通络方药调脂、抑炎及抗凝作用的分子机制研究
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摘要
论文Ⅰ通心络调脂、抑炎及抗凝作用的机制研究
背景
冠心病是一种严重危害人类健康的常见病、多发病,随着人们生活水平的提高和人口的老龄化,本病的发病率和死亡率有逐年上升趋势,其主要病理改变是动脉粥样硬化(AS)。AS是一种多因素参与的病理过程,其发病机制非常复杂,针对它的研究已很多。鉴于AS的易罹患性和终点事件的严重性,近年来,人们已经对本病有所认识,并且意识到及早进行防治的必要性。但已患病或其他因素(如遗传、女性绝经等)造成的动脉粥样硬化病人仍普遍存在,而且对动脉粥样硬化的防治缺乏有效的措施。因此,针对本病的发病机制,应用合理有效的药物,是延缓AS发生发展进程的根本原则。
脂质代谢紊乱对于致AS的作用已被大家所公认,脂质与冠心病的关系也已被证实。大量流行病学资料显示,动脉粥样硬化的严重程度随血浆胆固醇水平的升高呈线性加重,血浆胆固醇的浓度与冠心病死亡率呈正相关关系。过去认为血清总胆固醇是冠状动脉硬化的主要原因,现在已经明确低密度脂蛋白(LDL-C)更加重要。因为LDL-C的氧化损伤主要发生于动脉血管内膜,内皮细胞存在氧化低密度脂蛋白(ox-LDL)的特异性受体,内膜受到氧化损伤后会继发释放更多有害因子,导致内膜继续损伤的恶性循环。病变中脂质源于血浆脂蛋白的浸润,主要为游离胆固醇(FC)、胆固醇脂(CE),其次为甘油三酯(TG)、磷脂和载脂蛋白。所以,LDL-C水平升高是动脉粥样硬化及由动脉粥样硬化所引起的心脑血管疾病的最重要的危险因素。而高密度脂蛋白胆固醇(HDL-C)则是抗动脉硬化的,低HDL-C水平也是AS的风险因子之一。虽然循证研究认为HDL-C不能单独作为治疗靶点,但升高体内HDL-C的浓度确实能够减缓AS的病变进程。HDL-C水平每升高10 mg/L,冠脉疾病的危险就可能降低6%。低HDL-C水平与心血管疾病的相关性在老人和女性群体中表现更为显著。因此,降低LDL-C和升高HDL-C是防治动脉粥样硬化以至心脑血管病的重要目标。
虽然“脂质浸润”学说很好的解释了AS的发病机理,但很多研究者发现,粥样斑块的形成不仅是单纯的脂质沉积,20世纪70年代以来,越来越多的证据表明AS与血管损伤后发生的一系列生物过程有关,研究者们继“脂质浸润”学说之后又提出“内皮损伤”和“血栓形成”等多个假说。近年来Ross提出的AS“炎症反应”假说逐渐成为当前的主要学说。他认为AS斑块形成有两个途径:(1)各种炎症刺激或脂蛋白引起内皮细胞的损伤和由此引起的血小板聚集、活化、并导致血小板分泌血小板源性生长因子(PDGF),促进平滑肌细胞增生;(2)内皮受损,内皮细胞或巨噬细胞分泌生长因子,平滑肌细胞及内皮细胞也可产生PDGF样生长因子,这种相互作用导致纤维斑块形成,并继续发展。AS本身也是一种慢性炎症性疾病,炎症反应贯穿于AS发生、发展及斑块破裂、血栓形成的全过程。在炎性反应过程中,细胞黏附分子介导活化的白细胞结合到内皮细胞表面并迁徙至血管外的炎性组织中。在AS炎症发展过程中单核细胞与内皮细胞问的黏附主要靠细胞问黏附分子-1(intercellular adhesion molecules,sICAM-1)和血管细胞黏附分子-1(vascular cellular adhesion molecules,sVCAM-1)介导,Wallen等发现稳定型心绞痛患者血清中sICAM-1和sVCAM-1水平的升高程度与心源性死亡或急性心肌梗死发生率成正相关。这为粘附分子参与评估AS患者的预后提供了依据。
动脉粥样硬化性心脏病的最终结局往往是血管局部的血栓形成,侧枝循环建立不及时,进而导致心肌缺血引发急性冠脉综合症等严重心血管事件。而血栓形成离不开血液环境的改变,其中纤溶系统的功能是否正常至关重要。Hoffmeiste等报道冠心病患者纤溶活性降低,不正常的纤溶活性可能预示冠脉事件的发生。血浆中组织型纤溶酶原激活物(tissue plasminogen activator,t-PA)和纤溶酶原激活物抑制剂1(plasminogen activator inhibitor 1,PAI-1)是纤溶系统中重要的活性物质,PAI-1为t-PA的抑制物,它们之间的生理平衡对调节血流通畅,防止AS血栓形成起重要作用。
AS的发生发展及其复杂,脂质沉积和炎症反应以及血栓形成三者之间互相联系和影响。在AS早期阶段,多种刺激因素诱发动脉壁脂质聚集部位的炎症反应,引起细胞粘附,生成脂质斑块,引起平滑肌增殖、迁移;炎症还影响脂质代谢,加速LDL的氧化修饰,并且降低脂质斑块的稳定性,诱发斑块破裂血栓形成。所以,现在人们越来越清醒地认识到理想的策略应该是将药物治疗包括调脂、溶栓、抗凝以及抗炎等方法结合起来,发挥其协同作用,以达到最佳治疗效果。但若找出三者之间的共同作用靶点,则将为治疗提供捷径。
基础和临床研究证实,他汀类药物除具有强效的降低LDL-C作用外,还具有较好的抗炎症作用,通过降低C反应蛋白和粘附分子等炎症标记物的水平来改善患者的预后。同时,也有研究表明,他汀类药物能降低血浆PAI-1水平,明确改善机体纤溶功能。但Prove-It临床试验的结果显示,服用大剂量阿托伐他汀的治疗组两年后仍有22.4%的患者发生了急性冠状动脉事件,且服药后的肝功能异常使部分患者无法坚持用药。此外,使用单一药物进行二级预防效果很可能不如联合用药,作为心肌梗死二级预防最突出的范例就是2004年由2位英国学者提出的“复合制剂”想法。尽管文章发表后遭到了一定程度的置疑,但这一模式的转变意义重大。
中医药具有多途径、多环节、多靶点的特点,可通过保护血管内皮细胞、调节脂质代谢、抑制炎症反应和抗凝等多个角度稳定动脉粥样硬化斑块,在延缓AS进程方面具有潜在的疗效优势,且中药制剂药效缓和,药物之间因配伍而减毒增效,副作用较小,适宜作为二级预防用药长期服用。以往中药抗AS治疗研究中多只针对某一方面的病因或症状进行处理,缺少系统性的机制研究。通心络超微粉是近年应用络病理论与通络药物治疗心脑血管病的代表性方药,具有益气活血,搜风通络的功效,主要治疗冠心病心绞痛属心气虚乏,血瘀络阻者。本研究以辛伐他汀作为阳性对照药物,应用本实验室已成功构建的动脉粥样硬化动物模型方法,运用分子生物学及组织学等技术,观察通心络超微粉通过调脂、抑制炎症反应和抗凝作用防治动脉粥样硬化的疗效,并阐明其分子生物学机制及相互作用的联系靶点。
目的
(1)建立与人类动脉粥样硬化病变特征相似,易于观察药物疗效的动脉粥样硬化动物模型;
(2)应用血清学、组织学及分子生物学技术观察动脉粥样硬化病变过程中脂质、炎症及凝血机制的变化,并找出共同作用的靶点;
(3)对比不同剂量通心络超微粉和辛伐他汀治疗防治动脉粥样硬化的疗效及其分子生物学机制。
方法
1.动物模型的建立
健康雄性新西兰大白兔,在适应性饲养1周后,采用球囊损伤腹主动脉后,应用含有1%高胆固醇饲料喂养10周。
2.实验分组及药物干预
造模成功后随机分为对照组、小剂量、中剂量、大剂量通心络组和辛伐他汀组:
A对照组15只:造模成功后,给予普通饲料饲养8周;
B小剂量通心络组15只:造模成功后,普通饲料+通心络超微粉(0.15g/kg/d)饲养8周;
C中剂量通心络组15只:造模成功后,普通饲料+通心络超微粉(0.3g/kg/d)饲养8周;
D大剂量通心络组15只:造模成功后,普通饲料+通心络超微粉(0.6g/kg/d)饲养8周;
E辛伐他汀组15只:造模成功后,普通饲料+辛伐他汀(5mg/kg/d)饲养8周。
3.称量体重
实验前、10周造模后及18周末药物干预后分别对所有实验兔称量体重。
4.高频体表超声检查
分别于实验开始、10周末、18周末行腹主动脉高频体表超声检查。测量腹主动脉后壁内膜-中层厚度(intima-media thickness,IMT)、腹主动脉收缩期血流峰值速度(peak velocity,Vp)。
5.肝肾部分功能检查
分别于实验开始、10周末及18周末,采血作血液生化检查,主要包括ALT,GGT,TBIL,BUN和Cr。
6.血脂检测
分别于实验开始、10周末及18周末采血作血液生化检查。采用酶法测定血清总胆固醇(total cholesterol,TC)、甘油三酯(triglyceride,TG)、低密度脂蛋白胆固醇(low-density lipoprotein cholesterol,LDL-C)及高密度脂蛋白胆固醇(highdensitylipoprotein cholesterol,HDL-C)水平。
7.血清炎性因子的检测
分别于实验开始、10周末及18周末采血,应用酶联免疫吸附法(enzyme-linked immunosorbent assay,ELISA)检测氧化低密度脂蛋白(oxidizedlow-density lipoprotein,ox-LDL)、血清高敏C反应蛋白(high sensitivityC-reactive protein,hs-CRP)、血清sICAM-1及sVCAM-1的浓度。
8.血浆纤溶指标的检测
分别于实验开始、10周末及18周末采血,应用ELISA试剂盒检测血浆PAI-1、t-PA的浓度水平。
9.病理染色
留取球囊拉伤处腹主动脉标本,行苏木素—伊红(hematoxylin-eosin,HE)染色、油红O染色和Movat五色套染法染色。
10.免疫组织化学染色
对腹主动脉球囊损伤处血管段进行组织切片,行胆固醇酯转运蛋白(CETP)、低密度脂蛋白受体相关蛋白(LRP)、RAM11、VCAM-1、ICAM-1、及CRP免疫组织化学染色,此外并对肝脏组织切片行CETP及LRP的免疫组织化学染色。
11.Western blot
留取肝脏及血管标本,检测组织中CETP及LRP蛋白表达水平。
12.扫描电镜
各组均留取标本进行扫描电子显微镜检查,观察血管内膜损伤及脂质沉积的情况。
13.统计学分析
应用SPSS统计软件进行统计学处理(version 13.0;SPSS Inc),连续性数据用(?)±SD表示,P＜0.05有统计学差异。
结果
1.实验动物基本情况
进入药物干预阶段的实验兔中,辛伐他汀组(E组)死亡1只,大剂量通心络组(D组)死亡2只,中、小剂量通心络组各死亡1只,死亡原因分别为腹泻、呼吸道感染或原因不明。18周末各组存活实验兔为:对照组(A组)15只,小剂量通心络组(B组)14只,中等剂量通心络组(C组)14只,大剂量通心络组(D组)13只,辛伐他汀组(E组)14只。
2.实验动物体重的变化
10周末所有实验兔体重均较实验开始前加重,相比均有显著性差异(P均＜0.01),但治疗后各组实验兔之间无显著差异。
3.高频体表超声
10周末,所有实验兔IMT均显著升高(P均＜0.01),Vp值显著升高(P＜0.05);治疗后C、D、E三组IMT较对照组相比显著降低(P均＜0.05),D、E两组Vp值显著降低(P均＜0.05)。
4.肝肾功能检测
18周末,辛伐他汀组血清ALT、GGT两者浓度显著高于其他组(P均＜0.05),其他各组之间无显著差异。
5.血脂检测
组内比较:10周末,除HDL-C外,所有实验兔较造模前各项指标相比均显著升高(P均＜0.01);18周末,各组血脂含量除HDL-C外,与治疗前相比均显著降低(P均＜0.01)。
治疗后组间比较:与A组比较,各药物治疗组均可明显降低血清TC水平(P＜0.01),大剂量通心络组和辛伐他汀组组间无显著性差异(P＞0.05);大剂量通心络组TC水平明显低于小剂量通心络组;
与A组比较,各药物治疗组均可明显降低血清LDL-C水平(P＜0.01),辛伐他汀组降低水平最显著(P＜0.01),大剂量组次之(P＜0.01),中、小剂量通心络组间无显著性差异(P＞0.05);
与A组比较,各药物治疗组均可明显降低血清TG水平(P＜0.01),中剂量通心络组(C组)、大剂量通心络组(D组)和辛伐他汀组(E组)三组间无显著性差异(P＞0.05);大剂量通心络组TG水平明显低于小剂量通心络组(P＜0.01);
与A组比较,各药物治疗组均可明显升高血清HDL-C的值(P＜0.01～0.05),大剂量通心络组HDL-C的值明显高于中、小剂量通心络组,与辛伐他汀组比较无显著差异。
6.血清炎性因子的检测
组内比较:10周末造模成功实验兔血清ox-LDL、hs-CRP、sICAM-1及sVCAM-1水平较实验初始相比均显著升高(P＜0.01);18周末,C、D、E各组炎性因子血清水平均显著下降(P＜0.05)。
组间比较:与A组比较,治疗后中、大剂量通心络组和辛伐他汀组均可明显降低血清炎性因子hs-CRP的水平(P＜0.01),由低到高依次为E组、D组和C组。D组与E组降低hs-CRP水平无显著性差异(P＞0.05);
与A组比较,各药物治疗组血清可溶性粘附分子sVCAM-1的水平均显著下降(P＜0.01),辛伐他汀组sVCAM-1水平明显低于小、中、大三个剂量通心络治疗组(P＜0.05),各通心络治疗组间降低sVCAM-1水平无显著性差异(P＞0.05);
与A组比较,各药物治疗组血清可溶性粘附分子sICAM-1的水平均显著下降(P＜0.01),但辛伐他汀组降低水平最明显(P＜0.01);
与A组比较,各药物治疗组血清ox-LDL的水平均显著下降(P＜0.01～0.05),药物组间比较未见显著差异。
7.血浆纤溶指标的检测
组内比较:治疗后除对照组外,其他各组血浆PAI-1水平显著降低(P＜0.05～0.01),t-PA水平均显著升高(P＜0.05～0.01)。
组间比较:18周末,与对照组相比,B、C、D三组血浆PAI-1水平显著降低(P＜0.05～0.01),C、D两组最显著(P＜0.01);与对照组比较,C、D、E三组血浆t-PA水平显著升高(P＜0.05～0.01),但以D组差异最显著(P＜0.01)。
8.病理染色
HE染色显示,造模成功后各组血管斑块明显,治疗后各组间未见明显差异;油红“O”染色显示,对照组斑块内脂质含量显著高于药物组;Movat结果显示,对照组动物斑块内部基质和粘蛋白含量显著高于药物组,各药物组间未见差异。
9.免疫组织化学染色
五组斑块内均有CETP、LRP、RAM11、VCAM-1、ICAM-1、及CRP的局部表达;与对照组相比,药物组LRP、RAM11、VCAM-1、ICAM-1及CRP表达均显著减少,其中D、E两组较其他3组相比表达更低,而C组和B组间未见差异;E组CETP蛋白表达显著低于其他四组,四组间比较无显著差异。肝脏CETP及LRP免疫组织化学染色结果与其血管斑块内表达结果相似。
10.Western blot
各组肝脏及斑块中均有CETP、LRP的高度表达,其中D、E两组LRP蛋白表达显著低于其他三组(P＜0.01),两组间无显著差异;E组CETP蛋白表达水平显著低于其他四组(P＜0.05),其余四组间比较无显著差异。
11.扫描电镜
各组内皮细胞均有不同程度损伤,主要表现为内皮细胞排列紊乱,内膜较多区域有细胞脱落,暴露内皮下胶原,脱落区附近有大量红细胞、血小板及脂滴黏附,低倍镜下可见病变区域广泛,内皮隆起区域部分融合,形成表面粗糙的“斑块”状病变。A组内膜损伤显著甚于其他药物组,药物组间比较发现,D、E两组损伤显著较轻,C组和B组间未见显著差异。
结论
(1)应用球囊损伤加高脂饲养方法可建立与人类动脉粥样硬化病变特征相似、造模时间短及适于观察药物疗效的动物模型,结合血脂水平的检测,应用高频体表超声可判定模型的建立情况。
(2)通心络超微粉能够不通过抑制CETP升高HDL-C,保护了体内的正常胆固醇逆转运功能。
(3)大剂量通心络超微粉能有效降低血脂,抑制炎症反应及增强纤溶功能其作用的共同靶点可能是LRP。
论文Ⅱ薤白对氧化低密度脂蛋白诱导的内皮细胞组织因子及其抑制物表达的干预作用
背景
动脉粥样硬化(atherosclerosis,AS)是中老年人群心脑血管疾病高发的重要病理基础,有关AS的形成的一个主要高危因素就是高脂饮食导致的体内动脉内膜脂质沉积过多。近年来大量流行病学调查表明,体内血清胆固醇尤其是低密度脂蛋白(LDL-C)含量的增多更加促进了AS的发生发展,而氧化修饰后的LDL-C(ox-LDL)更是大为加强了病变程度,是导致粥样斑块破裂血栓形成的始动因素之一。随着人口老龄化的发展,我国冠心病病人的发病率呈明显上升趋势,已接近国际平均水平。尽管人们已对本病有一定的预防意识,但已患病或其他因素(如遗传、女性绝经等)造成的动脉粥样硬化病人仍普遍存在,因此,寻找合理有效的药物抑制ox-LDL对内皮细胞的损伤,降低促凝因子释放,使血管内环境保持稳定平衡的状态,是减少危险事件发生的有效途径。
动脉粥样硬化的发生发展贯穿冠心病病人整个病理过程,但是,从脂质沉积到粥样斑块形成,乃至最后的斑块破裂血栓形成造成危险事件的发生,内皮细胞都在其中起到重要作用。诸多学者都认可“损伤学说”是AS的始动机制的理论。因为LDL-C的氧化损伤主要发生于动脉血管内膜,内皮细胞存在ox-LDL的特异性受体,内膜受到氧化损伤后会继发释放更多有害因子,导致内膜继续损伤的恶性循环。文献报道,ox-LDL可使培养的人脐静脉内皮细胞的抗氧化酶谷胱甘肽过氧化物的活性下降,前列环素生成减少,脂质过氧化物产生增加,表明ox-LDL具有直接的内皮损伤作用。同时,内皮细胞表达的ox-LDL的特异性受体—血凝素样氧化型低密度脂蛋白受体1(Lox-1)增多,介导了内皮细胞对衰老细胞及凋亡细胞的吞噬,激活多条炎症反应的信号通路,比如p44/42MAPK、p38MAPK、CD40/CD40L等。此类信号通路被激活后可以诱发内皮细胞释放大量的促凝因子如组织因子(TF),并且减少了具有抗凝作用的组织因子途径抑制剂(TFPI)的表达,促使血管内凝血机制增强,有利于血栓形成。
目前临床治疗动脉粥样硬化多集中于调脂治疗,主要是他汀类调脂药物,其次是针对症状应用的抗血小板制剂、β-受体阻滞剂及血管紧张素转换酶抑制剂等。这些药物对于延缓动脉粥样硬化进程和改善临床症状都具有积极而肯定的疗效,并且由于高脂血症已经成为严重心血管疾病发病的高危因素,所以其中的他汀类调脂药已经成为治疗冠心病的基础用药。大量的临床实验研究表明,在对AS血管疾病的控制治疗中,能够保护内皮细胞,改善内皮功能,起到抗脂质氧化作用的药物越来越成为首选药物。但是大多数西药,比如他汀类调脂药,虽然有很好的耐受性和安全性,但是治疗的同时也带来较多副作用,比如肝功能异常及其他消化和皮肤疾病等。
近年来,对中草药降脂的研究颇为广泛。中药的优点在于成分纯天然,在抗氧化、降脂及抗炎抗凝方面都有良好功效。而且中药对人体毒副作用较小,在安全剂量时,对肝肾功能影响很小。薤白在以往中医药应用方面多采用复方制剂,在防治心血管疾病方面取得显著疗效。但是在细胞水平的研究方面,大多数研究者都集中于其挥发油的作用研究上。但由于挥发油成分不稳定极难保存,而且薤白还含有大量的皂苷类和多糖类化合物,所以对于薤白单方药物的系统研究有待更深入的进行。有关薤白挥发油的抗氧化、抗凝作用研究较多,但此作用的具体机制至今未见报道。而且之前的实验研究多采用静脉内皮细胞或牛动脉内皮细胞进行培养,所以为了更好地模拟临床现象,本实验采用原代人主动脉内皮细胞(human aortic endothelial cells,HAECs),以ox-LDL作为内皮细胞氧化损伤的诱导因子,观察了不同浓度及不同时间点ox-LDL对于体外培养HAECsLox-1、TF及TFPI蛋白表达的影响,并观察薤白对ox-LDL诱导的内皮细胞氧化损伤的防治作用。
目的
(1)建立人主动脉内皮细胞氧化损伤的模型;
(2)观察不同剂量浓度薤白超微粉抑制氧化低密度脂蛋白诱导的内皮细胞表达促凝因子的作用及其机制。
方法
为建立内皮细胞氧化损伤模型,确定ox-LDL最终刺激浓度及刺激时间,本实验通过体外培养人主动脉内皮细胞,观察不同浓度和不同时间ox-LDL刺激后,人主动脉内皮细胞Lox-1、TF及TFPI蛋白的表达变化,选定ox-LDL的最佳刺激条件。
不同浓度的薤白超微粉(0.25ug/ml、0.5ug/ml、1ug/ml)预孵育12h后再加ox-LDL刺激,同时应用Lox-1阻断剂-Poly(I:C)做对照组,倒置显微镜下观察细胞形态的变化,并且用western-blot方法检测各组Lox-1、TF及TFPI的蛋白表达水平。
结果
倒置显微镜下观察,正常血管内皮细胞生长状态良好,贴壁较牢,呈梭形或圆形,形态饱满,呈铺路石状生长,细胞间连接紧密,分裂相多,未见悬浮细胞。ox-LDL损伤组可见不同程度及范围的细胞收缩、变圆、胞间隙变宽、细胞脱壁悬浮;Lox-1阻断剂250ug/ml组及薤白超微粉1ug/ml组细胞形态与正常组比较无显著差异。
根据不同浓度及不同时间ox-LDL刺激后人主动脉内皮细胞Lox-1、TF及TFPI蛋白变化情况及本实验的研究目的,我们选定50ug/ml的ox-LDL培养液培养细胞24h为最佳刺激条件。50ug/ml ox-LDL培养细胞24h,Lox-1和TF的蛋白表达水平均显著升高,TFPI蛋白表达水平显著下降。
250ug/ml Poly(I:C)预孵育2h后再加入ox-LDL刺激细胞发现Lox-1蛋白表达水平较其他组显著降低,故可采用该浓度Poly(I:C)作为本实验的Lox-1阻断剂。
0.5ug/ml和1ug/ml薤白可以显著抑制Lox-1和TF蛋白表达,其中1ug/ml薤白组和Lox-1阻断剂组相比无显著差异;同时薤白增加了TFPI蛋白表达,和刺激组相比差异显著。
结论
(1) 50ug/ml ox-LDL培养人主动脉内皮细胞24h显著升高Lox-1蛋白表达水平,同时引起TF蛋白表达增强和TFPI蛋白的表达受到抑制,选为本实验的最佳刺激条件;
(2) 250ug/ml的Ploy(I:C)能显著抑制Lox-1蛋白表达,起到实验要求的阻断效果。
(3) 0.5ug/ml和1ug/ml薤白可以通过抑制Lox-1表达来减少内皮细胞的氧化损伤,进而减少促凝因子释放,稳定TF/TFPI的表达比例。
PAPERⅠTHE EFFECTS AND MECHANISM OF TONGXINLUO SUPERFINE ON LIPID-LOWERING,INHIBITING INFLAMMATION AND ANTICONGULATION
Coronary heart disease is the commonly encountered and frequently-occurring disease which harms health seriously.With the elevation of living standard and ageing of population,the morbidity and mortality rise year by year and the pathology change is atherosclerosis(AS).AS is the pathologic processes including many factors and the pathogenesis is very complex and the research about it is multiply.Because the AS is easy to happen and serious,so people have realized that is necessary to prevent and cure as soon as possible for the past few years.However,the AS patients are common and the effective intervention about its treatment is lacking.Therefore,to aim directly at the pathogenesis and make use of reasonable and effective drugs is the basic principle of delaying the development of AS.
The role of lipid metabolic disorder inducing the AS is generally acknowledged and the relation of lipid and CHD is confirmed.Lot's of epidemiological data showed the severity and death rate of AS aggravates with the increasing of serum cholesterol level.It is previously believed the serum cholesterol is the primary cause but now the low density lipoprotein(LDL-C) is deemed more important.The oxidative damage of LDL-C occurs on artery vascular intima and there is special receptor of oxidized low density lipoprotein(ox-LDL) in endothelial cell,and the cell would express more harmful substance because of sustained injuring,even which can bring the vicious cycle of tunica intima due to injured continually.The lipids about AS are from plasma lipoprotein like free cholesterol(TC),Triglyceride(TG),et al.The increasing of LDL-C level is the capital risk factors of AS or other cardiovascular and cerebrovascular diseases about AS.Conversely,the high density lipoprotein cholesferol(HDL-C) is beneficial to anti AS and the low level of HDL-C is one of risk factors of AS.Although the Cochrane doesn't recognize the HDL-C as simple curing target point,to increase the level of HDL-C really can slow down the process of AS.The correlation of low level of HDL-C and cardiovascular diseases is more significant in aged and female group.Therefore,to reduce the LDL-C and raise HDL-C level is the important target of curing AS.
Although the "lipid invasion theory" explains the pathologic mechanism of AS well,many researchers found that the form of plaque is not only because of lipid deposition but also the bioprocess after vascular injuring in 1970s.The hypothesis of "endothelium injured" and "thrombosis" were suggested by researchers on the base of "lipid invasion theory".For the past few years,the "inflammatory reaction theory" of Ross is becoming the major idea.Ross thought the form of AS had two pathways:(1) the injuring of endothelial cell because of various inflammation stimulus or lipoprotein and the platelet aggregation,activation,expression of platelet-derived growth factor(PDGF) and the proliferate of smooth muscle cell(SMC) since the injuring happened;(2) once the endothelium was injured the endothelial cell and macrophage would secrete kinds of factors and SMC also bring the PDGF,and then, the interaction of them will lead to the form and development of fibrous plaque.The AS is the chronic inflammation diseases which the inflammatory reaction penetrates in the all process of AS including the rupture of plaque and thrombosis.In the process of inflammatory reaction,the white blood cell activated by cellular adhesion molecule combines with endothelial cell surface and migrates to the inflammatory tissue out of vascular.In the development of AS,the adhesion of monocyte and endothelial cell is mediated by intercellular adhesion molecules(ICAM-1) and vascular cellular adhesion molecules(VCAM-1) chiefly.Wallen found the increase of serum ICAM-1 and VCAM-1 level in angina pectoris patients positively correlate to the incidence rate of cardiac death or acute myocardial infarction,which supplied the evidence for the adhesion molecule participating to evaluate the prognosis of AS patients.
The final result of atherosclerotic heart disease is the thrombosis of vascular and the collateral circulation isn't setup timely that result in myocardial ischemia and induce acute cardiovascular event.However,the form of thrombus relates with the change of blood environment and whether the function of fibrinolysis system is normal is important.Hoffmeiste reported the fibrinolysis activity of CHD patients decrease decreased and abnormal fibrinolysis activity might predict the occurring of coronary artery disorder.The plasma tissue plasminogen activator(t-PA) and plasminogen activator inhibitor 1(PAl-1) is the important active compound of fibrinolysis system and the physiologic equilibrium of them is fundamental to keep blood stream unobstructed and prevent thrombosis.
The pathomechanism of AS is very complicated and the lipid deposition, inflammatory reaction and thrombosis correlate and affect each other.In the early period,much stimulating factor induce the inflammatory reaction on the vessel wall lipid deposited,cell adhesion,lipid plaque,proliferate and immigration of SMC;the inflammation also affects the metabolism of lipid and promotes the oxidation of LDL-C,meanwhile,which can decrease the stability of lipid plaque and induce the rupture of plaque.Therefore,people realize ideal strategy about the treatment of AS is that can combine with the reducing blood fat,inhibiting inflammation and anticoagulation and play the joint action.However,to find the target point of them will be shortcut to treat AS.
The clinical research confirmed the statins could not only reduce LDL-C level but also inhibit the inflammation and improve outcomes by decreasing the inflammation markers like C reactive protein and adhesion molecules.Meanwhile, statins could reduce the plasma PAI-1 level and improve the function of fibrinolysis significantly.However,the clinical trial showed that 22.4%patients who summed the large dose of atorvastatin experienced acute coronary artery disease in 2 years. Moreover,some patients withdraw from statins treatment because of liver dysfunction. In addition,the drug combination could have better effect compared with one kind of medicine,so the "polypill" was preferred in secondary prevent treatment by two British scholars in 2004.Although the manuscript met with the doubt,the change of mode has important meaning.
Traditional Chinese medicines have the characteristic of much way,much link and much target point and stable the AS plaque by protecting the vascular endothelial cell,regulating the lipid metabolism,inhibiting the inflammatory reaction and anticoagulation.Traditional Chinese medicines have the potential curative effect in delaying the development of AS and are suitable to long-time drug taking of secondary treatment because of the moderate potency,less side effect and compatibility.Formerly,the research of traditional Chinese medicines about treating AS usually aim directly at the simple cause or symptom and the systemic research of mechanism is necessary.Tongxinluo superfine is the representative Chinese drug which treats cardiovascular and cerebrovascular disease with theory of collateral disorders and dredging collaterals herbs.Tongxinluo has the effects of benefiting vital energy and promoting blood flow and has been used in CHD that show off deficiency of heart-energy and blood stasis.The present study adopts the simvastatin to be positive control and uses the AS animal model made by laboratory and be aimed to observe the therapeutic effects of Tongxinluo on AS by regulating lipid,inhibiting inflammation and anticoagulation and elucidate the possible mechanism and relationship of them through the molecular biology and imageology technology.
Objective
(1) To establish an animal model of AS that is mimic to human pathological changes and convenient for intervention;
(2) To observe the changes of lipid,inflammation and clotting mechanism and find the functionary target point in common with serology histology Protocols in Molecular Biology.
(3) To compare the therapeutic effects of Tongxinluo and Simvastatin on preventing and curing AS and approach the possible mechanism.
Methods
1.Establishing the animal model:
75 rabbits were fed on an atherogenic diet containing 1%cholesterol for 10 weeks after abdominal aortic wall injuries were induced using an intravascular balloon.
2.Intervention methods:
Group A(control group):15 rabbits were fed normal diet for 8 weeks after the model was established.
Group B(low dose):15 rabbits were fed normal diet and Tongxinluo super (0.15g/kg/d) for 8 weeks after the model was established.
Group C(middle dose):15 rabbits were fed normal diet and Tongxinluo super (0.3g/kg/d) for 8 weeks after the model was established.
Group D(high dose):15 rabbits were fed normal diet and Tongxinluo super (0.6g/kg/d) for 8 weeks after the model was established.
Group E(simvastatin):15 rabbits were fed normal diet and simvastatin (5mg/kg/d) for 8 weeks after the model was established.
3.Body weight:
Body weights of all rabbits were measured at baseline,the end of the 10~(th) and 18~(th) week.
4.High frequency ultrasound:
At the baseline,end of the 10~(th) and 18~(th) week,the abdominal aorta was scanned using a high frequency duplex ultrasonographic system.The intima-media thickness (IMT) and peak velocity(Vp) were measured.
5.Liver and kidney function test:
Blood samples were collected from all rabbits at the baseline,the end of the 10~(th) and 18~(th) week.The serum ALT,GGT,TBIL,BUN and Cr were detected with automatic biochemical analyzer.
6.Lipid measurement:
Blood samples were collected from all rabbits at the baseline,the end of the 10~(th) and 18~(th) week.Serum levels of total cholesterol(TC),triglyceride(TG),low density lipoprotein cholesterol(LDL-C) and high density lipoprotein cholesterol(HDL-C) were measured by enzymic method.
7.Serum inflammatory factor:
Blood samples were collected from all rabbits at the baseline,the end of the 10~(th) and 18~(th) week.The serum high sensitivity C-reactive protein(hs-CRP)、ox-LDL、ICAM-1 and VCAM-1 were assayed using highly sensitive enzyme-linked immunosorbent assay(ELISA) kits.
8.Plasma fibrinolysis detection:
Blood samples were collected from all rabbits at the baseline,the end of the 10~(th) and 18~(th) week.The plasma PAI-1 and t-PA were assayed using ELISA kits.
9.Histopathological analysis:
The abdominal aorta was processed and examined by hematoxylin and eosin (HE),Movat and oil red O staining.
10.Immunohistochemical staining:
The expression of RAM11,VCAM-1,ICAM-1,CRP,cholesterol ester transfer protein(CETP) and low density lipoprotein receptor-related protein(LRP) were determined.
11.Western blot:
To detected the protein expression of CETP and LRP in liver.
12.Scanning electron microscope:
Scanning electron microscope was used to observe the the degree of endangium damage and lipids deposition.
13.Statistical analysis:
Data are expressed as mean±SD for continuous variables and by frequency count and percentage for qualitative variables.Incidence rate of plaque rupture was compared with Pearson Chi-Square test and other indexes were compared with One-Way ANOVA comparison test.The SPSS was version 13.0.P＜0.05 was considered statistically significant.
Results
1.General state of the experimental animals:
Five rabbits died of diarrhea,respiratory tract infection and so on,and there were 70 rabbits finished the study:15 rabbits in group A,14 rabbits in group B,14 rabbits in group C,13 rabbits in group D,14 rabbits in group E.
2.Body weight:
Body weights of all rabbits were significantly higher at the end of 10~(th) week than that in begin(P＜0.01);there were no difference among the groups after treatment.
3.High frequency ultrasound measurement:
At the end of 10~(th) week,the maximum IMT of the rabbits were significantly higher(P＜0.01),and the Vp was also higher(P＜0.05);after treatment,the IMT of group C,D and E were lower than group A(P＜0.05),the Vp of group C and E was lower(P＜0.05).
4.Detection of liver and kidney function:
At the end of 18~(th) week,the ALT and GGT in group E were higher than other groups(P＜0.05).
5.Lipid measurement:
Intra-group comparison:at the end of 10~(th) week,except the HDL-C,the TC,TG and LDL-C level were higher(P＜0.01);at the end of 18~(th) week,except the HDL-C, the TC,TG and LDL-C level were lower(P＜0.01).
Inter-group comparison:compared with group A,except the HDL-C,at the end of 18~(th) week,the lipid level in the drug groups decreased significantly especially in group D and E,but the serum HDL-C level of drug groups increased.
6.Detection of serum inflammatory factor:
Intra-group comparison:at the end of 10~(th) week,the serum ox-LDL,hs-CRP, ICAM-land VCAM-1 increased significantly(P＜0.01);at the end of 18~h week,the serum inflammatory factor of group C,D and E decreased markedly(P＜0.05).
Inter-group comparison:at the end of 18~(th) week,compared with group A,the serum ox-LDL,hs-CRP,ICAM- 1 and VCAM- 1 of group A was higher than group C, D and E significantly(P＜0.01～0.05).
7.Detection of plasma fibrinolysis factors:
Intra-group comparison:except the group A,at the end of 10~(th) week,the plasma PAI-1 level increased and t-PA decreased significantly(P＜0.05～0.01).
Inter-group comparison:at the end of 18~(th) week,compared with group A,the plasma PAI-1 level increased and t-PA decreased significantly in group D and E (P＜0.05～0.01).
8.Pathologic staining:
The H&E staining showed the obviously plaque in all groups;oil red "O" staining showed the lipid in plaque of group A was higher than group C,D and E; Movat showed the ground substance in plaque of group A was higher than in group C, D and E;there was no difference among the group D and E.
9.Inununohistochemical staining:
Compared with group A,the expressions of LRP,RAM11,VCAM-1,ICAM-1 and CRP in plaque of group C,D and E decreased significantly,and no difference was found between group D and E.There was no significantly difference of CETP among the Tongxinluo groups.
10.Western blot:
There was high expression of CETP and LRP in liver in all groups.The expression of LRP protein in group C,D and E is lower than in group A,The expression of CETP protein in group E is lower than in other groups.
11.Scanning electron microscope:
The endothelial cell of normal group was clostridial form,integrate,lining up in order and the long axis of cell was concordant with direction of blood stream,the nucleolus swelled up and orientated toward the lumens and there was no cell adhesion; the endothelial cell of other group was injured and the cell was arranged in disorder, and much cell of intima ablated and exposed the collagen,there adhered much red blood cell,platelet and lipid droplet near the deciduo-as area.Under the lower power lens,the endodermis swelled up and partly mixed together and the plaque formed. The damage of group A and B was more serious than other groups and there was no significant difference between group C,D and E.
Conclusions
(1) The rabbit AS model,which is established with balloon-induced abdominal aortic wall injury together with a cholesterol-rich diet is mimic to human disease and suitable for the observation of drug therapeutic effects.
(2) The Tongxinluo increased the HDL-C level and protected the normal function of reverse cholesterol transport not by inhibiting the CETP.
(3) The combination of Tongxinluo and simvastatin could effectively decrease the lipid,inhibit the inflammatory reaction and enhance the fibrinolysis function and the common target maybe was LRP.
PAPERⅡINTERVENTION OF B.MACROSTEMI ON TF/TFPI OF HAECs OX-LDL ACTIVATED
Background
The happen and development of AS run through the whole pathologic processes of CHD,however,from the lipid deposition to atheromatous plaque and even the plaque rupture and thrombosis inducing the danger incidence,the endothelial cell play the important role.Many professors consent the "injuring theory" is the initiate mechanism of AS.The oxidative damage of LDL-C betide artery vascular intima and there is specific receptor of ox-LDL in endothelial cell,and the cell would express more harmful substance because of sustained injuring,even which can bring the vicious cycle of tunica intima due to injured continually.Ox-LDL decrease the activity of glutathione peroxides and the generate prostacyclin of cultural HUVEC and increase the lipid peroxides that showed the ox-ldl can injure the endothelium directly.Meanwhile,the lectin-like oxidized LDL receptor-1(lox-1) that endothelial cell expressed increase and that promote the endothelial cell to swallow the senile and apoptosis cell,and activate much inflammatory reaction signal pathway,for example, p44/42MAPK、p38MAPK、CD40/CD40L,et al..The endothelial cell release more procoagulant factors like tissue factor(TF) and less anticoagulant substance like tissue factor pathway inhibitor(TFPI) when those pathways was activated,then,the clotting mechanism enhanced and thrombus form.
The important pathological basis of high incidence cardiovascular and cerebrovascular diseases of old people is atherosclerosis(AS),and the high risk factor of AS is too much lipid deposition in endarterium due to high fat diet.For the past few years,lots of epidemiological study showed the increase of serum cholesterol especially the low-density lipoprotein(LDL-C) promote the development of AS,and the oxidized low density lipoprotein(ox-LDL) more enhance the degree of pathological changes that it has become one of the initiating agent of plaque rupture and thrombosis.With the development of aging,the incidence of coronary heart disease(CHD) in our country was at the increased tendency and got close to the international average level.In spite of many patients have recognized the serious of CHD,the AS patients have been ill or other reason induced(smoking,heredity,and menopause) are still ubiquitous.Therefore,to find the effective drugs that can inhibit the injure of endothelial cell by ox-LDL,to decrease the release of procoagulant factor and keeping the balance of intravascular environment is good for reducing the danger incident.
Now the treatment of AS more often focus on regulating lipid,besides statins, there are antiplatelet drugs,β-receptor inhibitor,angiotensin converting enzyme inhibitor and so on that can improve clinic symptoms.The drugs play the positive therapeutic effect with regard to delay the process of AS.Moreover,because hyperlipidemia is the high risk of cardiovascular diseases,the statins have become the basic drugs to treat CHD.Much clinical trial indicated that those drugs which can protect endothelial cell,improve endothelium function and drugs have the role of anti-lipid peroxidation become choice drug.But most drugs like statins,although they have good survivability and safety,there are much side effects like dysfunction of liver,digestive system diseases and so on.
There is much study about herbs for the past few years,the component of herbs is natural and they are effective in antioxidation,reducing blood fat, anti-inflammatory and anticoagulation.Furthermore,because of little side effect of herbs,in the range of safe dose there is tiny dysfunction of liver and kidney.Bulbus allii macrostemonis(B.macrostemi) was often used in compound preparation in the past and it also showed noticeable effect.In the aspect of cellular level research,most researchers took their attention on the study of essential oil.But the essential oil is difficulty to preserve because of unstable component and B.macrostemi contain lots of saponins and polyoses compound,therefore,the more deeply systematic research about B.macrostemi is necessary.Although there are lots of research about anti-oxidation or anticoagulation of essential oil,the mechanism about that is absent. In addition,intravenous endothelial cells or bovine aortic endothelial cells were adopted usually in these researches.In order to simulate clinical diseases,the human aortic endothelial cells(HAECs) were cultured in vitro and ox-LDL was applied for inducing factor of oxidative damage to endothelial cell.We observed the effect of ox-LDL on expression of Lox-1,TF and TFPI and the protective effect of B. macrostemi on the endothelial cells damage induced by ox-LDL.
Objective
(1) To establish the model of human aortic endothelial cells oxidative damage;
(2) To investigate the effect and mechanism of different dose B.macrostemi inhibiting the expression of pro-coagulant factor in ox-LDL activated by HAECs.
Methods
To establish the model of human aortic endothelial cells oxidative damage,we observe the effect of ox-ldl with different concentration and different time on the expression of lox-1,TF and TFPI.
The cultured HAECs were pretreated with or without B.macrostemi(0.25ug/ml、0.5ug/ml、1ug/ml) for 12h and were observed under inverted microscope.Meanwhile, the control group that cell were pretreated with blocking agent of lox-1-Poly(I:C) for 2h was designed.The effects of B.macrostemi with different concentration on lox-1、TF and TFPI protein expression were investigated by western blot.
Results
Inverted microscope showed that the normal endothelial cells were at good condition,adherence stable,fusiform or round,tight cell-cell junction,more cleavage phase,less suspension cells;cell shrinkage,intercellular space broaden and more suspension cells in ox-LDL damage group;the shape and structure of endothelial cell in blocking agent and B.macrostemi 1ug/ml group was similar with normal cell.
Based on the aim of the present study and the effect of ox-LDL with different concentration and different time on the expression of Lox-1、TF and TFPI、50ug/ml ox-LDL for 24h is the optimized stimulus for the present study.
The Lox-1 protein expression of 250ug/ml Poly(I:C) group was less than ox-ldl group.
The Lox-1 and TF protein expression were markedly inhibited by B.macrostemi with dose of 0.5 and 1ug/ml,and the Lox-1 level of 1ug/ml group was similar with blocking agent group;comparing with ox-LDL group,the TFPI level increased obviously.
Conclusion
(1) 50ug/ml ox-LDL for 24h not only increased the Lox-1 and TF expression but also inhibited the TFPI expression.So 50ug/ml for 24h is the optimized stimulus for the present study.
(2) 250ug/ml Ploy(I:C) or inhibited lox-1 expression significantly and the effect of blocking was suitable for the present study.
(3) B.macrostemi with dose of 0.5 and 1ug/ml could decrease the oxidative damage of endothelial cell and the pro-coagulant factor level and keep the balance of TF/TFPI by inhibiting the expression of Lox-1.
背景
冠心病是一种严重危害人类健康的常见病、多发病,随着人们生活水平的提高和人口的老龄化,本病的发病率和死亡率有逐年上升趋势,其主要病理改变是动脉粥样硬化(AS)。AS是一种多因素参与的病理过程,其发病机制非常复杂,针对它的研究已很多。鉴于AS的易罹患性和终点事件的严重性,近年来,人们已经对本病有所认识,并且意识到及早进行防治的必要性。但已患病或其他因素(如遗传、女性绝经等)造成的动脉粥样硬化病人仍普遍存在,而且对动脉粥样硬化的防治缺乏有效的措施。因此,针对本病的发病机制,应用合理有效的药物,是延缓AS发生发展进程的根本原则。
脂质代谢紊乱对于致AS的作用已被大家所公认,脂质与冠心病的关系也已被证实。大量流行病学资料显示,动脉粥样硬化的严重程度随血浆胆固醇水平的升高呈线性加重,血浆胆固醇的浓度与冠心病死亡率呈正相关关系。过去认为血清总胆固醇是冠状动脉硬化的主要原因,现在已经明确低密度脂蛋白(LDL-C)更加重要。因为LDL-C的氧化损伤主要发生于动脉血管内膜,内皮细胞存在氧化低密度脂蛋白(ox-LDL)的特异性受体,内膜受到氧化损伤后会继发释放更多有害因子,导致内膜继续损伤的恶性循环。病变中脂质源于血浆脂蛋白的浸润,主要为游离胆固醇(FC)、胆固醇脂(CE),其次为甘油三酯(TG)、磷脂和载脂蛋白。所以,LDL-C水平升高是动脉粥样硬化及由动脉粥样硬化所引起的心脑血管疾病的最重要的危险因素。而高密度脂蛋白胆固醇(HDL-C)则是抗动脉硬化的,低HDL-C水平也是AS的风险因子之一。虽然循证研究认为HDL-C不能单独作为治疗靶点,但升高体内HDL-C的浓度确实能够减缓AS的病变进程。HDL-C水平每升高10 mg/L,冠脉疾病的危险就可能降低6%。低HDL-C水平与心血管疾病的相关性在老人和女性群体中表现更为显著。因此,降低LDL-C和升高HDL-C是防治动脉粥样硬化以至心脑血管病的重要目标。
虽然“脂质浸润”学说很好的解释了AS的发病机理,但很多研究者发现,粥样斑块的形成不仅是单纯的脂质沉积,20世纪70年代以来,越来越多的证据表明AS与血管损伤后发生的一系列生物过程有关,研究者们继“脂质浸润”学说之后又提出“内皮损伤”和“血栓形成”等多个假说。近年来Ross提出的AS“炎症反应”假说逐渐成为当前的主要学说。他认为AS斑块形成有两个途径:(1)各种炎症刺激或脂蛋白引起内皮细胞的损伤和由此引起的血小板聚集、活化、并导致血小板分泌血小板源性生长因子(PDGF),促进平滑肌细胞增生;(2)内皮受损,内皮细胞或巨噬细胞分泌生长因子,平滑肌细胞及内皮细胞也可产生PDGF样生长因子,这种相互作用导致纤维斑块形成,并继续发展。AS本身也是一种慢性炎症性疾病,炎症反应贯穿于AS发生、发展及斑块破裂、血栓形成的全过程。在炎性反应过程中,细胞黏附分子介导活化的白细胞结合到内皮细胞表面并迁徙至血管外的炎性组织中。在AS炎症发展过程中单核细胞与内皮细胞问的黏附主要靠细胞问黏附分子-1(intercellular adhesion molecules,sICAM-1)和血管细胞黏附分子-1(vascular cellular adhesion molecules,sVCAM-1)介导,Wallen等发现稳定型心绞痛患者血清中sICAM-1和sVCAM-1水平的升高程度与心源性死亡或急性心肌梗死发生率成正相关。这为粘附分子参与评估AS患者的预后提供了依据。
动脉粥样硬化性心脏病的最终结局往往是血管局部的血栓形成,侧枝循环建立不及时,进而导致心肌缺血引发急性冠脉综合症等严重心血管事件。而血栓形成离不开血液环境的改变,其中纤溶系统的功能是否正常至关重要。Hoffmeiste等报道冠心病患者纤溶活性降低,不正常的纤溶活性可能预示冠脉事件的发生。血浆中组织型纤溶酶原激活物(tissue plasminogen activator,t-PA)和纤溶酶原激活物抑制剂1(plasminogen activator inhibitor 1,PAI-1)是纤溶系统中重要的活性物质,PAI-1为t-PA的抑制物,它们之间的生理平衡对调节血流通畅,防止AS血栓形成起重要作用。
AS的发生发展及其复杂,脂质沉积和炎症反应以及血栓形成三者之间互相联系和影响。在AS早期阶段,多种刺激因素诱发动脉壁脂质聚集部位的炎症反应,引起细胞粘附,生成脂质斑块,引起平滑肌增殖、迁移;炎症还影响脂质代谢,加速LDL的氧化修饰,并且降低脂质斑块的稳定性,诱发斑块破裂血栓形成。所以,现在人们越来越清醒地认识到理想的策略应该是将药物治疗包括调脂、溶栓、抗凝以及抗炎等方法结合起来,发挥其协同作用,以达到最佳治疗效果。但若找出三者之间的共同作用靶点,则将为治疗提供捷径。
基础和临床研究证实,他汀类药物除具有强效的降低LDL-C作用外,还具有较好的抗炎症作用,通过降低C反应蛋白和粘附分子等炎症标记物的水平来改善患者的预后。同时,也有研究表明,他汀类药物能降低血浆PAI-1水平,明确改善机体纤溶功能。但Prove-It临床试验的结果显示,服用大剂量阿托伐他汀的治疗组两年后仍有22.4%的患者发生了急性冠状动脉事件,且服药后的肝功能异常使部分患者无法坚持用药。此外,使用单一药物进行二级预防效果很可能不如联合用药,作为心肌梗死二级预防最突出的范例就是2004年由2位英国学者提出的“复合制剂”想法。尽管文章发表后遭到了一定程度的置疑,但这一模式的转变意义重大。
中医药具有多途径、多环节、多靶点的特点,可通过保护血管内皮细胞、调节脂质代谢、抑制炎症反应和抗凝等多个角度稳定动脉粥样硬化斑块,在延缓AS进程方面具有潜在的疗效优势,且中药制剂药效缓和,药物之间因配伍而减毒增效,副作用较小,适宜作为二级预防用药长期服用。以往中药抗AS治疗研究中多只针对某一方面的病因或症状进行处理,缺少系统性的机制研究。通心络超微粉是近年应用络病理论与通络药物治疗心脑血管病的代表性方药,具有益气活血,搜风通络的功效,主要治疗冠心病心绞痛属心气虚乏,血瘀络阻者。本研究以辛伐他汀作为阳性对照药物,应用本实验室已成功构建的动脉粥样硬化动物模型方法,运用分子生物学及组织学等技术,观察通心络超微粉通过调脂、抑制炎症反应和抗凝作用防治动脉粥样硬化的疗效,并阐明其分子生物学机制及相互作用的联系靶点。
目的
(1)建立与人类动脉粥样硬化病变特征相似,易于观察药物疗效的动脉粥样硬化动物模型;
(2)应用血清学、组织学及分子生物学技术观察动脉粥样硬化病变过程中脂质、炎症及凝血机制的变化,并找出共同作用的靶点;
(3)对比不同剂量通心络超微粉和辛伐他汀治疗防治动脉粥样硬化的疗效及其分子生物学机制。
方法
1.动物模型的建立
健康雄性新西兰大白兔,在适应性饲养1周后,采用球囊损伤腹主动脉后,应用含有1%高胆固醇饲料喂养10周。
2.实验分组及药物干预
造模成功后随机分为对照组、小剂量、中剂量、大剂量通心络组和辛伐他汀组:
A对照组15只:造模成功后,给予普通饲料饲养8周;
B小剂量通心络组15只:造模成功后,普通饲料+通心络超微粉(0.15g/kg/d)饲养8周;
C中剂量通心络组15只:造模成功后,普通饲料+通心络超微粉(0.3g/kg/d)饲养8周;
D大剂量通心络组15只:造模成功后,普通饲料+通心络超微粉(0.6g/kg/d)饲养8周;
E辛伐他汀组15只:造模成功后,普通饲料+辛伐他汀(5mg/kg/d)饲养8周。
3.称量体重
实验前、10周造模后及18周末药物干预后分别对所有实验兔称量体重。
4.高频体表超声检查
分别于实验开始、10周末、18周末行腹主动脉高频体表超声检查。测量腹主动脉后壁内膜-中层厚度(intima-media thickness,IMT)、腹主动脉收缩期血流峰值速度(peak velocity,Vp)。
5.肝肾部分功能检查
分别于实验开始、10周末及18周末,采血作血液生化检查,主要包括ALT,GGT,TBIL,BUN和Cr。
6.血脂检测
分别于实验开始、10周末及18周末采血作血液生化检查。采用酶法测定血清总胆固醇(total cholesterol,TC)、甘油三酯(triglyceride,TG)、低密度脂蛋白胆固醇(low-density lipoprotein cholesterol,LDL-C)及高密度脂蛋白胆固醇(highdensitylipoprotein cholesterol,HDL-C)水平。
7.血清炎性因子的检测
分别于实验开始、10周末及18周末采血,应用酶联免疫吸附法(enzyme-linked immunosorbent assay,ELISA)检测氧化低密度脂蛋白(oxidizedlow-density lipoprotein,ox-LDL)、血清高敏C反应蛋白(high sensitivityC-reactive protein,hs-CRP)、血清sICAM-1及sVCAM-1的浓度。
8.血浆纤溶指标的检测
分别于实验开始、10周末及18周末采血,应用ELISA试剂盒检测血浆PAI-1、t-PA的浓度水平。
9.病理染色
留取球囊拉伤处腹主动脉标本,行苏木素—伊红(hematoxylin-eosin,HE)染色、油红O染色和Movat五色套染法染色。
10.免疫组织化学染色
对腹主动脉球囊损伤处血管段进行组织切片,行胆固醇酯转运蛋白(CETP)、低密度脂蛋白受体相关蛋白(LRP)、RAM11、VCAM-1、ICAM-1、及CRP免疫组织化学染色,此外并对肝脏组织切片行CETP及LRP的免疫组织化学染色。
11.Western blot
留取肝脏及血管标本,检测组织中CETP及LRP蛋白表达水平。
12.扫描电镜
各组均留取标本进行扫描电子显微镜检查,观察血管内膜损伤及脂质沉积的情况。
13.统计学分析
应用SPSS统计软件进行统计学处理(version 13.0;SPSS Inc),连续性数据用(?)±SD表示,P＜0.05有统计学差异。
结果
1.实验动物基本情况
进入药物干预阶段的实验兔中,辛伐他汀组(E组)死亡1只,大剂量通心络组(D组)死亡2只,中、小剂量通心络组各死亡1只,死亡原因分别为腹泻、呼吸道感染或原因不明。18周末各组存活实验兔为:对照组(A组)15只,小剂量通心络组(B组)14只,中等剂量通心络组(C组)14只,大剂量通心络组(D组)13只,辛伐他汀组(E组)14只。
2.实验动物体重的变化
10周末所有实验兔体重均较实验开始前加重,相比均有显著性差异(P均＜0.01),但治疗后各组实验兔之间无显著差异。
3.高频体表超声
10周末,所有实验兔IMT均显著升高(P均＜0.01),Vp值显著升高(P＜0.05);治疗后C、D、E三组IMT较对照组相比显著降低(P均＜0.05),D、E两组Vp值显著降低(P均＜0.05)。
4.肝肾功能检测
18周末,辛伐他汀组血清ALT、GGT两者浓度显著高于其他组(P均＜0.05),其他各组之间无显著差异。
5.血脂检测
组内比较:10周末,除HDL-C外,所有实验兔较造模前各项指标相比均显著升高(P均＜0.01);18周末,各组血脂含量除HDL-C外,与治疗前相比均显著降低(P均＜0.01)。
治疗后组间比较:与A组比较,各药物治疗组均可明显降低血清TC水平(P＜0.01),大剂量通心络组和辛伐他汀组组间无显著性差异(P＞0.05);大剂量通心络组TC水平明显低于小剂量通心络组;
与A组比较,各药物治疗组均可明显降低血清LDL-C水平(P＜0.01),辛伐他汀组降低水平最显著(P＜0.01),大剂量组次之(P＜0.01),中、小剂量通心络组间无显著性差异(P＞0.05);
与A组比较,各药物治疗组均可明显降低血清TG水平(P＜0.01),中剂量通心络组(C组)、大剂量通心络组(D组)和辛伐他汀组(E组)三组间无显著性差异(P＞0.05);大剂量通心络组TG水平明显低于小剂量通心络组(P＜0.01);
与A组比较,各药物治疗组均可明显升高血清HDL-C的值(P＜0.01～0.05),大剂量通心络组HDL-C的值明显高于中、小剂量通心络组,与辛伐他汀组比较无显著差异。
6.血清炎性因子的检测
组内比较:10周末造模成功实验兔血清ox-LDL、hs-CRP、sICAM-1及sVCAM-1水平较实验初始相比均显著升高(P＜0.01);18周末,C、D、E各组炎性因子血清水平均显著下降(P＜0.05)。
组间比较:与A组比较,治疗后中、大剂量通心络组和辛伐他汀组均可明显降低血清炎性因子hs-CRP的水平(P＜0.01),由低到高依次为E组、D组和C组。D组与E组降低hs-CRP水平无显著性差异(P＞0.05);
与A组比较,各药物治疗组血清可溶性粘附分子sVCAM-1的水平均显著下降(P＜0.01),辛伐他汀组sVCAM-1水平明显低于小、中、大三个剂量通心络治疗组(P＜0.05),各通心络治疗组间降低sVCAM-1水平无显著性差异(P＞0.05);
与A组比较,各药物治疗组血清可溶性粘附分子sICAM-1的水平均显著下降(P＜0.01),但辛伐他汀组降低水平最明显(P＜0.01);
与A组比较,各药物治疗组血清ox-LDL的水平均显著下降(P＜0.01～0.05),药物组间比较未见显著差异。
7.血浆纤溶指标的检测
组内比较:治疗后除对照组外,其他各组血浆PAI-1水平显著降低(P＜0.05～0.01),t-PA水平均显著升高(P＜0.05～0.01)。
组间比较:18周末,与对照组相比,B、C、D三组血浆PAI-1水平显著降低(P＜0.05～0.01),C、D两组最显著(P＜0.01);与对照组比较,C、D、E三组血浆t-PA水平显著升高(P＜0.05～0.01),但以D组差异最显著(P＜0.01)。
8.病理染色
HE染色显示,造模成功后各组血管斑块明显,治疗后各组间未见明显差异;油红“O”染色显示,对照组斑块内脂质含量显著高于药物组;Movat结果显示,对照组动物斑块内部基质和粘蛋白含量显著高于药物组,各药物组间未见差异。
9.免疫组织化学染色
五组斑块内均有CETP、LRP、RAM11、VCAM-1、ICAM-1、及CRP的局部表达;与对照组相比,药物组LRP、RAM11、VCAM-1、ICAM-1及CRP表达均显著减少,其中D、E两组较其他3组相比表达更低,而C组和B组间未见差异;E组CETP蛋白表达显著低于其他四组,四组间比较无显著差异。肝脏CETP及LRP免疫组织化学染色结果与其血管斑块内表达结果相似。
10.Western blot
各组肝脏及斑块中均有CETP、LRP的高度表达,其中D、E两组LRP蛋白表达显著低于其他三组(P＜0.01),两组间无显著差异;E组CETP蛋白表达水平显著低于其他四组(P＜0.05),其余四组间比较无显著差异。
11.扫描电镜
各组内皮细胞均有不同程度损伤,主要表现为内皮细胞排列紊乱,内膜较多区域有细胞脱落,暴露内皮下胶原,脱落区附近有大量红细胞、血小板及脂滴黏附,低倍镜下可见病变区域广泛,内皮隆起区域部分融合,形成表面粗糙的“斑块”状病变。A组内膜损伤显著甚于其他药物组,药物组间比较发现,D、E两组损伤显著较轻,C组和B组间未见显著差异。
结论
(1)应用球囊损伤加高脂饲养方法可建立与人类动脉粥样硬化病变特征相似、造模时间短及适于观察药物疗效的动物模型,结合血脂水平的检测,应用高频体表超声可判定模型的建立情况。
(2)通心络超微粉能够不通过抑制CETP升高HDL-C,保护了体内的正常胆固醇逆转运功能。
(3)大剂量通心络超微粉能有效降低血脂,抑制炎症反应及增强纤溶功能其作用的共同靶点可能是LRP。
论文Ⅱ薤白对氧化低密度脂蛋白诱导的内皮细胞组织因子及其抑制物表达的干预作用
背景
动脉粥样硬化(atherosclerosis,AS)是中老年人群心脑血管疾病高发的重要病理基础,有关AS的形成的一个主要高危因素就是高脂饮食导致的体内动脉内膜脂质沉积过多。近年来大量流行病学调查表明,体内血清胆固醇尤其是低密度脂蛋白(LDL-C)含量的增多更加促进了AS的发生发展,而氧化修饰后的LDL-C(ox-LDL)更是大为加强了病变程度,是导致粥样斑块破裂血栓形成的始动因素之一。随着人口老龄化的发展,我国冠心病病人的发病率呈明显上升趋势,已接近国际平均水平。尽管人们已对本病有一定的预防意识,但已患病或其他因素(如遗传、女性绝经等)造成的动脉粥样硬化病人仍普遍存在,因此,寻找合理有效的药物抑制ox-LDL对内皮细胞的损伤,降低促凝因子释放,使血管内环境保持稳定平衡的状态,是减少危险事件发生的有效途径。
动脉粥样硬化的发生发展贯穿冠心病病人整个病理过程,但是,从脂质沉积到粥样斑块形成,乃至最后的斑块破裂血栓形成造成危险事件的发生,内皮细胞都在其中起到重要作用。诸多学者都认可“损伤学说”是AS的始动机制的理论。因为LDL-C的氧化损伤主要发生于动脉血管内膜,内皮细胞存在ox-LDL的特异性受体,内膜受到氧化损伤后会继发释放更多有害因子,导致内膜继续损伤的恶性循环。文献报道,ox-LDL可使培养的人脐静脉内皮细胞的抗氧化酶谷胱甘肽过氧化物的活性下降,前列环素生成减少,脂质过氧化物产生增加,表明ox-LDL具有直接的内皮损伤作用。同时,内皮细胞表达的ox-LDL的特异性受体—血凝素样氧化型低密度脂蛋白受体1(Lox-1)增多,介导了内皮细胞对衰老细胞及凋亡细胞的吞噬,激活多条炎症反应的信号通路,比如p44/42MAPK、p38MAPK、CD40/CD40L等。此类信号通路被激活后可以诱发内皮细胞释放大量的促凝因子如组织因子(TF),并且减少了具有抗凝作用的组织因子途径抑制剂(TFPI)的表达,促使血管内凝血机制增强,有利于血栓形成。
目前临床治疗动脉粥样硬化多集中于调脂治疗,主要是他汀类调脂药物,其次是针对症状应用的抗血小板制剂、β-受体阻滞剂及血管紧张素转换酶抑制剂等。这些药物对于延缓动脉粥样硬化进程和改善临床症状都具有积极而肯定的疗效,并且由于高脂血症已经成为严重心血管疾病发病的高危因素,所以其中的他汀类调脂药已经成为治疗冠心病的基础用药。大量的临床实验研究表明,在对AS血管疾病的控制治疗中,能够保护内皮细胞,改善内皮功能,起到抗脂质氧化作用的药物越来越成为首选药物。但是大多数西药,比如他汀类调脂药,虽然有很好的耐受性和安全性,但是治疗的同时也带来较多副作用,比如肝功能异常及其他消化和皮肤疾病等。
近年来,对中草药降脂的研究颇为广泛。中药的优点在于成分纯天然,在抗氧化、降脂及抗炎抗凝方面都有良好功效。而且中药对人体毒副作用较小,在安全剂量时,对肝肾功能影响很小。薤白在以往中医药应用方面多采用复方制剂,在防治心血管疾病方面取得显著疗效。但是在细胞水平的研究方面,大多数研究者都集中于其挥发油的作用研究上。但由于挥发油成分不稳定极难保存,而且薤白还含有大量的皂苷类和多糖类化合物,所以对于薤白单方药物的系统研究有待更深入的进行。有关薤白挥发油的抗氧化、抗凝作用研究较多,但此作用的具体机制至今未见报道。而且之前的实验研究多采用静脉内皮细胞或牛动脉内皮细胞进行培养,所以为了更好地模拟临床现象,本实验采用原代人主动脉内皮细胞(human aortic endothelial cells,HAECs),以ox-LDL作为内皮细胞氧化损伤的诱导因子,观察了不同浓度及不同时间点ox-LDL对于体外培养HAECsLox-1、TF及TFPI蛋白表达的影响,并观察薤白对ox-LDL诱导的内皮细胞氧化损伤的防治作用。
目的
(1)建立人主动脉内皮细胞氧化损伤的模型;
(2)观察不同剂量浓度薤白超微粉抑制氧化低密度脂蛋白诱导的内皮细胞表达促凝因子的作用及其机制。
方法
为建立内皮细胞氧化损伤模型,确定ox-LDL最终刺激浓度及刺激时间,本实验通过体外培养人主动脉内皮细胞,观察不同浓度和不同时间ox-LDL刺激后,人主动脉内皮细胞Lox-1、TF及TFPI蛋白的表达变化,选定ox-LDL的最佳刺激条件。
不同浓度的薤白超微粉(0.25ug/ml、0.5ug/ml、1ug/ml)预孵育12h后再加ox-LDL刺激,同时应用Lox-1阻断剂-Poly(I:C)做对照组,倒置显微镜下观察细胞形态的变化,并且用western-blot方法检测各组Lox-1、TF及TFPI的蛋白表达水平。
结果
倒置显微镜下观察,正常血管内皮细胞生长状态良好,贴壁较牢,呈梭形或圆形,形态饱满,呈铺路石状生长,细胞间连接紧密,分裂相多,未见悬浮细胞。ox-LDL损伤组可见不同程度及范围的细胞收缩、变圆、胞间隙变宽、细胞脱壁悬浮;Lox-1阻断剂250ug/ml组及薤白超微粉1ug/ml组细胞形态与正常组比较无显著差异。
根据不同浓度及不同时间ox-LDL刺激后人主动脉内皮细胞Lox-1、TF及TFPI蛋白变化情况及本实验的研究目的,我们选定50ug/ml的ox-LDL培养液培养细胞24h为最佳刺激条件。50ug/ml ox-LDL培养细胞24h,Lox-1和TF的蛋白表达水平均显著升高,TFPI蛋白表达水平显著下降。
250ug/ml Poly(I:C)预孵育2h后再加入ox-LDL刺激细胞发现Lox-1蛋白表达水平较其他组显著降低,故可采用该浓度Poly(I:C)作为本实验的Lox-1阻断剂。
0.5ug/ml和1ug/ml薤白可以显著抑制Lox-1和TF蛋白表达,其中1ug/ml薤白组和Lox-1阻断剂组相比无显著差异;同时薤白增加了TFPI蛋白表达,和刺激组相比差异显著。
结论
(1) 50ug/ml ox-LDL培养人主动脉内皮细胞24h显著升高Lox-1蛋白表达水平,同时引起TF蛋白表达增强和TFPI蛋白的表达受到抑制,选为本实验的最佳刺激条件;
(2) 250ug/ml的Ploy(I:C)能显著抑制Lox-1蛋白表达,起到实验要求的阻断效果。
(3) 0.5ug/ml和1ug/ml薤白可以通过抑制Lox-1表达来减少内皮细胞的氧化损伤,进而减少促凝因子释放,稳定TF/TFPI的表达比例。
PAPERⅠTHE EFFECTS AND MECHANISM OF TONGXINLUO SUPERFINE ON LIPID-LOWERING,INHIBITING INFLAMMATION AND ANTICONGULATION
Coronary heart disease is the commonly encountered and frequently-occurring disease which harms health seriously.With the elevation of living standard and ageing of population,the morbidity and mortality rise year by year and the pathology change is atherosclerosis(AS).AS is the pathologic processes including many factors and the pathogenesis is very complex and the research about it is multiply.Because the AS is easy to happen and serious,so people have realized that is necessary to prevent and cure as soon as possible for the past few years.However,the AS patients are common and the effective intervention about its treatment is lacking.Therefore,to aim directly at the pathogenesis and make use of reasonable and effective drugs is the basic principle of delaying the development of AS.
The role of lipid metabolic disorder inducing the AS is generally acknowledged and the relation of lipid and CHD is confirmed.Lot's of epidemiological data showed the severity and death rate of AS aggravates with the increasing of serum cholesterol level.It is previously believed the serum cholesterol is the primary cause but now the low density lipoprotein(LDL-C) is deemed more important.The oxidative damage of LDL-C occurs on artery vascular intima and there is special receptor of oxidized low density lipoprotein(ox-LDL) in endothelial cell,and the cell would express more harmful substance because of sustained injuring,even which can bring the vicious cycle of tunica intima due to injured continually.The lipids about AS are from plasma lipoprotein like free cholesterol(TC),Triglyceride(TG),et al.The increasing of LDL-C level is the capital risk factors of AS or other cardiovascular and cerebrovascular diseases about AS.Conversely,the high density lipoprotein cholesferol(HDL-C) is beneficial to anti AS and the low level of HDL-C is one of risk factors of AS.Although the Cochrane doesn't recognize the HDL-C as simple curing target point,to increase the level of HDL-C really can slow down the process of AS.The correlation of low level of HDL-C and cardiovascular diseases is more significant in aged and female group.Therefore,to reduce the LDL-C and raise HDL-C level is the important target of curing AS.
Although the "lipid invasion theory" explains the pathologic mechanism of AS well,many researchers found that the form of plaque is not only because of lipid deposition but also the bioprocess after vascular injuring in 1970s.The hypothesis of "endothelium injured" and "thrombosis" were suggested by researchers on the base of "lipid invasion theory".For the past few years,the "inflammatory reaction theory" of Ross is becoming the major idea.Ross thought the form of AS had two pathways:(1) the injuring of endothelial cell because of various inflammation stimulus or lipoprotein and the platelet aggregation,activation,expression of platelet-derived growth factor(PDGF) and the proliferate of smooth muscle cell(SMC) since the injuring happened;(2) once the endothelium was injured the endothelial cell and macrophage would secrete kinds of factors and SMC also bring the PDGF,and then, the interaction of them will lead to the form and development of fibrous plaque.The AS is the chronic inflammation diseases which the inflammatory reaction penetrates in the all process of AS including the rupture of plaque and thrombosis.In the process of inflammatory reaction,the white blood cell activated by cellular adhesion molecule combines with endothelial cell surface and migrates to the inflammatory tissue out of vascular.In the development of AS,the adhesion of monocyte and endothelial cell is mediated by intercellular adhesion molecules(ICAM-1) and vascular cellular adhesion molecules(VCAM-1) chiefly.Wallen found the increase of serum ICAM-1 and VCAM-1 level in angina pectoris patients positively correlate to the incidence rate of cardiac death or acute myocardial infarction,which supplied the evidence for the adhesion molecule participating to evaluate the prognosis of AS patients.
The final result of atherosclerotic heart disease is the thrombosis of vascular and the collateral circulation isn't setup timely that result in myocardial ischemia and induce acute cardiovascular event.However,the form of thrombus relates with the change of blood environment and whether the function of fibrinolysis system is normal is important.Hoffmeiste reported the fibrinolysis activity of CHD patients decrease decreased and abnormal fibrinolysis activity might predict the occurring of coronary artery disorder.The plasma tissue plasminogen activator(t-PA) and plasminogen activator inhibitor 1(PAl-1) is the important active compound of fibrinolysis system and the physiologic equilibrium of them is fundamental to keep blood stream unobstructed and prevent thrombosis.
The pathomechanism of AS is very complicated and the lipid deposition, inflammatory reaction and thrombosis correlate and affect each other.In the early period,much stimulating factor induce the inflammatory reaction on the vessel wall lipid deposited,cell adhesion,lipid plaque,proliferate and immigration of SMC;the inflammation also affects the metabolism of lipid and promotes the oxidation of LDL-C,meanwhile,which can decrease the stability of lipid plaque and induce the rupture of plaque.Therefore,people realize ideal strategy about the treatment of AS is that can combine with the reducing blood fat,inhibiting inflammation and anticoagulation and play the joint action.However,to find the target point of them will be shortcut to treat AS.
The clinical research confirmed the statins could not only reduce LDL-C level but also inhibit the inflammation and improve outcomes by decreasing the inflammation markers like C reactive protein and adhesion molecules.Meanwhile, statins could reduce the plasma PAI-1 level and improve the function of fibrinolysis significantly.However,the clinical trial showed that 22.4%patients who summed the large dose of atorvastatin experienced acute coronary artery disease in 2 years. Moreover,some patients withdraw from statins treatment because of liver dysfunction. In addition,the drug combination could have better effect compared with one kind of medicine,so the "polypill" was preferred in secondary prevent treatment by two British scholars in 2004.Although the manuscript met with the doubt,the change of mode has important meaning.
Traditional Chinese medicines have the characteristic of much way,much link and much target point and stable the AS plaque by protecting the vascular endothelial cell,regulating the lipid metabolism,inhibiting the inflammatory reaction and anticoagulation.Traditional Chinese medicines have the potential curative effect in delaying the development of AS and are suitable to long-time drug taking of secondary treatment because of the moderate potency,less side effect and compatibility.Formerly,the research of traditional Chinese medicines about treating AS usually aim directly at the simple cause or symptom and the systemic research of mechanism is necessary.Tongxinluo superfine is the representative Chinese drug which treats cardiovascular and cerebrovascular disease with theory of collateral disorders and dredging collaterals herbs.Tongxinluo has the effects of benefiting vital energy and promoting blood flow and has been used in CHD that show off deficiency of heart-energy and blood stasis.The present study adopts the simvastatin to be positive control and uses the AS animal model made by laboratory and be aimed to observe the therapeutic effects of Tongxinluo on AS by regulating lipid,inhibiting inflammation and anticoagulation and elucidate the possible mechanism and relationship of them through the molecular biology and imageology technology.
Objective
(1) To establish an animal model of AS that is mimic to human pathological changes and convenient for intervention;
(2) To observe the changes of lipid,inflammation and clotting mechanism and find the functionary target point in common with serology histology Protocols in Molecular Biology.
(3) To compare the therapeutic effects of Tongxinluo and Simvastatin on preventing and curing AS and approach the possible mechanism.
Methods
1.Establishing the animal model:
75 rabbits were fed on an atherogenic diet containing 1%cholesterol for 10 weeks after abdominal aortic wall injuries were induced using an intravascular balloon.
2.Intervention methods:
Group A(control group):15 rabbits were fed normal diet for 8 weeks after the model was established.
Group B(low dose):15 rabbits were fed normal diet and Tongxinluo super (0.15g/kg/d) for 8 weeks after the model was established.
Group C(middle dose):15 rabbits were fed normal diet and Tongxinluo super (0.3g/kg/d) for 8 weeks after the model was established.
Group D(high dose):15 rabbits were fed normal diet and Tongxinluo super (0.6g/kg/d) for 8 weeks after the model was established.
Group E(simvastatin):15 rabbits were fed normal diet and simvastatin (5mg/kg/d) for 8 weeks after the model was established.
3.Body weight:
Body weights of all rabbits were measured at baseline,the end of the 10~(th) and 18~(th) week.
4.High frequency ultrasound:
At the baseline,end of the 10~(th) and 18~(th) week,the abdominal aorta was scanned using a high frequency duplex ultrasonographic system.The intima-media thickness (IMT) and peak velocity(Vp) were measured.
5.Liver and kidney function test:
Blood samples were collected from all rabbits at the baseline,the end of the 10~(th) and 18~(th) week.The serum ALT,GGT,TBIL,BUN and Cr were detected with automatic biochemical analyzer.
6.Lipid measurement:
Blood samples were collected from all rabbits at the baseline,the end of the 10~(th) and 18~(th) week.Serum levels of total cholesterol(TC),triglyceride(TG),low density lipoprotein cholesterol(LDL-C) and high density lipoprotein cholesterol(HDL-C) were measured by enzymic method.
7.Serum inflammatory factor:
Blood samples were collected from all rabbits at the baseline,the end of the 10~(th) and 18~(th) week.The serum high sensitivity C-reactive protein(hs-CRP)、ox-LDL、ICAM-1 and VCAM-1 were assayed using highly sensitive enzyme-linked immunosorbent assay(ELISA) kits.
8.Plasma fibrinolysis detection:
Blood samples were collected from all rabbits at the baseline,the end of the 10~(th) and 18~(th) week.The plasma PAI-1 and t-PA were assayed using ELISA kits.
9.Histopathological analysis:
The abdominal aorta was processed and examined by hematoxylin and eosin (HE),Movat and oil red O staining.
10.Immunohistochemical staining:
The expression of RAM11,VCAM-1,ICAM-1,CRP,cholesterol ester transfer protein(CETP) and low density lipoprotein receptor-related protein(LRP) were determined.
11.Western blot:
To detected the protein expression of CETP and LRP in liver.
12.Scanning electron microscope:
Scanning electron microscope was used to observe the the degree of endangium damage and lipids deposition.
13.Statistical analysis:
Data are expressed as mean±SD for continuous variables and by frequency count and percentage for qualitative variables.Incidence rate of plaque rupture was compared with Pearson Chi-Square test and other indexes were compared with One-Way ANOVA comparison test.The SPSS was version 13.0.P＜0.05 was considered statistically significant.
Results
1.General state of the experimental animals:
Five rabbits died of diarrhea,respiratory tract infection and so on,and there were 70 rabbits finished the study:15 rabbits in group A,14 rabbits in group B,14 rabbits in group C,13 rabbits in group D,14 rabbits in group E.
2.Body weight:
Body weights of all rabbits were significantly higher at the end of 10~(th) week than that in begin(P＜0.01);there were no difference among the groups after treatment.
3.High frequency ultrasound measurement:
At the end of 10~(th) week,the maximum IMT of the rabbits were significantly higher(P＜0.01),and the Vp was also higher(P＜0.05);after treatment,the IMT of group C,D and E were lower than group A(P＜0.05),the Vp of group C and E was lower(P＜0.05).
4.Detection of liver and kidney function:
At the end of 18~(th) week,the ALT and GGT in group E were higher than other groups(P＜0.05).
5.Lipid measurement:
Intra-group comparison:at the end of 10~(th) week,except the HDL-C,the TC,TG and LDL-C level were higher(P＜0.01);at the end of 18~(th) week,except the HDL-C, the TC,TG and LDL-C level were lower(P＜0.01).
Inter-group comparison:compared with group A,except the HDL-C,at the end of 18~(th) week,the lipid level in the drug groups decreased significantly especially in group D and E,but the serum HDL-C level of drug groups increased.
6.Detection of serum inflammatory factor:
Intra-group comparison:at the end of 10~(th) week,the serum ox-LDL,hs-CRP, ICAM-land VCAM-1 increased significantly(P＜0.01);at the end of 18~h week,the serum inflammatory factor of group C,D and E decreased markedly(P＜0.05).
Inter-group comparison:at the end of 18~(th) week,compared with group A,the serum ox-LDL,hs-CRP,ICAM- 1 and VCAM- 1 of group A was higher than group C, D and E significantly(P＜0.01～0.05).
7.Detection of plasma fibrinolysis factors:
Intra-group comparison:except the group A,at the end of 10~(th) week,the plasma PAI-1 level increased and t-PA decreased significantly(P＜0.05～0.01).
Inter-group comparison:at the end of 18~(th) week,compared with group A,the plasma PAI-1 level increased and t-PA decreased significantly in group D and E (P＜0.05～0.01).
8.Pathologic staining:
The H&E staining showed the obviously plaque in all groups;oil red "O" staining showed the lipid in plaque of group A was higher than group C,D and E; Movat showed the ground substance in plaque of group A was higher than in group C, D and E;there was no difference among the group D and E.
9.Inununohistochemical staining:
Compared with group A,the expressions of LRP,RAM11,VCAM-1,ICAM-1 and CRP in plaque of group C,D and E decreased significantly,and no difference was found between group D and E.There was no significantly difference of CETP among the Tongxinluo groups.
10.Western blot:
There was high expression of CETP and LRP in liver in all groups.The expression of LRP protein in group C,D and E is lower than in group A,The expression of CETP protein in group E is lower than in other groups.
11.Scanning electron microscope:
The endothelial cell of normal group was clostridial form,integrate,lining up in order and the long axis of cell was concordant with direction of blood stream,the nucleolus swelled up and orientated toward the lumens and there was no cell adhesion; the endothelial cell of other group was injured and the cell was arranged in disorder, and much cell of intima ablated and exposed the collagen,there adhered much red blood cell,platelet and lipid droplet near the deciduo-as area.Under the lower power lens,the endodermis swelled up and partly mixed together and the plaque formed. The damage of group A and B was more serious than other groups and there was no significant difference between group C,D and E.
Conclusions
(1) The rabbit AS model,which is established with balloon-induced abdominal aortic wall injury together with a cholesterol-rich diet is mimic to human disease and suitable for the observation of drug therapeutic effects.
(2) The Tongxinluo increased the HDL-C level and protected the normal function of reverse cholesterol transport not by inhibiting the CETP.
(3) The combination of Tongxinluo and simvastatin could effectively decrease the lipid,inhibit the inflammatory reaction and enhance the fibrinolysis function and the common target maybe was LRP.
PAPERⅡINTERVENTION OF B.MACROSTEMI ON TF/TFPI OF HAECs OX-LDL ACTIVATED
Background
The happen and development of AS run through the whole pathologic processes of CHD,however,from the lipid deposition to atheromatous plaque and even the plaque rupture and thrombosis inducing the danger incidence,the endothelial cell play the important role.Many professors consent the "injuring theory" is the initiate mechanism of AS.The oxidative damage of LDL-C betide artery vascular intima and there is specific receptor of ox-LDL in endothelial cell,and the cell would express more harmful substance because of sustained injuring,even which can bring the vicious cycle of tunica intima due to injured continually.Ox-LDL decrease the activity of glutathione peroxides and the generate prostacyclin of cultural HUVEC and increase the lipid peroxides that showed the ox-ldl can injure the endothelium directly.Meanwhile,the lectin-like oxidized LDL receptor-1(lox-1) that endothelial cell expressed increase and that promote the endothelial cell to swallow the senile and apoptosis cell,and activate much inflammatory reaction signal pathway,for example, p44/42MAPK、p38MAPK、CD40/CD40L,et al..The endothelial cell release more procoagulant factors like tissue factor(TF) and less anticoagulant substance like tissue factor pathway inhibitor(TFPI) when those pathways was activated,then,the clotting mechanism enhanced and thrombus form.
The important pathological basis of high incidence cardiovascular and cerebrovascular diseases of old people is atherosclerosis(AS),and the high risk factor of AS is too much lipid deposition in endarterium due to high fat diet.For the past few years,lots of epidemiological study showed the increase of serum cholesterol especially the low-density lipoprotein(LDL-C) promote the development of AS,and the oxidized low density lipoprotein(ox-LDL) more enhance the degree of pathological changes that it has become one of the initiating agent of plaque rupture and thrombosis.With the development of aging,the incidence of coronary heart disease(CHD) in our country was at the increased tendency and got close to the international average level.In spite of many patients have recognized the serious of CHD,the AS patients have been ill or other reason induced(smoking,heredity,and menopause) are still ubiquitous.Therefore,to find the effective drugs that can inhibit the injure of endothelial cell by ox-LDL,to decrease the release of procoagulant factor and keeping the balance of intravascular environment is good for reducing the danger incident.
Now the treatment of AS more often focus on regulating lipid,besides statins, there are antiplatelet drugs,β-receptor inhibitor,angiotensin converting enzyme inhibitor and so on that can improve clinic symptoms.The drugs play the positive therapeutic effect with regard to delay the process of AS.Moreover,because hyperlipidemia is the high risk of cardiovascular diseases,the statins have become the basic drugs to treat CHD.Much clinical trial indicated that those drugs which can protect endothelial cell,improve endothelium function and drugs have the role of anti-lipid peroxidation become choice drug.But most drugs like statins,although they have good survivability and safety,there are much side effects like dysfunction of liver,digestive system diseases and so on.
There is much study about herbs for the past few years,the component of herbs is natural and they are effective in antioxidation,reducing blood fat, anti-inflammatory and anticoagulation.Furthermore,because of little side effect of herbs,in the range of safe dose there is tiny dysfunction of liver and kidney.Bulbus allii macrostemonis(B.macrostemi) was often used in compound preparation in the past and it also showed noticeable effect.In the aspect of cellular level research,most researchers took their attention on the study of essential oil.But the essential oil is difficulty to preserve because of unstable component and B.macrostemi contain lots of saponins and polyoses compound,therefore,the more deeply systematic research about B.macrostemi is necessary.Although there are lots of research about anti-oxidation or anticoagulation of essential oil,the mechanism about that is absent. In addition,intravenous endothelial cells or bovine aortic endothelial cells were adopted usually in these researches.In order to simulate clinical diseases,the human aortic endothelial cells(HAECs) were cultured in vitro and ox-LDL was applied for inducing factor of oxidative damage to endothelial cell.We observed the effect of ox-LDL on expression of Lox-1,TF and TFPI and the protective effect of B. macrostemi on the endothelial cells damage induced by ox-LDL.
Objective
(1) To establish the model of human aortic endothelial cells oxidative damage;
(2) To investigate the effect and mechanism of different dose B.macrostemi inhibiting the expression of pro-coagulant factor in ox-LDL activated by HAECs.
Methods
To establish the model of human aortic endothelial cells oxidative damage,we observe the effect of ox-ldl with different concentration and different time on the expression of lox-1,TF and TFPI.
The cultured HAECs were pretreated with or without B.macrostemi(0.25ug/ml、0.5ug/ml、1ug/ml) for 12h and were observed under inverted microscope.Meanwhile, the control group that cell were pretreated with blocking agent of lox-1-Poly(I:C) for 2h was designed.The effects of B.macrostemi with different concentration on lox-1、TF and TFPI protein expression were investigated by western blot.
Results
Inverted microscope showed that the normal endothelial cells were at good condition,adherence stable,fusiform or round,tight cell-cell junction,more cleavage phase,less suspension cells;cell shrinkage,intercellular space broaden and more suspension cells in ox-LDL damage group;the shape and structure of endothelial cell in blocking agent and B.macrostemi 1ug/ml group was similar with normal cell.
Based on the aim of the present study and the effect of ox-LDL with different concentration and different time on the expression of Lox-1、TF and TFPI、50ug/ml ox-LDL for 24h is the optimized stimulus for the present study.
The Lox-1 protein expression of 250ug/ml Poly(I:C) group was less than ox-ldl group.
The Lox-1 and TF protein expression were markedly inhibited by B.macrostemi with dose of 0.5 and 1ug/ml,and the Lox-1 level of 1ug/ml group was similar with blocking agent group;comparing with ox-LDL group,the TFPI level increased obviously.
Conclusion
(1) 50ug/ml ox-LDL for 24h not only increased the Lox-1 and TF expression but also inhibited the TFPI expression.So 50ug/ml for 24h is the optimized stimulus for the present study.
(2) 250ug/ml Ploy(I:C) or inhibited lox-1 expression significantly and the effect of blocking was suitable for the present study.
(3) B.macrostemi with dose of 0.5 and 1ug/ml could decrease the oxidative damage of endothelial cell and the pro-coagulant factor level and keep the balance of TF/TFPI by inhibiting the expression of Lox-1.
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