高胆固醇血症大鼠心肌组织易损性增加的机制——心肌细胞膜流动性，髓过氧化物酶和部分G蛋白偶联受体自身抗体的可能作用
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摘要
研究背景
高胆固醇血症(hypercholesterolemia, HC)及其随后发生的动脉粥样硬化,是冠心病(coronary atherosclerotic heart disease, CAHD)时引起心肌缺血的始动因素之一,是缺血性心脏病(ischemic heart disease, IHD)最重要的危险因素之一,通过促进脂质堆积并进一步形成动脉粥样斑块、促进粥样硬化病变环绕造成冠脉痉挛、以及增加活性氧和活性氮产物并导致内皮机能障碍等机制在心肌缺血早期和缺血损伤的发展过程中发挥重要作用。目前,针对HC诱发的CAHD和心肌缺血损伤,临床治疗措施主要在于恢复缺血心肌血液灌流(再灌注)的同时降低血浆胆固醇,但是治疗效果明显低于无高胆固醇血症的心肌缺血患者。同时,临床流行病学研究提示,HC并发心肌缺血患者治疗过程中再灌注(心肌缺血/再灌注, myocardial ischemia/reperfusion, MI/R)损伤程度明显高于无HC心肌缺血患者,提示HC提高了心肌细胞对缺血/再灌注损伤的易感性,但具体机制复杂,仍有许多基本问题未被解决。
膜流动性是细胞膜脂质双分子层重要的动力学特征,在细胞的信号转导、物质运输、离子交换、能量传递以及酶活动性大小等方面起着重要的作用。稳定的膜流动性对于细胞内环境的维持和正常细胞功能的发挥至关重要,因此膜流动性的变化影响着许多疾病的发生和发展。在病理状态下,往往在细胞的一般形态学和生物学改变之前就能检测出膜流动性的改变;在治疗过程中,膜流动性趋向于正常的变化是反映细胞早期损伤、研究疾病过程的高灵敏度的分析指标。有研究发现,HC时红细胞膜的流动性降低,进而导致红细胞的功能改变。这些研究提示,HC时,由于脂代谢紊乱,有可能通过影响细胞膜脂类物质的比例,进而影响细胞膜的流动性。由于心肌细胞膜与红细胞膜具有同样的结构基础,那么当HC发生时,是否也会影响心肌细胞膜的流动性,进而影响心肌细胞跨膜信号的转导呢?如果这一可能存在,那么心肌细胞膜流动性的变化是否与HC致心肌细胞易损性增加有关呢?有关这些内容的研究,有助于我们从膜流动性的角度,分析HC致心肌对缺血/再灌注损伤易感性增加的机制。
髓过氧化物酶(myeloperoxidase, MPO)主要表达于中性粒细胞的过氧化物酶,是一种重要的炎性介质,也是CAHD发生发展的独立危险因子。MPO可以通过刺激血管内皮细胞凋亡,降解内皮源性舒张因子NO,降低NO生物学活性,引起内皮机能障碍,并增加粥样硬化斑块的易损性等作用促进心肌缺血的发展;活化和渗出的中性粒细胞释放的MPO催化同时由中性粒细胞和损伤组织释放的H2O2生成毒剂次氯酸和其他氧化剂(如3-氯化酪氨酸、硝基酪氨酸等),也可以导致心血管系统的氧化损伤;此外,有研究发现,在MI/R时,MPO大量聚集在血管内皮细胞与血管平滑肌之间的基底膜。我们的前期研究也发现,在MI/R时,渗出到心肌细胞的中性粒细胞发生了脱颗粒,将其中的MPO释放到心肌细胞浆中。以上研究强烈提示在MI/R时,MPO发生了游走和位移,但对其意义的解释还缺乏足够的证据。因此,MI/R时,释放入心肌细胞的MPO是否可以直接导致心肌细胞的损伤作用,是否参与HC所致的缺血/再灌注心肌易损性增加?如果是,又是如何发挥作用的,都有待于深入探究。而对这些问题的研究有助于我们进一步揭示HC缺血/再灌注心肌易损性增加的机制。
上世纪90年代以来,在心血管疾病的患者血清中检测到多种G蛋白偶联受体自身抗体,包括α1-肾上腺素受体自身抗体(α1- AA)、β1-肾上腺素受体自身抗体(β1-AA)、β3-肾上腺素受体自身抗体(β3-AA)、M2-胆碱受体自身抗体(M2-AA)和血管紧张素Ⅱ1型受体自身抗体(AT1-AA)等。研究证实,这些自身抗体都具有类激动剂样的作用,如:α1-AA长期存在可引起外周阻力升高,并导致心肌重构;β1-AA使心脏跳动频率增加,心肌收缩力增强,长期作用可发生肌原纤维的灶状溶解,诱导心肌重构发生,最终造成心功能下降;M2-AA使线粒体发生空泡样变性,导致心肌收缩力降低。这些研究提示,G蛋白偶联受体自身抗体在心血管疾病的发生发展过程中可能发挥重要作用。而HC病理发展过程中心肌超微结构有所改变并伴有血清补体升高等免疫反应,提示HC有可能诱发免疫系统功能紊乱。那么,这种免疫系统的功能紊乱,是否也会诱导G蛋白偶联受体自身抗体的产生?如果可以,这些自身抗体极有可能促使HC心肌易损性增加,进而加重心脏结构和功能的进一步恶化。因此,探查HC是否诱发G蛋白偶联受体自身抗体的产生,在心血管疾病的研究中是不容忽视的。
综上所述,本研究拟在食源性HC大鼠模型的基础上,探讨心肌细胞膜流动性、MPO和部分G蛋白偶联受体自身抗体在HC心肌易损性增加中的作用及其部分相关机制。
第一部分高胆固醇血症诱发大鼠心肌细胞膜流动性降低
目的
本部分实验在食源性HC大鼠模型上,给予吡格列酮(Pioglitazone, PIO)干预,观察并分析HC大鼠心肌细胞膜流动性和心肌损伤的变化,探讨心肌细胞膜流动性在HC大鼠心肌易损性增加中的作用及其病理机制。
材料与方法
1.材料
健康Wistar远交群大鼠,4～5 weeks,体重110±10 g,雄性。
2.实验分组
(1)食源性HC大鼠模型分组
①普通饮食大鼠组:普通饲料饲养10周;②Vehicle +高胆固醇饮食组:高胆固醇饮食饲养10周,后4周同时用色拉油(溶剂Vehicle: 0.5 ml/只)灌胃;③PIO +高胆固醇饮食组:高胆固醇饮食饲养10周,后4周同时用PIO(溶于色拉油: 10.00mg/kg?d)灌胃。
(2)在体大鼠心脏局部缺血/再灌注后心肌梗塞面积测定的实验分组
①伪手术组:仅在冠状动脉左前降支(Left Anterior Descending, LAD)下穿线,不结扎,持续24 h;②缺血/再灌注+普通饮食大鼠组:可逆性结扎LAD,心肌缺血30 min,再灌注24 h;③缺血/再灌注+ Vehicle+高胆固醇饮食组:可逆性结扎LAD,心肌缺血30 min,再灌注24 h;④缺血/再灌注+ PIO+高胆固醇饮食组:可逆性结扎LAD,心肌缺血30 min,再灌注24 h。
3.方法
(1)实验模型的建立及标本的留取
①食源性HC大鼠模型的建立及标本留取;②大鼠在体颈总动脉插管及标本留取;③单个心肌细胞的分离及标本留取;④大鼠在体心脏局部缺血/再灌注模型的建立及标本留取。
(2)检测指标的测定
①大鼠空腹血清总胆固醇(Total Cholesterol, TC)、甘油三酯(Triglyceride, TG)和低密度脂蛋白-胆固醇(Low Density Lipoprotein Cholesterol, LDL-cho)的测定;②心功能测定;③心肌细胞膜Na+-K+-ATPase活性的测定;④心肌细胞膜胆固醇含量、磷脂含量和胆固醇/磷脂比值(C/P)的测定;⑤心肌细胞膜流动性的测定;⑥心肌组织cAMP含量的测定;⑦大鼠心肌梗塞面积的测定;⑧心肌细胞膜流动性与心功能和缺血/再灌注后心肌梗塞面积的相关性分析。
结果
1.高胆固醇饮食后大鼠血脂变化和PIO对HC模型大鼠血脂的影响
1.1食源性HC大鼠模型的建立
给予高胆固醇饲料前,大鼠血脂水平无明显差异。高胆固醇饮食6周后,大鼠血脂水平明显升高,且高胆固醇饮食大鼠血清TC,TG和LDL-cho水平明显高于同期普通饮食饲养的大鼠(P<0.01);与0周大鼠血脂水平相比,高胆固醇饮食饲养10周后,大鼠血清血脂水平显著升高(TC: 3.44±0.86 mmol/l vs 1.24±0.26 mmol/l, P<0.01; TG: 1.13±0.34 mmol/l vs. 0.26±0.05 mmol/l, P<0.010; LDL-cho: 1.66±0.55 mmol/l vs 0.45±0.14 mmol/l, P<0.01),且较同期普通饮食大鼠显著升高(P<0.01)。说明食源性高胆固醇血症大鼠模型建立成功。
1.2 PIO干预后,HC大鼠血脂变化
高胆固醇饮食6周后,给予PIO干预,继续喂以高胆固醇饮食4周,大鼠血清TC,TG和LDL-cho水平较Vehicle高胆固醇饮食大鼠明显下降(TC: 2.16±0.33 mmol/l, P<0.01; TG: 0.59±0.18 mmol/l, P<0.05; LDL-cho: 1.24±0.28 mmol/l, P<0.05)。提示,PIO可以有效的改善HC大鼠血脂水平,纠正脂质紊乱。
2. HC大鼠心功能恶化,缺血/再灌注后心肌损伤增加
2.1 HC大鼠心功能障碍
与普通饮食大鼠相比,高胆固醇饮食大鼠LVSP,+dp/dtmax和-dp/dtmax显著降低(LVSP: 13.64±1.37 Kpa vs. 20.79±2.87 Kpa, P<0.01; +dp/dtmax: 537.43±102.24 Kpa/s vs. 953.38±171.12 Kpa/s, P<0.01; -dp/dtmax: 452.19±88.34 Kpa/s vs. 793.10±112.82 Kpa/s, P<0.01)。
2.2 HC大鼠缺血/再灌注后心肌梗塞面积增大
2.2.1危险区面积占左心室面积的百分比(AAR/LV比值)
以危险区面积占左心室面积的百分比(即AAR/LV比值)作为判别心肌缺血程度的指标,发现各实验组间的AAR/LV均无显著差异(P>0.05),表明各组动物模型的缺血程度大体相同,因而在此实验模型基础之上的实验结果具有可比性
2.2.2心肌梗塞面积占危险区面积的百分比(AN/AAR比值)
心肌梗塞面积不同程度的反应了心肌损伤状况以及心肌损伤易感性。高胆固醇血症大鼠心肌梗塞面积占危险区面积的百分比显著高于普通饮食大鼠缺血/再灌注组(56.81±6.45% vs. 35.25±2.52%, P<0.01)。
以上结果提示,HC可能引起大鼠心功能障碍,尤其是收缩功能受损,并可能进一步导致HC大鼠心肌易损性的升高。
3. HC对心肌细胞膜的影响
3.1 HC降低心肌细胞膜Na+-K+-ATPase活性
Na+-K+-ATPase仅存在于细胞膜,而不存在于细胞器膜,其活性通常作为细胞膜的标志。与普通饮食大鼠相比,HC大鼠Na+-K+-ATPase活性明显降低(5.99±1.69μmol Pi/mgprotein/hour vs. 7.88±1.25μmol Pi/mg protein/hour, P<0.01)。
3.2 HC大鼠心肌细胞膜流动性降低
与普通饮食大鼠相比,HC大鼠心肌细胞膜DPH荧光偏振度(P)和微粘度(η)明显升高(P: 0.34±0.07 vs. 0.24±0.05, P<0.01;η: 7.99±1.37 vs. 2.41±0.59, P<0.01),说明HC大鼠心肌细胞膜流动性下降。
3.3 HC大鼠心肌细胞膜C/P升高
与普通饮食大鼠相比,HC大鼠心肌细胞膜C/P升高(0.44±0.04 vs. 0.33±0.03, P<0.01)。
上述结果首先通过Na+-K+-ATPase活性的检测说明了心肌组织提取物为心肌细胞膜,其次发现HC大鼠心肌细胞膜流动性降低以及某些膜蛋白功能活性改变,而这种变化可能是由于HC大鼠心肌细胞膜脂质比例的失调而造成的。
4. HC降低心肌组织cAMP含量
为了进一步的阐述HC对心肌细胞易损性的影响,本实验检测了心肌组织cAMP含量,一种膜受体信号通路的下游产物。与普通饮食大鼠相比,HC大鼠心肌组织cAMP含量明显减少(53.77±22.17 pmol/mg protein vs. 127.50±19.72 pmol/mg protein, P<0.01)。进一步提示,HC可能导致心肌功能障碍,并引起心肌易损性升高。
5. PIO干预HC大鼠后,减轻HC大鼠心肌损伤,改善HC引起的大鼠心肌细胞膜的变化,恢复心肌组织cAMP含量
5.1 PIO改善HC大鼠心功能
与单纯的高胆固醇饮食大鼠相比,PIO干预HC大鼠后发挥显著的心血管保护效应,改善大鼠心功能,大鼠心肌收缩功能(LVSP: 20.40±2.83 Kpa, P<0.01; +dp/dtmax : 827.56±172.49 Kpa/s, P<0.01;–dp/dtmax: 684.01±113.43 Kpa/s, P<0.01)有所恢复。
5.2 PIO减小HC大鼠缺血/再灌注后心肌梗塞面积
AAR/LV %在各实验组大鼠无统计学差异(P>0.05)。与单纯的高胆固醇饮食大鼠相比,PIO干预HC大鼠后,大鼠缺血/再灌注后心肌梗塞面积明显减小(39.82±2.74, P<0.01)。
以上结果证实,PIO干预HC大鼠可以改善大鼠心功能,减小大鼠心肌梗塞面积;提示,PIO干预可能减小HC大鼠心肌对损伤的敏感性。
5.3 PIO增加HC大鼠心肌细胞膜Na+-K+-ATPase活性
与单纯高胆固醇饮食大鼠相比,PIO干预HC大鼠后,大鼠心肌细胞膜Na+-K+-ATPase活性明显增加(7.03±1.42μmol Pi/mg protein/hour, P<0.01)。
5.4 PIO改善HC大鼠心肌细胞膜流动性
与单纯高胆固醇饮食大鼠相比,PIO干预HC大鼠后,大鼠心肌细胞膜荧光偏振度(0.26±0.06, P<0.01)和微粘度(3.29±0.63, P<0.01)显著降低。说明PIO干预后HC大鼠心肌细胞膜流动性明显增加。
5.5 PIO降低HC大鼠心肌细胞膜C/P
与单纯高胆固醇饮食大鼠相比,PIO干预HC大鼠后,大鼠心肌细胞膜C/P明显降低(0.40±0.04, P<0.05)。
以上结果提示,PIO有可能通过改善HC大鼠心肌细胞膜脂质成分来恢复心肌细胞膜流动性,从而改善HC大鼠心肌细胞膜蛋白功能活性和心功能,降低HC大鼠心肌对损伤的敏感性。
5.6 PIO增加HC大鼠心肌组织cAMP含量
与单纯高胆固醇饮食大鼠相比,PIO干预HC大鼠后,大鼠心肌组织cAMP含量显著增加(102.05±25.51, P<0.05)。进一步说明,PIO可能在HC大鼠发挥心脏保护作用,减轻HC大鼠心肌对损伤的敏感性。
6.心肌细胞膜流动性与心功能和缺血/再灌注后心肌梗塞面积具有相关性
为了更深入的探讨心肌细胞膜流动性的改变在HC大鼠心功能下降和心肌损伤敏感性增加中的作用,以及PIO对HC大鼠心脏的保护效应是否涉及心肌细胞膜流动性,本实验对心肌细胞膜流动性和心功能、缺血/再灌注后心肌梗塞面积进行了相关性分析。
6.1心肌细胞膜流动性与心肌收缩功能呈正相关
通过对心肌细胞膜DPH荧光偏振度和心功能(LVSP,+dp/dtmax, -dp/dtmax)之间的直线相关分析,本实验发现心肌收缩功能与心肌细胞膜DPH荧光偏振度之间呈负相关(r=-0.191, P<0.05; r=-0.323, P<0.01; r=- 0.322, P<0.01),说明心肌细胞膜流动性与心肌收缩功能呈正相关。
6.2心肌细胞膜流动性与缺血/再灌注后心肌梗塞面积呈负相关
心肌细胞膜DPH荧光偏振度与缺血/再灌注后心肌梗塞面积直线相关分析显示,DPH荧光偏振度和心肌梗塞面积呈正相关(r=0.599, P<0.01)。说明心肌细胞膜流动性与缺血/再灌注后心肌梗塞面积呈负相关。
以上结果提示,HC诱发的大鼠心功能障碍和心肌损伤易感性增加可能与HC大鼠心肌细胞膜流动性下降有光,而PIO干预也可能通过或部分通过改善和恢复HC大鼠心肌细胞膜流动性来防御HC引起的大鼠心脏机能障碍。
小结
1. HC引起大鼠心功能下降,缺血/再灌注后心梗面积明显增大,提示在HC时心肌损伤易感性增加;
2. HC导致心肌细胞膜Na+-K+-ATPase活性降低,膜流动性下降,膜C/P比值升高,心肌细胞cAMP含量降低,而且膜流动性与心功能和心梗面积具有相关性,提示HC可能通过膜流动性降低,进而心肌细胞跨膜信号转导功能受阻,最终导致HC情况下心功能受神经体液调控能力下降而恶化心肌损伤;
3. PIO干预显著抑制HC诱导的心肌细胞膜C/P升高和膜流动性的下降以及膜Na+-K+-ATPase活性降低,以及细胞cAMP含量的减少,恢复心功能和减小缺血/再灌注后心梗面积。进一步提示HC大鼠心肌细胞膜流动性改变可能是HC大鼠心肌易损性增加的原因之一。
第二部分髓过氧化物酶对高胆固醇血症大鼠缺血/再灌注心肌易损性增加的作用
目的
本部分实验通过观察HC大鼠缺血/再灌注心肌组织MPO分布和活性的变化,以及这一变化与HC大鼠MI/R损伤之间的关系,说明MPO在HC大鼠缺血/再灌注心肌易损性增加中所发挥的作用并探讨其所涉及的部分损伤机制(如:NO有效利用率的下降等)。
材料和方法
1.材料
(1)健康Wistar远交群大鼠,4～5 weeks体重110±10 g,雄性;(2)H9c2(2-1)细胞株。
2.实验分组
(1)食源性HC大鼠模型分组
①普通饮食大鼠组:普通饲料饲养10周;②Vehicle +高胆固醇饮食组:高胆固醇饮食饲养10周,第9周开始10% DMSO(0.5 ml/只?d)腹腔注射一周;③ABAH+高胆固醇饮食组:高胆固醇饮食饲养10周,第9周开始ABAH(特异性MPO抑制剂,25.00mg/kg?d)腹腔注射一周。
(2)在体大鼠心脏局部缺血/再灌注后心肌梗塞面积测定的实验分组
①伪手术组:仅在冠状动脉左前降支(Left Anterior Descending, LAD)下穿线,不结扎,持续3/24 h;②缺血/再灌注+普通饮食大鼠组:普通饮食10周末,可逆性结扎LAD,心肌缺血30 min,再灌注3/24 h;③缺血/再灌注+ Vehicle+高胆固醇饮食组:高胆固醇饮食10周末,Vehicle大鼠可逆性结扎LAD,心肌缺血30 min,再灌注3/24 h;④缺血/再灌注+ ABAH +高胆固醇饮食组:高胆固醇饮食10周末,ABAH干预大鼠可逆性结扎LAD,心肌缺血30 min,再灌注3/24 h。
(3)H9c2(2-1)细胞株实验分组
①常氧组(Control):置于95%空气,5%CO2持续培养5 h;②ABAH+常氧组:加入100μΜABAH,摇匀后将细胞置于95%空气,5%CO2持续培养5 h;③缺氧/复氧组(H/R):置于充满95% N2和5% CO2的三气培养箱内缺氧培养3 h,然后再放入充满95%空气、5% CO2的常氧培养箱内复氧2 h;④MPO+缺氧/复氧组:加入0.1 u/ml MPO,摇匀后将细胞置于充满95% N2和5% CO2的三气培养箱内缺氧培养3 h,然后再放入充满95%空气、5% CO2的常氧培养箱内复氧2 h;⑤ABAH+缺氧/复氧组:加入100μΜABAH,摇匀后将细胞置于充满95% N2和5% CO2的三气培养箱内缺氧培养3 h,然后再放入充满95%空气、5% CO2的常氧培养箱内复氧2 h;⑥ABAH+MPO+缺氧/复氧组:先加入100μΜABAH摇匀后将细胞置于95%空气,5%CO2培养30 min后,再加入0.1 u/ml MPO,摇匀后将细胞置于充满95% N2和5% CO2的三气培养箱内缺氧培养3 h,然后再放入充满95%空气、5% CO2的常氧培养箱内复氧2 h。
3.方法
(1)实验模型的建立及标本的留取
①食源性HC大鼠模型的建立及标本的留取;②大鼠在体心脏局部缺血/再灌注模型的建立及标本的留取;③H9c2(2-1)细胞缺氧/复氧模型的建立及标本的留取。
(2)检测指标的测定
①大鼠空腹血清总胆固醇(Total Cholesterol, TC)、甘油三酯(Triglyceride, TG)和低密度脂蛋白-胆固醇(Low Density Lipoprotein Cholesterol, LDL-cho)的测定;②血清和培养液上清肌酸激酶(Creatine Kinase, CK)含量的测定;③血清和培养液上清乳酸脱氢酶(Lactate Dehydrogenase, LDH)含量的测定;④DNA原位末端缺口标记法(TUNEL)检测心肌细胞凋亡;⑤心肌细胞caspase-3相对活性检测;⑥大鼠缺血/再灌注后心肌梗塞面积的测定;⑦大鼠心功能监测;⑧免疫组织化学染色检测心肌组织MPO分布;⑨心肌组织MPO活性检测;⑩心肌组织MPO活性与血清CK含量、LDH含量、凋亡指数、caspase-3活性、心肌梗塞面积和心功能的相关性分析;○11心肌组织一氧化氮(nitric oxide, NO)含量测定;○12放射性免疫试剂盒测定心肌组织cGMP含量;○13Western-blot检测心肌组织一氧化氮合酶(nitric oxide synthase, NOS)的蛋白表达;○14实时定量PCR法检测心肌组织NOS的mRNA表达;○15细胞计数Kit-8检测H9c2(2-1)细胞增殖。
结果
1.食源性HC大鼠模型的建立
给予高胆固醇饮食饲养前(0周),各组大鼠的血脂水平无明显差异。高胆固醇饮食饲养10周后,与0周(TC: 1.30±0.32 mmol/L、TG: 0.29±0.16 mmol/L和LDL-cho: 0.39±0.12 mmol/L)和同期普通饮食饲养大鼠(TC; 1.44±0.14 mmol/L、TG: 0.26±0.05 mmol/L和LDL-cho: 0.45±0.14 mmol/L)相比,HC大鼠血脂水平显著升高(TC; 3.01±0.75 mmol/L、P<0.01; TG: 0.88±0.35 mmol/L, P<0.01和LDL-cho: 1.53±0.65 mmol/L, P<0.01),说明食源性HC大鼠模型建立成功。ABAH干预后,与Vehicle组相比血脂水平未发生明显改变。
2. HC大鼠缺血/再灌注心肌易损性增加
2.1 HC大鼠MI/R后心肌损伤加重
2.1.1 HC大鼠MI/R后血清CK含量增加
与伪手术组相比,MI/R组大鼠血清CK含量明显增高(0.17±0.01 U/ml vs. 0.05±0.02 U/ml, P<0.01)。与普通饮食大鼠MI/R组相比,HC大鼠MI/R组血清CK含量显著升高(0.70±0.08 U/ml, P<0.01)。
2.1.2 HC大鼠MI/R后血清LDH含量增加
与伪手术组相比,普通饮食MI/R组大鼠血清LDH含量明显增高(3091.59±33.69 U/L vs. 1854.45±422.97 U/L, P<0.01)。与普通饮食大鼠MI/R组相比,HC大鼠MI/R组血清LDH含量显著升高(3596.18±99.81 U/L, P<0.01)。
2.2 HC大鼠缺血/再灌注后心肌细胞凋亡增加
2.2.1 TUNEL检测
普通饮食大鼠缺血/再灌注危险区心肌组织凋亡阳性核显著增多,凋亡指数较伪手术组显著升高(16.07±1.02% vs. 1.44±0.14%,P<0.01)。与普通饮食缺血/再灌注大鼠相比,HC大鼠缺血/再灌注危险区心肌组织凋亡指数明显升高(22.63±1.02%, P<0.01)。
2.2.2 caspase-3活性分析
与伪手术组大鼠相比,普通饮食大鼠缺血/再灌注危险区心肌组织caspase-3活性显著升高(6.66±0.61 vs. 1.00±0.08,P<0.01);HC大鼠缺血/再灌注危险区心肌组织caspase-3活性较普通饮食大鼠明显升高(12.29±0.92,P<0.01)。
2.3 HC大鼠缺血/再灌注后心肌梗塞面积增加
各组大鼠危险区面积占左心室面积的百分比(AAR/LV比值)无统计学差异(P>0.05),表明在进行缺血/再灌注实验过程中,各组动物模型的缺血程度大致相当,排除了由此而造成的误差。实验结果显示,与普通饮食大鼠缺血/再灌注组相比,HC大鼠缺血/再灌注后心肌梗塞面积显著增大(56.05±3.91% vs 34.90±2.52%, P<0.01)。
2.4 HC大鼠MI/R心功能恶化
大鼠MI/R 3 h末,与伪手术组相比,普通饮食大鼠MI/R组LVSP、±dP/dtmax显著下降(LVSP: 15.12±1.38 Kpa vs. 18.28±3.02 Kpa, P<0.05; +dp/dtmax: 610.43±110.26 Kpa/s vs. 785.22±162.22 Kpa/s, P<0.01; -dp/dtmax: 500.57±116.20 Kpa/s vs. 667.00±234.83 Kpa/s, P<0.05);HC大鼠MI/R组LVSP、±dP/dtmax较普通饮食大鼠降低明显(LVSP: 9.95±1.85 Kpa, P<0.01; +dp/dtmax: 386.43±100.76 Kpa/s, P<0.01; -dp/dtmax: 264.53±98.76 Kpa/s, P<0.01)。
以上的结果显示,给予MI/R实验干预后,与普通饮食大鼠相比,HC大鼠血清CK含量和LDH含量增加,危险区心肌组织凋亡指数和caspase-3活性升高,心肌梗塞面积增大,心功能(LVSP和±dP/dtmax)降低。这些结果说明,HC大鼠MI/R损伤加重,提示HC大鼠缺血/再灌注心肌易损性增加。
3. HC大鼠缺血/再灌注心肌组织MPO的变化
3.1 HC大鼠缺血/再灌注心肌组织MPO活性分析
与伪手术组相比,MI/R引起普通饮食大鼠缺血/再灌注心肌组织MPO活性显著增加(13.48±4.58 U/g protein vs. 8.29±2.80 U/g protein, P<0.05);HC大鼠缺血/再灌注心肌组织MPO活性进一步升高(19.44±2.35 U/g protein, P<0.05)。
3.2 HC大鼠缺血/再灌注心肌组织MPO的分布
大鼠心肌组织MPO免疫组化观察结果显示,心脏局部缺血/再灌注后,大鼠非缺血区心肌组织仅可见少量散在的MPO免疫染色,而在缺血/再灌注区血管组织和心肌组织内可见大量密集的MPO染色。这一结果提示,缺血/再灌注心脏浸润的中性粒细胞释放大量MPO,这些MPO可能通过主动或被动的方式侵入缺血/再灌注心肌细胞,进而通过多种机制引起心肌细胞损伤。
而且,与普通饮食大鼠相比,HC大鼠缺血/再灌注区心肌组织MPO免疫染色显著增强,这与上述结果显示的HC进一步增加缺血/再灌注心肌组织MPO活性相一致。
以上结果说明,HC大鼠缺血/再灌注区心肌组织中MPO含量和活性升高,提示MPO可能参与了HC大鼠缺血/再灌注心肌损伤易感性的增加。
4.缺血/再灌注心肌组织MPO活性与缺血/再灌注心肌损伤之间的相关性分析
为了探讨MPO是否参与了HC大鼠缺血/再灌注心肌损伤易感性的增加,本实验采用SPSS 13.0软件对有关实验数据进行了以下相关性分析。
4.1缺血/再灌注心肌组织MPO活性与缺血/再灌注后大鼠血清CK含量和LDH含量呈正相关(CK: r = 0.618 , P<0.01; LDH: r = 0.64, P<0.01)。
4.2缺血/再灌注心肌组织MPO活性与缺血/再灌注心肌组织凋亡指数和caspase-3活性呈正相关(凋亡指数:r = 0.651, P<0.01; caspase-3: r = 0.619, P<0.01)。
4.3缺血/再灌注心肌组织MPO活性与缺血/再灌注后心肌梗塞面积呈正相关(r = 0.663, P< 0.01)。
4.4缺血/再灌注心肌组织MPO活性与大鼠缺血/再灌注后心肌收缩功能(LVSP和±dP/dtmax)呈负相关(LVSP: r = -0.555, P<0.01; +dP/dtmax: r = -0.794, P< 0.01; -dP/dtmax: r =-0.748, P<0.01)。
以上结果说明,大鼠缺血/再灌注心肌组织MPO活性与HC大鼠缺血/再灌注心脏损伤加重密切相关;提示,HC大鼠缺血/再灌注心肌组织MPO的变化可能与HC大鼠缺血/再灌注心肌易损性的增加有直接关系。
5. MPO抑制剂干预后,HC大鼠缺血/再灌注心肌组织MPO和心脏损伤的变化
为了进一步探讨MPO是否参与了HC大鼠MI/R心肌易损性的增加,二者之间是否存在直接作用,本实验应用MPO抑制剂ABAH在体干预HC大鼠,观察相应变化。
5.1 ABAH对H9c2(2-1)细胞的影响
ABAH干预后,H9c2(2-1)细胞常氧培养细胞存活率与常氧对照组组相比(0.98±0.01 vs. 1.01±0.05),无统计学差异;给予缺氧/复氧干预后,ABAH组H9c2(2-1)细胞存活率与缺氧/复氧组相比(0.63±0.06 vs. 0.60±0.09),也无统计学意义。说明,ABAH本身对心肌细胞以及给予缺氧/复氧干预后心肌细胞无影响,排除了由药物干预而形成的实验误差。
5.2 ABAH干预后,HC大鼠缺血/再灌注心肌组织MPO分布和活性的变化
与HC大鼠MI/R组相比,ABAH干预HC大鼠后,缺血/再灌注心肌组织MPO免疫染色明显减少,MPO活性显著降低(13.66±2.63 U/g protein, P<0.05)。
5.3 ABAH干预后,HC大鼠缺血/再灌注心肌损伤减轻
与HC大鼠MI/R组相比,ABAH干预HC大鼠后,缺血/再灌注后血清CK含量(0.31±0.06 U/ml, P<0.01)和LDH含量(3286.65±40.04 U/L, P<0.01)明显减少。
5.4 ABAH干预后,HC大鼠缺血/再灌注心肌细胞凋亡减轻
与HC大鼠MI/R组相比,ABAH干预HC大鼠后,缺血/再灌注心肌组织细胞凋亡指数(18.55±1.29%, P<0.01)和caspase-3活性(7.97±0.29, P<0.01)明显降低。
5.5 ABAH干预后,HC大鼠缺血/再灌注后心肌梗塞面积减小
经测定,各组大鼠危险区面积占左心室面积的百分比(AAR/LV比值)无统计学差异(P>0.05)。与HC大鼠MI/R组相比,ABAH干预HC大鼠后,缺血/再灌注后心肌梗塞面积明显减小(38.89±2.47%, P<0.01)。
5.6 ABAH干预后,HC大鼠缺血/再灌注心功能障碍减轻
与HC大鼠MI/R组相比,ABAH干预HC大鼠后,缺血/再灌注3 h末LVSP、±dP/dtmax有所恢复(LVSP: 13.73±2.20 Kpa, P<0.05; +dP/dtmax: 564.00±128.56 Kpa/s, P<0.05; -dP/dtmax: 444.00±96.08 Kpa/s, P<0.05)。
以上结果显示:首先,ABAH药物本身对心肌细胞基本无影响;其次,ABAH干预HC大鼠后,大鼠缺血/再灌注区心肌组织MPO分布和活性均减少;而且,ABAH干预HC大鼠后,HC大鼠MI/R损伤减轻。进一步提示,HC大鼠缺血/再灌注区心肌组织MPO分布与活性可能与HC大鼠缺血/再灌注心肌损伤易感性的增加有密切关系,但二者之间是否具有直接关系仍有待于进一步证实。
6. MPO对H9c2(2-1)细胞的影响
为了排除在体神经、体液调节等因素对实验结果的多方面的影响,本实验利用H9c2(2-1)细胞来观察MPO对心肌细胞的直接效应。
6.1 H9c2(2-1)细胞缺氧/复氧模型建立成功
与常氧对照组(Control)相比(CK: 0.02±0.01 U/ml; LDH: 33.11±21.18 U/L),缺氧/复氧(H/R)组细胞培养液上清中CK含量和LDH含量显著升高(0.08±0.03 U/ml, P<0.01; 856.22±28.91 U/L, P<0.01)。说明,成功地建立了H9c2(2-1)细胞缺氧/复氧损伤模型
6.2 MPO加重H9c2(2-1)细胞缺氧/复氧损伤
与H/R组相比,0.1 u/ml MPO+H/R组培养液上清CK含量和LDH含量均明显升高(0.42±0.11 U/ml, P<0.01; 1362.78±106.89 U/L, P<0.01)。说明,MPO不仅可以引起心肌细胞的损伤,而且可以进一步加重心肌细胞缺氧/复氧损失。提示, MPO分布与活性可能直接参与了心肌细胞的损伤,并进一步加重了缺氧/复氧心肌细胞损伤。
6.3 ABAH干预后,H9c2(2-1)细胞缺氧/复氧损伤的变化
与0.1 u/ml MPO+H/R组相比,ABAH+0.1 u/ml MPO+H/R组培养液上清CK含量和LDH含量均明显减少(0.13±0.07 U/ml, P<0.01; 880.81±80.86 U/L, P<0.01)。更进一步提示,MPO的分布和活性可能直接参与了心肌细胞的损伤,而且可能与HC大鼠缺血/再灌注心肌易损性的增加有直接关系。
7. HC大鼠缺血/再灌注心肌组织NO-cGMP信号通路受损
7.1 HC大鼠缺血/再灌注心肌组织NOx含量增加
与普通饮食大鼠MI/R组相比,HC大鼠缺血/再灌注心肌组织NOx含量显著增加(972.78±77.45μmol/g protein vs. 421.44±58.96μmol/g protein, P<0.01)。ABAH干预后,可以有效的减少HC大鼠缺血/再灌注心肌组织NOx含量(615.76±50.54μmol/g protein, P<0.01),但未完全恢复至正常生理状态。
7.2 HC大鼠缺血/再灌注心肌组织cGMP含量降低
经测定,与伪手术组大鼠相比,普通饮食大鼠缺血/再灌注心肌组织cGMP含量明显减少(31.53±11.37 pmol/mg protein vs. 74.22±22.23 pmol/mg protein, P<0.01);与普通饮食MI/R组大鼠相比,HC大鼠缺血/再灌注心肌组织cGMP含量明显减少(7.35±2.47 pmol/mg protein, P<0.05);ABAH干预后,与HC + MI/R组大鼠相比,HC大鼠缺血/再灌注心肌组织cGMP含量有所恢复(28.84±9.08 pmol/mg protein, P<0.01)。
7.3 HC大鼠缺血/再灌注心肌组织NOS mRNA表达和蛋白表达的变化
7.3.1 HC大鼠缺血/再灌注心肌组织NOS mRNA表达的变化
与伪手术组相比,普通饮食大鼠MI/R组危险区心肌组织iNOS mRNA表达明显升高(4.80±1.73 vs. 1±0.70, P<0.01);与普通饮食大鼠MI/R组相比,HC大鼠MI/R组危险区心肌组织心肌组织iNOS mRNA表达明显升高(10.09±2.15, P<0.01);ABAH干预后,HC大鼠MI/R危险区心肌组织iNOS mRNA表达较HC大鼠MI/R组降低(3.41±1.50, P<0.01)。
与普通饮食未手术大鼠相比,HC未手术大鼠心肌eNOS mRNA表达明显降低(0.87±0.02 vs. 1.00±0.04, P<0.01)。
各组大鼠心肌组织nNOS mRNA表达无统计学差异。
7.3.2 HC大鼠缺血/再灌注心肌组织NOS蛋白表达的变化
与伪手术组相比,普通饮食大鼠MI/R组危险区心肌组织iNOS蛋白表达明显升高(2.22±0.44 vs.1.00±0.26, P<0.05);与普通饮食大鼠MI/R组相比,HC大鼠MI/R组危险区心肌组织心肌组织iNOS蛋白表达明显升高(3.17±0.93, P<0.05);ABAH干预后,HC大鼠MI/R危险区心肌组织iNOS蛋白表达较HC大鼠MI/R组降低(2.28±0.46, P<0.05)。
与普通饮食未手术大鼠相比,HC未手术大鼠心肌eNOS蛋白表达明显降低(0.36±0.14vs. 1.00±0.37, P<0.05)。
各组大鼠心肌组织nNOS蛋白表达无统计学差异。
以上结果显示,普通饮食大鼠缺血/再灌注心肌组织NOx含量增加,HC大鼠缺血/再灌注后危险区心肌组织NOx含量进一步增加;普通饮食大鼠缺血/再灌注心肌组织cGMP含量减少,HC大鼠缺血/再灌注后危险区心肌组织cGMP含量进一步减少;普通饮食大鼠缺血/再灌注心肌组织iNOS mRNA表达和蛋白表达均升高,HC大鼠缺血/再灌注后危险区心肌组织iNOS mRNA表达和蛋白表达均进一步升高;HC大鼠心肌组织eNOS mRNA表达和蛋白表达均降低;各组nNOS mRNA表达和蛋白表达无统计学差异。提示,HC大鼠MI/R后,虽然危险区心肌组织NOx含量大量增加,但NO生物利用度降低,NO/cGMP信号通路可能受损,应用MPO抑制剂干预后,NO/cGMP信号通路损伤有所减轻;MPO可能通过降低心肌组织NO的生物利用度、损伤NO/cGMP信号通路,从而部分参与大鼠缺血/再灌注心肌损伤,以及在HC大鼠缺血/再灌注心肌易损性增加中发挥作用。而且,HC大鼠缺血/再灌注心肌组织大量生成的NO可能来自于iNOS催化生成。
小结
1. HC大鼠心肌组织MPO含量和活性明显升高,提示MPO活性的改变可能是HC使缺血/再灌注后心肌损伤恶化的原因之一;
2. HC大鼠缺血/再灌注后心肌组织MPO含量和活性显著升高的同时NO/cGMP通路受损,NO生物利用率下降,进一步提示我们HC缺血/再灌注后心肌损伤易感性增加可能部分是通过MPO活性的改变影响NO-cGMP信号通路来实现。
第三部分高胆固醇血症诱导大鼠血清α1-、β1-肾上腺素受体和M2-胆碱受体自身抗体的产生
目的
本部分实验首先选择α1 -AA、β1 -AA、β3 -AA、M2 -AA和AT1-AA阴性的健康雄性大鼠建立食源性HC大鼠模型;进而观察食源性HC是否会诱发G蛋白偶联受体自身抗体的产生;如果会,则进一步了解自身抗体的产生情况及其作用。
材料和方法
1.材料
健康Wistar远交群大鼠,4～5 weeks体重110±10 g,雄性。
2.实验分组
食源性HC大鼠模型分组
①普通饮食大鼠组:普通饲料饲养10周;②高胆固醇饮食6周组:高胆固醇饮食饲养6周;③高胆固醇饮食10周组:高胆固醇饮食饲养10周。
3.方法
(1)实验模型的建立及标本的留取
食源性HC大鼠模型的建立及标本的留取
(2)检测指标的测定
①大鼠空腹血清总胆固醇(Total Cholesterol, TC)、甘油三酯(Triglyceride, TG)和低密度脂蛋白-胆固醇(Low Density Lipoprotein Cholesterol, LDL-cho)的测定;②心肌组织石蜡切片的HE染色;③心肌组织超微结构电镜观察;④血清受体自身抗体(α1- AA、β1- AA、β3 -AA、M2- AA和AT1-AA)的测定;⑤流式细胞仪检测大鼠脾脏淋巴细胞CD4+/CD8+和淋巴细胞含量。
(3)肽段合成及自身抗体的测定:
5种抗原肽段由吉尔生化上海有限公司合成,分别相当于α1- AR、β1- AR、β3 -AR、M2- AR和AT1-AR细胞外第二环氨基酸序列的特异性抗原决定簇,合成肽段纯度为90%~95%。用ELISA法检测血清中各自身抗体的含量。
结果
1食源性HC大鼠模型建立成功
高胆固醇饮食饲养前,各组大鼠血脂水平无明显差异。高胆固醇饮食6周后,大鼠血脂水平显著升高(TC: 2.64±0.22, P<0.01; TG: 0.89±0.33, P<0.01; LDL-cho: 1.06±0.23, P<0.01)。高胆固醇饮食10周后血脂水平进一步升高(TC: 3.46±0.68 mmol/l, P<0.01; TG: 1.17±0.39 mmol/l, P<0.01; LDL-cho: 1.67±0.35 mmol/l, P<0.01)。说明食源性HC大鼠模型建立成功。
2. HC大鼠血清α1-AA、β1-AA和M2-AA的OD值升高
与正常饮食大鼠相比,高胆固醇饮食6周大鼠血清α1-AA(0.60±0.05 vs 0.27±0.06,P<0.01)、β1-AA(0.57±0.06 vs 0.18±0.02,P<0.01)和M2-AA(0.53±0.07 vs 0.16±0.04,P<0.01)的OD值明显升高,高胆固醇饮食10周大鼠血清抗体OD值有略有下降的趋势。HC大鼠血清β3-AA和AT1-AA与正常饮食大鼠相比无明显改变。提示,HC可能诱发部分G蛋白偶联受体自身抗体的产生。
3. HC大鼠心肌组织形态学观察
3.1 HC大鼠心肌组织病理形态学观察
普通饮食大鼠,心肌纤维完整,排列整齐,呈束状,结构清楚,着色均匀,细胞核形态正常,细胞膜完整,细胞没有水肿,无变性坏死,心肌间质未见炎性细胞浸润。高胆固醇饮食6周和10周的HC大鼠心肌形态发生了改变,同时伴随程度不同的炎性细胞浸润。
3.2 HC大鼠心肌组织超微结构观察
普通饮食大鼠,心肌肌原纤维排列整齐,肌节长短一致,明带、暗带清晰可见;线粒体丰富,结构清晰完整,大小形态正常,无肿胀,嵴无断裂,嵴排列整齐,线粒体间糖原颗粒丰富;毛细血管内皮细胞结构正常,细胞膜完整。
高胆固醇饮食6周HC大鼠心肌细胞间质微血管官腔狭窄,内皮细胞水肿,胞质中胞饮泡增多、散在较多的脂泡,肌浆网扩张,可见等电子密度云絮状物质,个别细胞可见肌丝溶解。高胆固醇饮食10周HC大鼠心肌细胞核染色质小灶性溶解,胞质内脂泡增多,可见大面积脂质区域,部分细胞肌丝溶解面积较大,肌丝疏松散在,肌浆网明显扩张,个别心肌细胞膜溶解,心肌细胞间质中微血管内皮细胞胞饮泡增生,血管腔内散在血小板和红细胞。
以上结果说明HC大鼠心肌细胞结构受损,超微结构发生改变,提示HC可能造成大鼠心肌组织超微结构改变,并可能引起大鼠心肌组织重构,激活HC大鼠免疫系统,从而诱发HC大鼠G蛋白偶联受体自身抗体的产生。
4. HC大鼠脾脏淋巴细胞CD4~+/CD8~+升高,淋巴细胞含量升高
脾脏是人体免疫系统最重要的免疫器官之一。流式细胞技术检测大鼠脾脏淋巴细胞结果显示,HC大鼠与同期普通饮食大鼠相比,CD4~+/CD8~+(2.34±0.157 vs 1.61±0.21,P<0.05)明显升高,淋巴细胞(DC45R~+细胞)含量(25.34±3.18 vs 7.64±3.47,P<0.01)升高。进一步提示,血脂水平升高可能激活HC大鼠免疫系统,从而诱发HC大鼠G蛋白偶联受体自身抗体的产生。
小结
1.本研究首次在HC大鼠血清中发现了α1-AA、β1-AA和M2-AA等G蛋白偶联受体自身抗体。
2. HC大鼠心肌超微结构和脾脏免疫系统功能发生改变,提示HC有可能通过引起心肌重构并激活机体免疫系统,从而诱发部分G蛋白偶联受体自身抗体产生,并进一步影响HC大鼠心脏功能。
结论
1. HC可以诱发心肌细胞膜脂质比例失调,降低心肌细胞膜流动性,引起心肌细胞功能障碍,从而导致心肌损伤加重;有效的改善心肌细胞膜流动性可以减轻心肌细胞损伤。提示心肌细胞膜流动性可能是HC大鼠心肌易损性增加的影响因素之一。
2. MPO可能通过降低心肌组织NO生物利用率,损伤NO/cGMP/cGK信号通路,参与HC大鼠缺血/再灌注心肌易损性增加,导致HC大鼠MI/R损伤加重。
3.本研究首次在HC大鼠血清中发现了α1-、β1-和M2-受体的自身抗体,提示部分G蛋白偶联受体自身抗体的产生可能也是HC大鼠心肌易损性增加的病理生理机制之一。
Background
Hypercholesterolemia and the corresponding artherosclerosis are the pathological foundation of coronary atherosclerotic heart disease (CAHD) and heart muscle ischemia, and are critical risk factors of ischemic heart disease. Hypercholesterolemia plays an important role in both the beginning of ischemia and the development of the injury. We and other researchers have found the myocardium has increased susceptibility to ischemia/reperfusion injury in hypercholesterolemia, but the mechanisms are not clear. We know that lipid can accumulate and later form artherosclerosis plaques. When such plaque covers the coronary, it will cause spasm. So do the increase of active oxygen production and reactive nitrogen production, which induce dysfunction of endothelium. However, these initial factors can not explain the phenomenon of increased susceptibility. There are still lots of questions.
Membrane fluidity is the dynamical character of lipid bilayer, which is important in signal transduction, chemical transportation, ion exchange, energy transmission and activity of enzymes. Stable membrane fluidity is so significant to the maintenance of internal environment and normal function of cells that it affects the development of many diseases. The change of membrane fluidity comes earlier than the morphological and biological changes of cells in a disease. The changes of membrane fluidity can also reflect the early cell injury and the development of disease. Some studies found that membrane fluidity of red blood cell decreases in hypercholesterolemia. This indicates that the disorder of lipid metabolism may change the lipid proportion of the membrane and further affect its fluidity. Will hypercholesterolemia change the fluidity of myocardium and affect its signal transduction? If so, is there any relationship between the changes of myocardium membrane fluidity and heart muscle increased that susceptibility caused by hypercholesterolemia? Study considering these questions will help us to investigate the mechanism of increased susceptibility of myocardium to ischemia/reperfusion injury caused by hypercholesterolemia.
Myeloperoxidase (MPO), which expresses in neutrophil, is an important cytokine and an independent risk factor of CAHD. MPO stimulates the apoptosis of endothelium, degrades the relaxing factor NO, lowers its biological activity, causes endothelium dysfunction and increases the vulnerability of atherosclerotic plaque, which enhances the development of heart muscle ischemia. Moreover, MPO released by activated and effused neutrophils could catalyze H2O2 produced by neutrophils and injured tissue forms hypochloric acid and other oxidants, which are toxic and will cause cardiovascular injury. However, inflammation reaction plays an important role in myocardium ischemic/reperfusion injury. So, whether MPO plays a direct function of injury to myocardium? Does it take part in the increased susceptibility of ischemia/reperfusion injury heart muscle in hypercholesterolemia? And how does it work?
From 1990, researchers detected many G protein-coupled receptor autoantibodies from serum of patients with cardiovascular diseases, includingα1-AA、β1-AA、β3-AA、M2-AA and AT1-AA etc. Some study indicatesα1-AA existence would cause increased peripheral resistance and myocardium remodel.β1-AA induces heart rate and later exacerbates heart burden and then results in injury. M2-AA destroys mitochondria, which would lead to myofibrilla dissolve and heart injury. These results suggest that G protein-coupled Receptors autoantibody may play an important role in the development of cardiovascular diseases. Evidence proved that hypercholesterolemia causes ultrastructure changes of myocardium and inflammation response in cardiovascular diseases. These indicate that hypercholesterolemia probably induce the production of G protein-coupled receptors autoantibody by the dysfunction of immune system. If this hypothesis is true, these autoantibodies have high probability to increase the susceptibility of myocardium in hypercholesterolemia and aggravate heart injury. As a result, investigation of the production of G protein-coupled receptors autoantibody in hypercholesterolemia is critical in cardiovascular diseases.
Overall, basing on a dietetic hypercholesterolemia rat model, current research aim to discuss the functions and some related mechanisms of cardiac muscle cell fluidity, myeloperoxidase and certain portion of G protein-coupled receptors autoantibody in hypercholesterolemia increased susceptibility of heart muscle.
PART 1 Hypercholesterolemia-induced myocardial cell membrane fluidity decline in rats
Objective:
To determine whether decreased membrane fluidity of myocardial cells may contribute to enhanced myocardial injury in hypercholesterolemia.
Materials and Methods:
1.1 Materials:
Male Wistar rats (4-5 weeks) weighing110±10g, provided by Shanxi Medical University Laboratory Animal Center.
1.2 Experimental groups
1.2.1 The groups of diet-derived HC rats
(1) Control group: Rats were given a normal diet for 10 weeks;
(2) Vehicle + high cholesterol group: Rats were given a HC diet for 10 weeks and receive vehicle for 4 week in the 6th week;
(3) PIO + high cholesterol group: Rats were given a HC diet for 10 weeks and receive PIO (10 mg/kg/day, oral gavage) for 4 week in the 6th week.
1.2.2 The groups of rats’regional myocardial ischemia/ reperfusion (MI/R) in vivo
(1) Sham group: Left Anterior Descending (LAD) without occlusion, total time course is 24 hours;
(2) MI/R+ normal diet group: LAD with reversible occlusion, 30min ischemia followed by 24 hours reperfusion;
(3) MI/R+ Vehicle + HC diet group: LAD with reversible occlusion, 30min ischemia followed by 24 hours reperfusion;
(4) MI/R+ PIO + HC diet group: LAD with reversible occlusion, 30min ischemia followed by 24 hours reperfusion.
1.3 Methods
1.3.1 Establishment of experimental model and sample collection:
(1) The establishment of diet-derived HC rat model and tissue collection;
(2) The establishment of rat via a polyethylene catheter inserted into the left ventricular cavity through the right carotid artery in vivo and tissue collection;
(3) Isolation of cardiac myocyte and sample collection;
(4) The establishment of rat regional MI/R model in vivo and tissue collection
1.3.2 Index determination
(1) Measurement of Total Cholesterol (TC), Triglyceride (TG) and Low Density Lipoprotein Cholesterol (LDL-cho) in serum of rat on an empty stomach;
(2) Determination of myocardial function;
(3) Detection of membrane Na~+-K~+-ATPase activity;
(4) Detection of cholesterol and phospholipid content in myocardial cell membrane;
(5) Membrane fluidity measurements
(6) Determination of cAMP with radioimmunoassay
(7) Determination of myocardial infarct size
(8) Correlation analysis between membrane fluidity and myocardial contractile function or cardiac infarct size
Results:
2.1 PIO had effects on plasma lipid profiles after high cholesterol diet
Ten weeks of HC diet resulted in a dramatic increase in plasma cholesterol (TC) (3.44±0.86 mmol/l vs. 1.24±0.26 mmol/l, P<0.01), triglyerde (TG) (1.13±0.34 mmol/l vs. 0.26±0.05 mmol/l, P<0.01) and low-density lipoprotein (LDL-cho) (1.66±0.55 mmol/l vs. 0.45±0.14 mmol/l, P<0.01). There were no significant differences between groups in plasma lipid profiles before the onset of the study (time 0) or after 6 weeks of a HC diet (before PIO treatment). After an additional 4 weeks of high cholesterol diet and treated with vehicle, plasma TC, TG and LDL levels further increased. At all these post-cholesterol times, PIO-treated rats on the HC diet exhibited significantly lower plasma TC (2.16±0.33 mmol/l, P<0.01), TG (0.59±0.18 mmol/l, P<0.05) and LDL-cho (1.24±0.28 mmol/l, P<0.05) than the vehicle group. These results indicate that PIO improved plasma lipid profiles in this diet-induced HC model, and therefore, any cardiovascular effects observed could be attributed to this mechanism.
2.2 Hypercholesterolemia worsens cardiac functional and increases post-ischemic myocardial injury
HC diet markedly aggravated myocardial contractile function. The values of LVSP, +dp/dtmax and–dp/dtmax were significantly decreased in the HC diet group compared with the normal diet group (LVSP: 13.64±1.37 Kpa vs. 20.79±2.87 Kpa, P<0.01; +dp/dtmax: 537.43±102.24 Kpa/s vs. 953.38±171.12 Kpa/s, P<0.01; -dp/dtmax: 452.19±88.34 Kpa/s vs. 793.10±112.82 Kpa/s, P<0.01). Additionally, ischemia/reperfusion-induced myocardial infarct size in the HC rats (i.e. vehicle) was significantly larger than the normal rats (56.81±6.45% vs 35.25±2.52%, P<0.01). There was no difference in ischemic area (AAR) expressed as percent of left ventricle, indicating a comparable degree of ischemic insult between sham-, normal diet-, vehicle- and PIO-treated groups after left anterior descending artery occlusion.
2.3 Hypercholesterolemia decreased membrane Na~+-K~+-ATPase activity, increased C/P and reduced the membrane fluidity of cardiac myocytes
Na~+-K~+-ATPase reside in cell membrane nullity of the organelle membrane, which activity is usually to identify the cell membrane. A clear decrease in Na~+-K~+-ATPase activity was observed in HC rats compared with that in normal diet rats (5.99±1.69μmol Pi/mg protein/hour vs. 7.88±1.25μmol Pi/mg protein/hour, P<0.01). The C/P of cardiac myocytes membrane was largely higher in rats with HC diet than normal diet (0.44±0.04 vs. 0.33±0.03, P<0.01). Most importantly, in myocardial cells from HC rats, the DPH fluorescence polarization (P) and microviscosity (η) were markedly higher than that seen in the normal diet group (0.34±0.07 vs 0.24±0.05, P<0.01; 7.99±1.37 vs 2.41±0.59, P<0.01, respectively), indicating that hypercholesterolemia decreased cardiac myocytes membrane fluidity.
2.4 Hypercholesterolemia decreased myocardial cAMP content
To further elucidate the effect by which hypercholesterolemia may disturb myocardial function, myocardial cAMP content, a downstream product in signal transduction pathway of membrane receptor, was determined. Hypercholesterolemia markedly decreased myocardial cAMP content (53.77±22.17 pmol/mg protein vs. 127.50±19.72 pmol/mg protein, P<0.01). The result demonstrated that hypercholesterolemia may cause myocardial function injury and thus contribute to increased myocardial vulnerability after ischemia and reperfusion.
2.5 PIO treatment improved myocardial contractile function, attenuated myocardial infarct size, increased Na~+-K~+-ATPase activity and cAMP content, reduced C/P, and restored membrane fluidity
Previous studies have demonstrated that PPARγagonists, such as rosiglitazone (RSG), exert beneficial cardiovascular effects in diabetic patients. In recent studies, researchers have demonstrated that treatment with RSG significantly improved endothelial and myocardial function in hypercholesterolemic rabbits. To determine whether the PPARγsignaling pathway may improve cardiac function by restoring membrane fluidity in a non-diabetic model, 18 rats fed with a high cholesterol diet were treated with PIO for 4 weeks. Treatment with PIO in hypercholesterolemic rats exerted significant protective effects as evidenced by markedly improveded cardiac contractile dysfunction (LVSP: 20.40±2.83 Kpa, P<0.01; +dp/dtmax : 827.56±172.49 Kpa/s, P<0.01;–dp/dtmax: 684.01±113.43 Kpa/s, P<0.01) and attenuated myocardial infarct size (39.82±2.74, P<0.01). PIO treatment tended to significantly increase Na+-K+-ATPase activity (7.43±1.42μmol Pi/mg protein/hour, P<0.01) and decrease C/P (0.40±0.04, P<0.05) in cardiac myocytes membrane. In addition, treatment with PIO markedly decreased both DPH fluorescence polarization (0.26±0.06, P<0.01) and microviscosity (3.29±0.63, P<0.01), a parameter that primarily reflects membrane fluidity. To further determine the effect by which PIO may exert its cardiac protective properties, the cAMP content in myocardial cell of PIO treatment was determined. Treatment with PIO in hypercholesterolemia significantly increased myocardial cAMP content (102.05±25.51, P<0.05).
2.6 Negative correlation between myocardial contractile function and DPH fluorescence polarization, positive correlation between cardiac infarct size and DPH fluorescence polarization
To determine the direct relation between membrane fluidity and myocardial function or cardiac injury, correlation between DPH fluorescence polarization and LVSP, +dp/dt_(max), -dp/dt_(max) and infarct size were analyzed. LVSP, +dp/dt_(max) and -dp/dt_(max) negatively correlated to DPH fluorescence polarization respectively (r=-0.191, P<0.05; r=-0.323, P<0.01; r=- 0.322, P<0.01, respectively.). Cardiac infarct size positively correlated to DPH fluorescence polarization (r=0.599, P<0.01). In other words, membrane fluidity positively correlated to myocardial contractile function and negatively correlated to cardiac infarct size. These results suggest that hypercholesterolemia-induced cardiac impairment may be associated with membrane fluidity decrease, and PIO treatment protects hypercholesterolemia-induced myocardial dysfunction by preserving the integrity of membrane fluidity.
Summary
1. Hypercholesterolemia impairs cardiac function, and increases myocardial infarct size after ischemic/reperfusion, suggesting that hypercholesterolemia may enhance cardiac vulnerability.
2. Hypercholesterolemia increases C/P in the myocardial cell membrane, decreases membrane fluidity and myocardial cAMP content, as well as membrane fluidity positively correlates to myocardial contractile function and negatively correlates to cardiac infarct size, suggesting that hypercholesterolemia may exacerbate cardiac injury by decreased membrane fluidity-induced myocardial dysfunction.
3. PIO treatment markedly inhibited hypercholesterolemia-induced C/P increase, membrane fluidity decrease of cardiac myocyte membrane, and myocardial cAMP content decrease, thus recovered cardiac function and decreased infarct size after myocardial ischemia/reperfusion.
PART 2 The role of MPO in the increased susceptibility of ischemic-reperfusion injury in hypercholesterolemia rats
Objective:
To confirm the role of MPO in the increased susceptibility of ischemic-reperfusion injury in hypercholesterolemia rats and to explore the mechanism (such as the reduction in the effective utilization of NO etc.) by observing the changes of MPO activity and distribution in myocardial tissue of ischemia/reperfusion HC rats.
Materials and Methods:
1.1 Materials:
(1) Male Wistar rats (4-5 weeks) weighing110±10g, provided by Shanxi Medical University Laboratory Animal Center.
(2) H9c2 (2-1) cell line
1.2 Experimental groups
1.2.1 The groups of diet-derived HC rats
(1) Control group: Rats were given a normal diet for 10 weeks;
(2) Vehicle + high cholesterol group: Rats were given a HC diet for 10 weeks and intraperitoneal injection of 10% DMSO for 1 week in the 9th week;
(3) ABAH (MPO inhibitor) + high cholesterol group: Rats were given a HC diet for 10 weeks and intraperitoneal injection of ABAH (25.00mg/kg?d) for 1 week in the 9th week.
1.2.2 The groups of rats’regional myocardial ischemia/ reperfusion (MI/R) in vivo
(1) Sham group: Left Anterior Descending (LAD) without occlusion, total time course is 3 or 24 hours;
(2) MI/R+ normal diet group: LAD with reversible occlusion, 30min ischemia followed by 3 or 24 hours reperfusion;
(3) MI/R+ Vehicle + HC diet group: LAD with reversible occlusion, 30min ischemia followed by 3 or 24 hours reperfusion;
(4) MI/R+ ABAH + HC diet group: LAD with reversible occlusion, 30min ischemia followed by 3 or 24 hours reperfusion.
1.2.3 The groups of H9c2 (2-1) cell lines
(1) Normoxia group: cultured in atmosphere of 5%CO2, 95% air for 5h;
(2) ABAH+ Normoxia group: added 100μM ABAH into the cultured medium then cultured in atmosphere of 5%CO_2, 95% air for 5h;
(3) H/R group: hypoxia with 95% N_2 and 5% CO_2 for 3h then followed by 2h reoxygenation in 5% CO_2 and 95% air;
(4) MPO+H/R group: added 0.1 u/ml MPO into the cultured medium then operated as H/R group;
(5) ABAH+H/R group: added 100μM ABAH into the cultured medium then operated as H/R group;
(6) ABAH+MPO+ H/R group: added 100 mΜABAH into the cultured medium for 30 min before adding 0.1 u/ml MPO then operated as H/R group.
1.3 Methods
1.3.1 Establishment of experimental model and sample collection
(1) The establishment of diet-derived HC rat model and tissue collection;
(2) The establishment of rat regional MI/R model in vivo and tissue collection;
(3) The establishment of H9c2 (2-1) H/R model and cell collection.
1.3.2 Index determination
(1) Measurement of Total Cholesterol (TC), Triglyceride (TG) and Low Density Lipoprotein Cholesterol (LDL-cho) in serum of rat on an empty stomach;
(2) Measurement of Creatine Kinase (CK) content in serum and culture medium supernatant;
(3) Measurement of Lactate Dehydrogenase (LDH) content in serum and culture medium supernatant;
(4) Myocardial apoptosis is detected by TdT-mediated dUTP nick end labeling (TUNEL);
(5) Caspase-3 relative activity assay;
(6) Determination of myocardial infarct size;
(7) Measurement of cardiac function;
(8) Determination of myocardial tissue MPO distribution by immunohistochemistry;
(9) Myocardial tissue MPO activity assay;
(10) Correlation analysis of myocardial tissue MPO activity , serum CK concentration, LDH leakage, apoptotic index, caspase-3 activity , myocardial infarction size and cardiac function;
(11) Measurement of nitric oxide (NO) content in myocardial tissue;
(12) Determination of myocardial cGMP content by Radioimmunoassay kit;
(13) Detection of myocardial nitric oxide synthase (NOS) protein expression by Western-blot;
(14) Detection of the mRNA expression of NOS in myocardial tissue by real time PCR;
(15) Detected H9c2 (2-1) cell proliferation by Cell counting Kit-8.
Results:
2.1 The establishment of Diet-derived HC rat model
The lipid levels (TC, TG and LDL-cho) in rats of each group had no significant difference before given HC diet feeding (0 week). The lipid levels of vehicle + high cholesterol group was significantly higher than control group after feeding with high-cholesterol diet for 10 weeks (TC: 3.01±0.75 mmol/L vs. 1.44±0.14 mmol/L, P<0.01; TG: 0.88±0.35 mmol/L vs. 0.26±0.05 mmol/L, P<0.01; LDL-cho: 1.53±0.65 mmol/L vs. 0.45±0.14 mmol/L, P<0.01, respectively.). All these demonstrated that diet-derived HC rat model was set up successfully.
2.2 HC rats had increased vulnerability of MI/R
2.2.1 A significantly exacerbated myocardial MI/R injury was found of HC rats
2.2.1.1 CK content in serum of HC rats were increased
Compared with sham group, the CK content in MI/R group increased significantly (0.17±0.01 U/ml vs. 0.05±0.02 U/ml, P<0.01); Compared with normal diet MI/R group, the CK content in HC MI/R group increased significantly (0.70±0.08 U/ml, P<0.01).
2.2.1.2 LDH content in serum of HC rats were increased
Compared with sham group, the LDH content in MI/R group increased significantly (3091.59±33.69 U/L vs. 1854.45±422.97 U/L, P<0.01); Compared with normal diet MI/R group, the LDH content in HC MI/R group increased significantly (3596.18±99.81 U/L, P<0.01).
2.2.2 Increased myocardial apoptosis in HC rats with ischemia/reperfusion
2.2.2.1 TUNEL detection
The apoptotic index in MI/R group was significantly higher than that in sham group (16.07±1.02% vs. 1.44±0.14%,P<0.01); the apoptotic index in HC MI/R group was significantly higher than that in normal diet MI/R group (22.63±1.02%, P<0.01).
2.2.2.2 Caspase3 activity assay:
Caspase3 activity in MI/R group was also significantly increased compared with sham group (6.66±0.61 vs. 1.00±0.08,P<0.01); caspase3 activity in HC MI/R group was also significantly increased compared with normal diet MI/R group (12.29±0.92,P<0.01).
2.2.3 Myocardial infarct size in HC group enhanced after MI/R
The ratio of area at risk to left ventricular area (AAR/LV): no significant differences were observed among these groups (P>0.05), which indicated that the ischemic area induced by coronary ligation is roughly identical among each experimental group, so that these groups are comparable. The myocardial infarct size of HC MI/R group was significantly enhanced as compared with normal diet MI/R group (56.05±3.91% vs. 34.90±2.52%, P<0.01).
2.2.4 The cardiac function in HC MI/R group were aggravated
At the end of 3 h reperfusion, compared with the sham group, the LVSP and±dP/dtmax in MI/R group decreased obviously (LVSP: 15.12±1.38 Kpa vs. 18.28±3.02 Kpa, P<0.05; +dp/dtmax: 610.43±110.26 Kpa/s vs. 785.22±162.22 Kpa/s, P<0.01; -dp/dtmax: 500.57±116.20 Kpa/s vs. 667.00±234.83 Kpa/s, P<0.05, respectively.);compared with the normal diet MI/R group,the LVSP and±dP/dtmax in HC MI/R group decreased obviously (LVSP: 9.95±1.85 Kpa, P<0.01; +dp/dtmax: 386.43±100.76 Kpa/s, P<0.01; -dp/dtmax: 264.53±98.76 Kpa/s, P<0.01, respectively.).
These results confirmed that, compared with the normal diet group, the MI/R injury in HC group increased, which indicated the increased susceptibility of ischemic-reperfusion injury in hypercholesterolemia rats.
2.3 Myocardial tissue MPO changes in HC rats with ischemia/reperfusion
2.3.1 Myocardial tissue MPO activity assay in HC rats with ischemia/reperfusion
Compared with the sham group, the myocardial tissue MPO activity in MI/R group increased obviously (13.48±4.58 U/g protein vs. 8.29±2.80 U/g protein, P<0.05), and further increased in HC MI/R group (19.44±2.35 U/g protein, P<0.05).
2.3.2 Myocardial tissue MPO distribution of HC rats with ischemia/reperfusion
The result of immunohistochemistry indicated that rare MPO was detected in the area not at risk (ANAR) of myocardial tissue from normal diet rats, but clear staining was observed in the vascular tissue and cardiac myocytes from ischemia/reperfusion area. The results suggested that infiltrating neutrophils in ischemia/reperfusion heart release a large number of MPO, these MPO possibly invasive ischemia/reperfusion myocardial cells through active or passive manner then cause myocardial cell injury through a variety of mechanisms. Moreover, the MPO immunostaining further enhanced in HC MI/R group than the normal diet MI/R group.This is consistent with the above-mentioned results that MPO activity increased in HC MI/R group.
Prompted MPO may be involved in the increased susceptibility of ischemia/reperfusion myocardial injury in HC rats.
2.4 Correlation analysis of myocardial tissue MPO activity with MI/R injury.
2.4.1 Positive correlation between MPO activity in ischemia/reperfusion myocardial tissue and serum CK and LDH levels (CK: r = 0.618, P<0.01; LDH: r = 0.64, P<0.01, respectively.).
2.4.2 Positive correlation between MPO activity and apoptotic index and caspase-3 activity in ischemia/reperfusion myocardial tissue (apoptotic index: r = 0.651, P<0.01; caspase-3: r = 0.619, P<0.01, respectively.).
2.4.3 Positive correlation between MPO activity and myocardial infarct size in ischemia/reperfusion myocardial tissue (r = 0.663, P< 0.01).
2.4.4 Negative correlation between MPO activity in ischemia/reperfusion myocardial tissue and myocardial contractile function (LVSP and±dP/dtmax) of ischemia/reperfusion heart (LVSP: r = -0.555, P<0.01; +dP/dtmax: r = -0.794, P< 0.01; -dP/dtmax: r =-0.748, P<0.01, respectively.).
These results confirmed the closely correlation between MPO activity in ischemia/reperfusion myocardial tissue and increased susceptibility of ischemia/reperfusion injury in hypercholesterolemic rats. Changes of MPO in myocardial tissue of HC rats with ischemia/reperfusion were directly related to ischemia/reperfusion myocardial vulnerability of HC rats.
2.5 MI/R HC rats’myocardial tissue MPO and cardiac injury changed after the intervention with MPO inhibitors
In order to further explore whether or not MPO participate in and directly affect the HC rat MI / R myocardial vulnerability increased, ABAH was applicated to HC rats in vivo to observe the corresponding changes.
2.5.1 Effect of ABAH on the H9c2 (2-1) cell
There was no statistical difference of cell survival rate between ABAH + normoxia group and normoxia group (0.98±0.01 vs. 1.01±0.05); there was no statistical difference of cell survival rate between ABAH+H/R group and H/R group too (0.63±0.06 vs. 0.60±0.09). The results above showed that ABAH itself had no effect on myocardial cells and removed the experimental error which forms by the medicine intervention.
2.5.2 The change of MPO activity and distribution in myocardial tissue, after ABAH intervention
Compared with the MI/R+ Vehicle + HC diet group, the MPO immunostaining and MPO activity (13.66±2.63 U/g protein, P<0.05) in MI/R+ ABAH + HC diet group significantly decreased.
2.5.3 After ABAH intervention, the myocardial ischemia/reperfusion injury of HC rats mitigated.
Compared with the MI/R+ Vehicle + HC diet group CK (0.31±0.06 U/ml, P<0.01) and LDH (3286.65±40.04 U/L, P<0.01) content in MI/R+ ABAH + HC diet group significantly decreased.
2.5.4 After ABAH intervention, cardiomyocyte apoptosis of HC rats with ischemia/reperfusion mitigated.
Compared with the MI/R + Vehicle + HC diet group, apoptotic index (18.55±1.29%, P<0.01) and Caspase3 activity (7.97±0.29, P<0.01) in MI/R+ ABAH + HC diet group significantly decreased.
2.5.5 After ABAH intervention, the myocardial infarct size of HC rats decreased
The ratio of AAR/LV: no significant differences were observed among these groups (P>0.05). The myocardial infarct size of MI/R+ ABAH + HC diet group was significantly decreased as compared with MI/R+ Vehicle + HC diet group (38.89±2.47%, P<0.01).
2.5.6 After ABAH intervention, HC rats with ischemia/reperfusion cardiac dysfunction mitigated Compared with the MI/R + Vehicle + HC diet group, the cardiac dysfunction in MI/R+ ABAH + HC diet group had some degrees recovery (LVSP: 13.73±2.20 Kpa, P<0.05; +dP/dtmax: 564.00±128.56 Kpa/s, P<0.05; -dP/dt_(max): 444.00±96.08 Kpa/s, P<0.05, respectively.).
These results indicated: First, ABAH itself had no effect on myocardial cells. Second, after ABAH intervention, MPO activity and distribution in myocardial tissue of HC rats with ischemia/reperfusion decreased and ischemia/reperfusion injury mitigated. Third, MPO changes in myocardial tissue of HC rats with ischemia/reperfusion had close related to ischemia/reperfusion myocardial vulnerability of HC rats. Howerve, it needs to be further confirmed that whether there is direct relation between them.
2.6 Effect of MPO on the H9c2 (2-1) cell
In order to remove various influence of neural and humoral regulation to experimental result in vivo, the experimental use of H9c2 (2-1) cells to observe the MPO on the direct effects of myocardial cells.
2.6.1 H9c2 (2-1) H/R model was established successfully
Compared with the normoxia group (control group), the CK and LDH content in culture medium supernatant in H/R group significantly increased (CK: 0.08±0.03 U/ml vs. 0.02±0.01 U/ml, P<0.01; LDH: 856.22±28.91 U/L vs. 33.11±21.18 U/L, P<0.01, respectively.), showing that H9c2 (2-1) H/R model had been established successfully.
2.6.2 MPO aggravated hypoxia/reoxygenation injury in H9c2 (2-1) cells
Compared with H/R group, the CK and LDH content in culture medium supernatant in 0.1u/ml MPO+H/R group significantly increased (0.42±0.11 U/ml, P<0.01; 1362.78±106.89 U/L, P<0.01, respectively.), showing that MPO could not only cause myocardial cells' damage but also further aggravate the myocardial cell hypoxia/reoxygenation injury, which prompted that the MPO distribution and activity possibly directly participated in myocardial cell's damage, and further aggravated the myocardial cell hypoxia/reoxygenation injury.
2.6.3 After ABAH intervention, H9c2 (2-1) cell H/R damage change
Compared with 0.1 u/ml MPO+H/R group, the CK and LDH content in ABAH+0.1 u/ml MPO+H/R group significantly decreased (0.13±0.07 U/ml, P<0.01; 880.81±80.86 U/L, P<0.01, respectively.), further prompted that MPO distribution and activity possibly directly participated in myocardial cells’damage, and had directly relationship with ischemia/reperfusion myocardial vulnerability of HC rat.
2.7 HC rats with ischemia/reperfusion myocardial tissue NO-cGMP signaling pathway impaired
2.7.1 NOx content increased in myocardial tissue of HC rats with ischemia/reperfusion
Compared with MI/R + normal diet group, the NOx content in MI/R + Vehicle + HC diet group significantly increased (972.78±77.45μmol/g protein vs. 421.44±58.96μmol/g protein, P<0.01). ABAH could effectively reduce the NOx content in HC rat ischemia/reperfusion myocardial tissue (615.76±50.54μmol/g protein, P<0.01), but had not restored completely to the normal physiological condition.
2.7.2 The cGMP content in myocardial tissue of HC rats with ischemia/reperfusion decreased
Compared to sham group the cGMP content in MI/R+ normal diet group decreased (31.53±11.37 pmol/mg protein vs. 74.22±22.23 pmol/mg protein, P<0.01); compared to MI/R+ normal diet group, the cGMP content in MI/R+ Vehicle + HC diet group decreased (7.35±2.47 pmol/mg protein, P<0.01); compared to MI/R+ Vehicle + HC diet group, the cGMP content in MI/R+ ABAH + HC diet group restored partly (28.84±9.08 pmol/mg protein, P<0.01).
2.7.3 The changes of mRNA and protein expression of NOS in myocardial tissue of HC rats with ischemia/reperfusion
2.7.3.1 The change of mRNA expression of NOS in myocardial tissue of HC rats with ischemia/reperfusion
Compared with sham group, the mRNA expression of iNOS in MI/R+ normal diet group increased (4.80±1.73 vs. 1±0.70, P<0.01); compared with MI/R+ normal diet group, the mRNA expression of iNOS in MI/R+ Vehicle + HC diet group increased (10.09±2.15, P<0.01); compared with MI/R+ Vehicle + HC diet group the mRNA expression of iNOS in I/R+ ABAH + HC diet group decreased (3.41±1.50, P<0.01).
Compared with control group the mRNA expression of eNOS in vehicle + high cholesterol group decreased significantly (0.87±0.02 vs. 1.00±0.04, P<0.01). No significant differences of nNOS mRNA expression were observed among these groups.
2.7.3.2 The change of protein expression of NOS in myocardial tissue of HC rats with ischemia/reperfusion
Compared with sham group, the protein expression of iNOS in MI/R+ normal diet group increased (2.22±0.44 vs.1.00±0.26, P<0.05); compared with MI/R+ normal diet group, the protein expression of iNOS in MI/R+ Vehicle + HC diet group increased (3.17±0.93, P<0.05); compared with I/R+ Vehicle + HC diet group, the protein expression of iNOS in MI/R+ ABAH + HC diet group decreased (2.28±0.46, P<0.05).
Compared with control group, the protein expression of eNOS in vehicle + high cholesterol group decreased significantly (0.36±0.14 vs. 1.00±0.37, P<0.05). No significant differences of nNOS protein expression were observed among these groups.
These results indicated that NOx content increased in myocardial tissue of HC rats and it further increased in AAR of HC rats with ischemia/reperfusion; cGMP content decreased in myocardial tissue of HC rats and it further decreased in AAR of HC rats with ischemia/reperfusion; both mRNA and protein expression of iNOS increased in myocardial tissue of HC rats and it further increased in AAR of HC rats with ischemia/reperfusion; both mRNA and protein expression of eNOS decreased significantly among control group and vehicle + high cholesterol group; both mRNA and protein expression of nNOS had no significant differences among these groups. These data demonstrated that although NOx content in AAR of HC rats with ischemia/reperfusion increased significantly, nitric oxide bioavailability decreased and NO-cGMP signaling pathway maybe impaired; MPO may impaired NO-cGMP signaling pathway in myocardial tissue by reduced nitric oxide bioavailability, and so to participate in the rat ischemia/rep
高胆固醇血症(hypercholesterolemia, HC)及其随后发生的动脉粥样硬化,是冠心病(coronary atherosclerotic heart disease, CAHD)时引起心肌缺血的始动因素之一,是缺血性心脏病(ischemic heart disease, IHD)最重要的危险因素之一,通过促进脂质堆积并进一步形成动脉粥样斑块、促进粥样硬化病变环绕造成冠脉痉挛、以及增加活性氧和活性氮产物并导致内皮机能障碍等机制在心肌缺血早期和缺血损伤的发展过程中发挥重要作用。目前,针对HC诱发的CAHD和心肌缺血损伤,临床治疗措施主要在于恢复缺血心肌血液灌流(再灌注)的同时降低血浆胆固醇,但是治疗效果明显低于无高胆固醇血症的心肌缺血患者。同时,临床流行病学研究提示,HC并发心肌缺血患者治疗过程中再灌注(心肌缺血/再灌注, myocardial ischemia/reperfusion, MI/R)损伤程度明显高于无HC心肌缺血患者,提示HC提高了心肌细胞对缺血/再灌注损伤的易感性,但具体机制复杂,仍有许多基本问题未被解决。
膜流动性是细胞膜脂质双分子层重要的动力学特征,在细胞的信号转导、物质运输、离子交换、能量传递以及酶活动性大小等方面起着重要的作用。稳定的膜流动性对于细胞内环境的维持和正常细胞功能的发挥至关重要,因此膜流动性的变化影响着许多疾病的发生和发展。在病理状态下,往往在细胞的一般形态学和生物学改变之前就能检测出膜流动性的改变;在治疗过程中,膜流动性趋向于正常的变化是反映细胞早期损伤、研究疾病过程的高灵敏度的分析指标。有研究发现,HC时红细胞膜的流动性降低,进而导致红细胞的功能改变。这些研究提示,HC时,由于脂代谢紊乱,有可能通过影响细胞膜脂类物质的比例,进而影响细胞膜的流动性。由于心肌细胞膜与红细胞膜具有同样的结构基础,那么当HC发生时,是否也会影响心肌细胞膜的流动性,进而影响心肌细胞跨膜信号的转导呢?如果这一可能存在,那么心肌细胞膜流动性的变化是否与HC致心肌细胞易损性增加有关呢?有关这些内容的研究,有助于我们从膜流动性的角度,分析HC致心肌对缺血/再灌注损伤易感性增加的机制。
髓过氧化物酶(myeloperoxidase, MPO)主要表达于中性粒细胞的过氧化物酶,是一种重要的炎性介质,也是CAHD发生发展的独立危险因子。MPO可以通过刺激血管内皮细胞凋亡,降解内皮源性舒张因子NO,降低NO生物学活性,引起内皮机能障碍,并增加粥样硬化斑块的易损性等作用促进心肌缺血的发展;活化和渗出的中性粒细胞释放的MPO催化同时由中性粒细胞和损伤组织释放的H2O2生成毒剂次氯酸和其他氧化剂(如3-氯化酪氨酸、硝基酪氨酸等),也可以导致心血管系统的氧化损伤;此外,有研究发现,在MI/R时,MPO大量聚集在血管内皮细胞与血管平滑肌之间的基底膜。我们的前期研究也发现,在MI/R时,渗出到心肌细胞的中性粒细胞发生了脱颗粒,将其中的MPO释放到心肌细胞浆中。以上研究强烈提示在MI/R时,MPO发生了游走和位移,但对其意义的解释还缺乏足够的证据。因此,MI/R时,释放入心肌细胞的MPO是否可以直接导致心肌细胞的损伤作用,是否参与HC所致的缺血/再灌注心肌易损性增加?如果是,又是如何发挥作用的,都有待于深入探究。而对这些问题的研究有助于我们进一步揭示HC缺血/再灌注心肌易损性增加的机制。
上世纪90年代以来,在心血管疾病的患者血清中检测到多种G蛋白偶联受体自身抗体,包括α1-肾上腺素受体自身抗体(α1- AA)、β1-肾上腺素受体自身抗体(β1-AA)、β3-肾上腺素受体自身抗体(β3-AA)、M2-胆碱受体自身抗体(M2-AA)和血管紧张素Ⅱ1型受体自身抗体(AT1-AA)等。研究证实,这些自身抗体都具有类激动剂样的作用,如:α1-AA长期存在可引起外周阻力升高,并导致心肌重构;β1-AA使心脏跳动频率增加,心肌收缩力增强,长期作用可发生肌原纤维的灶状溶解,诱导心肌重构发生,最终造成心功能下降;M2-AA使线粒体发生空泡样变性,导致心肌收缩力降低。这些研究提示,G蛋白偶联受体自身抗体在心血管疾病的发生发展过程中可能发挥重要作用。而HC病理发展过程中心肌超微结构有所改变并伴有血清补体升高等免疫反应,提示HC有可能诱发免疫系统功能紊乱。那么,这种免疫系统的功能紊乱,是否也会诱导G蛋白偶联受体自身抗体的产生?如果可以,这些自身抗体极有可能促使HC心肌易损性增加,进而加重心脏结构和功能的进一步恶化。因此,探查HC是否诱发G蛋白偶联受体自身抗体的产生,在心血管疾病的研究中是不容忽视的。
综上所述,本研究拟在食源性HC大鼠模型的基础上,探讨心肌细胞膜流动性、MPO和部分G蛋白偶联受体自身抗体在HC心肌易损性增加中的作用及其部分相关机制。
第一部分高胆固醇血症诱发大鼠心肌细胞膜流动性降低
目的
本部分实验在食源性HC大鼠模型上,给予吡格列酮(Pioglitazone, PIO)干预,观察并分析HC大鼠心肌细胞膜流动性和心肌损伤的变化,探讨心肌细胞膜流动性在HC大鼠心肌易损性增加中的作用及其病理机制。
材料与方法
1.材料
健康Wistar远交群大鼠,4～5 weeks,体重110±10 g,雄性。
2.实验分组
(1)食源性HC大鼠模型分组
①普通饮食大鼠组:普通饲料饲养10周;②Vehicle +高胆固醇饮食组:高胆固醇饮食饲养10周,后4周同时用色拉油(溶剂Vehicle: 0.5 ml/只)灌胃;③PIO +高胆固醇饮食组:高胆固醇饮食饲养10周,后4周同时用PIO(溶于色拉油: 10.00mg/kg?d)灌胃。
(2)在体大鼠心脏局部缺血/再灌注后心肌梗塞面积测定的实验分组
①伪手术组:仅在冠状动脉左前降支(Left Anterior Descending, LAD)下穿线,不结扎,持续24 h;②缺血/再灌注+普通饮食大鼠组:可逆性结扎LAD,心肌缺血30 min,再灌注24 h;③缺血/再灌注+ Vehicle+高胆固醇饮食组:可逆性结扎LAD,心肌缺血30 min,再灌注24 h;④缺血/再灌注+ PIO+高胆固醇饮食组:可逆性结扎LAD,心肌缺血30 min,再灌注24 h。
3.方法
(1)实验模型的建立及标本的留取
①食源性HC大鼠模型的建立及标本留取;②大鼠在体颈总动脉插管及标本留取;③单个心肌细胞的分离及标本留取;④大鼠在体心脏局部缺血/再灌注模型的建立及标本留取。
(2)检测指标的测定
①大鼠空腹血清总胆固醇(Total Cholesterol, TC)、甘油三酯(Triglyceride, TG)和低密度脂蛋白-胆固醇(Low Density Lipoprotein Cholesterol, LDL-cho)的测定;②心功能测定;③心肌细胞膜Na+-K+-ATPase活性的测定;④心肌细胞膜胆固醇含量、磷脂含量和胆固醇/磷脂比值(C/P)的测定;⑤心肌细胞膜流动性的测定;⑥心肌组织cAMP含量的测定;⑦大鼠心肌梗塞面积的测定;⑧心肌细胞膜流动性与心功能和缺血/再灌注后心肌梗塞面积的相关性分析。
结果
1.高胆固醇饮食后大鼠血脂变化和PIO对HC模型大鼠血脂的影响
1.1食源性HC大鼠模型的建立
给予高胆固醇饲料前,大鼠血脂水平无明显差异。高胆固醇饮食6周后,大鼠血脂水平明显升高,且高胆固醇饮食大鼠血清TC,TG和LDL-cho水平明显高于同期普通饮食饲养的大鼠(P<0.01);与0周大鼠血脂水平相比,高胆固醇饮食饲养10周后,大鼠血清血脂水平显著升高(TC: 3.44±0.86 mmol/l vs 1.24±0.26 mmol/l, P<0.01; TG: 1.13±0.34 mmol/l vs. 0.26±0.05 mmol/l, P<0.010; LDL-cho: 1.66±0.55 mmol/l vs 0.45±0.14 mmol/l, P<0.01),且较同期普通饮食大鼠显著升高(P<0.01)。说明食源性高胆固醇血症大鼠模型建立成功。
1.2 PIO干预后,HC大鼠血脂变化
高胆固醇饮食6周后,给予PIO干预,继续喂以高胆固醇饮食4周,大鼠血清TC,TG和LDL-cho水平较Vehicle高胆固醇饮食大鼠明显下降(TC: 2.16±0.33 mmol/l, P<0.01; TG: 0.59±0.18 mmol/l, P<0.05; LDL-cho: 1.24±0.28 mmol/l, P<0.05)。提示,PIO可以有效的改善HC大鼠血脂水平,纠正脂质紊乱。
2. HC大鼠心功能恶化,缺血/再灌注后心肌损伤增加
2.1 HC大鼠心功能障碍
与普通饮食大鼠相比,高胆固醇饮食大鼠LVSP,+dp/dtmax和-dp/dtmax显著降低(LVSP: 13.64±1.37 Kpa vs. 20.79±2.87 Kpa, P<0.01; +dp/dtmax: 537.43±102.24 Kpa/s vs. 953.38±171.12 Kpa/s, P<0.01; -dp/dtmax: 452.19±88.34 Kpa/s vs. 793.10±112.82 Kpa/s, P<0.01)。
2.2 HC大鼠缺血/再灌注后心肌梗塞面积增大
2.2.1危险区面积占左心室面积的百分比(AAR/LV比值)
以危险区面积占左心室面积的百分比(即AAR/LV比值)作为判别心肌缺血程度的指标,发现各实验组间的AAR/LV均无显著差异(P>0.05),表明各组动物模型的缺血程度大体相同,因而在此实验模型基础之上的实验结果具有可比性
2.2.2心肌梗塞面积占危险区面积的百分比(AN/AAR比值)
心肌梗塞面积不同程度的反应了心肌损伤状况以及心肌损伤易感性。高胆固醇血症大鼠心肌梗塞面积占危险区面积的百分比显著高于普通饮食大鼠缺血/再灌注组(56.81±6.45% vs. 35.25±2.52%, P<0.01)。
以上结果提示,HC可能引起大鼠心功能障碍,尤其是收缩功能受损,并可能进一步导致HC大鼠心肌易损性的升高。
3. HC对心肌细胞膜的影响
3.1 HC降低心肌细胞膜Na+-K+-ATPase活性
Na+-K+-ATPase仅存在于细胞膜,而不存在于细胞器膜,其活性通常作为细胞膜的标志。与普通饮食大鼠相比,HC大鼠Na+-K+-ATPase活性明显降低(5.99±1.69μmol Pi/mgprotein/hour vs. 7.88±1.25μmol Pi/mg protein/hour, P<0.01)。
3.2 HC大鼠心肌细胞膜流动性降低
与普通饮食大鼠相比,HC大鼠心肌细胞膜DPH荧光偏振度(P)和微粘度(η)明显升高(P: 0.34±0.07 vs. 0.24±0.05, P<0.01;η: 7.99±1.37 vs. 2.41±0.59, P<0.01),说明HC大鼠心肌细胞膜流动性下降。
3.3 HC大鼠心肌细胞膜C/P升高
与普通饮食大鼠相比,HC大鼠心肌细胞膜C/P升高(0.44±0.04 vs. 0.33±0.03, P<0.01)。
上述结果首先通过Na+-K+-ATPase活性的检测说明了心肌组织提取物为心肌细胞膜,其次发现HC大鼠心肌细胞膜流动性降低以及某些膜蛋白功能活性改变,而这种变化可能是由于HC大鼠心肌细胞膜脂质比例的失调而造成的。
4. HC降低心肌组织cAMP含量
为了进一步的阐述HC对心肌细胞易损性的影响,本实验检测了心肌组织cAMP含量,一种膜受体信号通路的下游产物。与普通饮食大鼠相比,HC大鼠心肌组织cAMP含量明显减少(53.77±22.17 pmol/mg protein vs. 127.50±19.72 pmol/mg protein, P<0.01)。进一步提示,HC可能导致心肌功能障碍,并引起心肌易损性升高。
5. PIO干预HC大鼠后,减轻HC大鼠心肌损伤,改善HC引起的大鼠心肌细胞膜的变化,恢复心肌组织cAMP含量
5.1 PIO改善HC大鼠心功能
与单纯的高胆固醇饮食大鼠相比,PIO干预HC大鼠后发挥显著的心血管保护效应,改善大鼠心功能,大鼠心肌收缩功能(LVSP: 20.40±2.83 Kpa, P<0.01; +dp/dtmax : 827.56±172.49 Kpa/s, P<0.01;–dp/dtmax: 684.01±113.43 Kpa/s, P<0.01)有所恢复。
5.2 PIO减小HC大鼠缺血/再灌注后心肌梗塞面积
AAR/LV %在各实验组大鼠无统计学差异(P>0.05)。与单纯的高胆固醇饮食大鼠相比,PIO干预HC大鼠后,大鼠缺血/再灌注后心肌梗塞面积明显减小(39.82±2.74, P<0.01)。
以上结果证实,PIO干预HC大鼠可以改善大鼠心功能,减小大鼠心肌梗塞面积;提示,PIO干预可能减小HC大鼠心肌对损伤的敏感性。
5.3 PIO增加HC大鼠心肌细胞膜Na+-K+-ATPase活性
与单纯高胆固醇饮食大鼠相比,PIO干预HC大鼠后,大鼠心肌细胞膜Na+-K+-ATPase活性明显增加(7.03±1.42μmol Pi/mg protein/hour, P<0.01)。
5.4 PIO改善HC大鼠心肌细胞膜流动性
与单纯高胆固醇饮食大鼠相比,PIO干预HC大鼠后,大鼠心肌细胞膜荧光偏振度(0.26±0.06, P<0.01)和微粘度(3.29±0.63, P<0.01)显著降低。说明PIO干预后HC大鼠心肌细胞膜流动性明显增加。
5.5 PIO降低HC大鼠心肌细胞膜C/P
与单纯高胆固醇饮食大鼠相比,PIO干预HC大鼠后,大鼠心肌细胞膜C/P明显降低(0.40±0.04, P<0.05)。
以上结果提示,PIO有可能通过改善HC大鼠心肌细胞膜脂质成分来恢复心肌细胞膜流动性,从而改善HC大鼠心肌细胞膜蛋白功能活性和心功能,降低HC大鼠心肌对损伤的敏感性。
5.6 PIO增加HC大鼠心肌组织cAMP含量
与单纯高胆固醇饮食大鼠相比,PIO干预HC大鼠后,大鼠心肌组织cAMP含量显著增加(102.05±25.51, P<0.05)。进一步说明,PIO可能在HC大鼠发挥心脏保护作用,减轻HC大鼠心肌对损伤的敏感性。
6.心肌细胞膜流动性与心功能和缺血/再灌注后心肌梗塞面积具有相关性
为了更深入的探讨心肌细胞膜流动性的改变在HC大鼠心功能下降和心肌损伤敏感性增加中的作用,以及PIO对HC大鼠心脏的保护效应是否涉及心肌细胞膜流动性,本实验对心肌细胞膜流动性和心功能、缺血/再灌注后心肌梗塞面积进行了相关性分析。
6.1心肌细胞膜流动性与心肌收缩功能呈正相关
通过对心肌细胞膜DPH荧光偏振度和心功能(LVSP,+dp/dtmax, -dp/dtmax)之间的直线相关分析,本实验发现心肌收缩功能与心肌细胞膜DPH荧光偏振度之间呈负相关(r=-0.191, P<0.05; r=-0.323, P<0.01; r=- 0.322, P<0.01),说明心肌细胞膜流动性与心肌收缩功能呈正相关。
6.2心肌细胞膜流动性与缺血/再灌注后心肌梗塞面积呈负相关
心肌细胞膜DPH荧光偏振度与缺血/再灌注后心肌梗塞面积直线相关分析显示,DPH荧光偏振度和心肌梗塞面积呈正相关(r=0.599, P<0.01)。说明心肌细胞膜流动性与缺血/再灌注后心肌梗塞面积呈负相关。
以上结果提示,HC诱发的大鼠心功能障碍和心肌损伤易感性增加可能与HC大鼠心肌细胞膜流动性下降有光,而PIO干预也可能通过或部分通过改善和恢复HC大鼠心肌细胞膜流动性来防御HC引起的大鼠心脏机能障碍。
小结
1. HC引起大鼠心功能下降,缺血/再灌注后心梗面积明显增大,提示在HC时心肌损伤易感性增加;
2. HC导致心肌细胞膜Na+-K+-ATPase活性降低,膜流动性下降,膜C/P比值升高,心肌细胞cAMP含量降低,而且膜流动性与心功能和心梗面积具有相关性,提示HC可能通过膜流动性降低,进而心肌细胞跨膜信号转导功能受阻,最终导致HC情况下心功能受神经体液调控能力下降而恶化心肌损伤;
3. PIO干预显著抑制HC诱导的心肌细胞膜C/P升高和膜流动性的下降以及膜Na+-K+-ATPase活性降低,以及细胞cAMP含量的减少,恢复心功能和减小缺血/再灌注后心梗面积。进一步提示HC大鼠心肌细胞膜流动性改变可能是HC大鼠心肌易损性增加的原因之一。
第二部分髓过氧化物酶对高胆固醇血症大鼠缺血/再灌注心肌易损性增加的作用
目的
本部分实验通过观察HC大鼠缺血/再灌注心肌组织MPO分布和活性的变化,以及这一变化与HC大鼠MI/R损伤之间的关系,说明MPO在HC大鼠缺血/再灌注心肌易损性增加中所发挥的作用并探讨其所涉及的部分损伤机制(如:NO有效利用率的下降等)。
材料和方法
1.材料
(1)健康Wistar远交群大鼠,4～5 weeks体重110±10 g,雄性;(2)H9c2(2-1)细胞株。
2.实验分组
(1)食源性HC大鼠模型分组
①普通饮食大鼠组:普通饲料饲养10周;②Vehicle +高胆固醇饮食组:高胆固醇饮食饲养10周,第9周开始10% DMSO(0.5 ml/只?d)腹腔注射一周;③ABAH+高胆固醇饮食组:高胆固醇饮食饲养10周,第9周开始ABAH(特异性MPO抑制剂,25.00mg/kg?d)腹腔注射一周。
(2)在体大鼠心脏局部缺血/再灌注后心肌梗塞面积测定的实验分组
①伪手术组:仅在冠状动脉左前降支(Left Anterior Descending, LAD)下穿线,不结扎,持续3/24 h;②缺血/再灌注+普通饮食大鼠组:普通饮食10周末,可逆性结扎LAD,心肌缺血30 min,再灌注3/24 h;③缺血/再灌注+ Vehicle+高胆固醇饮食组:高胆固醇饮食10周末,Vehicle大鼠可逆性结扎LAD,心肌缺血30 min,再灌注3/24 h;④缺血/再灌注+ ABAH +高胆固醇饮食组:高胆固醇饮食10周末,ABAH干预大鼠可逆性结扎LAD,心肌缺血30 min,再灌注3/24 h。
(3)H9c2(2-1)细胞株实验分组
①常氧组(Control):置于95%空气,5%CO2持续培养5 h;②ABAH+常氧组:加入100μΜABAH,摇匀后将细胞置于95%空气,5%CO2持续培养5 h;③缺氧/复氧组(H/R):置于充满95% N2和5% CO2的三气培养箱内缺氧培养3 h,然后再放入充满95%空气、5% CO2的常氧培养箱内复氧2 h;④MPO+缺氧/复氧组:加入0.1 u/ml MPO,摇匀后将细胞置于充满95% N2和5% CO2的三气培养箱内缺氧培养3 h,然后再放入充满95%空气、5% CO2的常氧培养箱内复氧2 h;⑤ABAH+缺氧/复氧组:加入100μΜABAH,摇匀后将细胞置于充满95% N2和5% CO2的三气培养箱内缺氧培养3 h,然后再放入充满95%空气、5% CO2的常氧培养箱内复氧2 h;⑥ABAH+MPO+缺氧/复氧组:先加入100μΜABAH摇匀后将细胞置于95%空气,5%CO2培养30 min后,再加入0.1 u/ml MPO,摇匀后将细胞置于充满95% N2和5% CO2的三气培养箱内缺氧培养3 h,然后再放入充满95%空气、5% CO2的常氧培养箱内复氧2 h。
3.方法
(1)实验模型的建立及标本的留取
①食源性HC大鼠模型的建立及标本的留取;②大鼠在体心脏局部缺血/再灌注模型的建立及标本的留取;③H9c2(2-1)细胞缺氧/复氧模型的建立及标本的留取。
(2)检测指标的测定
①大鼠空腹血清总胆固醇(Total Cholesterol, TC)、甘油三酯(Triglyceride, TG)和低密度脂蛋白-胆固醇(Low Density Lipoprotein Cholesterol, LDL-cho)的测定;②血清和培养液上清肌酸激酶(Creatine Kinase, CK)含量的测定;③血清和培养液上清乳酸脱氢酶(Lactate Dehydrogenase, LDH)含量的测定;④DNA原位末端缺口标记法(TUNEL)检测心肌细胞凋亡;⑤心肌细胞caspase-3相对活性检测;⑥大鼠缺血/再灌注后心肌梗塞面积的测定;⑦大鼠心功能监测;⑧免疫组织化学染色检测心肌组织MPO分布;⑨心肌组织MPO活性检测;⑩心肌组织MPO活性与血清CK含量、LDH含量、凋亡指数、caspase-3活性、心肌梗塞面积和心功能的相关性分析;○11心肌组织一氧化氮(nitric oxide, NO)含量测定;○12放射性免疫试剂盒测定心肌组织cGMP含量;○13Western-blot检测心肌组织一氧化氮合酶(nitric oxide synthase, NOS)的蛋白表达;○14实时定量PCR法检测心肌组织NOS的mRNA表达;○15细胞计数Kit-8检测H9c2(2-1)细胞增殖。
结果
1.食源性HC大鼠模型的建立
给予高胆固醇饮食饲养前(0周),各组大鼠的血脂水平无明显差异。高胆固醇饮食饲养10周后,与0周(TC: 1.30±0.32 mmol/L、TG: 0.29±0.16 mmol/L和LDL-cho: 0.39±0.12 mmol/L)和同期普通饮食饲养大鼠(TC; 1.44±0.14 mmol/L、TG: 0.26±0.05 mmol/L和LDL-cho: 0.45±0.14 mmol/L)相比,HC大鼠血脂水平显著升高(TC; 3.01±0.75 mmol/L、P<0.01; TG: 0.88±0.35 mmol/L, P<0.01和LDL-cho: 1.53±0.65 mmol/L, P<0.01),说明食源性HC大鼠模型建立成功。ABAH干预后,与Vehicle组相比血脂水平未发生明显改变。
2. HC大鼠缺血/再灌注心肌易损性增加
2.1 HC大鼠MI/R后心肌损伤加重
2.1.1 HC大鼠MI/R后血清CK含量增加
与伪手术组相比,MI/R组大鼠血清CK含量明显增高(0.17±0.01 U/ml vs. 0.05±0.02 U/ml, P<0.01)。与普通饮食大鼠MI/R组相比,HC大鼠MI/R组血清CK含量显著升高(0.70±0.08 U/ml, P<0.01)。
2.1.2 HC大鼠MI/R后血清LDH含量增加
与伪手术组相比,普通饮食MI/R组大鼠血清LDH含量明显增高(3091.59±33.69 U/L vs. 1854.45±422.97 U/L, P<0.01)。与普通饮食大鼠MI/R组相比,HC大鼠MI/R组血清LDH含量显著升高(3596.18±99.81 U/L, P<0.01)。
2.2 HC大鼠缺血/再灌注后心肌细胞凋亡增加
2.2.1 TUNEL检测
普通饮食大鼠缺血/再灌注危险区心肌组织凋亡阳性核显著增多,凋亡指数较伪手术组显著升高(16.07±1.02% vs. 1.44±0.14%,P<0.01)。与普通饮食缺血/再灌注大鼠相比,HC大鼠缺血/再灌注危险区心肌组织凋亡指数明显升高(22.63±1.02%, P<0.01)。
2.2.2 caspase-3活性分析
与伪手术组大鼠相比,普通饮食大鼠缺血/再灌注危险区心肌组织caspase-3活性显著升高(6.66±0.61 vs. 1.00±0.08,P<0.01);HC大鼠缺血/再灌注危险区心肌组织caspase-3活性较普通饮食大鼠明显升高(12.29±0.92,P<0.01)。
2.3 HC大鼠缺血/再灌注后心肌梗塞面积增加
各组大鼠危险区面积占左心室面积的百分比(AAR/LV比值)无统计学差异(P>0.05),表明在进行缺血/再灌注实验过程中,各组动物模型的缺血程度大致相当,排除了由此而造成的误差。实验结果显示,与普通饮食大鼠缺血/再灌注组相比,HC大鼠缺血/再灌注后心肌梗塞面积显著增大(56.05±3.91% vs 34.90±2.52%, P<0.01)。
2.4 HC大鼠MI/R心功能恶化
大鼠MI/R 3 h末,与伪手术组相比,普通饮食大鼠MI/R组LVSP、±dP/dtmax显著下降(LVSP: 15.12±1.38 Kpa vs. 18.28±3.02 Kpa, P<0.05; +dp/dtmax: 610.43±110.26 Kpa/s vs. 785.22±162.22 Kpa/s, P<0.01; -dp/dtmax: 500.57±116.20 Kpa/s vs. 667.00±234.83 Kpa/s, P<0.05);HC大鼠MI/R组LVSP、±dP/dtmax较普通饮食大鼠降低明显(LVSP: 9.95±1.85 Kpa, P<0.01; +dp/dtmax: 386.43±100.76 Kpa/s, P<0.01; -dp/dtmax: 264.53±98.76 Kpa/s, P<0.01)。
以上的结果显示,给予MI/R实验干预后,与普通饮食大鼠相比,HC大鼠血清CK含量和LDH含量增加,危险区心肌组织凋亡指数和caspase-3活性升高,心肌梗塞面积增大,心功能(LVSP和±dP/dtmax)降低。这些结果说明,HC大鼠MI/R损伤加重,提示HC大鼠缺血/再灌注心肌易损性增加。
3. HC大鼠缺血/再灌注心肌组织MPO的变化
3.1 HC大鼠缺血/再灌注心肌组织MPO活性分析
与伪手术组相比,MI/R引起普通饮食大鼠缺血/再灌注心肌组织MPO活性显著增加(13.48±4.58 U/g protein vs. 8.29±2.80 U/g protein, P<0.05);HC大鼠缺血/再灌注心肌组织MPO活性进一步升高(19.44±2.35 U/g protein, P<0.05)。
3.2 HC大鼠缺血/再灌注心肌组织MPO的分布
大鼠心肌组织MPO免疫组化观察结果显示,心脏局部缺血/再灌注后,大鼠非缺血区心肌组织仅可见少量散在的MPO免疫染色,而在缺血/再灌注区血管组织和心肌组织内可见大量密集的MPO染色。这一结果提示,缺血/再灌注心脏浸润的中性粒细胞释放大量MPO,这些MPO可能通过主动或被动的方式侵入缺血/再灌注心肌细胞,进而通过多种机制引起心肌细胞损伤。
而且,与普通饮食大鼠相比,HC大鼠缺血/再灌注区心肌组织MPO免疫染色显著增强,这与上述结果显示的HC进一步增加缺血/再灌注心肌组织MPO活性相一致。
以上结果说明,HC大鼠缺血/再灌注区心肌组织中MPO含量和活性升高,提示MPO可能参与了HC大鼠缺血/再灌注心肌损伤易感性的增加。
4.缺血/再灌注心肌组织MPO活性与缺血/再灌注心肌损伤之间的相关性分析
为了探讨MPO是否参与了HC大鼠缺血/再灌注心肌损伤易感性的增加,本实验采用SPSS 13.0软件对有关实验数据进行了以下相关性分析。
4.1缺血/再灌注心肌组织MPO活性与缺血/再灌注后大鼠血清CK含量和LDH含量呈正相关(CK: r = 0.618 , P<0.01; LDH: r = 0.64, P<0.01)。
4.2缺血/再灌注心肌组织MPO活性与缺血/再灌注心肌组织凋亡指数和caspase-3活性呈正相关(凋亡指数:r = 0.651, P<0.01; caspase-3: r = 0.619, P<0.01)。
4.3缺血/再灌注心肌组织MPO活性与缺血/再灌注后心肌梗塞面积呈正相关(r = 0.663, P< 0.01)。
4.4缺血/再灌注心肌组织MPO活性与大鼠缺血/再灌注后心肌收缩功能(LVSP和±dP/dtmax)呈负相关(LVSP: r = -0.555, P<0.01; +dP/dtmax: r = -0.794, P< 0.01; -dP/dtmax: r =-0.748, P<0.01)。
以上结果说明,大鼠缺血/再灌注心肌组织MPO活性与HC大鼠缺血/再灌注心脏损伤加重密切相关;提示,HC大鼠缺血/再灌注心肌组织MPO的变化可能与HC大鼠缺血/再灌注心肌易损性的增加有直接关系。
5. MPO抑制剂干预后,HC大鼠缺血/再灌注心肌组织MPO和心脏损伤的变化
为了进一步探讨MPO是否参与了HC大鼠MI/R心肌易损性的增加,二者之间是否存在直接作用,本实验应用MPO抑制剂ABAH在体干预HC大鼠,观察相应变化。
5.1 ABAH对H9c2(2-1)细胞的影响
ABAH干预后,H9c2(2-1)细胞常氧培养细胞存活率与常氧对照组组相比(0.98±0.01 vs. 1.01±0.05),无统计学差异;给予缺氧/复氧干预后,ABAH组H9c2(2-1)细胞存活率与缺氧/复氧组相比(0.63±0.06 vs. 0.60±0.09),也无统计学意义。说明,ABAH本身对心肌细胞以及给予缺氧/复氧干预后心肌细胞无影响,排除了由药物干预而形成的实验误差。
5.2 ABAH干预后,HC大鼠缺血/再灌注心肌组织MPO分布和活性的变化
与HC大鼠MI/R组相比,ABAH干预HC大鼠后,缺血/再灌注心肌组织MPO免疫染色明显减少,MPO活性显著降低(13.66±2.63 U/g protein, P<0.05)。
5.3 ABAH干预后,HC大鼠缺血/再灌注心肌损伤减轻
与HC大鼠MI/R组相比,ABAH干预HC大鼠后,缺血/再灌注后血清CK含量(0.31±0.06 U/ml, P<0.01)和LDH含量(3286.65±40.04 U/L, P<0.01)明显减少。
5.4 ABAH干预后,HC大鼠缺血/再灌注心肌细胞凋亡减轻
与HC大鼠MI/R组相比,ABAH干预HC大鼠后,缺血/再灌注心肌组织细胞凋亡指数(18.55±1.29%, P<0.01)和caspase-3活性(7.97±0.29, P<0.01)明显降低。
5.5 ABAH干预后,HC大鼠缺血/再灌注后心肌梗塞面积减小
经测定,各组大鼠危险区面积占左心室面积的百分比(AAR/LV比值)无统计学差异(P>0.05)。与HC大鼠MI/R组相比,ABAH干预HC大鼠后,缺血/再灌注后心肌梗塞面积明显减小(38.89±2.47%, P<0.01)。
5.6 ABAH干预后,HC大鼠缺血/再灌注心功能障碍减轻
与HC大鼠MI/R组相比,ABAH干预HC大鼠后,缺血/再灌注3 h末LVSP、±dP/dtmax有所恢复(LVSP: 13.73±2.20 Kpa, P<0.05; +dP/dtmax: 564.00±128.56 Kpa/s, P<0.05; -dP/dtmax: 444.00±96.08 Kpa/s, P<0.05)。
以上结果显示:首先,ABAH药物本身对心肌细胞基本无影响;其次,ABAH干预HC大鼠后,大鼠缺血/再灌注区心肌组织MPO分布和活性均减少;而且,ABAH干预HC大鼠后,HC大鼠MI/R损伤减轻。进一步提示,HC大鼠缺血/再灌注区心肌组织MPO分布与活性可能与HC大鼠缺血/再灌注心肌损伤易感性的增加有密切关系,但二者之间是否具有直接关系仍有待于进一步证实。
6. MPO对H9c2(2-1)细胞的影响
为了排除在体神经、体液调节等因素对实验结果的多方面的影响,本实验利用H9c2(2-1)细胞来观察MPO对心肌细胞的直接效应。
6.1 H9c2(2-1)细胞缺氧/复氧模型建立成功
与常氧对照组(Control)相比(CK: 0.02±0.01 U/ml; LDH: 33.11±21.18 U/L),缺氧/复氧(H/R)组细胞培养液上清中CK含量和LDH含量显著升高(0.08±0.03 U/ml, P<0.01; 856.22±28.91 U/L, P<0.01)。说明,成功地建立了H9c2(2-1)细胞缺氧/复氧损伤模型
6.2 MPO加重H9c2(2-1)细胞缺氧/复氧损伤
与H/R组相比,0.1 u/ml MPO+H/R组培养液上清CK含量和LDH含量均明显升高(0.42±0.11 U/ml, P<0.01; 1362.78±106.89 U/L, P<0.01)。说明,MPO不仅可以引起心肌细胞的损伤,而且可以进一步加重心肌细胞缺氧/复氧损失。提示, MPO分布与活性可能直接参与了心肌细胞的损伤,并进一步加重了缺氧/复氧心肌细胞损伤。
6.3 ABAH干预后,H9c2(2-1)细胞缺氧/复氧损伤的变化
与0.1 u/ml MPO+H/R组相比,ABAH+0.1 u/ml MPO+H/R组培养液上清CK含量和LDH含量均明显减少(0.13±0.07 U/ml, P<0.01; 880.81±80.86 U/L, P<0.01)。更进一步提示,MPO的分布和活性可能直接参与了心肌细胞的损伤,而且可能与HC大鼠缺血/再灌注心肌易损性的增加有直接关系。
7. HC大鼠缺血/再灌注心肌组织NO-cGMP信号通路受损
7.1 HC大鼠缺血/再灌注心肌组织NOx含量增加
与普通饮食大鼠MI/R组相比,HC大鼠缺血/再灌注心肌组织NOx含量显著增加(972.78±77.45μmol/g protein vs. 421.44±58.96μmol/g protein, P<0.01)。ABAH干预后,可以有效的减少HC大鼠缺血/再灌注心肌组织NOx含量(615.76±50.54μmol/g protein, P<0.01),但未完全恢复至正常生理状态。
7.2 HC大鼠缺血/再灌注心肌组织cGMP含量降低
经测定,与伪手术组大鼠相比,普通饮食大鼠缺血/再灌注心肌组织cGMP含量明显减少(31.53±11.37 pmol/mg protein vs. 74.22±22.23 pmol/mg protein, P<0.01);与普通饮食MI/R组大鼠相比,HC大鼠缺血/再灌注心肌组织cGMP含量明显减少(7.35±2.47 pmol/mg protein, P<0.05);ABAH干预后,与HC + MI/R组大鼠相比,HC大鼠缺血/再灌注心肌组织cGMP含量有所恢复(28.84±9.08 pmol/mg protein, P<0.01)。
7.3 HC大鼠缺血/再灌注心肌组织NOS mRNA表达和蛋白表达的变化
7.3.1 HC大鼠缺血/再灌注心肌组织NOS mRNA表达的变化
与伪手术组相比,普通饮食大鼠MI/R组危险区心肌组织iNOS mRNA表达明显升高(4.80±1.73 vs. 1±0.70, P<0.01);与普通饮食大鼠MI/R组相比,HC大鼠MI/R组危险区心肌组织心肌组织iNOS mRNA表达明显升高(10.09±2.15, P<0.01);ABAH干预后,HC大鼠MI/R危险区心肌组织iNOS mRNA表达较HC大鼠MI/R组降低(3.41±1.50, P<0.01)。
与普通饮食未手术大鼠相比,HC未手术大鼠心肌eNOS mRNA表达明显降低(0.87±0.02 vs. 1.00±0.04, P<0.01)。
各组大鼠心肌组织nNOS mRNA表达无统计学差异。
7.3.2 HC大鼠缺血/再灌注心肌组织NOS蛋白表达的变化
与伪手术组相比,普通饮食大鼠MI/R组危险区心肌组织iNOS蛋白表达明显升高(2.22±0.44 vs.1.00±0.26, P<0.05);与普通饮食大鼠MI/R组相比,HC大鼠MI/R组危险区心肌组织心肌组织iNOS蛋白表达明显升高(3.17±0.93, P<0.05);ABAH干预后,HC大鼠MI/R危险区心肌组织iNOS蛋白表达较HC大鼠MI/R组降低(2.28±0.46, P<0.05)。
与普通饮食未手术大鼠相比,HC未手术大鼠心肌eNOS蛋白表达明显降低(0.36±0.14vs. 1.00±0.37, P<0.05)。
各组大鼠心肌组织nNOS蛋白表达无统计学差异。
以上结果显示,普通饮食大鼠缺血/再灌注心肌组织NOx含量增加,HC大鼠缺血/再灌注后危险区心肌组织NOx含量进一步增加;普通饮食大鼠缺血/再灌注心肌组织cGMP含量减少,HC大鼠缺血/再灌注后危险区心肌组织cGMP含量进一步减少;普通饮食大鼠缺血/再灌注心肌组织iNOS mRNA表达和蛋白表达均升高,HC大鼠缺血/再灌注后危险区心肌组织iNOS mRNA表达和蛋白表达均进一步升高;HC大鼠心肌组织eNOS mRNA表达和蛋白表达均降低;各组nNOS mRNA表达和蛋白表达无统计学差异。提示,HC大鼠MI/R后,虽然危险区心肌组织NOx含量大量增加,但NO生物利用度降低,NO/cGMP信号通路可能受损,应用MPO抑制剂干预后,NO/cGMP信号通路损伤有所减轻;MPO可能通过降低心肌组织NO的生物利用度、损伤NO/cGMP信号通路,从而部分参与大鼠缺血/再灌注心肌损伤,以及在HC大鼠缺血/再灌注心肌易损性增加中发挥作用。而且,HC大鼠缺血/再灌注心肌组织大量生成的NO可能来自于iNOS催化生成。
小结
1. HC大鼠心肌组织MPO含量和活性明显升高,提示MPO活性的改变可能是HC使缺血/再灌注后心肌损伤恶化的原因之一;
2. HC大鼠缺血/再灌注后心肌组织MPO含量和活性显著升高的同时NO/cGMP通路受损,NO生物利用率下降,进一步提示我们HC缺血/再灌注后心肌损伤易感性增加可能部分是通过MPO活性的改变影响NO-cGMP信号通路来实现。
第三部分高胆固醇血症诱导大鼠血清α1-、β1-肾上腺素受体和M2-胆碱受体自身抗体的产生
目的
本部分实验首先选择α1 -AA、β1 -AA、β3 -AA、M2 -AA和AT1-AA阴性的健康雄性大鼠建立食源性HC大鼠模型;进而观察食源性HC是否会诱发G蛋白偶联受体自身抗体的产生;如果会,则进一步了解自身抗体的产生情况及其作用。
材料和方法
1.材料
健康Wistar远交群大鼠,4～5 weeks体重110±10 g,雄性。
2.实验分组
食源性HC大鼠模型分组
①普通饮食大鼠组:普通饲料饲养10周;②高胆固醇饮食6周组:高胆固醇饮食饲养6周;③高胆固醇饮食10周组:高胆固醇饮食饲养10周。
3.方法
(1)实验模型的建立及标本的留取
食源性HC大鼠模型的建立及标本的留取
(2)检测指标的测定
①大鼠空腹血清总胆固醇(Total Cholesterol, TC)、甘油三酯(Triglyceride, TG)和低密度脂蛋白-胆固醇(Low Density Lipoprotein Cholesterol, LDL-cho)的测定;②心肌组织石蜡切片的HE染色;③心肌组织超微结构电镜观察;④血清受体自身抗体(α1- AA、β1- AA、β3 -AA、M2- AA和AT1-AA)的测定;⑤流式细胞仪检测大鼠脾脏淋巴细胞CD4+/CD8+和淋巴细胞含量。
(3)肽段合成及自身抗体的测定:
5种抗原肽段由吉尔生化上海有限公司合成,分别相当于α1- AR、β1- AR、β3 -AR、M2- AR和AT1-AR细胞外第二环氨基酸序列的特异性抗原决定簇,合成肽段纯度为90%~95%。用ELISA法检测血清中各自身抗体的含量。
结果
1食源性HC大鼠模型建立成功
高胆固醇饮食饲养前,各组大鼠血脂水平无明显差异。高胆固醇饮食6周后,大鼠血脂水平显著升高(TC: 2.64±0.22, P<0.01; TG: 0.89±0.33, P<0.01; LDL-cho: 1.06±0.23, P<0.01)。高胆固醇饮食10周后血脂水平进一步升高(TC: 3.46±0.68 mmol/l, P<0.01; TG: 1.17±0.39 mmol/l, P<0.01; LDL-cho: 1.67±0.35 mmol/l, P<0.01)。说明食源性HC大鼠模型建立成功。
2. HC大鼠血清α1-AA、β1-AA和M2-AA的OD值升高
与正常饮食大鼠相比,高胆固醇饮食6周大鼠血清α1-AA(0.60±0.05 vs 0.27±0.06,P<0.01)、β1-AA(0.57±0.06 vs 0.18±0.02,P<0.01)和M2-AA(0.53±0.07 vs 0.16±0.04,P<0.01)的OD值明显升高,高胆固醇饮食10周大鼠血清抗体OD值有略有下降的趋势。HC大鼠血清β3-AA和AT1-AA与正常饮食大鼠相比无明显改变。提示,HC可能诱发部分G蛋白偶联受体自身抗体的产生。
3. HC大鼠心肌组织形态学观察
3.1 HC大鼠心肌组织病理形态学观察
普通饮食大鼠,心肌纤维完整,排列整齐,呈束状,结构清楚,着色均匀,细胞核形态正常,细胞膜完整,细胞没有水肿,无变性坏死,心肌间质未见炎性细胞浸润。高胆固醇饮食6周和10周的HC大鼠心肌形态发生了改变,同时伴随程度不同的炎性细胞浸润。
3.2 HC大鼠心肌组织超微结构观察
普通饮食大鼠,心肌肌原纤维排列整齐,肌节长短一致,明带、暗带清晰可见;线粒体丰富,结构清晰完整,大小形态正常,无肿胀,嵴无断裂,嵴排列整齐,线粒体间糖原颗粒丰富;毛细血管内皮细胞结构正常,细胞膜完整。
高胆固醇饮食6周HC大鼠心肌细胞间质微血管官腔狭窄,内皮细胞水肿,胞质中胞饮泡增多、散在较多的脂泡,肌浆网扩张,可见等电子密度云絮状物质,个别细胞可见肌丝溶解。高胆固醇饮食10周HC大鼠心肌细胞核染色质小灶性溶解,胞质内脂泡增多,可见大面积脂质区域,部分细胞肌丝溶解面积较大,肌丝疏松散在,肌浆网明显扩张,个别心肌细胞膜溶解,心肌细胞间质中微血管内皮细胞胞饮泡增生,血管腔内散在血小板和红细胞。
以上结果说明HC大鼠心肌细胞结构受损,超微结构发生改变,提示HC可能造成大鼠心肌组织超微结构改变,并可能引起大鼠心肌组织重构,激活HC大鼠免疫系统,从而诱发HC大鼠G蛋白偶联受体自身抗体的产生。
4. HC大鼠脾脏淋巴细胞CD4~+/CD8~+升高,淋巴细胞含量升高
脾脏是人体免疫系统最重要的免疫器官之一。流式细胞技术检测大鼠脾脏淋巴细胞结果显示,HC大鼠与同期普通饮食大鼠相比,CD4~+/CD8~+(2.34±0.157 vs 1.61±0.21,P<0.05)明显升高,淋巴细胞(DC45R~+细胞)含量(25.34±3.18 vs 7.64±3.47,P<0.01)升高。进一步提示,血脂水平升高可能激活HC大鼠免疫系统,从而诱发HC大鼠G蛋白偶联受体自身抗体的产生。
小结
1.本研究首次在HC大鼠血清中发现了α1-AA、β1-AA和M2-AA等G蛋白偶联受体自身抗体。
2. HC大鼠心肌超微结构和脾脏免疫系统功能发生改变,提示HC有可能通过引起心肌重构并激活机体免疫系统,从而诱发部分G蛋白偶联受体自身抗体产生,并进一步影响HC大鼠心脏功能。
结论
1. HC可以诱发心肌细胞膜脂质比例失调,降低心肌细胞膜流动性,引起心肌细胞功能障碍,从而导致心肌损伤加重;有效的改善心肌细胞膜流动性可以减轻心肌细胞损伤。提示心肌细胞膜流动性可能是HC大鼠心肌易损性增加的影响因素之一。
2. MPO可能通过降低心肌组织NO生物利用率,损伤NO/cGMP/cGK信号通路,参与HC大鼠缺血/再灌注心肌易损性增加,导致HC大鼠MI/R损伤加重。
3.本研究首次在HC大鼠血清中发现了α1-、β1-和M2-受体的自身抗体,提示部分G蛋白偶联受体自身抗体的产生可能也是HC大鼠心肌易损性增加的病理生理机制之一。
Background
Hypercholesterolemia and the corresponding artherosclerosis are the pathological foundation of coronary atherosclerotic heart disease (CAHD) and heart muscle ischemia, and are critical risk factors of ischemic heart disease. Hypercholesterolemia plays an important role in both the beginning of ischemia and the development of the injury. We and other researchers have found the myocardium has increased susceptibility to ischemia/reperfusion injury in hypercholesterolemia, but the mechanisms are not clear. We know that lipid can accumulate and later form artherosclerosis plaques. When such plaque covers the coronary, it will cause spasm. So do the increase of active oxygen production and reactive nitrogen production, which induce dysfunction of endothelium. However, these initial factors can not explain the phenomenon of increased susceptibility. There are still lots of questions.
Membrane fluidity is the dynamical character of lipid bilayer, which is important in signal transduction, chemical transportation, ion exchange, energy transmission and activity of enzymes. Stable membrane fluidity is so significant to the maintenance of internal environment and normal function of cells that it affects the development of many diseases. The change of membrane fluidity comes earlier than the morphological and biological changes of cells in a disease. The changes of membrane fluidity can also reflect the early cell injury and the development of disease. Some studies found that membrane fluidity of red blood cell decreases in hypercholesterolemia. This indicates that the disorder of lipid metabolism may change the lipid proportion of the membrane and further affect its fluidity. Will hypercholesterolemia change the fluidity of myocardium and affect its signal transduction? If so, is there any relationship between the changes of myocardium membrane fluidity and heart muscle increased that susceptibility caused by hypercholesterolemia? Study considering these questions will help us to investigate the mechanism of increased susceptibility of myocardium to ischemia/reperfusion injury caused by hypercholesterolemia.
Myeloperoxidase (MPO), which expresses in neutrophil, is an important cytokine and an independent risk factor of CAHD. MPO stimulates the apoptosis of endothelium, degrades the relaxing factor NO, lowers its biological activity, causes endothelium dysfunction and increases the vulnerability of atherosclerotic plaque, which enhances the development of heart muscle ischemia. Moreover, MPO released by activated and effused neutrophils could catalyze H2O2 produced by neutrophils and injured tissue forms hypochloric acid and other oxidants, which are toxic and will cause cardiovascular injury. However, inflammation reaction plays an important role in myocardium ischemic/reperfusion injury. So, whether MPO plays a direct function of injury to myocardium? Does it take part in the increased susceptibility of ischemia/reperfusion injury heart muscle in hypercholesterolemia? And how does it work?
From 1990, researchers detected many G protein-coupled receptor autoantibodies from serum of patients with cardiovascular diseases, includingα1-AA、β1-AA、β3-AA、M2-AA and AT1-AA etc. Some study indicatesα1-AA existence would cause increased peripheral resistance and myocardium remodel.β1-AA induces heart rate and later exacerbates heart burden and then results in injury. M2-AA destroys mitochondria, which would lead to myofibrilla dissolve and heart injury. These results suggest that G protein-coupled Receptors autoantibody may play an important role in the development of cardiovascular diseases. Evidence proved that hypercholesterolemia causes ultrastructure changes of myocardium and inflammation response in cardiovascular diseases. These indicate that hypercholesterolemia probably induce the production of G protein-coupled receptors autoantibody by the dysfunction of immune system. If this hypothesis is true, these autoantibodies have high probability to increase the susceptibility of myocardium in hypercholesterolemia and aggravate heart injury. As a result, investigation of the production of G protein-coupled receptors autoantibody in hypercholesterolemia is critical in cardiovascular diseases.
Overall, basing on a dietetic hypercholesterolemia rat model, current research aim to discuss the functions and some related mechanisms of cardiac muscle cell fluidity, myeloperoxidase and certain portion of G protein-coupled receptors autoantibody in hypercholesterolemia increased susceptibility of heart muscle.
PART 1 Hypercholesterolemia-induced myocardial cell membrane fluidity decline in rats
Objective:
To determine whether decreased membrane fluidity of myocardial cells may contribute to enhanced myocardial injury in hypercholesterolemia.
Materials and Methods:
1.1 Materials:
Male Wistar rats (4-5 weeks) weighing110±10g, provided by Shanxi Medical University Laboratory Animal Center.
1.2 Experimental groups
1.2.1 The groups of diet-derived HC rats
(1) Control group: Rats were given a normal diet for 10 weeks;
(2) Vehicle + high cholesterol group: Rats were given a HC diet for 10 weeks and receive vehicle for 4 week in the 6th week;
(3) PIO + high cholesterol group: Rats were given a HC diet for 10 weeks and receive PIO (10 mg/kg/day, oral gavage) for 4 week in the 6th week.
1.2.2 The groups of rats’regional myocardial ischemia/ reperfusion (MI/R) in vivo
(1) Sham group: Left Anterior Descending (LAD) without occlusion, total time course is 24 hours;
(2) MI/R+ normal diet group: LAD with reversible occlusion, 30min ischemia followed by 24 hours reperfusion;
(3) MI/R+ Vehicle + HC diet group: LAD with reversible occlusion, 30min ischemia followed by 24 hours reperfusion;
(4) MI/R+ PIO + HC diet group: LAD with reversible occlusion, 30min ischemia followed by 24 hours reperfusion.
1.3 Methods
1.3.1 Establishment of experimental model and sample collection:
(1) The establishment of diet-derived HC rat model and tissue collection;
(2) The establishment of rat via a polyethylene catheter inserted into the left ventricular cavity through the right carotid artery in vivo and tissue collection;
(3) Isolation of cardiac myocyte and sample collection;
(4) The establishment of rat regional MI/R model in vivo and tissue collection
1.3.2 Index determination
(1) Measurement of Total Cholesterol (TC), Triglyceride (TG) and Low Density Lipoprotein Cholesterol (LDL-cho) in serum of rat on an empty stomach;
(2) Determination of myocardial function;
(3) Detection of membrane Na~+-K~+-ATPase activity;
(4) Detection of cholesterol and phospholipid content in myocardial cell membrane;
(5) Membrane fluidity measurements
(6) Determination of cAMP with radioimmunoassay
(7) Determination of myocardial infarct size
(8) Correlation analysis between membrane fluidity and myocardial contractile function or cardiac infarct size
Results:
2.1 PIO had effects on plasma lipid profiles after high cholesterol diet
Ten weeks of HC diet resulted in a dramatic increase in plasma cholesterol (TC) (3.44±0.86 mmol/l vs. 1.24±0.26 mmol/l, P<0.01), triglyerde (TG) (1.13±0.34 mmol/l vs. 0.26±0.05 mmol/l, P<0.01) and low-density lipoprotein (LDL-cho) (1.66±0.55 mmol/l vs. 0.45±0.14 mmol/l, P<0.01). There were no significant differences between groups in plasma lipid profiles before the onset of the study (time 0) or after 6 weeks of a HC diet (before PIO treatment). After an additional 4 weeks of high cholesterol diet and treated with vehicle, plasma TC, TG and LDL levels further increased. At all these post-cholesterol times, PIO-treated rats on the HC diet exhibited significantly lower plasma TC (2.16±0.33 mmol/l, P<0.01), TG (0.59±0.18 mmol/l, P<0.05) and LDL-cho (1.24±0.28 mmol/l, P<0.05) than the vehicle group. These results indicate that PIO improved plasma lipid profiles in this diet-induced HC model, and therefore, any cardiovascular effects observed could be attributed to this mechanism.
2.2 Hypercholesterolemia worsens cardiac functional and increases post-ischemic myocardial injury
HC diet markedly aggravated myocardial contractile function. The values of LVSP, +dp/dtmax and–dp/dtmax were significantly decreased in the HC diet group compared with the normal diet group (LVSP: 13.64±1.37 Kpa vs. 20.79±2.87 Kpa, P<0.01; +dp/dtmax: 537.43±102.24 Kpa/s vs. 953.38±171.12 Kpa/s, P<0.01; -dp/dtmax: 452.19±88.34 Kpa/s vs. 793.10±112.82 Kpa/s, P<0.01). Additionally, ischemia/reperfusion-induced myocardial infarct size in the HC rats (i.e. vehicle) was significantly larger than the normal rats (56.81±6.45% vs 35.25±2.52%, P<0.01). There was no difference in ischemic area (AAR) expressed as percent of left ventricle, indicating a comparable degree of ischemic insult between sham-, normal diet-, vehicle- and PIO-treated groups after left anterior descending artery occlusion.
2.3 Hypercholesterolemia decreased membrane Na~+-K~+-ATPase activity, increased C/P and reduced the membrane fluidity of cardiac myocytes
Na~+-K~+-ATPase reside in cell membrane nullity of the organelle membrane, which activity is usually to identify the cell membrane. A clear decrease in Na~+-K~+-ATPase activity was observed in HC rats compared with that in normal diet rats (5.99±1.69μmol Pi/mg protein/hour vs. 7.88±1.25μmol Pi/mg protein/hour, P<0.01). The C/P of cardiac myocytes membrane was largely higher in rats with HC diet than normal diet (0.44±0.04 vs. 0.33±0.03, P<0.01). Most importantly, in myocardial cells from HC rats, the DPH fluorescence polarization (P) and microviscosity (η) were markedly higher than that seen in the normal diet group (0.34±0.07 vs 0.24±0.05, P<0.01; 7.99±1.37 vs 2.41±0.59, P<0.01, respectively), indicating that hypercholesterolemia decreased cardiac myocytes membrane fluidity.
2.4 Hypercholesterolemia decreased myocardial cAMP content
To further elucidate the effect by which hypercholesterolemia may disturb myocardial function, myocardial cAMP content, a downstream product in signal transduction pathway of membrane receptor, was determined. Hypercholesterolemia markedly decreased myocardial cAMP content (53.77±22.17 pmol/mg protein vs. 127.50±19.72 pmol/mg protein, P<0.01). The result demonstrated that hypercholesterolemia may cause myocardial function injury and thus contribute to increased myocardial vulnerability after ischemia and reperfusion.
2.5 PIO treatment improved myocardial contractile function, attenuated myocardial infarct size, increased Na~+-K~+-ATPase activity and cAMP content, reduced C/P, and restored membrane fluidity
Previous studies have demonstrated that PPARγagonists, such as rosiglitazone (RSG), exert beneficial cardiovascular effects in diabetic patients. In recent studies, researchers have demonstrated that treatment with RSG significantly improved endothelial and myocardial function in hypercholesterolemic rabbits. To determine whether the PPARγsignaling pathway may improve cardiac function by restoring membrane fluidity in a non-diabetic model, 18 rats fed with a high cholesterol diet were treated with PIO for 4 weeks. Treatment with PIO in hypercholesterolemic rats exerted significant protective effects as evidenced by markedly improveded cardiac contractile dysfunction (LVSP: 20.40±2.83 Kpa, P<0.01; +dp/dtmax : 827.56±172.49 Kpa/s, P<0.01;–dp/dtmax: 684.01±113.43 Kpa/s, P<0.01) and attenuated myocardial infarct size (39.82±2.74, P<0.01). PIO treatment tended to significantly increase Na+-K+-ATPase activity (7.43±1.42μmol Pi/mg protein/hour, P<0.01) and decrease C/P (0.40±0.04, P<0.05) in cardiac myocytes membrane. In addition, treatment with PIO markedly decreased both DPH fluorescence polarization (0.26±0.06, P<0.01) and microviscosity (3.29±0.63, P<0.01), a parameter that primarily reflects membrane fluidity. To further determine the effect by which PIO may exert its cardiac protective properties, the cAMP content in myocardial cell of PIO treatment was determined. Treatment with PIO in hypercholesterolemia significantly increased myocardial cAMP content (102.05±25.51, P<0.05).
2.6 Negative correlation between myocardial contractile function and DPH fluorescence polarization, positive correlation between cardiac infarct size and DPH fluorescence polarization
To determine the direct relation between membrane fluidity and myocardial function or cardiac injury, correlation between DPH fluorescence polarization and LVSP, +dp/dt_(max), -dp/dt_(max) and infarct size were analyzed. LVSP, +dp/dt_(max) and -dp/dt_(max) negatively correlated to DPH fluorescence polarization respectively (r=-0.191, P<0.05; r=-0.323, P<0.01; r=- 0.322, P<0.01, respectively.). Cardiac infarct size positively correlated to DPH fluorescence polarization (r=0.599, P<0.01). In other words, membrane fluidity positively correlated to myocardial contractile function and negatively correlated to cardiac infarct size. These results suggest that hypercholesterolemia-induced cardiac impairment may be associated with membrane fluidity decrease, and PIO treatment protects hypercholesterolemia-induced myocardial dysfunction by preserving the integrity of membrane fluidity.
Summary
1. Hypercholesterolemia impairs cardiac function, and increases myocardial infarct size after ischemic/reperfusion, suggesting that hypercholesterolemia may enhance cardiac vulnerability.
2. Hypercholesterolemia increases C/P in the myocardial cell membrane, decreases membrane fluidity and myocardial cAMP content, as well as membrane fluidity positively correlates to myocardial contractile function and negatively correlates to cardiac infarct size, suggesting that hypercholesterolemia may exacerbate cardiac injury by decreased membrane fluidity-induced myocardial dysfunction.
3. PIO treatment markedly inhibited hypercholesterolemia-induced C/P increase, membrane fluidity decrease of cardiac myocyte membrane, and myocardial cAMP content decrease, thus recovered cardiac function and decreased infarct size after myocardial ischemia/reperfusion.
PART 2 The role of MPO in the increased susceptibility of ischemic-reperfusion injury in hypercholesterolemia rats
Objective:
To confirm the role of MPO in the increased susceptibility of ischemic-reperfusion injury in hypercholesterolemia rats and to explore the mechanism (such as the reduction in the effective utilization of NO etc.) by observing the changes of MPO activity and distribution in myocardial tissue of ischemia/reperfusion HC rats.
Materials and Methods:
1.1 Materials:
(1) Male Wistar rats (4-5 weeks) weighing110±10g, provided by Shanxi Medical University Laboratory Animal Center.
(2) H9c2 (2-1) cell line
1.2 Experimental groups
1.2.1 The groups of diet-derived HC rats
(1) Control group: Rats were given a normal diet for 10 weeks;
(2) Vehicle + high cholesterol group: Rats were given a HC diet for 10 weeks and intraperitoneal injection of 10% DMSO for 1 week in the 9th week;
(3) ABAH (MPO inhibitor) + high cholesterol group: Rats were given a HC diet for 10 weeks and intraperitoneal injection of ABAH (25.00mg/kg?d) for 1 week in the 9th week.
1.2.2 The groups of rats’regional myocardial ischemia/ reperfusion (MI/R) in vivo
(1) Sham group: Left Anterior Descending (LAD) without occlusion, total time course is 3 or 24 hours;
(2) MI/R+ normal diet group: LAD with reversible occlusion, 30min ischemia followed by 3 or 24 hours reperfusion;
(3) MI/R+ Vehicle + HC diet group: LAD with reversible occlusion, 30min ischemia followed by 3 or 24 hours reperfusion;
(4) MI/R+ ABAH + HC diet group: LAD with reversible occlusion, 30min ischemia followed by 3 or 24 hours reperfusion.
1.2.3 The groups of H9c2 (2-1) cell lines
(1) Normoxia group: cultured in atmosphere of 5%CO2, 95% air for 5h;
(2) ABAH+ Normoxia group: added 100μM ABAH into the cultured medium then cultured in atmosphere of 5%CO_2, 95% air for 5h;
(3) H/R group: hypoxia with 95% N_2 and 5% CO_2 for 3h then followed by 2h reoxygenation in 5% CO_2 and 95% air;
(4) MPO+H/R group: added 0.1 u/ml MPO into the cultured medium then operated as H/R group;
(5) ABAH+H/R group: added 100μM ABAH into the cultured medium then operated as H/R group;
(6) ABAH+MPO+ H/R group: added 100 mΜABAH into the cultured medium for 30 min before adding 0.1 u/ml MPO then operated as H/R group.
1.3 Methods
1.3.1 Establishment of experimental model and sample collection
(1) The establishment of diet-derived HC rat model and tissue collection;
(2) The establishment of rat regional MI/R model in vivo and tissue collection;
(3) The establishment of H9c2 (2-1) H/R model and cell collection.
1.3.2 Index determination
(1) Measurement of Total Cholesterol (TC), Triglyceride (TG) and Low Density Lipoprotein Cholesterol (LDL-cho) in serum of rat on an empty stomach;
(2) Measurement of Creatine Kinase (CK) content in serum and culture medium supernatant;
(3) Measurement of Lactate Dehydrogenase (LDH) content in serum and culture medium supernatant;
(4) Myocardial apoptosis is detected by TdT-mediated dUTP nick end labeling (TUNEL);
(5) Caspase-3 relative activity assay;
(6) Determination of myocardial infarct size;
(7) Measurement of cardiac function;
(8) Determination of myocardial tissue MPO distribution by immunohistochemistry;
(9) Myocardial tissue MPO activity assay;
(10) Correlation analysis of myocardial tissue MPO activity , serum CK concentration, LDH leakage, apoptotic index, caspase-3 activity , myocardial infarction size and cardiac function;
(11) Measurement of nitric oxide (NO) content in myocardial tissue;
(12) Determination of myocardial cGMP content by Radioimmunoassay kit;
(13) Detection of myocardial nitric oxide synthase (NOS) protein expression by Western-blot;
(14) Detection of the mRNA expression of NOS in myocardial tissue by real time PCR;
(15) Detected H9c2 (2-1) cell proliferation by Cell counting Kit-8.
Results:
2.1 The establishment of Diet-derived HC rat model
The lipid levels (TC, TG and LDL-cho) in rats of each group had no significant difference before given HC diet feeding (0 week). The lipid levels of vehicle + high cholesterol group was significantly higher than control group after feeding with high-cholesterol diet for 10 weeks (TC: 3.01±0.75 mmol/L vs. 1.44±0.14 mmol/L, P<0.01; TG: 0.88±0.35 mmol/L vs. 0.26±0.05 mmol/L, P<0.01; LDL-cho: 1.53±0.65 mmol/L vs. 0.45±0.14 mmol/L, P<0.01, respectively.). All these demonstrated that diet-derived HC rat model was set up successfully.
2.2 HC rats had increased vulnerability of MI/R
2.2.1 A significantly exacerbated myocardial MI/R injury was found of HC rats
2.2.1.1 CK content in serum of HC rats were increased
Compared with sham group, the CK content in MI/R group increased significantly (0.17±0.01 U/ml vs. 0.05±0.02 U/ml, P<0.01); Compared with normal diet MI/R group, the CK content in HC MI/R group increased significantly (0.70±0.08 U/ml, P<0.01).
2.2.1.2 LDH content in serum of HC rats were increased
Compared with sham group, the LDH content in MI/R group increased significantly (3091.59±33.69 U/L vs. 1854.45±422.97 U/L, P<0.01); Compared with normal diet MI/R group, the LDH content in HC MI/R group increased significantly (3596.18±99.81 U/L, P<0.01).
2.2.2 Increased myocardial apoptosis in HC rats with ischemia/reperfusion
2.2.2.1 TUNEL detection
The apoptotic index in MI/R group was significantly higher than that in sham group (16.07±1.02% vs. 1.44±0.14%,P<0.01); the apoptotic index in HC MI/R group was significantly higher than that in normal diet MI/R group (22.63±1.02%, P<0.01).
2.2.2.2 Caspase3 activity assay:
Caspase3 activity in MI/R group was also significantly increased compared with sham group (6.66±0.61 vs. 1.00±0.08,P<0.01); caspase3 activity in HC MI/R group was also significantly increased compared with normal diet MI/R group (12.29±0.92,P<0.01).
2.2.3 Myocardial infarct size in HC group enhanced after MI/R
The ratio of area at risk to left ventricular area (AAR/LV): no significant differences were observed among these groups (P>0.05), which indicated that the ischemic area induced by coronary ligation is roughly identical among each experimental group, so that these groups are comparable. The myocardial infarct size of HC MI/R group was significantly enhanced as compared with normal diet MI/R group (56.05±3.91% vs. 34.90±2.52%, P<0.01).
2.2.4 The cardiac function in HC MI/R group were aggravated
At the end of 3 h reperfusion, compared with the sham group, the LVSP and±dP/dtmax in MI/R group decreased obviously (LVSP: 15.12±1.38 Kpa vs. 18.28±3.02 Kpa, P<0.05; +dp/dtmax: 610.43±110.26 Kpa/s vs. 785.22±162.22 Kpa/s, P<0.01; -dp/dtmax: 500.57±116.20 Kpa/s vs. 667.00±234.83 Kpa/s, P<0.05, respectively.);compared with the normal diet MI/R group,the LVSP and±dP/dtmax in HC MI/R group decreased obviously (LVSP: 9.95±1.85 Kpa, P<0.01; +dp/dtmax: 386.43±100.76 Kpa/s, P<0.01; -dp/dtmax: 264.53±98.76 Kpa/s, P<0.01, respectively.).
These results confirmed that, compared with the normal diet group, the MI/R injury in HC group increased, which indicated the increased susceptibility of ischemic-reperfusion injury in hypercholesterolemia rats.
2.3 Myocardial tissue MPO changes in HC rats with ischemia/reperfusion
2.3.1 Myocardial tissue MPO activity assay in HC rats with ischemia/reperfusion
Compared with the sham group, the myocardial tissue MPO activity in MI/R group increased obviously (13.48±4.58 U/g protein vs. 8.29±2.80 U/g protein, P<0.05), and further increased in HC MI/R group (19.44±2.35 U/g protein, P<0.05).
2.3.2 Myocardial tissue MPO distribution of HC rats with ischemia/reperfusion
The result of immunohistochemistry indicated that rare MPO was detected in the area not at risk (ANAR) of myocardial tissue from normal diet rats, but clear staining was observed in the vascular tissue and cardiac myocytes from ischemia/reperfusion area. The results suggested that infiltrating neutrophils in ischemia/reperfusion heart release a large number of MPO, these MPO possibly invasive ischemia/reperfusion myocardial cells through active or passive manner then cause myocardial cell injury through a variety of mechanisms. Moreover, the MPO immunostaining further enhanced in HC MI/R group than the normal diet MI/R group.This is consistent with the above-mentioned results that MPO activity increased in HC MI/R group.
Prompted MPO may be involved in the increased susceptibility of ischemia/reperfusion myocardial injury in HC rats.
2.4 Correlation analysis of myocardial tissue MPO activity with MI/R injury.
2.4.1 Positive correlation between MPO activity in ischemia/reperfusion myocardial tissue and serum CK and LDH levels (CK: r = 0.618, P<0.01; LDH: r = 0.64, P<0.01, respectively.).
2.4.2 Positive correlation between MPO activity and apoptotic index and caspase-3 activity in ischemia/reperfusion myocardial tissue (apoptotic index: r = 0.651, P<0.01; caspase-3: r = 0.619, P<0.01, respectively.).
2.4.3 Positive correlation between MPO activity and myocardial infarct size in ischemia/reperfusion myocardial tissue (r = 0.663, P< 0.01).
2.4.4 Negative correlation between MPO activity in ischemia/reperfusion myocardial tissue and myocardial contractile function (LVSP and±dP/dtmax) of ischemia/reperfusion heart (LVSP: r = -0.555, P<0.01; +dP/dtmax: r = -0.794, P< 0.01; -dP/dtmax: r =-0.748, P<0.01, respectively.).
These results confirmed the closely correlation between MPO activity in ischemia/reperfusion myocardial tissue and increased susceptibility of ischemia/reperfusion injury in hypercholesterolemic rats. Changes of MPO in myocardial tissue of HC rats with ischemia/reperfusion were directly related to ischemia/reperfusion myocardial vulnerability of HC rats.
2.5 MI/R HC rats’myocardial tissue MPO and cardiac injury changed after the intervention with MPO inhibitors
In order to further explore whether or not MPO participate in and directly affect the HC rat MI / R myocardial vulnerability increased, ABAH was applicated to HC rats in vivo to observe the corresponding changes.
2.5.1 Effect of ABAH on the H9c2 (2-1) cell
There was no statistical difference of cell survival rate between ABAH + normoxia group and normoxia group (0.98±0.01 vs. 1.01±0.05); there was no statistical difference of cell survival rate between ABAH+H/R group and H/R group too (0.63±0.06 vs. 0.60±0.09). The results above showed that ABAH itself had no effect on myocardial cells and removed the experimental error which forms by the medicine intervention.
2.5.2 The change of MPO activity and distribution in myocardial tissue, after ABAH intervention
Compared with the MI/R+ Vehicle + HC diet group, the MPO immunostaining and MPO activity (13.66±2.63 U/g protein, P<0.05) in MI/R+ ABAH + HC diet group significantly decreased.
2.5.3 After ABAH intervention, the myocardial ischemia/reperfusion injury of HC rats mitigated.
Compared with the MI/R+ Vehicle + HC diet group CK (0.31±0.06 U/ml, P<0.01) and LDH (3286.65±40.04 U/L, P<0.01) content in MI/R+ ABAH + HC diet group significantly decreased.
2.5.4 After ABAH intervention, cardiomyocyte apoptosis of HC rats with ischemia/reperfusion mitigated.
Compared with the MI/R + Vehicle + HC diet group, apoptotic index (18.55±1.29%, P<0.01) and Caspase3 activity (7.97±0.29, P<0.01) in MI/R+ ABAH + HC diet group significantly decreased.
2.5.5 After ABAH intervention, the myocardial infarct size of HC rats decreased
The ratio of AAR/LV: no significant differences were observed among these groups (P>0.05). The myocardial infarct size of MI/R+ ABAH + HC diet group was significantly decreased as compared with MI/R+ Vehicle + HC diet group (38.89±2.47%, P<0.01).
2.5.6 After ABAH intervention, HC rats with ischemia/reperfusion cardiac dysfunction mitigated Compared with the MI/R + Vehicle + HC diet group, the cardiac dysfunction in MI/R+ ABAH + HC diet group had some degrees recovery (LVSP: 13.73±2.20 Kpa, P<0.05; +dP/dtmax: 564.00±128.56 Kpa/s, P<0.05; -dP/dt_(max): 444.00±96.08 Kpa/s, P<0.05, respectively.).
These results indicated: First, ABAH itself had no effect on myocardial cells. Second, after ABAH intervention, MPO activity and distribution in myocardial tissue of HC rats with ischemia/reperfusion decreased and ischemia/reperfusion injury mitigated. Third, MPO changes in myocardial tissue of HC rats with ischemia/reperfusion had close related to ischemia/reperfusion myocardial vulnerability of HC rats. Howerve, it needs to be further confirmed that whether there is direct relation between them.
2.6 Effect of MPO on the H9c2 (2-1) cell
In order to remove various influence of neural and humoral regulation to experimental result in vivo, the experimental use of H9c2 (2-1) cells to observe the MPO on the direct effects of myocardial cells.
2.6.1 H9c2 (2-1) H/R model was established successfully
Compared with the normoxia group (control group), the CK and LDH content in culture medium supernatant in H/R group significantly increased (CK: 0.08±0.03 U/ml vs. 0.02±0.01 U/ml, P<0.01; LDH: 856.22±28.91 U/L vs. 33.11±21.18 U/L, P<0.01, respectively.), showing that H9c2 (2-1) H/R model had been established successfully.
2.6.2 MPO aggravated hypoxia/reoxygenation injury in H9c2 (2-1) cells
Compared with H/R group, the CK and LDH content in culture medium supernatant in 0.1u/ml MPO+H/R group significantly increased (0.42±0.11 U/ml, P<0.01; 1362.78±106.89 U/L, P<0.01, respectively.), showing that MPO could not only cause myocardial cells' damage but also further aggravate the myocardial cell hypoxia/reoxygenation injury, which prompted that the MPO distribution and activity possibly directly participated in myocardial cell's damage, and further aggravated the myocardial cell hypoxia/reoxygenation injury.
2.6.3 After ABAH intervention, H9c2 (2-1) cell H/R damage change
Compared with 0.1 u/ml MPO+H/R group, the CK and LDH content in ABAH+0.1 u/ml MPO+H/R group significantly decreased (0.13±0.07 U/ml, P<0.01; 880.81±80.86 U/L, P<0.01, respectively.), further prompted that MPO distribution and activity possibly directly participated in myocardial cells’damage, and had directly relationship with ischemia/reperfusion myocardial vulnerability of HC rat.
2.7 HC rats with ischemia/reperfusion myocardial tissue NO-cGMP signaling pathway impaired
2.7.1 NOx content increased in myocardial tissue of HC rats with ischemia/reperfusion
Compared with MI/R + normal diet group, the NOx content in MI/R + Vehicle + HC diet group significantly increased (972.78±77.45μmol/g protein vs. 421.44±58.96μmol/g protein, P<0.01). ABAH could effectively reduce the NOx content in HC rat ischemia/reperfusion myocardial tissue (615.76±50.54μmol/g protein, P<0.01), but had not restored completely to the normal physiological condition.
2.7.2 The cGMP content in myocardial tissue of HC rats with ischemia/reperfusion decreased
Compared to sham group the cGMP content in MI/R+ normal diet group decreased (31.53±11.37 pmol/mg protein vs. 74.22±22.23 pmol/mg protein, P<0.01); compared to MI/R+ normal diet group, the cGMP content in MI/R+ Vehicle + HC diet group decreased (7.35±2.47 pmol/mg protein, P<0.01); compared to MI/R+ Vehicle + HC diet group, the cGMP content in MI/R+ ABAH + HC diet group restored partly (28.84±9.08 pmol/mg protein, P<0.01).
2.7.3 The changes of mRNA and protein expression of NOS in myocardial tissue of HC rats with ischemia/reperfusion
2.7.3.1 The change of mRNA expression of NOS in myocardial tissue of HC rats with ischemia/reperfusion
Compared with sham group, the mRNA expression of iNOS in MI/R+ normal diet group increased (4.80±1.73 vs. 1±0.70, P<0.01); compared with MI/R+ normal diet group, the mRNA expression of iNOS in MI/R+ Vehicle + HC diet group increased (10.09±2.15, P<0.01); compared with MI/R+ Vehicle + HC diet group the mRNA expression of iNOS in I/R+ ABAH + HC diet group decreased (3.41±1.50, P<0.01).
Compared with control group the mRNA expression of eNOS in vehicle + high cholesterol group decreased significantly (0.87±0.02 vs. 1.00±0.04, P<0.01). No significant differences of nNOS mRNA expression were observed among these groups.
2.7.3.2 The change of protein expression of NOS in myocardial tissue of HC rats with ischemia/reperfusion
Compared with sham group, the protein expression of iNOS in MI/R+ normal diet group increased (2.22±0.44 vs.1.00±0.26, P<0.05); compared with MI/R+ normal diet group, the protein expression of iNOS in MI/R+ Vehicle + HC diet group increased (3.17±0.93, P<0.05); compared with I/R+ Vehicle + HC diet group, the protein expression of iNOS in MI/R+ ABAH + HC diet group decreased (2.28±0.46, P<0.05).
Compared with control group, the protein expression of eNOS in vehicle + high cholesterol group decreased significantly (0.36±0.14 vs. 1.00±0.37, P<0.05). No significant differences of nNOS protein expression were observed among these groups.
These results indicated that NOx content increased in myocardial tissue of HC rats and it further increased in AAR of HC rats with ischemia/reperfusion; cGMP content decreased in myocardial tissue of HC rats and it further decreased in AAR of HC rats with ischemia/reperfusion; both mRNA and protein expression of iNOS increased in myocardial tissue of HC rats and it further increased in AAR of HC rats with ischemia/reperfusion; both mRNA and protein expression of eNOS decreased significantly among control group and vehicle + high cholesterol group; both mRNA and protein expression of nNOS had no significant differences among these groups. These data demonstrated that although NOx content in AAR of HC rats with ischemia/reperfusion increased significantly, nitric oxide bioavailability decreased and NO-cGMP signaling pathway maybe impaired; MPO may impaired NO-cGMP signaling pathway in myocardial tissue by reduced nitric oxide bioavailability, and so to participate in the rat ischemia/rep
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