-r．B活性抑制炎症。
本研究的第一部分证实，炎症状态是MS发病和MS心脏结构和功能改变的重要机制之一，炎症因子IL-1 8可能通过JNK／AP．1信号转导通路在MS心肌纤维化和左室舒张功能障碍的发生和发展中起重要作用，上述直接和间接的证据提示CCBs具有一定的抗炎作用。因此，二氢吡啶类CCB非洛地平能否通过抑制炎症反应而对MS状态下心脏结构和功能改变起到有益的保护作用?我们将以MS大鼠模型为研究对象，给予非洛地平进行干预治疗，观察非洛地平对MS大鼠心脏结构和功能的保护作用及其对炎症反应的影响。目的
(1)评价非洛地平对MS大鼠体重、尾动脉收缩压和血液生化指标的影响；
(2)采用超声心动图技术和心导管技术全面评价非洛地平对MS大鼠心脏结构和功能的作用；
(3)观察非洛地平对MS大鼠心肌组织病理学及超微结构的干预作用；
(4)观察非洛地平对MS大鼠血液和心肌组织IL-18表达的作用；
(5)观察非洛地平对“IL-18／JNK／AP-1”信号转导通路各关键分子表达的影响以阐明非洛地平心血管保护作用的可能机制。
方法
雄性Wistar大鼠33只，随机分为两组：正常对照组(，l=12只)，MS组(n=21只)。正常对照组以标准大鼠饲料喂养，并给予普通饮水；MS组大鼠以标准大鼠饲料喂养，并给予10％高果糖饮水。高果糖喂养32周后，将MS组大鼠随机分为两组：MS组(n=9只)，继续给予高果糖饮水；非洛地平组(n=9只)，继续给予高果糖饮水的同时每日给予5mg／kg非洛地平灌胃，正常对照组及MS组给予同等剂量的生理盐水。治疗6周后，处死动物，取大鼠心肌组织进行实验。实验过程中。进行以下检测：(1)各组大鼠每两周测量体重及尾动脉血压1次；(2)实验前。高果糖喂养32周后，治疗第6周抽取各组大鼠颈静脉窦血2ml检测血清IL-18浓度。空腹血糖、空腹胰岛素、甘油三酯、胆固醇，并计算胰岛素抵抗指数(HOMA)：(3)分别于实验开始前、高果糖喂养32周、实验末进行超声心动图检查；(4)于实验末记录各组大鼠心电图．以心导管法进行血流动力学测定；(5)心肌组织超微结构和病理学检查：(6)应用Masson染色观察心肌纤维化程度;(7)免疫组织化学检测IL-18、I型胶原、III型胶原的蛋白表达；(8)实时定量RT-PCR法检测IL-18、I型胶原、III型胶原的raRNA表达；(9)Western-blot检测JNK和磷酸化JNK蛋白的表达；(10)凝胶电泳迁移分析检测转录因子AP-1活性。
结果
1体重、尾动脉收缩压及生化指标
自非洛地平治疗后2周，血压即降至正常水平(P<0．001)。体重在非洛地平组与MS组间无统计学意义。与MS组比较，非洛地平组血胰岛素水平及HOMA降低(P<0．001)；而甘油三酯、胆固醇、血糖在非洛地平组与MS组间无统计学意义。
2非洛地平治疗后三组大鼠血清IL-18水平变化
非洛地平治疗后6周，与MS组相比，非洛地平组大鼠血清IL-18水平降低(P<0．05)。与正常对照组相比，非洛地平组大鼠血清IL-18水平仍显著升高(p<0．001)。
3超声心动图检测
二、三尖瓣返流情况比较：非洛地平组9只大鼠，轻度二尖瓣返流1只，中度二尖瓣返流0只，重度二尖瓣返流0只，轻度三尖瓣返流1只，中度三尖瓣返流O只，重度三尖瓣返流0只，无二尖瓣、三尖瓣均存在返流，二尖瓣、三尖瓣均无返流7只。非洛地平组瓣膜返流发生率为22．22％。Fisher检验显示：与MS组相比，非洛地平组大鼠瓣膜返流发生率减少，差异有统计学意义(P<0．01)：与正常对照组相比，非洛地平组大鼠瓣膜返流发生率增加，但无统计学意义。
与MS相比，非洛地平组左室舒张末期内径(LVDd)缩小(P<0．01)，舒张末期室间隔(IVSd)、左室后壁(LVPWd)同步肥厚减轻(P<0．05)，左房内径(LA)明显缩小(P<0．001)。非洛地平E峰、Ea峰明显升高(P<0．001)，等容舒张时间(IVRT)缩短(P<0．01)，Tei指数减少(P<0．001)，左室射血分数(EF)、短轴缩短率(FS)及E／Ea峰比值无统计学意义。
4心电图变化
非洛地平组心电图异常比较轻微，9只大鼠中5只心电图基本正常，3只出现QRS波正负交替变化，无复杂的心律失常。非洛地平组心律失常发生率是33．33％。Fisher检验显示：与正常对照组相比，MS组大鼠心律失常发生率明显增加，差异有统计学意义(P<0．001)；与MS组相比，非洛地平组大鼠心律失常发生率明显减少，差异有统计学意义(P<0．01)。5血液动力学
非洛地平组较MS组左室收缩压(LVSP)、左室舒张末压(LVEDP)均明显降低(P<0．001)：左室压力最大下降速率(-dp／dt)回升(P<0．01)，-dp／dt／LVSP明显回升(P<0．001)；左室松弛时间常数(T)明显缩短(P<0．001)，且与正常对照组相比无统计学意义。．
6透射电镜观察超微结构的改变
非洛地平组心肌组织电镜观察，可见心肌细胞捧列较整齐；肌原纤维断裂，溶解现象好转；线粒体大小较一致，排列较规整；间质内胶原纤维明显减少。
7组织病理观察
HE染色显示，非洛地平组可见心肌细胞肥大程度减轻，排列规整，未见明显的肌纤维增粗、断裂，细胞核较规则，胞浆染色均匀；壁内小动脉管腔、管壁比例较正常，血管周围及肌间隙的胶原明显减少。
8 Masson染色胶原含量的检测
心肌细胞染红色，胶原纤维呈绿色。非洛地平组可见胶原纤维较MS组明显减少，排列较规整。
非洛地平对左室心肌组织胶原含量的影响如下：非洛地平组左室心肌组织间(P<0．01)和血管周围(P<0．01)胶原含量较MS组明显降低；与正常对照组相比,非洛地平组左室血管周围(P<0.05)胶原含量有升高趋势。
9免疫组织化学检测
IL-18：染色阳性信号为棕色颗粒，定位于心肌细胞胞浆内。与MS组比较，非洛地平组心肌细胞胞浆内棕色颗粒明显减少；与正常对照组比较，非洛地平组心肌细胞胞浆内棕色颗粒增多。以各视野IOD值作为反应IL-18蛋白表达的定量指标结果显示，与MS组比较，非洛地平组IOD值降低，差异有统计学意义(P<0．001)；与正常对照组比较，非洛地平组IOD值升高，差异有统计学意义(P<0．05)。
I型胶原：染色阳性信号为棕色条索状纤维，定位于心肌细胞问质和血管周围。非洛地平组心肌细胞间和血管周围棕色条索状纤维较MS组明显减少。以各视野IOD值作为反应I型胶原蛋蛋白表达的定量指标结果显示，与MS比较，非洛地平组IOD值降低，差异有统计学意义(P<0．001)；与对照组比较，非洛地平组IOD值无统计学意义。
III型胶原：染色阳性信号为棕色条索状纤维，定位于心肌细胞问质和血管周围。非洛地平组心肌细胞间和血管周围棕色条索状纤维较MS组明显减少。以各视野IOD值作为反应III型胶原蛋蛋白表达的定量指标结果显示，与MS比较，非洛地平组IOD值降低，差异有统计学意义(P<0．05)；与对照组比较，非洛地平组IOD值无统计学意义。
10实时定量RT-PCR检测各关键分子mRNA表达
与MS组比较，非洛地平组大鼠左室心肌组织IL-18mRNA表达减弱，差异有统计学意义(P<0．01)。与正常对照组比较，非洛地平组大鼠左室心肌组织IL-18mRNA表达增强，差异有统计学意义(P<0．001)。
与MS组比较，非洛地平组大鼠左室心肌组织I型胶原(P<0．001)、III型胶原(P<0．01)mRNA表达降低：与正常对照组比较，非洛地平组大鼠左室心肌组织I、III型胶原mRNA表达无统计学意义。
11各组大鼠关键分子蛋白质表达水平的比较
各组大鼠左室心肌组织JNK总蛋白水平比较：三组大鼠左室心肌组织JNK总蛋白质表达水平无统计学意义。
各组大鼠左室心肌组织磷酸化JNK蛋白表达水平比较：与MS组比较，非洛地平组磷酸化JNK蛋白含量下降，差异有统计学意义(P<0．01)；与对照组比较，非洛地平组磷酸化JNK蛋白含量略升高，差异有统计学意义(P<0．01)。
12 AP-l转录因子活性的比较
以EMSA法检测各组大鼠左室心肌组织AP．1活性，结果显示与MS组比较，非洛地平组AP-1活性减弱，具体表现为探针与核蛋白结合产生的条带变窄，条带颜色变浅；与对照组比较，非洛地平组AP-1活性稍增强，具体表现为探针与核蛋白结合产生的条带变宽，条带颜色略深。
结论
(1)非洛地平显著降低MS大鼠尾动脉收缩压和血液中胰岛素水平，改善胰岛素抵抗状态；
(2)超声心动图检测显示，非洛地平干预治疗的MS大鼠心脏E峰、Ea峰升高，IVRT缩短，Tei指数减低，心导管检查显示LVSP、LVEDP下降，-dp／dt、-dp／dt／LVSP升高，T缩短，提示非洛地平能够改善MS导致的心脏功能损伤；
(3)非洛地平干预后，Masson染色发现心肌间质及血管周围胶原含量减少；免疫组织化学染色和实时定量RT-PCR证实I型胶原和III型胶原蛋白和mRNA表达水平降低，提示非洛地平能够降低心肌纤维化；
(4)非洛地平能够降低血中IL-18浓度，降低心肌组织中IL-18蛋白和mRNA表达，提示非洛地平具有抗炎作用：
(5)非洛地平干预后心肌组织JNK磷酸化水平下降，AP-1活性减低，提示非洛地平可能通过抑制炎症因子IL-18对“JNK／AP-1”信号转导通路的作用，减轻心肌纤维化程度，改善心脏的舒张功能。
Background
Over the past two decades,a striking increase in the number of people with the metabolic syndrome(MS)worldwide has taken place.This increase is associated with the global epidemic of obesity and diabetes.With the elevated risk not only of diabetes but also of cardiovascular disease from the MS,there is urgent need for strategies to prevent the emerging global epidemic.The constellation of metabolic abnormalities includes glucose intolerance,insulin resistance,central obesity, dyslipidaemia,and hypertension.Insulin resistance is considered central to the pathophysiology of this metabolic and cardiovascular syndrome.A proinflammatory state probably contributes to the syndrome.
In clinical study,the patients with MS have cardiac diastolic dysfunction, characterized by decreased relaxation and increased stiffness.Cardiac fibrosis is the major cause of cardiac diastolic dysfunction,but the mechanism in MS is still unclear. Many studies have shown that inflammation is the third fibrosis-triggering pathway, not independent of the hormonal control and/or blood pressure,but related to the inflammatory cytokines that invariably infiltrate the overloaded cardiac tissue. Chronic subclinical inflammation is thought to be part of the MS.Serum level of high sensitive-C reactive protein(hs-CRP),interluekin-6(IL-6)and IL-18 are increased.It known that IL-18,a member of the IL-1 family of ligands,induces a variety of responses via JNK pathway.The JNK pathway is a cascade of serine/threonine kinases that transduce the signals from the cell surface to the nucleus in response to growth factors and cellular stress.AP-1,which contains the c-Jun/c-fos protein, occurs downstream of JNK.Activated JNK phosphorylates AP-1 and binds to a specific DNA sequence known as the 'AP-1 binding site'.The gene promoters related to cardiac fibrosis such as collagenⅠ,collagenⅢ,matrix metalloproteinase-2 (MMP-2)and MMP-9 all have AP-1 binding sites.This suggests that IL-18 promoted cardiac fibrosis is related to the AP-1 complex via JNK activation.
In this study,molecular biology,cellular biology,histopathology and echocardiography were used to qualitatively observe and quantitatively analysize the morphology and ultrastructure of cardiac fibrosis in MS rats.The effect of IL-18 and JNK/AP-1 pathway on the pathogenesis of cardiac fibrosis were studied.
Objectives
(1)To establish an animal model of MS used fructose;
(2)To explore the changes of myocardial histopathotogy and ultrastructure;
(3)To define the myocardial expressions of key molecules of the IL-18/JNK/ AP-1 signal pathway;
(4)To delineate the role of IL-18/JNK/AP-1 signal pathway in cardiac fibrosis used IL-18 adenovirus administered;
(5)To explore the changes of cardiac structure and function used echocardiographie and hemodynamic methods.
Methods
50 male Wistar rats were randomly assigned to two groups:control group(n=12) and MS group(n=38).Rats in the control group were allowed to drink tap water and were fed standard rat chow ad libitum.Rats in the MS group were given 10%fructose water and standard rat chow ad libitum.After being fed for 32 weeks,rats in the MS group were randomly assigned to three groups:the MS group(n=9)which were given 10%fructose water continuedly;the AdGFP group(n=9)which were administered adenovirus containing GFP through tail vein at a dose of 1×10~(10)PFU and were given 10%fructose water meanwhile;the AdIL-18 group(n=13)which were administered adenovirus containing murine IL-18 through tail vein at a dose of 1×10~(10)PFU and were given 10%fructose water meanwhile.The control and MS group were given an equal volume of normal saline solution administered.After 6-week-treatment,rats were kill and the heart was rapidly excised for further study.Following observations on animals were performed during the whole experiments:(1)Body weight(BW)and systolic blood pressure(SBP)were documented every two weeks;(2)Fast blood glutose,insulin,triglyceride,total cholesterol and serum IL-18 were analysis at baseline,32 weeks after and at the end of the study,with HOMA calculated; (3)Echocardiography was performed at baseline,32 weeks after and at the end of the study to assess cardiac structure and function;(4)In vivo hemodynamie measurements was performed to evaluate cardiac function;(5)The changes of myocardial ultrastructure and histopathology were observed;(6)Cardiac fibrosis was evaluated quantified and qualitatively by Masson staining;(7)The mRNA expression of IL-18, collagenⅠand collagenⅢwere detected by quantification real-time PCR;(8)The protein level of IL-18,collagenⅠand collagenⅢwere determined by immunohistochemistry;(9)The protein level of JNK and phosphate-JNK were determined by Western-Blot;(10)The activity of AP-1 was determined by electrophoretic mobility shift assay(EMSA).
Results
1 BW,SBP and biochemical indices measurement
MS rats exhibited significant increases in BW(P＜0.01)and SBP(P＜0.001) compared with those of controls;Serum insulin levels(P＜0.001),triglycerides (P＜0.01)and HOMA index(P＜0.001)were also increased in the MS group(P＜0.001). However,there were no differences in glucose,cholesterol levels.Compared with the AdGFP group,serum insulin levels(P＜0.001)and HOMA index(P＜0.05)were also increased in the AdIL-18 group.However,there were no differences in BW,SBP, glucose and cholesterol levels.
2 Serum IL-18 levels
Before IL-18 adenovirus administered,serum IL-18 levels of the MS rats were higher(P＜0.001)compared with those of the controls.On the first week after IL-18 adenovirus administered,serum IL-18 levels in the AdIL-18 group were higher (P＜0.001)compared with those in the AdGFP group.On the second week,serum IL-18 levels in the AdIL-18 group were four times higher(P＜0.001)than those in the AdGFP group.On the third week,serum IL-18 levels in the AdIL-18 group began decreased,but were also higher(P＜0.001)than those in the AdGFP group.On the forth week,the levels of serum IL-18 in the AdIL-18 and AdGFP groups were similar.
3 Echocardiographic detection
Mitral and tricuspid valvular regurgition detection:none of 12 rats in the control group showed mitral and tricuspid valvular regurgitation.In the MS group,mild mitral valvular regurgitation was occurred in 2 rat,moderate mitral valvular regurgitation was occurred in 1 rat,mild tricuspid valvular regurgitation was occurred in 2 rats,severe tricuspid valvular regurgitation was occurred in 1 rat;2 rats of the MS group showed both mitral and tricuspid valvular simultaneous regurgition;5 rats of the MS group did not show valvular regurgition.In the AdGFP group,mild mitral valvular regurgitation was occurred in 1 rat,moderate mitral valvular regurgitation was occurred in 2 rat,severe mitral valvular regurgitation was occurred in 1 rat;mild tricuspid valvular regurgitation was occurred in 1 rats,severe tricuspid valvular regurgitation was occurred in 1 rat,2 rats of the AdGFP group showed both mitral and tricuspid valvular simultaneous regurgition;5 rats of the AdGFP group did not show valvular regurgition.In the AdIL-18 group,mild mitral valvular regurgitation was occurred in 3 rat,moderate mitral valvular regurgitation was occurred in 2 rat,severe mitral valvular regurgitation was occurred in 1 rat;mild tricuspid valvular regurgitation was occurred in 2 rats,moderate tricuspid valvular regurgitation was occurred in 1 rat,severe tricuspid valvular regurgitation was occurred in 1 rat;3 rats of the AdIL-18 group showed both mitral and tricuspid valvular simultaneous regurgition;6 rats of the AdIL-18 group did not show valvular regurgition.There were no rats with valvular regurgitation in the control group.The incidence of valvular regurgitionin the MS group was 44.44%and 53.85%in the AdIL-18 group.Fisher analysis:compared with that of the control group,the incidence of valvular regurgitation in the MS group was significantly increased(P＜0.01).Compared with that of the MS and the AdGFP groups,the incidence of valvular regurgitation in the AdIL-18 group was significantly increased(P＜0.05).
Compared with the controls,LVEDD(P＜0.01),LAD(P＜0.001),PW and SW (P＜0.05)were greater in the MS rats.E and Ea were lower(P＜0.001)in the MS rats. IVRT(P＜0.01)and Tei index(P＜0.001)were higher in the MS rats.No significant differences in EF,FS,RWth and E/Ea ratio were found between the control group and the MS group.
There was no differences in LVEDD,LAD,PW,SW and RWth among the MS, AdGFP and AdIL-18 groups.Compared with the rats in the MS and AdGFP groups, IVRT and Tei index were higher(P＜0.01)in the AdIL-18 group.No significant differences in EF,FS and E/Ea ratio were found among the three groups.
4 Electrocardiogram recordation
Electrocardiograms(ECG)of 12 rats in the control group were all normal.6 rats of the MS group showed characteristic QRS wave interchanges and 1 rats showed complex arrhythmia.The AdGFP group was similar to the MS group.10 rats of the AdIL-18 group showed QRS wave interchanges ECG,and 2 rats showed complex arrhythmia.There were no rats with arrhythmia in the control group.The incidence of arrhythmia was 77.78%in the MS group and 92.31%in the AdIL-18 group.Fisher analysis:compared with that of the control group,the incidence of arrhythmia in the MS group was significantly increased(P＜0.001).Compared with those of the MS group and the AdGFP group,the incidence of arrhythinia in the AdIL-18 group was significantly increased(P＜0.01).
5 In vivo hemodynamic measurements
Compared with the controls,LVSP,LVEDP and tau were significantly increased (P＜0.001),and -dp/dt was more negative(P＜0.01)in the MS rats.The load-independent index of relaxation,namely,-dp/dt/LVSP,was significantly lower (P＜0.001)in the MS group.There was no significant difference in HR and +dp/dt.
Compared with the rats in the MS group and AdGFP group,LVEDP(P＜0.001) and tau(P＜0.05)were increased,and -dp/dt was more negative in the AdIL-18 group. -dp/dt/LVSP(P＜0.05)was also lower.HR and +dp/dt were similar during invasive hemodynamic study.
6 Ultrastructural change observation by transmission electron microscopy
The left ventricular myocytes from the control group arranged regularly.The pericellular membrane was uninterrupted and intact.The thick and thin myofilament arranged regularly.The sarcomere and light dark band were clear.The uniformly sized mitochondrial was abundant and showed round or oval shape.A little fibroblast and collagenous fibers distributed in extra-cellular matrix.
The left ventricular myocytes from the MS and AdGFP groups arranged irregularly.The pericellular membrane was interrupted partly.The local myofibril was disintegrated partly.The myofilament was distorted and interrupted.The sarcomere was in a bad apposition.The swelling mitochondrial increased and accumulated. Collagenous fibers proliferated in extra-cellular matrix.
The left ventricular myocytes from the AdIL-18 group arranged irregularly.The pericellular membrane was interrupted and unclear.The local myofibril was disintegrated.The myofilament was distorted and interrupted.The sarcomere was in a bad apposition.The swelling mitochondrial increased and accumulated.A lot of collagenous fibers distributed in extra-cellular matrix.
7 Pathological detecion
HE staining showed that the myocytes from the control group arranged regularly. The size of the nuclear was uniform.The staining cytoplasm was homogeneous.The myocytes from the MS group arranged irregularly.The nuclear was irregular and interrupted myofibril arranged irregularly.The AdGFP group was same with the MS group.The myocytes from the AdIL-18 group arranged more irregularly.The nuclear was more irregular and interrupted myofibril arranged more irregularly compared with the AdGFP group.
8 The detection of collagen content by Masson-staining
The myocyte was red or yellow and the collagen was green or blue by Masson-staining.The collagen tissue was appropriate arranged among cardiomyocytes in the control group.However,collagen tissue increased markedly, and disrupted in some area in the MS group.The AdGFP group was same with the MS group.Collagen tissue increased and disrupted in the AdIL-18 group compared with the AdGFP group.Quantitative analysis results:the CVF(P＜0.001)and PVCA (P＜0.01)were higher in the MS group compared with the control group.The CVF and PVCA were higher in the AdIL-18 group compared with the other three groups(P＜0.05).
Correlation analysis demonstrated that,in the MS group,serum IL-18 level was positively correlated with CVF(P＜0.05)and PCVA(P＜0.01).
9 Immunohiatochemistry detection
IL-18:The positive reaction of IL-18 protein were stained brown and localized in myocytes.Brown granules were not seen in the control group,but thick brown granules in the MS group.A lot of thicker brown granules were observed in the AdIL-18 group.Compared with the control group,the level of IL-18 IOD was increased(P＜0.001)in the MS group.Compared with the the MS and the AdGFP groups,the level of IL-18 IOD was increased(P＜0.001)in the AdIL-18 group.
CollagenⅠ:The positive reaction of collagenⅠprotein were stained brown and localized among cardiomyocytes.Weak brown collagen tissue were uniformly and sparsely distributed in the control group,but thick brown collagen tissue in the MS group.A lot of thicker brown collagen tissue were observed in the AdIL-18 group. Compared with the control group,the level of collagenⅠIOD was increased(P＜0.001) in the MS group.Compared with the MS and the AdGFP groups,the level of collagenⅠIOD was increased(P＜0.001)in the AdIL-18 group.
CollagenⅢ:The positive reaction of collagenⅢprotein were stained brown and localized among cardiomyocytes.Weak brown collagen tissue were uniformly and sparsely distributed in the control group,but thick brown collagen tissue in the MS group.A lot of thicker brown collagen tissue were observed in the AdIL-18 group. Compared with the control group,the level of collagenⅢIOD was increased(P＜0.05) in the MS group.Compared with the MS and the AdGFP groups,the level of collagenⅢIOD was increased(P＜0.05)in the AdIL-18 group.
Correlation analysis demonstrated that,in the MS group,serum IL-18 level was positively correlated with the ratio of collagenⅠandⅢprotein(P＜0.05);the IL-18 protein was positively correlated with the ratio of collagenⅠandⅢprotein(P＜0.05); the IL-18 protein was positively correlated with CVF(P＜0.05)and PCVA(P＜0.05).
10 The mRNA experession of key molecules
In comparison with the controls,the expression of IL-18(P＜0.001),collagenⅠ(P＜0.001),collagenⅢ(P＜0.01)mRNA in the MS rats were significantly higher.In comparison with the MS and the AdGFP groups,the expression of IL-18(P＜0.001), collagenⅠ,collagenⅢmRNA(P＜0.01)in the AdIL-18 group were significantly higher.
Correlation analysis demonstrated that,in the MS group,serum IL-18 level was positively correlated with the ratio of collagenⅠandⅢmRNA(P＜0.05);the IL-18 mRNA was positively correlated with the ratio of collagenⅠandⅢmRNA(P＜0.05); the IL-18 mRNA was positively correlated with CVF(P＜0.05)and PCVA(P＜0.01).
11 The protein expression of several factors
The expression of P-JNK protein in the MS group was higher significantly than that of the control group(P＜0.001).The expression of P-JNK protein in the AdIL-18 group was higher significantly than those of the MS and the AdGFP groups(P＜0.01). However,there was no significant differences in the expression of JNK protein among the four groups.
12 The activity of AP-1
The activity of AP-1 assessed by EMSA was enhanced in the MS group compared with the control group.The activity of AP-1 was enhanced in the AdIL-18 group compared with the MS and the AdGFP groups.
Conclusions
(1)An animal model of MS was established by high-fructose water.After fed fructose for 32 weeks,the MS rats had higher SBP and BW compared with the controls.And serum triglyceride concentrations,insulin levels and HOMA index were also increased.This model is valuable to investigate cardiovascular and metabolic abnormalities similar to human MS;
(2)Echocardiography showed E and Ea were lower,IVRT and Tei index were higher in the MS rats.Hemodynamics showed LVSP,LVEDP and tau were increased, and -dp/dt and -dp/dt/LVSP were lower in the MS rats.Those findings suggested the MS rats had cardiac diastolic dysfunction;
(3)The collagen content was increased in the MS rats detected by Masson-staining.The expressions of collagenⅠandⅢmRNA as well as protein detected by real-time RT-PCR and immunohistochemistry were increased.Those changes suggested the MS rats had cardiac fibrosis,which plays an important role in the pathogenesis of diastolic dysfunction;
(4)Serum IL-18 levels as well as the myocardial expression of IL-18 were increased in the MS rats.Moreover,collagen content was positively correlated with them,which suggested IL-18 may be play an important role in cardiac fibrosis.The expression of P-JNK protein and the activity of AP-1 were increased,which suggested IL-18 could induce cardiac fibrosis through JNK/AP-1 pathway;
(5)After IL-18 adenovirus was administered,cardiac fibrosis and diastolic dysfunction were more aggravating.IL-18-induced the upregulation of JNK and activiation of AP-1 represent an important mechanism leading to cardiac fibrosis and diastolic dysfunction.
Background
Metabolic syndrome (MS) has many major risks for type 2 diabetes and cardiovascular disease. The constellation of metabolic abnormalities includes glucose intolerance, insulin resistance, central obesity, dyslipidaemia, and hypertension. For the WHO and NCEP:ATPIII definitions, the prevalence of MS is 23.7% in the USA and 43.5% for those aged over 60 years. The risk for coronary heart disease and stroke was increased mreefold in subjects with the MS, and cardiovascular mortality was also markedly increased in those subjects. Cardiovascular disease and all-cause mortality are increased in men with the MS, even in the absence of baseline CVD and diabetes. This means that sub-clinical cardiovascular impairments are the fundamental reason for higher cardiovascular complications in those patients. Therefore, to finding the drug which reverses or retards cardiovascular impairments in patients with the MS is of great importance.
Calcium channel blockers (CCBs) are widely used to treat patients with hypertension and coronary heart disease. Clinical study demonstrated that CCBs decrease cardiovascular events in patients with coronary heart disease. The results of CAMELOT study were administration of amlodipine to patients with CAD and normal blood pressure resulted in reduced adverse cardiovascular events. For amlodipine, IVUS showed evidence of slowing of atherosclerosis progression. The INSIGHT study showed nifedipine was as effective as diuretic therapy in reducing cardiovascular complications in hypertensive diabetics. Many clinical studies suggest that CCBs could be considered as first-line therapy for hypertensive diabetics. Moreover, CCBs improve ventricular systolic and diastolic function in hypertensive patients. Cell culture studies have shown that CCBs attenuate the AngII-mediated increases in intracellular calcium levels of adult cardiac fibroblasts, thereby abolishing an Angll-mediated increase in collagen synthesis. Although the mechanisms underlying the direct protective effects of calcium channel blockades on cardiovascular injury are not fully understood, many studies revealed that CCBs decrease cardiovascular events is related to its anti-inflammation effects. A recent clinical study has demonstrated that amlodipine seemed to improve IR. and decrease TNF-alpha levels besides reducing BP, but further interventions are needed. Furthermore, amlodipine attenuated arteriosclerosis through inhibition of inflammatory disorders in a rat model of long-term inhibition of NO synthesis. The anti-inflammatory effects of amlodipine may be mediated by the inhibition of local factors, such as MCP-1, TGF-β1, and oxidative stress. Recently many findings suggest that some dihydropyridine-type CCBs, such as nifedipine, nilvadipine, and benidipine, have a direct beneficial effect by inhibiting the nuclear translocation and DNA-binding activity of NF-kappaB, which plays a central role in this process by regulating the expression of several pro-inflammatory genes.
From the first part of the study, we found inflammation was a key player in the pathogenesis of the MS, and was related to the ventricular remodeling of MS. IL-18 played an important role in cardiac fibrosis and diastolic dysfunction of MS through 'JNK/AP-1' pathway. Moreover, CCBs has novel anti-inflammation actions beyond blood pressure lowering. So the aim of this study was to observe the effect of felodipine on inflammation in rat model of MS and to study the possible mechanism of felodipine on cardiac structure and function of the MS rats. Objectives
(1) To evaluate the effect of felodipine on the body weight, systolic blood pressure and biochemical indices of the MS rats;
(2) To evaluate the effect of felodipine on the changes of cardiac structure and function used echocardiographic and hemodynamic methods;
(3) To explore the effect of felodipine on the changes of myocardial histopathology and ultrastructure of the MS rats;
(4) To explore the effect of felodipine on serum IL-18 levels and cardiac IL-18 expressions;
(5) To explore the effect of felodipine on IL-18/JNK/AP-1 signal pathway in cardiac fibrosis and to delineate the pathogenesis of the beneficial effect of felodipine on the cardiovascular diseases.
Methods
33 male Wistar rats were randomly assigned to two groups: control group (n=12) and MS group (n=21). Rats in the control group were allowed to drink tap water and were fed standard rat chow ad libitum. Rats in the MS group were given 10% fructose water and standard rat chow ad libitum. After being fed for 8 months, rats in the MS group were randomly assigned to two groups: the MS group (n=9) which were given 10% fructose water continuedly; the felodipine group (n=9) which were given a daily dose of 5mg/kg felodipine by intubation with a stomach tube and were given 10% fructose water meanwhile. The control and MS group were given an equal volume of normal saline solution. After 6-week-treatment, rats were kill and the heart was rapidly excised for further study. Following observations on animals were performed during the whole experiments: (1)Body weight (BW) and systolic blood pressure (SBP) were documented every two weeks; (2)Serum IL-18 level, fast blood glutose, insulin, triglyceride and cholesterol were analysis at baseline, 32 weeks after and at the end of the study, with HOMA calculated; (3)Echocardiography was performed at baseline, 32 weeks after and at the end of the study to evaluate cardiac function; (4)In vivo hemodynamic measurements was performed to evaluate cardiac function; (5)The changes of mvocardial ultrastructure and histopathologv were observed: (6)Cardiac fibrosis was evaluated quantified and qualitatively by Masson staining; (7)The mRNA expression of IL-18, collagen I and collagen III were detected by quantification real-time PCR; (8)The protein level of IL-18, collagen I and collagen III were determined by immunohistochemistry, (9)The protein level of JNK and P-JNK were determined by western blot; (10)The activity of AP-1 was determined by electrophoretic mobility shift assay (EMSA). Results
1 BW, SBP and biochemical indices measurement
The SBP of the rats treated with felodipine significant decreased (P<0.001) compared with those in the MS group. However, there were no differences in BW. Compared with the MS group, serum insulin levels and HOMA index (P<0.001) were decreased in the felodipine group. However, there were no differences in glucose and cholesterol levels among the three groups.
2 Serum IL-18 level
After six-week treatment of felodipine, serum IL-18 levels in the felodipine group were lower (P<0.05) compared with those in the MS group, however, were higher significantly (P<0.001) compared with those in the control group.
3 Echocardiographic detection
Mitral and tricuspid valvular regurgition detection: In the felodipine group, mild mitral valvular regurgitation was occurred in 1 rat, moderate mitral valvular regurgitation was occurred in 0 rat, severe mitral valvular regurgitation was occurred in 0 rat; mild tricuspid valvular regurgitation was occurred in 2 rats, moderate tricuspid valvular regurgitation was occurred in 0 rats, severe tricuspid valvular regurgitation was occurred in 0 rat; 1 rats of the felodipine group showed both mitral and tricuspid valvular simultaneous regurgition; 7 rats of the MS group did not show valvular regurgition. The incidence of valvular regurgitionin the felodipine group was 22.22%. Fisher analysis: compared with that of the MS group, the incidence of valvular regurgitation in the felodipine group was significantly decreased (P<0.01).
Compared with the MS rats, LVEDD (P<0.01), LAD (P<0.001), PW and SW (P<0.05) decreased in the rats treated with felodipine. E, Ea (P<0.001) were higher and IVRT (P<0.01), Tei (P<0.001) index were lower in the rats treated with felodipine.
4 Electrocardiogram recordation
Electrocardiograms (ECG) of 5 rats in the felodipine group were normal. 3 rats of the felodipine group showed characteristic QRS wave interchanges and none rat showed complex arrhythmia. The incidence of arrhythmia was 33.33% in the felodipine group. Fisher analysis: compared with that of the MS group, the incidence of arrhythmia in the felodipine group was significantly decreased (P<0.01). Compared with those of the control group, the incidence of arrhythmia in the felodipine group was significantly increased (P<0.001).
5 In vivo hemodynamic measurements
Compared with the MS rats, LVSP, LVEDP and tau were significantly decreased (P<0.001), and -dp/dt was more positive (P<0.01) in the rats treated with felodipine. The load-independent index of relaxation, namely, -dp/dt/LVSP, was significantly higher (P<0.001) in the rats treated with felodipine. HR and +dp/dt were similar during invasive hemodynamic study.
6 Ultrastructural change observation by transmission electron microscopy
The myocytes from the felodipine group arranged more regularly than the MS group. The phenomenon that the local myofibril was disintegrated was decreased. The mitochondrial was more regular than the MS group. The nuclear shape was more regular than the MS group. The collagenous fibers in extra-cellular matrix decreased obviously.
7 Pathological detecion
HE staining showed that the myocytes from the felodipine group arranged more regularly. The size of the nuclear was more uniform. The staining cytoplasm was more homogeneous compared with the MS group.
8 The content of collagen detection by Masson-stainingThe myocyte was red or yellow and the collagen was green or blue. The collagen tissue was more appropriate arranged among cardiomyocytes in the felodpine grouptissue was more appropriate arranged among cardiomyocytes in the felodpine group compared with the MS group. Quantitative analysis results: the CVF and PVCA were lower in the felodipine group compared with the MS group(P<0.01).
9 Immunohiatochemistry detection
IL-18: The positive reaction of IL-18 protein were stained brown and localized in myocytes. Weak brown granules were uniformly and sparsely distributed in the felodipine group, but thick brown granules in the MS group. Compared with the control group, the level of IL-18 IOD was increased (P<0.01) in the felodipine group. Compared with the MS group, the level of IL-18 IOD was decreased (P<0.001) in the felodipine group.
Collagen I: The positive reaction of collagen I protein were stained brown and localized among cardiomyocytes. Weak brown collagen tissue were uniformly and sparsely distributed in the felodipine group, but thick brown collagen tissue in the MS group. Compared with the control group, the level of collagen I IOD was increased (P<0.001) in the felodipine group. Compared with the MS group, the level of collagen I IOD was decreased (P<0.001) in the felodipine group.
Collagen III: The positive reaction of collagen III protein were stained brown and localized among cardiomyocytes. Weak brown collagen tissue were uniformly and -sparsely distributed in the felodipine group, but thick brown collagen tissue in the MS group. Compared with the control group, the level of collagen III IOD was increased in the felodipine group. Compared with the MS group, the level of collagen III IOD was decreased (P<0.01) in the felodipine group.
10 The mRN A experession of key molecules
In comparison with the controls, the expression of IL-18 (P<0.001), collagen I, collagen III mRNA (P<0.0l) in FFRs treated with felodipine were higher. In comparison with the MS group, the expression of IL-18 (P<0.001), collagen I, collagen III mRNA (P<0.001) in the felodipine group were significantly lower.
11 The expression of several factors
The expression of P-JNK protein in the felodipine group was lower significantly than that of the MS group (P<0.01). However, there was no significant differences in the expression of JNK protein among the three groups.
12 The activity of AP-1
The activity of AP-1 assessed by EMSA was enhanced in the felodipine group compared with the control group. The activity of AP-1 was weakened in the felodipine group compared with the MS group. Conclusions
(1) Felodipine treatment decrease SBP and the insulin level and improve insulin resistance;
(2) Echocardiography showed E and Ea were higher, IVRT and Tei index were lower in the rats treated with felodipine. Hemodynamics showed LVSP, LVEDP and tau were decreased, and -dp/dt and -dp/dt/LVSP were higher in the rats treated with felodipine. Those findings suggested felodipine treatment ameliorates diastolic dysfunction in the MS rats;
(3) The collagen content detected by Masson-staining was decreased in the rats treated with felodipine. The expressions of collagen I and III mRNA as well as protein detected by real-time RT-PCR and immunohistochemistry were also decreased. Those changes suggested felodipine had beneficial effects on cardiac fibrosis;
(4) Serum IL-18 levels as well as the myocardial expressions of IL-18 were decreased in the felodipine group, which suggested felodipine had anti-inflammation effect;
(5) The expression of P-JNK protein and the activity of AP-1 were decreased in the felodipine group, which suggested felodipine could decrease cardiac fibrosis and ameliorate diastolic dysfunction through IL-18/JNK/AP-1 pathway.
过去的二十年里,全球代谢综合征(MS)的患病率逐年递增,糖尿病及肥胖人数也随之增加。随着MS带来的心血管病发病率的上升,我们认识到防治这一全球性疾病迫在眉睫。MS是包括糖耐量异常、胰岛素抵抗、腹型肥胖、血脂异常及高血压等心血管疾病危险因素的一组症候群。胰岛素抵抗被认为是该综合征的核心的病理生理学基础。而炎症可能在MS中起重要作用。
临床研究证实MS患者有不同程度的左室舒张功能障碍,表现为心肌松弛性减低和僵硬度增大。心肌纤维化是左室舒张功能障碍发生发展的重要机制,但在MS中的发生机制尚不明确。研究表明炎症是除肾素-血管紧张素-醛固酮(RASS)系统和高血压之外致心肌纤维化的重要因素,许多炎症因子参与了心肌纤维化的形成和发展。而MS是一种慢性炎症状态,血中炎症因子高敏C反应蛋白(hs-CRP)、白介素-6(IL-6)、白介素-18(IL-18)等都有升高。IL-18作为IL-1家族成员之一,可以通过激活JNK信号转导通路参与一些反应。JNK信号转导通路是将信息从细胞表面传到细胞核内的一系列丝氨酸/苏氨酸酶,主要对细胞的生长发育起介导作用。位于JNK信号转导通路下游的核因子AP-1包含c-jun/c-fos蛋白,JNK通过激活AP-1使存在AP-1结合位点的基因表达增强。与心肌纤维化有关的基因,例如Ⅰ型胶原、Ⅲ型胶原、基质金属蛋白酶-2(MMP-2)、MMP-9基因,都存在AP-1的结合位点。因此我们推断IL-18可以通过激活JNK/AP-1信号转导通路加重心肌纤维化参与MS的发生发展,这在国内外均未见研究报道。
本研究在综合分析心肌纤维化研究动态和细胞信号转导途径的基础上,提出“IL-18/JNK/AP-1”信号转导通路在MS心肌纤维化发病中可能起重要作用的假说,利用转染IL-18腺病毒载体技术,深入研究IL-18在MS心肌纤维化发生发展中的作用。
目的
(1)建立果糖喂养Wistar大鼠MS的动物模型;
(2)观察MS大鼠模型心肌组织病理学和超微结构的变化,明确心肌纤维化存在及分布特点;
(3)明确“IL-18/JNK/AP-1”信号转导通路各关键分子在MS大鼠心肌组织的表达;
(4)构建IL-18腺病毒载体并转染MS大鼠,验证“IL-18/JNK/AP-1”信号转导途径在MS心肌纤维化发生发展的作用;
(5)采用超声心动图技术和心导管技术全面评价MS大鼠模型以及转染IL-18腺病毒载体后MS大鼠心脏结构和功能的变化。
方法
雄性Wistar大鼠50只,随机分为两组:正常对照组(n=12只),MS组(n=38只)。正常对照组以标准大鼠饲料喂养,并给予普通饮水:MS组大鼠以标准大鼠饲料喂养,并给予10%高果糖饮水。高果糖喂养32周后,将MS组大鼠随机分为三组:MS组(n=9只),继续给予高果糖饮水;空载体组(n=9只),继续给予高果糖饮水的同时通过尾静脉注射1×10~(10)PFU含GFP腺病毒载体;IL-18载体组(n=13只),继续给予高果糖饮水的同时通过尾静脉注射1×10~(10)PFU IL-18腺病毒载体;正常对照组及MS组给予等体积的生理盐水尾静脉注射。转染腺病毒载体6周后,处死动物,取大鼠心肌组织进行实验。实验过程中,进行以下检测:(1)各组大鼠每两周测量体重及尾动脉血压一次;(2)分别于实验前,高果糖喂养32周后,转染载体后1周、2周、3周、4周、6周抽取各组大鼠颈静脉窦血2ml检测空腹血糖、空腹胰岛素、甘油三酯、总胆固醇、血中IL-18浓度,并计算胰岛素抵抗指数(HOMA);(3)分别于实验前、高果糖喂养32周后、实验末进行超声心动图检查;(4)于实验末记录各组大鼠心电图,以心导管法进行血流动力学测定;(5)心肌组织超微结构和病理学检查;(6)应用Masson染色观察心肌纤维化程度:(7)免疫组织化学检测IL-18、Ⅰ型胶原、Ⅲ型胶原的蛋白表达;(8)实时定量RT-PCR法检测IL-18、Ⅰ型胶原、Ⅲ型胶原的mRNA表达;(9)Western-blot检测JNK和磷酸化JNK蛋白的表达;(10)凝胶电泳迁移分析检测转录因子AP-1活性。
结果
1体重、尾动脉收缩压及生化指标
高果糖喂养32周后,与正常对照组相比,MS组大鼠体重增加(P＜0.01),尾动脉收缩压升高(P＜0.001);血清甘油三酯(P＜0.01)及空腹胰岛素(P＜0.001)升高,HOMA增加(P＜0.001),而总胆固醇及空腹血糖无统计学意义。
转染IL-18腺病毒载体后6周,与MS组和空载体组比较,IL-18载体组血胰岛素水平(P＜0.001)及HOMA(P＜0.05)升高;而体重、尾动脉收缩压、甘油三酯、总胆固醇在IL-18载体组与MS组和空载体组间无统计学意义。
2血清IL-18水平变化
转染前MS组大鼠血IL-18水平较正常对照组明显升高(P＜0.001)。IL-18腺病毒转染后1周,IL-18载体组大鼠血清IL-18水平较空载体组显著升高(P＜0.001),转染后2周达到最高水平,约为空载体组血清IL-18水平的4倍(P＜0.001),于转染后3周开始降低,但仍高于空载体组IL-18水平(P＜0.001),4周时与空载体组IL-18水平相当。
3超声心动图检测
正常对照组12只大鼠,均未见二尖瓣或三尖瓣返流。MS组9只大鼠,轻度二尖瓣返流2只,中度二尖瓣返流1只,重度二尖瓣返流0只;轻度三尖瓣返流2只,中度三尖瓣返流0只,重度三尖瓣返流1只;其中2只二尖瓣、三尖瓣均存在返流;二尖瓣、三尖瓣均无返流5只。空载体组9只大鼠,轻度二尖瓣返流1只,中度二尖瓣返流2只,重度二尖瓣返流1只;轻度三尖瓣返流1只,中度三尖瓣返流0只,重度三尖瓣返流1只;其中2只二尖瓣、三尖瓣均存在返流;二尖瓣、三尖瓣均无返流5只。IL-18载体组13只大鼠,轻度二尖瓣返流3只,中度二尖瓣返流2只,重度二尖瓣返流1只;轻度三尖瓣返流2只,中度三尖瓣返流1只,重度三尖瓣返流1只;其中3只二尖瓣、三尖瓣均存在返流;二尖瓣、三尖瓣均无返流6只。正常对照组大鼠无瓣膜返流发生,MS组及空载体组大鼠瓣膜返流发生率均为44.44%,IL-18载体组大鼠瓣膜返流发生率为53.85%。Fisher检验显示:与对照组相比,MS组大鼠瓣膜返流发生率增加,差异有统计学意义(P＜0.01)。与空载体组相比,IL-18载体组大鼠瓣膜返流发生率增加,差异有统计学意义(P＜0.05)。
与正常对照组相比,MS组大鼠左室舒张末期内径(LVDd)明显扩大(P＜0.01),舒张末期室间隔(IVSd)、左室后壁同步增厚(LVPWd)(P＜0.05),左房内径(LA)明显扩大(P＜0.001)。与正常对照组相比,MS组大鼠E峰、Ea峰明显降低(P＜0.001),等容舒张时间(IVRT)延长(P＜0.01),Tei指数增加(P＜0.001),左室射血分数(EF)、短轴缩短率(FS)、相对室壁厚度(RWth)及E/Ea峰比值无统计学意义。
与MS组和空载体组相比,IL-18载体组大鼠LVDd、IVSd、LVPWd、LA及RWth均无统计学意义。与MS组和空载体组相比,IL-18载体组大鼠E峰、Ea峰降低,但无统计学意义;IVRT(P＜0.01);Tei指数增加(P＜0.01):EF、FS及E/Ea峰比值无统计学意义。
4心电图变化
正常对照组12只大鼠心电图均基本正常。MS组和空载体组各9只大鼠中6只心电图出现QRS波正负交替变化为特征的心电图改变,1只大鼠出现复杂心律失常(两种以上的心电图异常或宽大畸形的QRS波),2只大鼠心电图基本正常。IL-18载体组13只大鼠中10只心电图出现QRS波正负交替变化为特征的心电图改变,2只大鼠出现复杂心律失常,1只大鼠心电图基本正常。正常对照组无心律失常发生,MS组和空载体组心律失常发生率是77.78%,IL-18载体组心律失常发生率是92.31%。Fisher检验显示:与正常对照组相比,MS组大鼠心律失常发生率明显增加,差异有统计学意义(P＜0.001);与MS组和空载体组相比,IL-18载体组大鼠心律失常发生率明显增加,差异有统计学意义(P＜0.01)。
5血液动力学
与正常对照组相比,MS组大鼠左室收缩压(LVSP)、左室舒张末压(LVEDP)升高(P＜0.001);左室压力最大下降速率(-dp/dt)下降(P＜0.01),-dp/dt/LVSP明显下降(P＜0.001);左室松弛时间常数(T)延长(P＜0.001)。心率(HR)及左室压力最大上升速率(+dp/dt)均无统计学意义。
与MS组和空载体组相比,IL-18载体组大鼠LVEDP升高(P＜0.001);-dp/dt、-dp/dt/LVSP下降(P＜0.05);T延长(P＜0.05)。心率及+dp/dt均无统计学意义。
6透射电镜观察超微结构的改变
正常对照组可见心肌细胞排列整齐,质膜连续;粗细肌丝排列规整,肌小节及明暗各带清晰可见;线粒体丰富,大小均一;细胞间质可见成纤维细胞和少量胶原纤维。
MS组可见心肌细胞排列紊乱,质膜部分断裂;粗细肌丝部分溶解丧失;增生的线粒体呈多形性,大小不一;间质充满了增生的胶原纤维,胶原纤维走形紊乱,断裂;成纤维细胞核不规则,胞浆内质网肿胀,线粒体嵴减少。
空载体组可见心肌细胞排列紊乱,质膜部分断裂;粗细肌丝部分溶解丧失;增生的线粒体呈多形性,大小不一;间质充满了增生的胶原纤维,胶原纤维走形紊乱,断裂。
IL-18载体组可见心肌细胞排列紊乱,质膜断裂;肌原纤维呈灶性溶解,肌丝扭曲、断裂,肌节对位不齐;线粒体明显增多,大小不一呈堆积状;间质可见大量的胶原纤维。
7组织病理观察
HE染色显示,正常对照组心肌细胞排列整齐,细胞核大小均一,胞浆染色均匀。MS组及空载体组心肌细胞排列紊乱,细胞内可见肌纤维增粗、变长、有较多分支,细胞核增大、深染蓝色,胞质丰富红染。IL-18载体组心肌细胞肥大,排列紊乱更为严重,细胞内可见肌纤维断裂、排列疏松紊乱,细胞核大小不甚规则,胞浆染色不均匀。
8 Masson染色胶原含量的检测
心肌细胞呈红色,胶原纤维呈绿色。正常对照组胶原组织分布均匀,相邻细胞和血管周围的胶原纤维网完好:MS组和空载体组心肌间和血管周围的胶原组织增多,围绕心肌细胞的胶原纤维网部分断裂;IL-18载体组心肌间和血管周围的胶原组织明显增多,围绕心肌细胞的胶原纤维网断裂、排列紊乱。
与正常对照组相比,MS组左室心肌组织间(P＜0.001)和血管周围(P＜0.01)胶原含量升高。与MS组和空载体组相比,IL-18载体组左室心肌组织间(P＜0.05)和血管周围(P＜0.05)胶原含量升高。
相关性分析显示,MS组大鼠左室心肌间质和血管周围胶原含量与IL-18腺病毒载体转染后第二周血清IL-18浓度呈正相关(CVF:P＜0.01;PCVA:P＜0.05)。
9免疫组织化学检测
IL-18:染色阳性信号为棕色颗粒,定位于心肌细胞胞浆内。正常对照组心肌细胞胞浆内几乎未见IL-18表达;MS组与空载体组心肌细胞内可见分布均匀、稀疏的浅棕色颗粒;IL-18载体组心肌细胞内可见浓密的深棕色颗粒。以各视野累计光密度值(IOD)作为反应IL-18蛋白表达的定量指标结果显示,与正常对照组比较,MS组IOD值升高(P＜0.001);与MS组和空载体组比较,IL-18载体组IOD值升高(P＜0.001)。
Ⅰ型胶原:染色阳性信号为棕色条索状纤维,定位于心肌细胞间质和血管周围。正常对照组心肌细胞间和血管周围可见少数纤细的浅棕色条索状纤维;MS组与空载体组心肌细胞间和血管周围可见较多粗大、排列紊乱的棕色条索状纤维;IL-18载体组心肌细胞间和血管周围可见充满粗大、排列紊乱的深棕色条索状纤维。以各视野IOD值作为反应Ⅰ型胶原蛋蛋白表达的定量指标结果显示,与正常对照组比较,MS组IOD值升高(P＜0.001);与MS组和空载体组比较,IL-18载体组IOD值升高(P＜0.001)。
Ⅲ型胶原:染色阳性信号为棕色条索状纤维,定位于心肌细胞间质和血管周围。正常对照组心肌细胞间和血管周围可见少数纤细的浅棕色条索状纤维;MS组与空载体组心肌细胞间和血管周围可见较多粗大、排列紊乱的棕色条索状纤维;IL-18载体组心肌细胞间和血管周围可见充满粗大、排列紊乱的深棕色条索状纤维。以各视野IOD值作为反应Ⅲ型胶原蛋白表达的定量指标结果显示,与正常对照组比较,MS组IOD值升高(P＜0.05);与MS组和空载体组比较,IL-18载体组IOD值升高(P＜0.05)。
相关性分析显示,MS组大鼠左室Ⅰ、Ⅲ型胶原蛋白表达水平比值与IL-18腺病毒载体转染后第二周血清IL-18浓度呈正相关(P＜0.05);MS组大鼠左室Ⅰ、Ⅱ型胶原蛋白表达水平比值与IL-18蛋白表达水平呈正相关(P＜0.05):MS组大鼠左室IL-18蛋白表达水平与左室心肌间质和血管周围胶原含量呈正相关(P＜0.05)。
10实时定量RT-PCR检测各关键分子mRNA表达
与正常对照组比较,MS组大鼠左室心肌组织IL-18 mRNA表达增加(P＜0.001);MS组大鼠左室心肌组织Ⅰ型胶原(P＜0.001)及Ⅲ型胶原(P＜0.01)mRNA表达增加。
与MS组和空载体组比较,IL-18载体组大鼠左室心肌组织IL-18mRNA表达增加(P＜0.001);IL-18载体组大鼠心肌组织Ⅰ、Ⅲ型胶原mRNA表达增加(P＜0.01)。
相关性分析显示,MS组大鼠左室Ⅰ、Ⅲ型胶原mRNA表达水平比值与IL-18腺病毒载体转染后第二周血清IL-18浓度呈正相关(P＜0.05);MS组大鼠左室Ⅰ、Ⅲ型胶原mRNA表达水平比值与IL-18mRNA表达水平呈正相关(P＜0.05);MS组大鼠左室IL-18mRNA表达水平与左室心肌间质和血管周围胶原含量呈正相关(CVF:P＜0.05:PCVA:P＜0.01)。
11各组大鼠关键分子蛋白质表达水平的比较
各组大鼠左室心肌组织总JNK蛋白水平比较:四组大鼠左室心肌组织JNK总蛋白质表达水平无统计学意义。
各组大鼠左室心肌组织磷酸化JNK蛋白表达水平比较:与正常对照组比较,MS组磷酸化JNK蛋白含量升高(P＜0.001);与MS组和空载体组比较,IL-18载体组磷酸化JNK蛋白含量升高(P＜0.01)。
12 AP-1转录因子活性的比较
以EMSA法检测各组大鼠左室心肌组织AP-1活性,结果显示与正常对照组比较,MS组AP-1活性增强,具体表现为探针与核蛋白结合产生的条带变宽,条带颜色加深;与MS组和空载体组相比,IL-18载体组AP-1活性增强,具体表现为探针与核蛋白结合产生的条带变宽,条带颜色加深。
结论
(1)给予Wistar大鼠10%的高果糖饮水32周后,大鼠出现尾动脉收缩压升高,体重增加,血液中甘油三酯、胰岛素水平升高,反应胰岛素抵抗的指标HOMA增加,提示成功建立了符合人类MS特点的大鼠模型;
(2)超声心动图检测显示,MS大鼠心脏E峰、Ea均降低,IVRT延长,Tei指数增加,心导管检查显示LVSP、LVEDP升高。-dp/dt、-dp/dt/LVSP下降,T延长,提示MS大鼠存在心脏舒张功能减低;
(3)Masson染色证实心肌间质及血管周围胶原含量增加,免疫组织化学染色和实时定量RT-PCR证实Ⅰ型胶原和Ⅲ型胶原蛋白和mRNA表达水平升高,提示MS大鼠存在心肌纤维化,这可能是MS心脏舒张功能的障碍的主要病理学和分子生物学基础;
(4)MS大鼠血液中IL-18浓度升高,免疫组织化学染色和实时定量RT-PCR证实心肌组织中炎症因子IL-18蛋白和mRNA表达水平升高,且与心肌胶原含量及Ⅰ、Ⅲ型胶原蛋白和mRNA表达水平呈显著相关关系,提示IL-18可能是MS心肌纤维化的重要机制之一。MS大鼠心肌组织磷酸化JNK水平升高,转录因子AP-1活性增强,提示在MS大鼠模型中,IL-18可能通过“JNK/AP-1”信号途径导致心肌纤维化;
(5)构建IL-18腺病毒载体并转染MS大鼠,血液中和心肌组织中IL-18水平显著增加,同时伴有心肌纤维化程度加重,出现左室舒张功能进一步减低,心肌组织磷酸化JNK蛋白表达水平和转录因子AP-1活性进一步增强,提示“IL-18/JNK/AP-1”信号转导通路是引起MS心肌纤维化和心脏舒张功能减低的重要分子机制。
背景
代谢综合征(MS)是严重威胁人类健康的疾病。MS包括高血压、腹型肥胖、糖耐量异常、高甘油三脂血症、低高密度脂蛋白血症、胰岛素抵抗等一组心血管危险因素。以世界卫生组织(WHO)、美国国家胆固醇教育计划成人治疗组(NCEP-ATPIII)MS诊断标准及美国国家健康和营养普查III(NHANESIII)结果判断，美国MS的发病率为23．7％，60岁以上者发病率达43．5％。研究表明MS患者发生冠心病及中风的危险性增加3倍，心血管死亡率也显著增加，即使在无基础心血管疾病及糖尿病患者中，MS也显著增加心血管疾病的发生率和死亡率。提示MS潜在的心血管损害可能是其心血管并发症高发的原因。因此，寻找能逆转或延缓MS患者心血管损害的药物对于更好的治疗MS患者至关重要。
二氢吡啶类钙通道阻滞剂(CCB)已被广泛用于治疗高血压、心绞痛等疾病。临床与基础研究提示CCBs在降压的同时还具有一定的靶器官保护作用。CAMELOT研究显示氨氯地平能降低血压正常的冠心病患者心血管事件发生率，血管内超声提示氨氯地平能延缓动脉粥样硬化的进展。INSIGHT研究表明CCBs能降低伴糖尿病的高血压患者总死亡率，不增加新发糖尿病的发生率。对多项大规模临床试验结果进行荟萃分析示CCBs能有效降低伴糖尿病的高血压患者心血管事件死亡率。给予高血压患者CCBs治疗能显著改善患者心脏收缩及舒张功能。动物实验结果显示。长效CCBs能降低血管源性生长因子的表达并能阻止心肌重构。体内培养心肌成纤维细胞发现，CCBs可减少血管紧张素II介导的Ca2+内流，从而阻止血管紧张素II介导的心肌纤维化。因此，有学者建议要重视CCBs在代谢紊乱中的应用。但CCBs的的靶器官保护作用的机制尚不清楚，一些间接和直接的证据提示可能与CCBs的抗炎作用有关。研究发现，氨氯地平能改善肥胖的2型糖尿病患者胰岛素抵抗，降低血TNF"G水平。动物试验证实氨氯地平能够通过持续抑制NO合成而抑制炎症，改善动脉粥样硬化，同时降低MCP-1、TGF-[11等炎症因子，改善氧化应激。国外一些研究证实硝苯地平、尼伐地平、贝尼地平能通过减弱NF-r．B活性抑制炎症。
本研究的第一部分证实，炎症状态是MS发病和MS心脏结构和功能改变的重要机制之一，炎症因子IL-1 8可能通过JNK／AP．1信号转导通路在MS心肌纤维化和左室舒张功能障碍的发生和发展中起重要作用，上述直接和间接的证据提示CCBs具有一定的抗炎作用。因此，二氢吡啶类CCB非洛地平能否通过抑制炎症反应而对MS状态下心脏结构和功能改变起到有益的保护作用?我们将以MS大鼠模型为研究对象，给予非洛地平进行干预治疗，观察非洛地平对MS大鼠心脏结构和功能的保护作用及其对炎症反应的影响。目的
(1)评价非洛地平对MS大鼠体重、尾动脉收缩压和血液生化指标的影响；
(2)采用超声心动图技术和心导管技术全面评价非洛地平对MS大鼠心脏结构和功能的作用；
(3)观察非洛地平对MS大鼠心肌组织病理学及超微结构的干预作用；
(4)观察非洛地平对MS大鼠血液和心肌组织IL-18表达的作用；
(5)观察非洛地平对“IL-18／JNK／AP-1”信号转导通路各关键分子表达的影响以阐明非洛地平心血管保护作用的可能机制。
方法
雄性Wistar大鼠33只，随机分为两组：正常对照组(，l=12只)，MS组(n=21只)。正常对照组以标准大鼠饲料喂养，并给予普通饮水；MS组大鼠以标准大鼠饲料喂养，并给予10％高果糖饮水。高果糖喂养32周后，将MS组大鼠随机分为两组：MS组(n=9只)，继续给予高果糖饮水；非洛地平组(n=9只)，继续给予高果糖饮水的同时每日给予5mg／kg非洛地平灌胃，正常对照组及MS组给予同等剂量的生理盐水。治疗6周后，处死动物，取大鼠心肌组织进行实验。实验过程中。进行以下检测：(1)各组大鼠每两周测量体重及尾动脉血压1次；(2)实验前。高果糖喂养32周后，治疗第6周抽取各组大鼠颈静脉窦血2ml检测血清IL-18浓度。空腹血糖、空腹胰岛素、甘油三酯、胆固醇，并计算胰岛素抵抗指数(HOMA)：(3)分别于实验开始前、高果糖喂养32周、实验末进行超声心动图检查；(4)于实验末记录各组大鼠心电图．以心导管法进行血流动力学测定；(5)心肌组织超微结构和病理学检查：(6)应用Masson染色观察心肌纤维化程度;(7)免疫组织化学检测IL-18、I型胶原、III型胶原的蛋白表达；(8)实时定量RT-PCR法检测IL-18、I型胶原、III型胶原的raRNA表达；(9)Western-blot检测JNK和磷酸化JNK蛋白的表达；(10)凝胶电泳迁移分析检测转录因子AP-1活性。
结果
1体重、尾动脉收缩压及生化指标
自非洛地平治疗后2周，血压即降至正常水平(P<0．001)。体重在非洛地平组与MS组间无统计学意义。与MS组比较，非洛地平组血胰岛素水平及HOMA降低(P<0．001)；而甘油三酯、胆固醇、血糖在非洛地平组与MS组间无统计学意义。
2非洛地平治疗后三组大鼠血清IL-18水平变化
非洛地平治疗后6周，与MS组相比，非洛地平组大鼠血清IL-18水平降低(P<0．05)。与正常对照组相比，非洛地平组大鼠血清IL-18水平仍显著升高(p<0．001)。
3超声心动图检测
二、三尖瓣返流情况比较：非洛地平组9只大鼠，轻度二尖瓣返流1只，中度二尖瓣返流0只，重度二尖瓣返流0只，轻度三尖瓣返流1只，中度三尖瓣返流O只，重度三尖瓣返流0只，无二尖瓣、三尖瓣均存在返流，二尖瓣、三尖瓣均无返流7只。非洛地平组瓣膜返流发生率为22．22％。Fisher检验显示：与MS组相比，非洛地平组大鼠瓣膜返流发生率减少，差异有统计学意义(P<0．01)：与正常对照组相比，非洛地平组大鼠瓣膜返流发生率增加，但无统计学意义。
与MS相比，非洛地平组左室舒张末期内径(LVDd)缩小(P<0．01)，舒张末期室间隔(IVSd)、左室后壁(LVPWd)同步肥厚减轻(P<0．05)，左房内径(LA)明显缩小(P<0．001)。非洛地平E峰、Ea峰明显升高(P<0．001)，等容舒张时间(IVRT)缩短(P<0．01)，Tei指数减少(P<0．001)，左室射血分数(EF)、短轴缩短率(FS)及E／Ea峰比值无统计学意义。
4心电图变化
非洛地平组心电图异常比较轻微，9只大鼠中5只心电图基本正常，3只出现QRS波正负交替变化，无复杂的心律失常。非洛地平组心律失常发生率是33．33％。Fisher检验显示：与正常对照组相比，MS组大鼠心律失常发生率明显增加，差异有统计学意义(P<0．001)；与MS组相比，非洛地平组大鼠心律失常发生率明显减少，差异有统计学意义(P<0．01)。5血液动力学
非洛地平组较MS组左室收缩压(LVSP)、左室舒张末压(LVEDP)均明显降低(P<0．001)：左室压力最大下降速率(-dp／dt)回升(P<0．01)，-dp／dt／LVSP明显回升(P<0．001)；左室松弛时间常数(T)明显缩短(P<0．001)，且与正常对照组相比无统计学意义。．
6透射电镜观察超微结构的改变
非洛地平组心肌组织电镜观察，可见心肌细胞捧列较整齐；肌原纤维断裂，溶解现象好转；线粒体大小较一致，排列较规整；间质内胶原纤维明显减少。
7组织病理观察
HE染色显示，非洛地平组可见心肌细胞肥大程度减轻，排列规整，未见明显的肌纤维增粗、断裂，细胞核较规则，胞浆染色均匀；壁内小动脉管腔、管壁比例较正常，血管周围及肌间隙的胶原明显减少。
8 Masson染色胶原含量的检测
心肌细胞染红色，胶原纤维呈绿色。非洛地平组可见胶原纤维较MS组明显减少，排列较规整。
非洛地平对左室心肌组织胶原含量的影响如下：非洛地平组左室心肌组织间(P<0．01)和血管周围(P<0．01)胶原含量较MS组明显降低；与正常对照组相比,非洛地平组左室血管周围(P<0.05)胶原含量有升高趋势。
9免疫组织化学检测
IL-18：染色阳性信号为棕色颗粒，定位于心肌细胞胞浆内。与MS组比较，非洛地平组心肌细胞胞浆内棕色颗粒明显减少；与正常对照组比较，非洛地平组心肌细胞胞浆内棕色颗粒增多。以各视野IOD值作为反应IL-18蛋白表达的定量指标结果显示，与MS组比较，非洛地平组IOD值降低，差异有统计学意义(P<0．001)；与正常对照组比较，非洛地平组IOD值升高，差异有统计学意义(P<0．05)。
I型胶原：染色阳性信号为棕色条索状纤维，定位于心肌细胞问质和血管周围。非洛地平组心肌细胞间和血管周围棕色条索状纤维较MS组明显减少。以各视野IOD值作为反应I型胶原蛋蛋白表达的定量指标结果显示，与MS比较，非洛地平组IOD值降低，差异有统计学意义(P<0．001)；与对照组比较，非洛地平组IOD值无统计学意义。
III型胶原：染色阳性信号为棕色条索状纤维，定位于心肌细胞问质和血管周围。非洛地平组心肌细胞间和血管周围棕色条索状纤维较MS组明显减少。以各视野IOD值作为反应III型胶原蛋蛋白表达的定量指标结果显示，与MS比较，非洛地平组IOD值降低，差异有统计学意义(P<0．05)；与对照组比较，非洛地平组IOD值无统计学意义。
10实时定量RT-PCR检测各关键分子mRNA表达
与MS组比较，非洛地平组大鼠左室心肌组织IL-18mRNA表达减弱，差异有统计学意义(P<0．01)。与正常对照组比较，非洛地平组大鼠左室心肌组织IL-18mRNA表达增强，差异有统计学意义(P<0．001)。
与MS组比较，非洛地平组大鼠左室心肌组织I型胶原(P<0．001)、III型胶原(P<0．01)mRNA表达降低：与正常对照组比较，非洛地平组大鼠左室心肌组织I、III型胶原mRNA表达无统计学意义。
11各组大鼠关键分子蛋白质表达水平的比较
各组大鼠左室心肌组织JNK总蛋白水平比较：三组大鼠左室心肌组织JNK总蛋白质表达水平无统计学意义。
各组大鼠左室心肌组织磷酸化JNK蛋白表达水平比较：与MS组比较，非洛地平组磷酸化JNK蛋白含量下降，差异有统计学意义(P<0．01)；与对照组比较，非洛地平组磷酸化JNK蛋白含量略升高，差异有统计学意义(P<0．01)。
12 AP-l转录因子活性的比较
以EMSA法检测各组大鼠左室心肌组织AP．1活性，结果显示与MS组比较，非洛地平组AP-1活性减弱，具体表现为探针与核蛋白结合产生的条带变窄，条带颜色变浅；与对照组比较，非洛地平组AP-1活性稍增强，具体表现为探针与核蛋白结合产生的条带变宽，条带颜色略深。
结论
(1)非洛地平显著降低MS大鼠尾动脉收缩压和血液中胰岛素水平，改善胰岛素抵抗状态；
(2)超声心动图检测显示，非洛地平干预治疗的MS大鼠心脏E峰、Ea峰升高，IVRT缩短，Tei指数减低，心导管检查显示LVSP、LVEDP下降，-dp／dt、-dp／dt／LVSP升高，T缩短，提示非洛地平能够改善MS导致的心脏功能损伤；
(3)非洛地平干预后，Masson染色发现心肌间质及血管周围胶原含量减少；免疫组织化学染色和实时定量RT-PCR证实I型胶原和III型胶原蛋白和mRNA表达水平降低，提示非洛地平能够降低心肌纤维化；
(4)非洛地平能够降低血中IL-18浓度，降低心肌组织中IL-18蛋白和mRNA表达，提示非洛地平具有抗炎作用：
(5)非洛地平干预后心肌组织JNK磷酸化水平下降，AP-1活性减低，提示非洛地平可能通过抑制炎症因子IL-18对“JNK／AP-1”信号转导通路的作用，减轻心肌纤维化程度，改善心脏的舒张功能。
Background
Over the past two decades,a striking increase in the number of people with the metabolic syndrome(MS)worldwide has taken place.This increase is associated with the global epidemic of obesity and diabetes.With the elevated risk not only of diabetes but also of cardiovascular disease from the MS,there is urgent need for strategies to prevent the emerging global epidemic.The constellation of metabolic abnormalities includes glucose intolerance,insulin resistance,central obesity, dyslipidaemia,and hypertension.Insulin resistance is considered central to the pathophysiology of this metabolic and cardiovascular syndrome.A proinflammatory state probably contributes to the syndrome.
In clinical study,the patients with MS have cardiac diastolic dysfunction, characterized by decreased relaxation and increased stiffness.Cardiac fibrosis is the major cause of cardiac diastolic dysfunction,but the mechanism in MS is still unclear. Many studies have shown that inflammation is the third fibrosis-triggering pathway, not independent of the hormonal control and/or blood pressure,but related to the inflammatory cytokines that invariably infiltrate the overloaded cardiac tissue. Chronic subclinical inflammation is thought to be part of the MS.Serum level of high sensitive-C reactive protein(hs-CRP),interluekin-6(IL-6)and IL-18 are increased.It known that IL-18,a member of the IL-1 family of ligands,induces a variety of responses via JNK pathway.The JNK pathway is a cascade of serine/threonine kinases that transduce the signals from the cell surface to the nucleus in response to growth factors and cellular stress.AP-1,which contains the c-Jun/c-fos protein, occurs downstream of JNK.Activated JNK phosphorylates AP-1 and binds to a specific DNA sequence known as the 'AP-1 binding site'.The gene promoters related to cardiac fibrosis such as collagenⅠ,collagenⅢ,matrix metalloproteinase-2 (MMP-2)and MMP-9 all have AP-1 binding sites.This suggests that IL-18 promoted cardiac fibrosis is related to the AP-1 complex via JNK activation.
In this study,molecular biology,cellular biology,histopathology and echocardiography were used to qualitatively observe and quantitatively analysize the morphology and ultrastructure of cardiac fibrosis in MS rats.The effect of IL-18 and JNK/AP-1 pathway on the pathogenesis of cardiac fibrosis were studied.
Objectives
(1)To establish an animal model of MS used fructose;
(2)To explore the changes of myocardial histopathotogy and ultrastructure;
(3)To define the myocardial expressions of key molecules of the IL-18/JNK/ AP-1 signal pathway;
(4)To delineate the role of IL-18/JNK/AP-1 signal pathway in cardiac fibrosis used IL-18 adenovirus administered;
(5)To explore the changes of cardiac structure and function used echocardiographie and hemodynamic methods.
Methods
50 male Wistar rats were randomly assigned to two groups:control group(n=12) and MS group(n=38).Rats in the control group were allowed to drink tap water and were fed standard rat chow ad libitum.Rats in the MS group were given 10%fructose water and standard rat chow ad libitum.After being fed for 32 weeks,rats in the MS group were randomly assigned to three groups:the MS group(n=9)which were given 10%fructose water continuedly;the AdGFP group(n=9)which were administered adenovirus containing GFP through tail vein at a dose of 1×10~(10)PFU and were given 10%fructose water meanwhile;the AdIL-18 group(n=13)which were administered adenovirus containing murine IL-18 through tail vein at a dose of 1×10~(10)PFU and were given 10%fructose water meanwhile.The control and MS group were given an equal volume of normal saline solution administered.After 6-week-treatment,rats were kill and the heart was rapidly excised for further study.Following observations on animals were performed during the whole experiments:(1)Body weight(BW)and systolic blood pressure(SBP)were documented every two weeks;(2)Fast blood glutose,insulin,triglyceride,total cholesterol and serum IL-18 were analysis at baseline,32 weeks after and at the end of the study,with HOMA calculated; (3)Echocardiography was performed at baseline,32 weeks after and at the end of the study to assess cardiac structure and function;(4)In vivo hemodynamie measurements was performed to evaluate cardiac function;(5)The changes of myocardial ultrastructure and histopathology were observed;(6)Cardiac fibrosis was evaluated quantified and qualitatively by Masson staining;(7)The mRNA expression of IL-18, collagenⅠand collagenⅢwere detected by quantification real-time PCR;(8)The protein level of IL-18,collagenⅠand collagenⅢwere determined by immunohistochemistry;(9)The protein level of JNK and phosphate-JNK were determined by Western-Blot;(10)The activity of AP-1 was determined by electrophoretic mobility shift assay(EMSA).
Results
1 BW,SBP and biochemical indices measurement
MS rats exhibited significant increases in BW(P＜0.01)and SBP(P＜0.001) compared with those of controls;Serum insulin levels(P＜0.001),triglycerides (P＜0.01)and HOMA index(P＜0.001)were also increased in the MS group(P＜0.001). However,there were no differences in glucose,cholesterol levels.Compared with the AdGFP group,serum insulin levels(P＜0.001)and HOMA index(P＜0.05)were also increased in the AdIL-18 group.However,there were no differences in BW,SBP, glucose and cholesterol levels.
2 Serum IL-18 levels
Before IL-18 adenovirus administered,serum IL-18 levels of the MS rats were higher(P＜0.001)compared with those of the controls.On the first week after IL-18 adenovirus administered,serum IL-18 levels in the AdIL-18 group were higher (P＜0.001)compared with those in the AdGFP group.On the second week,serum IL-18 levels in the AdIL-18 group were four times higher(P＜0.001)than those in the AdGFP group.On the third week,serum IL-18 levels in the AdIL-18 group began decreased,but were also higher(P＜0.001)than those in the AdGFP group.On the forth week,the levels of serum IL-18 in the AdIL-18 and AdGFP groups were similar.
3 Echocardiographic detection
Mitral and tricuspid valvular regurgition detection:none of 12 rats in the control group showed mitral and tricuspid valvular regurgitation.In the MS group,mild mitral valvular regurgitation was occurred in 2 rat,moderate mitral valvular regurgitation was occurred in 1 rat,mild tricuspid valvular regurgitation was occurred in 2 rats,severe tricuspid valvular regurgitation was occurred in 1 rat;2 rats of the MS group showed both mitral and tricuspid valvular simultaneous regurgition;5 rats of the MS group did not show valvular regurgition.In the AdGFP group,mild mitral valvular regurgitation was occurred in 1 rat,moderate mitral valvular regurgitation was occurred in 2 rat,severe mitral valvular regurgitation was occurred in 1 rat;mild tricuspid valvular regurgitation was occurred in 1 rats,severe tricuspid valvular regurgitation was occurred in 1 rat,2 rats of the AdGFP group showed both mitral and tricuspid valvular simultaneous regurgition;5 rats of the AdGFP group did not show valvular regurgition.In the AdIL-18 group,mild mitral valvular regurgitation was occurred in 3 rat,moderate mitral valvular regurgitation was occurred in 2 rat,severe mitral valvular regurgitation was occurred in 1 rat;mild tricuspid valvular regurgitation was occurred in 2 rats,moderate tricuspid valvular regurgitation was occurred in 1 rat,severe tricuspid valvular regurgitation was occurred in 1 rat;3 rats of the AdIL-18 group showed both mitral and tricuspid valvular simultaneous regurgition;6 rats of the AdIL-18 group did not show valvular regurgition.There were no rats with valvular regurgitation in the control group.The incidence of valvular regurgitionin the MS group was 44.44%and 53.85%in the AdIL-18 group.Fisher analysis:compared with that of the control group,the incidence of valvular regurgitation in the MS group was significantly increased(P＜0.01).Compared with that of the MS and the AdGFP groups,the incidence of valvular regurgitation in the AdIL-18 group was significantly increased(P＜0.05).
Compared with the controls,LVEDD(P＜0.01),LAD(P＜0.001),PW and SW (P＜0.05)were greater in the MS rats.E and Ea were lower(P＜0.001)in the MS rats. IVRT(P＜0.01)and Tei index(P＜0.001)were higher in the MS rats.No significant differences in EF,FS,RWth and E/Ea ratio were found between the control group and the MS group.
There was no differences in LVEDD,LAD,PW,SW and RWth among the MS, AdGFP and AdIL-18 groups.Compared with the rats in the MS and AdGFP groups, IVRT and Tei index were higher(P＜0.01)in the AdIL-18 group.No significant differences in EF,FS and E/Ea ratio were found among the three groups.
4 Electrocardiogram recordation
Electrocardiograms(ECG)of 12 rats in the control group were all normal.6 rats of the MS group showed characteristic QRS wave interchanges and 1 rats showed complex arrhythmia.The AdGFP group was similar to the MS group.10 rats of the AdIL-18 group showed QRS wave interchanges ECG,and 2 rats showed complex arrhythmia.There were no rats with arrhythmia in the control group.The incidence of arrhythmia was 77.78%in the MS group and 92.31%in the AdIL-18 group.Fisher analysis:compared with that of the control group,the incidence of arrhythmia in the MS group was significantly increased(P＜0.001).Compared with those of the MS group and the AdGFP group,the incidence of arrhythinia in the AdIL-18 group was significantly increased(P＜0.01).
5 In vivo hemodynamic measurements
Compared with the controls,LVSP,LVEDP and tau were significantly increased (P＜0.001),and -dp/dt was more negative(P＜0.01)in the MS rats.The load-independent index of relaxation,namely,-dp/dt/LVSP,was significantly lower (P＜0.001)in the MS group.There was no significant difference in HR and +dp/dt.
Compared with the rats in the MS group and AdGFP group,LVEDP(P＜0.001) and tau(P＜0.05)were increased,and -dp/dt was more negative in the AdIL-18 group. -dp/dt/LVSP(P＜0.05)was also lower.HR and +dp/dt were similar during invasive hemodynamic study.
6 Ultrastructural change observation by transmission electron microscopy
The left ventricular myocytes from the control group arranged regularly.The pericellular membrane was uninterrupted and intact.The thick and thin myofilament arranged regularly.The sarcomere and light dark band were clear.The uniformly sized mitochondrial was abundant and showed round or oval shape.A little fibroblast and collagenous fibers distributed in extra-cellular matrix.
The left ventricular myocytes from the MS and AdGFP groups arranged irregularly.The pericellular membrane was interrupted partly.The local myofibril was disintegrated partly.The myofilament was distorted and interrupted.The sarcomere was in a bad apposition.The swelling mitochondrial increased and accumulated. Collagenous fibers proliferated in extra-cellular matrix.
The left ventricular myocytes from the AdIL-18 group arranged irregularly.The pericellular membrane was interrupted and unclear.The local myofibril was disintegrated.The myofilament was distorted and interrupted.The sarcomere was in a bad apposition.The swelling mitochondrial increased and accumulated.A lot of collagenous fibers distributed in extra-cellular matrix.
7 Pathological detecion
HE staining showed that the myocytes from the control group arranged regularly. The size of the nuclear was uniform.The staining cytoplasm was homogeneous.The myocytes from the MS group arranged irregularly.The nuclear was irregular and interrupted myofibril arranged irregularly.The AdGFP group was same with the MS group.The myocytes from the AdIL-18 group arranged more irregularly.The nuclear was more irregular and interrupted myofibril arranged more irregularly compared with the AdGFP group.
8 The detection of collagen content by Masson-staining
The myocyte was red or yellow and the collagen was green or blue by Masson-staining.The collagen tissue was appropriate arranged among cardiomyocytes in the control group.However,collagen tissue increased markedly, and disrupted in some area in the MS group.The AdGFP group was same with the MS group.Collagen tissue increased and disrupted in the AdIL-18 group compared with the AdGFP group.Quantitative analysis results:the CVF(P＜0.001)and PVCA (P＜0.01)were higher in the MS group compared with the control group.The CVF and PVCA were higher in the AdIL-18 group compared with the other three groups(P＜0.05).
Correlation analysis demonstrated that,in the MS group,serum IL-18 level was positively correlated with CVF(P＜0.05)and PCVA(P＜0.01).
9 Immunohiatochemistry detection
IL-18:The positive reaction of IL-18 protein were stained brown and localized in myocytes.Brown granules were not seen in the control group,but thick brown granules in the MS group.A lot of thicker brown granules were observed in the AdIL-18 group.Compared with the control group,the level of IL-18 IOD was increased(P＜0.001)in the MS group.Compared with the the MS and the AdGFP groups,the level of IL-18 IOD was increased(P＜0.001)in the AdIL-18 group.
CollagenⅠ:The positive reaction of collagenⅠprotein were stained brown and localized among cardiomyocytes.Weak brown collagen tissue were uniformly and sparsely distributed in the control group,but thick brown collagen tissue in the MS group.A lot of thicker brown collagen tissue were observed in the AdIL-18 group. Compared with the control group,the level of collagenⅠIOD was increased(P＜0.001) in the MS group.Compared with the MS and the AdGFP groups,the level of collagenⅠIOD was increased(P＜0.001)in the AdIL-18 group.
CollagenⅢ:The positive reaction of collagenⅢprotein were stained brown and localized among cardiomyocytes.Weak brown collagen tissue were uniformly and sparsely distributed in the control group,but thick brown collagen tissue in the MS group.A lot of thicker brown collagen tissue were observed in the AdIL-18 group. Compared with the control group,the level of collagenⅢIOD was increased(P＜0.05) in the MS group.Compared with the MS and the AdGFP groups,the level of collagenⅢIOD was increased(P＜0.05)in the AdIL-18 group.
Correlation analysis demonstrated that,in the MS group,serum IL-18 level was positively correlated with the ratio of collagenⅠandⅢprotein(P＜0.05);the IL-18 protein was positively correlated with the ratio of collagenⅠandⅢprotein(P＜0.05); the IL-18 protein was positively correlated with CVF(P＜0.05)and PCVA(P＜0.05).
10 The mRNA experession of key molecules
In comparison with the controls,the expression of IL-18(P＜0.001),collagenⅠ(P＜0.001),collagenⅢ(P＜0.01)mRNA in the MS rats were significantly higher.In comparison with the MS and the AdGFP groups,the expression of IL-18(P＜0.001), collagenⅠ,collagenⅢmRNA(P＜0.01)in the AdIL-18 group were significantly higher.
Correlation analysis demonstrated that,in the MS group,serum IL-18 level was positively correlated with the ratio of collagenⅠandⅢmRNA(P＜0.05);the IL-18 mRNA was positively correlated with the ratio of collagenⅠandⅢmRNA(P＜0.05); the IL-18 mRNA was positively correlated with CVF(P＜0.05)and PCVA(P＜0.01).
11 The protein expression of several factors
The expression of P-JNK protein in the MS group was higher significantly than that of the control group(P＜0.001).The expression of P-JNK protein in the AdIL-18 group was higher significantly than those of the MS and the AdGFP groups(P＜0.01). However,there was no significant differences in the expression of JNK protein among the four groups.
12 The activity of AP-1
The activity of AP-1 assessed by EMSA was enhanced in the MS group compared with the control group.The activity of AP-1 was enhanced in the AdIL-18 group compared with the MS and the AdGFP groups.
Conclusions
(1)An animal model of MS was established by high-fructose water.After fed fructose for 32 weeks,the MS rats had higher SBP and BW compared with the controls.And serum triglyceride concentrations,insulin levels and HOMA index were also increased.This model is valuable to investigate cardiovascular and metabolic abnormalities similar to human MS;
(2)Echocardiography showed E and Ea were lower,IVRT and Tei index were higher in the MS rats.Hemodynamics showed LVSP,LVEDP and tau were increased, and -dp/dt and -dp/dt/LVSP were lower in the MS rats.Those findings suggested the MS rats had cardiac diastolic dysfunction;
(3)The collagen content was increased in the MS rats detected by Masson-staining.The expressions of collagenⅠandⅢmRNA as well as protein detected by real-time RT-PCR and immunohistochemistry were increased.Those changes suggested the MS rats had cardiac fibrosis,which plays an important role in the pathogenesis of diastolic dysfunction;
(4)Serum IL-18 levels as well as the myocardial expression of IL-18 were increased in the MS rats.Moreover,collagen content was positively correlated with them,which suggested IL-18 may be play an important role in cardiac fibrosis.The expression of P-JNK protein and the activity of AP-1 were increased,which suggested IL-18 could induce cardiac fibrosis through JNK/AP-1 pathway;
(5)After IL-18 adenovirus was administered,cardiac fibrosis and diastolic dysfunction were more aggravating.IL-18-induced the upregulation of JNK and activiation of AP-1 represent an important mechanism leading to cardiac fibrosis and diastolic dysfunction.
Background
Metabolic syndrome (MS) has many major risks for type 2 diabetes and cardiovascular disease. The constellation of metabolic abnormalities includes glucose intolerance, insulin resistance, central obesity, dyslipidaemia, and hypertension. For the WHO and NCEP:ATPIII definitions, the prevalence of MS is 23.7% in the USA and 43.5% for those aged over 60 years. The risk for coronary heart disease and stroke was increased mreefold in subjects with the MS, and cardiovascular mortality was also markedly increased in those subjects. Cardiovascular disease and all-cause mortality are increased in men with the MS, even in the absence of baseline CVD and diabetes. This means that sub-clinical cardiovascular impairments are the fundamental reason for higher cardiovascular complications in those patients. Therefore, to finding the drug which reverses or retards cardiovascular impairments in patients with the MS is of great importance.
Calcium channel blockers (CCBs) are widely used to treat patients with hypertension and coronary heart disease. Clinical study demonstrated that CCBs decrease cardiovascular events in patients with coronary heart disease. The results of CAMELOT study were administration of amlodipine to patients with CAD and normal blood pressure resulted in reduced adverse cardiovascular events. For amlodipine, IVUS showed evidence of slowing of atherosclerosis progression. The INSIGHT study showed nifedipine was as effective as diuretic therapy in reducing cardiovascular complications in hypertensive diabetics. Many clinical studies suggest that CCBs could be considered as first-line therapy for hypertensive diabetics. Moreover, CCBs improve ventricular systolic and diastolic function in hypertensive patients. Cell culture studies have shown that CCBs attenuate the AngII-mediated increases in intracellular calcium levels of adult cardiac fibroblasts, thereby abolishing an Angll-mediated increase in collagen synthesis. Although the mechanisms underlying the direct protective effects of calcium channel blockades on cardiovascular injury are not fully understood, many studies revealed that CCBs decrease cardiovascular events is related to its anti-inflammation effects. A recent clinical study has demonstrated that amlodipine seemed to improve IR. and decrease TNF-alpha levels besides reducing BP, but further interventions are needed. Furthermore, amlodipine attenuated arteriosclerosis through inhibition of inflammatory disorders in a rat model of long-term inhibition of NO synthesis. The anti-inflammatory effects of amlodipine may be mediated by the inhibition of local factors, such as MCP-1, TGF-β1, and oxidative stress. Recently many findings suggest that some dihydropyridine-type CCBs, such as nifedipine, nilvadipine, and benidipine, have a direct beneficial effect by inhibiting the nuclear translocation and DNA-binding activity of NF-kappaB, which plays a central role in this process by regulating the expression of several pro-inflammatory genes.
From the first part of the study, we found inflammation was a key player in the pathogenesis of the MS, and was related to the ventricular remodeling of MS. IL-18 played an important role in cardiac fibrosis and diastolic dysfunction of MS through 'JNK/AP-1' pathway. Moreover, CCBs has novel anti-inflammation actions beyond blood pressure lowering. So the aim of this study was to observe the effect of felodipine on inflammation in rat model of MS and to study the possible mechanism of felodipine on cardiac structure and function of the MS rats. Objectives
(1) To evaluate the effect of felodipine on the body weight, systolic blood pressure and biochemical indices of the MS rats;
(2) To evaluate the effect of felodipine on the changes of cardiac structure and function used echocardiographic and hemodynamic methods;
(3) To explore the effect of felodipine on the changes of myocardial histopathology and ultrastructure of the MS rats;
(4) To explore the effect of felodipine on serum IL-18 levels and cardiac IL-18 expressions;
(5) To explore the effect of felodipine on IL-18/JNK/AP-1 signal pathway in cardiac fibrosis and to delineate the pathogenesis of the beneficial effect of felodipine on the cardiovascular diseases.
Methods
33 male Wistar rats were randomly assigned to two groups: control group (n=12) and MS group (n=21). Rats in the control group were allowed to drink tap water and were fed standard rat chow ad libitum. Rats in the MS group were given 10% fructose water and standard rat chow ad libitum. After being fed for 8 months, rats in the MS group were randomly assigned to two groups: the MS group (n=9) which were given 10% fructose water continuedly; the felodipine group (n=9) which were given a daily dose of 5mg/kg felodipine by intubation with a stomach tube and were given 10% fructose water meanwhile. The control and MS group were given an equal volume of normal saline solution. After 6-week-treatment, rats were kill and the heart was rapidly excised for further study. Following observations on animals were performed during the whole experiments: (1)Body weight (BW) and systolic blood pressure (SBP) were documented every two weeks; (2)Serum IL-18 level, fast blood glutose, insulin, triglyceride and cholesterol were analysis at baseline, 32 weeks after and at the end of the study, with HOMA calculated; (3)Echocardiography was performed at baseline, 32 weeks after and at the end of the study to evaluate cardiac function; (4)In vivo hemodynamic measurements was performed to evaluate cardiac function; (5)The changes of mvocardial ultrastructure and histopathologv were observed: (6)Cardiac fibrosis was evaluated quantified and qualitatively by Masson staining; (7)The mRNA expression of IL-18, collagen I and collagen III were detected by quantification real-time PCR; (8)The protein level of IL-18, collagen I and collagen III were determined by immunohistochemistry, (9)The protein level of JNK and P-JNK were determined by western blot; (10)The activity of AP-1 was determined by electrophoretic mobility shift assay (EMSA). Results
1 BW, SBP and biochemical indices measurement
The SBP of the rats treated with felodipine significant decreased (P<0.001) compared with those in the MS group. However, there were no differences in BW. Compared with the MS group, serum insulin levels and HOMA index (P<0.001) were decreased in the felodipine group. However, there were no differences in glucose and cholesterol levels among the three groups.
2 Serum IL-18 level
After six-week treatment of felodipine, serum IL-18 levels in the felodipine group were lower (P<0.05) compared with those in the MS group, however, were higher significantly (P<0.001) compared with those in the control group.
3 Echocardiographic detection
Mitral and tricuspid valvular regurgition detection: In the felodipine group, mild mitral valvular regurgitation was occurred in 1 rat, moderate mitral valvular regurgitation was occurred in 0 rat, severe mitral valvular regurgitation was occurred in 0 rat; mild tricuspid valvular regurgitation was occurred in 2 rats, moderate tricuspid valvular regurgitation was occurred in 0 rats, severe tricuspid valvular regurgitation was occurred in 0 rat; 1 rats of the felodipine group showed both mitral and tricuspid valvular simultaneous regurgition; 7 rats of the MS group did not show valvular regurgition. The incidence of valvular regurgitionin the felodipine group was 22.22%. Fisher analysis: compared with that of the MS group, the incidence of valvular regurgitation in the felodipine group was significantly decreased (P<0.01).
Compared with the MS rats, LVEDD (P<0.01), LAD (P<0.001), PW and SW (P<0.05) decreased in the rats treated with felodipine. E, Ea (P<0.001) were higher and IVRT (P<0.01), Tei (P<0.001) index were lower in the rats treated with felodipine.
4 Electrocardiogram recordation
Electrocardiograms (ECG) of 5 rats in the felodipine group were normal. 3 rats of the felodipine group showed characteristic QRS wave interchanges and none rat showed complex arrhythmia. The incidence of arrhythmia was 33.33% in the felodipine group. Fisher analysis: compared with that of the MS group, the incidence of arrhythmia in the felodipine group was significantly decreased (P<0.01). Compared with those of the control group, the incidence of arrhythmia in the felodipine group was significantly increased (P<0.001).
5 In vivo hemodynamic measurements
Compared with the MS rats, LVSP, LVEDP and tau were significantly decreased (P<0.001), and -dp/dt was more positive (P<0.01) in the rats treated with felodipine. The load-independent index of relaxation, namely, -dp/dt/LVSP, was significantly higher (P<0.001) in the rats treated with felodipine. HR and +dp/dt were similar during invasive hemodynamic study.
6 Ultrastructural change observation by transmission electron microscopy
The myocytes from the felodipine group arranged more regularly than the MS group. The phenomenon that the local myofibril was disintegrated was decreased. The mitochondrial was more regular than the MS group. The nuclear shape was more regular than the MS group. The collagenous fibers in extra-cellular matrix decreased obviously.
7 Pathological detecion
HE staining showed that the myocytes from the felodipine group arranged more regularly. The size of the nuclear was more uniform. The staining cytoplasm was more homogeneous compared with the MS group.
8 The content of collagen detection by Masson-stainingThe myocyte was red or yellow and the collagen was green or blue. The collagen tissue was more appropriate arranged among cardiomyocytes in the felodpine grouptissue was more appropriate arranged among cardiomyocytes in the felodpine group compared with the MS group. Quantitative analysis results: the CVF and PVCA were lower in the felodipine group compared with the MS group(P<0.01).
9 Immunohiatochemistry detection
IL-18: The positive reaction of IL-18 protein were stained brown and localized in myocytes. Weak brown granules were uniformly and sparsely distributed in the felodipine group, but thick brown granules in the MS group. Compared with the control group, the level of IL-18 IOD was increased (P<0.01) in the felodipine group. Compared with the MS group, the level of IL-18 IOD was decreased (P<0.001) in the felodipine group.
Collagen I: The positive reaction of collagen I protein were stained brown and localized among cardiomyocytes. Weak brown collagen tissue were uniformly and sparsely distributed in the felodipine group, but thick brown collagen tissue in the MS group. Compared with the control group, the level of collagen I IOD was increased (P<0.001) in the felodipine group. Compared with the MS group, the level of collagen I IOD was decreased (P<0.001) in the felodipine group.
Collagen III: The positive reaction of collagen III protein were stained brown and localized among cardiomyocytes. Weak brown collagen tissue were uniformly and -sparsely distributed in the felodipine group, but thick brown collagen tissue in the MS group. Compared with the control group, the level of collagen III IOD was increased in the felodipine group. Compared with the MS group, the level of collagen III IOD was decreased (P<0.01) in the felodipine group.
10 The mRN A experession of key molecules
In comparison with the controls, the expression of IL-18 (P<0.001), collagen I, collagen III mRNA (P<0.0l) in FFRs treated with felodipine were higher. In comparison with the MS group, the expression of IL-18 (P<0.001), collagen I, collagen III mRNA (P<0.001) in the felodipine group were significantly lower.
11 The expression of several factors
The expression of P-JNK protein in the felodipine group was lower significantly than that of the MS group (P<0.01). However, there was no significant differences in the expression of JNK protein among the three groups.
12 The activity of AP-1
The activity of AP-1 assessed by EMSA was enhanced in the felodipine group compared with the control group. The activity of AP-1 was weakened in the felodipine group compared with the MS group. Conclusions
(1) Felodipine treatment decrease SBP and the insulin level and improve insulin resistance;
(2) Echocardiography showed E and Ea were higher, IVRT and Tei index were lower in the rats treated with felodipine. Hemodynamics showed LVSP, LVEDP and tau were decreased, and -dp/dt and -dp/dt/LVSP were higher in the rats treated with felodipine. Those findings suggested felodipine treatment ameliorates diastolic dysfunction in the MS rats;
(3) The collagen content detected by Masson-staining was decreased in the rats treated with felodipine. The expressions of collagen I and III mRNA as well as protein detected by real-time RT-PCR and immunohistochemistry were also decreased. Those changes suggested felodipine had beneficial effects on cardiac fibrosis;
(4) Serum IL-18 levels as well as the myocardial expressions of IL-18 were decreased in the felodipine group, which suggested felodipine had anti-inflammation effect;
(5) The expression of P-JNK protein and the activity of AP-1 were decreased in the felodipine group, which suggested felodipine could decrease cardiac fibrosis and ameliorate diastolic dysfunction through IL-18/JNK/AP-1 pathway.
引文
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