芪参益气滴丸对心肌梗死二级预防的作用及其机制研究
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摘要
背景
心血管疾病是严重威胁人类生命健康的常见病和多发病,全球每年大约有2000万人死于急性心血管事件,其中,半数以上死于急性心肌梗死(acutemyocardial infarction,AMI)。随着人口老龄化的发展,我国急性心肌梗死的发病率呈明显上升趋势,已接近国际平均水平。近年来,由于监控和治疗水平的提高,心肌梗死的死亡率明显降低,但存活患者再次发生心肌梗死、充血性心力衰竭和猝死等急性心血管事件的危险性仍很高。因此,除在急性期应积极治疗外,还应加强心肌梗死后的二级预防。
心肌梗死的二级预防是指心肌梗死发生后,预防再梗死和猝死,大量研究业已证明,抗血小板制剂、β-受体阻滞剂、他汀类调脂药物及血管紧张素转换酶抑制剂等对心肌梗死后的长期二级预防具有积极而肯定的疗效,并且其疗效不受患者其他情况(如年龄、性别等)的影响。自从第一个标志性试验显示他汀类药物能够降低心肌梗死后患者冠心病危险事件的发生率和总病死率以来,该类药物已经成为预防治疗的基础。调脂治疗虽只能使冠状动脉狭窄的病变减轻1%～4%,但可显著降低心肌梗死的再发率和病死率,其原因在于心肌梗死多发生于轻或中度狭窄的冠状动脉,导致心肌坏死的主要原因为粥样斑块的破裂、出血及继发形成的血栓与血管腔闭塞,而非冠状动脉的固定性狭窄。调脂治疗在防止斑块破裂和出血方面的功效,正是其用于心肌梗死二级预防的基础和依据。
冠状动脉粥样硬化斑块不稳定或易于破裂者及血栓形成倾向严重者易发生再梗死,因此,心肌梗死后急性心血管事件的发生与否取决于斑块的易损性,与防治血栓相比,稳定易损斑块具有更大的临床价值,是心肌梗死二级预防的最终目标。近年来的研究发现,急性冠脉综合征(acute coronary syndrome,ACS)患者常具有多个易损斑块,因此,全身性的药物治疗成为稳定易损斑块的首选方法。虽然基础研究证明,他汀类药物可以通过改善血脂、抑制斑块炎症及增加纤维帽厚度等作用稳定易损斑块。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。
炎症反应在不稳定斑块形成及破裂中起着重要的作用,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、抑制炎症反应和抑制胶原纤维降解等多个角度稳定动脉粥样硬化斑块,在稳定易损斑块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。近年来,中医对心肌梗死的研究很多,进展很快,但有关心肌梗死二级预防与斑块稳定性的相关研究甚少。芪参益气滴丸是天士力集团研制的治疗心血管疾病的现代中药,具有益气活血、通络止痛的功效,在冠心病防治中疗效肯定,但对于不稳定斑块的干预作用如何,尚未见系统研究报道。近年来,血管生物学和分子生物学技术的发展为深入研究中医药稳定动脉粥样斑块的机制提供了新的手段。大量循证医学研究证明,他汀类降脂药物具有一定稳定斑块的作用,可显著降低急性心血管事件的发生率和死亡率,因此,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易损斑块动物模型,运用分子生物学及影像学等技术,观察芪参益气滴丸消退和稳定动脉粥样硬化斑块的疗效,并阐明其分子生物学机制。
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易损斑块动物模型;
(2)应用血管内超声显像技术观察易损斑块影像学特征,评价其动态观察动脉粥样硬化斑块变化的价值;
(3)对比芪参益气滴丸和辛伐他汀稳定易损斑块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70只,随机分为正常对照组和实验组。正常对照组10只,给予普通颗粒饲料;实验组60只,在适应性饲养1周后,球囊损伤腹主动脉+高胆固醇饲料喂养,造模期为12周。
2.实验分组及药物干预
球囊损伤手术中2只实验兔麻醉意外死亡,造模过程中1只实验兔死于腹泻,8只作为AS验证组,49只动脉粥样硬化模型兔停止高胆固醇饲料饲养,随机分为3组。对照组15只,喂以普通饲料;中药组(芪参益气滴丸组)17只,喂以普通饲料加芪参益气滴丸1.5g/kg/d;西药组(辛伐他汀组)17只,喂以普通饲料加辛伐他汀5mg/kg/d,持续12周。
3.斑块局部的p53基因转染
24周末对3组实验兔进行斑块局部的基因转染,向转染部位的管腔中注入10μl携带人野生型p53基因的复制缺陷型重组腺病毒载体(Ad5-p53,滴度为1.5×10~(10)pfu/ml)。所有实验兔继续普通颗粒饲料喂养2周。
4.药物触发
26周末,对实验兔进行药物触发:中国斑点蝰蛇毒0.15mg/kg腹膜下注射,30min后耳缘静脉注射组胺0.02mg/kg,于处死动物前24h、48h给予两次药物触发。
5.称量体重
实验前、12周造模后及26周末药物干预后分别对所有实验兔称量体重。
6.血脂检测
分别于实验开始、12周末及24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、12周末及26周末采血,采用速率散射光比浊法测定血浆纤维蛋白原(fibrinogen,Fib)水平;应用酶联免疫吸附法(enzyme-linkedimmunosorbent assay,ELISA)检测触发前后血清高敏C反应蛋白(highsensitivity C-reactive protein,hs-CRP)、可溶性血管间细胞黏附分子-1(solublevascular cell adhesion molecule-1,sVCAM-1)和氧化低密度脂蛋白(oxidizedlow-density lipoprotein,ox-LDL)的水平。
8.高频体表超声检查
分别于实验开始、12周末、24周末行腹主动脉高频体表超声检查。测量腹主动脉舒张末期内径(end-diastolic luminal diameter,Dd)、收缩末期内径(endsystolicluminal diameter,Ds)、腹主动脉后壁内膜-中层厚度(intima-mediathickness,IMT)、腹主动脉收缩期血流峰值速度(peak velocity,Vp)、收缩期平均速度(mean velocity,Vm)和收缩期流速积分(velocity integral,VTI)。将内-中膜平均回声强度AⅡ-Ⅰ与外膜平均回声强度AⅡ-A的比值作为内-中膜校正的AD值(AⅡc%),AⅡc%=AⅡ-Ⅰ/(AⅡ-A)×100%。
9.高频血管内超声显像检查
分别于实验开始、12周末及26周末处死动物前行腹主动脉血管内超声(IVUS)检查。测量血管外弹力膜面积(external elastic membrane area,EEMA)、管腔面积(lumen area,LA)、斑块面积(plaque area,PA)、血管内径(vesseldiameter,VD)、管腔内径(lumen diameter,LD),并计算偏心指数(eccentricityindex,EI)和血管重构指数(remodeling index,RI)。
10.病理染色
留取腹主动脉标本,苏木素—伊红(hematoxylin-eosin,HE)染色、Masson三色染色、油红O染色和Movat五色套染法染色。
11.免疫组织化学染色
行p53、RAM11、a-actin、VCAM-1、MCP-1、MMP-9及TIMP-1免疫组织化学染色。
12.电镜检查
留取标本进行扫描电子显微镜和透射电子显微镜检查,观察血管超微结构的变化。
13.Real-time PCR
检测血管组织中p53、VCAM-1、MCP-1、MMP-9及TIMP-1的mRNA表达水平,并以管家基因GAPDH作为参照。
14.Western blot
检测血管组织中p53、VCAM-1、MCP-1、MMP-9及TIMP-1的蛋白表达水平。
15.统计学分析
连续性数据用(?)±SD表示,离散数据用例数和百分数表示。三组间斑块破裂率的比较应用Pearson Chi-Square检验,其他指标应用单因素方差分析。应用SPSS统计软件进行统计学处理(version 13.0;SPSS Inc),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49只实验兔中,中药组和西药组各有1只实验兔死亡,分别死于腹泻和呼吸道感染。最终完成药物干预的实验兔共47只,其中,对照组15只,中药组和西药组各16只。本文对完成药物干预的47只实验兔的资料进行分析。
2.实验动物体重的变化
基础状态和12周末各组实验兔体重的比较均无显著性差异(P均＞0.05),26周末中药组实验兔体重较对照组显著降低(P＜0.05),而西药组和对照组之间无显著差异。
3.斑块破裂率的比较
大体病理形态学特征结合病理学及血管内超声显像证实对照组、中药组和西药组分别有12只,6只和7只实验兔出现斑块破裂、血栓形成。破裂率分别为80.00%,37.50%和43.75%。与对照组比较,中药组和西药组斑块破裂率均显著降低(P均＜0.05),而两药物组间斑块破裂率无显著差异。
4.血脂检测
与对照组相比,中药组和西药组LDL-C水平均显著降低(P均＜0.01),且西药组水平显著低于中药组(P＜0.01);
与对照组相比,中药组的TG水平显著降低(P＜0.01),西药组虽有所降低,但未达统计学意义;
与对照组相比,西药组的TC水平显著降低(P＜0.01),中药组虽有所降低,但未达统计学意义,且西药组水平显著低于中药组(P＜0.01);
三组间HDL-C水平无显著差异。
5.血清炎性因子的检测
与对照组相比,中药组和西药组hs-CRP水平均显著降低(P均＜0.01),且西药组显著低于中药组(P＜0.05);
与对照组相比,中药组和西药组纤维蛋白原水平均显著降低(P＜0.05和P＜0.01),且西药组显著低于中药组(P＜0.01);
与对照组相比,中药组和西药组ox-LDL水平均显著降低(P＜0.01和P＜0.05),且中药组显著低于西药组(P＜0.01);
与对照组相比,中药组sVCAM-1显著降低(P＜0.05),西药组虽有所降低,但未达统计学意义。
6.高频体表超声
与对照组相比,中药组和西药组IMT均显著降低(P均＜0.01),且西药组显著低于中药组(P＜0.05);
与对照组相比,中药组和西药组AⅡc%均显著升高(P均＜0.01),而两药物组之间无显著差异;
各组间血流速度测值无显著差异(P均＞0.05)。
7.血管内超声显像检查
与对照组相比,中药组和西药组PA均显著减小(P均＜0.01),且西药组显著低于中药组(P＜0.05);
与对照组相比,西药组LAS%和EEMA均显著降低(P＜0.05和P＜0.01),中药组虽有所降低,但未达统计学意义;
三组间其他测值无显著性差异(P均＞0.05)。
8.病理染色
病理染色显示,对照组斑块内脂质含量较高,多数斑块破裂,且有血栓形成,斑块破裂处及血栓内可见大量炎性细胞浸润,血栓与血管壁附着紧密,中膜明显萎缩。中药组和西药组泡沫细胞减少,纤维组织增生明显。
9.免疫组织化学染色
免疫组化染色显示转染Ad5-p53兔腹主动脉p53表达明显较之未转染兔明显增强。三组斑块内均有a-actin(平滑肌细胞)、RAM11(巨噬细胞)、VCAM-1、MCP-1、MMP-9及TIMP-1的局部表达,且与对照组相比,两药物组a-actin的表达明显增强,其余炎症因子的表达明显减弱。
10.电镜检查
对照组大部分实验兔发生斑块破裂、血栓形成,内皮细胞不完整,平滑肌细胞核固缩,胞质浓染,胶原纤维排列紊乱,线粒体空泡状变性。中药组和西药组泡沫细胞减少,平滑肌形态基本正常,细胞间连接较紧密,胶原纤维排列稍紊乱,异染色质轻微边聚。
11.Real-time PCR
与对照组比,中药组和西药组腹主动脉VCAM-1、MCP-1、MMP-9及TIMP-1mRNA水平均显著降低(P＜0.01或P＜0.05),其中,中药组VCAM-1 mRNA表达水平显著低于西药组(P＜0.01),而西药组MMP-9 mRNA表达显著低于中药组(P＜0.05),两药物组间其余炎症因子mRNA表达水平无显著差异;
三组间p53 mRNA表达水平无显著差异。
12.Western blot
与对照组比,中药组和西药组腹主动脉MCP-1、MMP-9和TIMP-1蛋白表达均显著降低(P均＜0.01),而两药物组间无显著差异;
与对照组比,中药组和西药组腹主动脉VCAM-1蛋白表达水平均显著降低(P均＜0.01),且中药组水平显著低于西药组(P＜0.01);
三组间p53蛋白表达水平无显著差异。
结论
(1)应用内皮损伤、高脂饲养、斑块局部转染p53基因和药物触发的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易损斑块的动物模型;
(2)血管内超声成像技术是识别易损斑块的可靠影像学技术,可用于动态观察AS斑块变化及判断药物疗效;
(3)芪参益气滴丸和辛伐他汀均能通过降低血脂和血清炎症因子水平、抗脂质过氧化、减少斑块局部的脂质含量及抑制斑块局部炎性反应等作用稳定易损斑块,辛伐他汀的降血脂及抗炎效果明显优于芪参益气滴丸,而芪参益气滴丸的抗脂质过氧化作用强于辛伐他汀,两药合用可能在心肌梗死二级预防方面起到更好的疗效。
背景
内皮功能障碍与动脉粥样硬化(atherosclerosis,AS)等心血管疾病有密切关系,血管内皮细胞损伤被认为是AS发展的一个早期关键性环节,持续、轻度的内皮细胞损伤可使内皮细胞的调节功能出现异常,且急性冠脉综合征(acutecoronary syndrome,ACS)患者体内亦有不同程度的血管内皮细胞功能紊乱和损伤。一氧化氮(Nitric oxide,NO)是影响内皮细胞功能的一种重要生物活性物质,由L-精氨酸在一氧化氮合酶(Nitric oxide synthase,NOS)作用下氧化生成,具有维持血管的舒张状态、抑制血小板和单核巨噬细胞的粘附和聚集、抑制血管平滑肌细胞分裂增殖等作用。NO的减少可增加血管紧张度、使血管易于发生炎性改变、促使斑块不稳定及诱发血栓形成。由于内皮细胞直接和血液接触,因此,很易受到血液中有害物质如细菌内毒素脂多糖(lipopolysaccharide,LPS)、炎症介质和自由基等的刺激而受损。其中,LPS由于其广泛的生物学作用,越来越引起人们的关注。大量研究表明,内毒素参与AS的形成过程,特别是AS血管炎症的初始过程。
内皮型一氧化氮合酶(Endothelial nitric oxide synthase,eNOS)的表达或活性改变均可直接影响内皮NO生成,从而影响内皮细胞功能,eNOS磷酸化/去磷酸化对于机体的eNOS活化具有重要作用。在eNOS众多的磷酸化位点中,丝氨酸-1177(Ser1177)和苏氨酸-495(Thr495)是2个最重要的磷酸化位点,前者磷酸化可以增强其活性,而后者磷酸化可以抑制其活性。在生理状态下,内皮释放的NO可减少血小板聚集、抑制单核细胞黏附和平滑肌细胞增殖;假如内皮功能障碍,NO合成和释放减少,未受伤但功能失调的内皮细胞通过在其表面表达的黏附分子与各种类型的白细胞结合,激活炎症细胞进入动脉壁,释放水解酶、细胞因子、化学因子和生长因子等,引起斑块的生长,启动AS的发生。因此,eNOS功能障碍在AS发生和发展中发挥着重要作用。
改善内皮细胞功能、加强对内皮功能障碍的预防性治疗是冠心病治疗领域的一个新的发展趋势。冠心病发病与NO的生物利用度降低有关,因此,增强eNOS活性和表达可能会对心血管系统有保护作用。芪参益气滴丸经前期体内实验已证实具有调节血脂及抗炎作用,方中主要代表药物黄芪的现代药理研究表明,该药物对血管内皮细胞具有保护作用,可能与其防止细胞凋亡、改变血管通透性、抗氧化、调节血管舒缩等作用有关。黄芪多糖(Astragalus polysaccharides,APS)是黄芪的主要有效成分之一,具有保护血管内皮细胞的功能,但这种作用与上调eNOS的表达与活性是否有关还不甚清楚。以往研究多采用静脉内皮细胞或牛动脉内皮细胞进行培养,且研究多关注LPS对iNOS的影响,为更好地模拟人动脉内皮细胞的功能,本实验采用原代人主动脉内皮细胞(human aortic endothelial cells,HAECs),以LPS作为形成内皮细胞损伤的诱导因子,观察不同浓度及不同时间点LPS对于体外培养人主动脉内皮细胞eNOS及Phospho-eNOS蛋白表达的影响,并观察黄芪多糖对LPS诱导的内皮细胞损伤的防治疗效。
目的
(1)建立人主动脉内皮细胞损伤的模型;
(2)观察不同浓度黄芪多糖对脂多糖所致的内皮细胞损伤的保护作用及其机制。
方法
为建立内皮细胞损伤模型,确定LPS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LPS刺激后,人主动脉内皮细胞eNOS及Phospho-eNOS表达变化,选定LPS的最佳刺激条件。
不同浓度的黄芪多糖(0.025mg/ml、0.25mg/ml、2.5mg/ml)预孵育12h后再加LPS,倒置显微镜下观察细胞形态的变化,并且用RT-PCR方法检测各组eNOS的mRNA表达水平,western blot方法测定各组eNOS及Phospho-eNOS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。LPS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;黄芪多糖2.5mg/ml治疗组细胞形态与正常组差别不明显。
根据不同浓度及不同时间LPS刺激后人主动脉内皮细胞eNOS及Phospho-eNOS变化情况及本实验的研究目的,我们选定100ng/ml的LPS培养液培养细胞24h为最佳刺激条件。
100ng/ml LPS培养细胞24h,eNOS的mRNA及蛋白表达水平均显著下降,Phospho-eNOS(Ser1177)蛋白表达水平显著下降,而Phospho-eNOS(Thr495)蛋白表达水平显著上升。
0.25mg/ml和2.5mg/ml黄芪多糖可以显著缓解LPS对eNOS mRNA表达、eNOS和Phospho-eNOS(Ser1177)蛋白表达的抑制作用,但对于Phospho-eNOS(Thr495)蛋白表达无明显影响。
结论
(1)100ng/ml LPS培养人主动脉内皮细胞24h不仅可以降低eNOS表达水平,还可以通过降低Phospho-eNOS(Ser1177)表达水平及升高Phospho-eNOS(Thr495)表达水平,使eNOS活性降低,为适合本实验的最佳刺激条件;
(2)0.25mg/ml和2.5mg/ml黄芪多糖可以通过上调eNOS表达水平及Phospho-eNOS(Ser1177)蛋白表达水平,从而改善内皮细胞功能;
(3)黄芪多糖对Phospho-eNOS(Thr495)蛋白表达无明显影响。
Background
Acute cardiovascular diseases are common and multi-onsct diseases,which have serious effects on the hcalth of human beings.It has been reported that 20 million patients died of acute cardiovascular events,and over ten thousands patients occurred acute myocardial infarction(AMI).With the development of aging,the incidence of AMI in our country was at the increased tendency and got close to thc international average level.In recent years,although the advances in monitoring and therapy have improved the survival of patients who cxpcrience myocardial infarction,patients who survive an MI are still at increased risk of subsequent re-infarction,congestive heart failure,and sudden death.So much more attention should be paid to the secondary treatment of MI.
The secondary treatment of MI is thc prevention of re-infarction and sudden death after MI.Several pharmacological agents arc known to improve prognosis in these patients,i.e.beta-blockers,antiplatelet agents,statins,and angiotensin converting enzyme inhibitors(ACEI).Statins is the basis of preventive treatment since the first trial which demonstrated that statins could decrease the risk and the total mortality of coronary incidences of the patients who survived from MI. Improving the blood lipid could decrease the rate of recurrence and mortality of MI despite it only reduced the coronary stenosis by 1%-4%because the most AMI occurred at slight or moderate stenotic coronary artery,and the lesion which induced myocardiolysis is the rupture and hemorrhage of plaques and subsequent thrombus formation and occlusive blood vessel not the fixed stenosis.The secondary treatment of MI for statins is based on the prevention of plaques ruptures and hemorrhage.
The plaques unstable or prone to rupture and thrombosis were at increased risk of reinfarction,so the stablization of vulnerable plaques has more clinical values compared with the prevention of thrombis,and it is the final target of secondary treatment of MI.Recently,many studies have discovered that there are multiple vulnerable piques in patients with ACS.So the general drug therapy is the basis of stabilizing vulnerable plques.Many basic researchs have demonstrated statins could significantly stabilize the vulnerable plaques by improving the blood lipid,inhibiting the inflammatory reaction in plaques and thickening the fibrous caps of plaques. However,the Prove-It Trial showed that 22.4%of patients experienced acute coronary event in 2 years despite an intensive statin therapy.Moreover,some patients withdraw from statin treatment because of liver dysfunction.In addition,the drug combination could have better effect compared with one kind of medicine,so the "polypill" was prefered in secondary prevent treatment by two British scholars in 2004.Although the manuscription met with the doubt,the change of mode has important meaning.
The inflammatory reaction plays an important role in the formation and rupture M of unstable plaque.Chinese herb medicine could protect vascular endothelial cells, improve the lipid metabolism,inhibit the inflammatory reaction to stabilize the vulnerable plaques and might have potential therapeutic effect on stabilization of vulnerable plaques.In addition,Chinese drugs pharmaceutics is moderate and have less toxicant and more effect when compatibility with other Chinese drugs,so it could be used for a long time in secondery treatment of MI.In recent years,the researchs on MI were much and made rapid advancement,however,there were very few studies in the relationship between the secondary treatment of MI and plaque stabilization. QiShenYiQi drop pills(QSYQ drop pills)made by TianShiLi Group has the effects of benefiting vital energy and promoting blood flow and has been used in cardiovascular system diseases with positive effects,however the effects on stabilizing unstable plaques is unknown.The present study was aimed to observe the therapeutic effects QSYQ drop pills on vulnerable plaques through the molecular biology and imageology technology and to elucidate the possible mechanism.
Objective
(1)To establish an animal model of vulnerable plaques that is mimic to human pathological changes and convenient for intervention;
(2)To observe the imaging feature of vulnerable plaques using intravascular ultrasound technique and to assess the value of this technique in evaluating the development of atherosclerotic plaques;
(3)To compare the therapeutic effects of QSYQ drop pills and Simvastatin on stabilizing vulnerable plaques and approach the possible mechanism.
Methods
1.Establishing the animal model:70 rabbits were randomly divided into the normal group and the experiment group.10 rabbits in the normal group were fed normal diet,60 rabbits in the experiment group were fed on an atherogenic diet containing 1%cholesterol for 12 weeks after abdominal aortic wall injuries were induced using an intravascular balloon.
2.Intervention methods:Two rabbits died from the anesthetic accident in the operation of abdominal aortic wall injuries induced by an intravascular balloon and one rabbit died of diarrhea.Eight rabbit were used to prove the establishment of the animal model.At the end of 12~(th)week,49 rabbits were randomly divided into three groups and given drugs for another 12 weeks.15 rabbits in the control group were fed with a normal diet plus 20ml distilled water.17 rabbits in QSYQ Drop pills group were given the same diet as in the first group plus QSYQ Drop pills(1.5g/kg/d).17 rabbits in Simvastatin group of rabbits were fed the same diet as the other two groups but supplemented with Simvastatin(5mg/kg/d).
3.Gene transfection:At the end of the 24~(th)week,laparotomy was conducted in all rabbits after having been anesthetized with an intravenous injection of sodium pentobarbital(30mg/kg)and an adenoviral vector containing recombinant p53 was transfected into the location of plaques of these rabbits.These rabbits were maintained on a normal diet for additional 2 weeks.
4.Pharmacological triggering:At the end of the 26~(th)week 26,plaque rupture was induced by pharmacological triggering using Chinese Russell's Viper Venom and histamine.Rabbits were euthanized 24-48 hours after the above procedure.
5.Body weight:Body weights of all rabbits were measured at baseline,the end of the 12~(th)and 26~(th)week.
6.Lipid measurement:blood samples were collected from all rabbits at the baseline,the end of the 12~(th)and 26~(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.Biochemical studies:The blood samples were collected from all rabbits at the baseline,the end of the 12~(th)and 26~(th)week.Plasma fibrinogen was measured by rate-nephelometry.Serum high sensitivity C-reactive protein(hsCRP),soluble vascular cell adhesion molecule-1(sVCAM-1)and oxidized low-density lipoprotein (ox-LDL)were assayed using highly sensitive enzyme-linked immunosorbent assay (ELISA)kits.
8.High frequency ultrasound:At the baseline,end of the 12~(th)and 24~(th)week,the abdominal aorta was scanned using a high frequency duplex ultrasonographic system. The intima-media thickness(IMT),the end-diastolic luminal diameters(Dd),the end-systolic luminal diameter(Ds),the mean velocity(Vm),peak velocity(Vp), velocity integral(VTI)and the average image intensity(AⅡ)of the abdominal aorta were measured.
9.Intravascular ultrasound(IVUS):IVUS studies were performed at the baseline, the end of the 12~(th)and 26~(th)week.The external elastic membrane area(EEMA),lumen area(LA),plaque area(PA)and vessel diameter(VD)were measured.The EI and RI were calculated.
10.Histopathological analysis:the abdominal aorta was processed and examined by hematoxylin and eosin,Masson and oil red O staining.
11.Immunohistochemical staining:The expression of RAM11,a-actin,p53, VCAM-1,MCP-1,MMP-9 and TIMP-1 were determined.
12.Electron microscope:Scanning electron microscope and transmission electron microscope were used to observe the ultrastructure of the abdominal aorta.
13.Real-time PCR:The mRNA expressions of p53,VCAM-1,MCP-1,MMP-9 and TIMP-1 in the abdominal aorta tissue were analyzed.
14.Western blot:The protein expressions of p53,VCAM-1,MCP-1,MMP-9 and TIMP-1 in the abdominal aorta tissue were analyzed.
15.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:15 rabbits in the control group,16 rabbits in QSYQ Drop pills group and Simvastatin group completed the study.
2.Body weight:Body weights in the three groups were essentially equal. However,body weight in QSYQ Drop pills group was significantly lower than that in the control group(P＜0.05).But there was no difference between control group and Simvastatin group.
3.Incidence of Plaque Rupture:12 rabbits in the control group(80.00%),6 in QSYQ Drop pills group(37.50%)and 7 in Simvastatin group(43.75%)had ruptured atherosclerotic plaques in the abdominal aorta.The incidence of rupture in QSYQ Drop pills group and Simvastatin group was significantly lower than that in the control group(both P＜0.05),while there were no differences in the plaque rupture rates between QSYQ Drop pills group and Simvastatin group.
4.Lipid measurement:Compared with the control group,the levels of LDL-C decreased significantly in QSYQ Drop pills group and Simvastatin group(both P＜0.01),and the LDL-C levels were lower in Simvastatin group than those in QSYQ Drop pills group.In addition,Simvastatin decreased the levels of TC(P＜0.01)and QSYQ Drop pills decreased TG levels significantly(P＜0.01).
5.Inflammatory markers:Compared with the control group,the serum hs-CRP levels decreased significantly in QSYQ Drop pills group and Simvastatin group at the end of the 26~(th)week(both P＜0.01),and the hs-CRP levels in Simvastatin group were significantly lower than those in QSYQ Drop pills group(P＜0.05).The plasma fibrinogen increased in QSYQ Drop pills group and Simvastatin group compared with the control group(P＜0.05 and P＜0.01),and the fibrinogen levels in Simvastatin group were significantly lower than those in QSYQ Drop pills group(P＜0.01).The serum sVCAM-1 levels in QSYQ Drop pills group were significantly lower than those in the control group(P＜0.05).
6.High frequency ultrasound measurement:The maximum IMT of the abdominal aorta in QSYQ Drop pills group and Simvastatin group were significantly lower than that in the control group(both P＜0.01),and the value of the maximum IMT in Simvastatin group was lower than that in QSYQ Drop pills group(P＜0.05).In addition,AIIc%values were increased in QSYQ Drop pills group and Simvastatin group compared with the control group(both P＜0.01).
7.IVUS measurement:The values of PA in QSYQ Drop pills group and Simvastatin group were significantly lower than those in the control group(both P＜0.01),and the values of PA in Simvastatin group were lower than those in QSYQ Drop pills group.The LAS%and EEMA values decreased in Simvastatin group compared with the control group(P＜0.05 and P＜0.01).
8.Pathologic staining:Most rabbits in the control group developed plaque rupture and intravascular thrombosis with a dense infiltration of inflammatory cells. In contrast,the atherosclerotic lesions in QSYQ Drop pills group and Simvastatin group were similar in that plaque thickness was diminished,foam cells decreased and collagen proliferation present.
9.Inununohistochemical staining:The expressions of RAM11,VCAM-1, MCP-1,MMP-9 and TIMP-1 in plaques were lower in the two drug group than those in the control group.The expressions of a-actin in control group were lower than those in the two drug groups.
10.Electron microscopy examination:Most rabbits in the control group developed plaque rupture and intravascular thrombosis.Transmission electron microscopy showed karyopyknosis,heavy dyed-cytochylema,vacuolar degeneration chondriosome,and chaoticly alined muscle fiber in the control group and less disorderly-alined SMC,mainly normal structure of SMC and collagenous fibers in QSYQ Drop pills group and simvastatin group.
11.Real-time PCR:The mRNA expression levels of VCAM-1,MCP-1,MMP-9 and TIMP-1 in the atherosclerostic lesions of the abdominal aorta in the two drug groups were significantly lower than those in the control group(P＜0.01 or P＜0.05). The expression levels of VCAM-1 in QSYQ Drop pills group were lower than those in Simvastatin group(P＜0.01),and the expression levels of MMP-9 in Simvastatin group is lower than those in QSYQ Drop pills group(P＜0.05).
12.Western blot:The protein expression levels of MCP-1,MMP-9 and TIMP-1 in the atherosclerostic lesions of the abdominal aorta in the two drug groups were significantly lower than those in the control group(both P＜0.01).Compared with the control group,the protein expression levels of VCAM-1 in the atherosclerostic lesions of the abdominal aorta in the two drug groups decreased(P＜0.01 or P＜0.05),and the expression levels of VCAM-1 in QSYQ Drop pills group than those in Simvastatin group(P＜0.01).
Conclusions
(1)The rabbit vulnerable plaque model,which is established with balloon-induced abdominal aortic wall injury together with a cholesterol-rich diet,gene transfection and drug triggering,is mimic to human disease and suitable for the observation of drug therapeutic effects.
(2)Intravascular unltrasound technique is a realiable technique which can be used evaluate vulnerable plaques quantificationally to monitor the atherosclerotic plaque progression and evaluate the therapeutic effects.
(3)QSYQ Drop pills and Simvastatin could stabilize the vulnerable plaque by improving the lipid metabolism,decreasing the levels of blood inflammatory factors, inhibiting the oxidation of lipid,decreasing the lipid contents in plaques and inhibiting the inflammatory reaction in plaques.The effects of Simvastatin about improving the lipid metabolism and anti-inflammation are better,and the anti-oxidation of QSYQ Drop pills is powerful.The combination of the two drugs might have better therapeutic effects on the secondary prevention of MI.
Background
Endothelial dysfunction was associated with cardiovascular disease such as atherosclerosis(AS).The vascular endothelial cell damage is the critical stage of AS, and the continuous endothelial cell injury led to the regulation dysfunction.In addition,the endothelial functional disorder was also in patients with acute coronary syndrome(ACS).Endothelium-derived nitric oxide(NO)is synthesized from L-arginine by endothelial nitric oxide synthase(eNOS)and plays a critical role in the regulation of endothelial cell function.NO possesses complex cardiovascular actions such as regulating vascular tone,protecting the intima from platelet aggregation and mononuclear macrophage adhesion,and inhibiting vascular smooth muscle cell proliferation.A relative deficiency in vascular NO influences the vascular tone, increases vascular wall inflammation,plaque instability and thrombosis.Endothelial cells line the inner wall of blood vessels,so it is sensitive to injury by the stimulus in the blood such as endotoxin(i.e.lipopolysaccharide,LPS),mediators of inflammation, free radical.LPS was paid close attention for its extensive biological effect.Many investigations have demonstrated that the endotoxin was involved in the AS formation especially the initial procedure of the vascular inflammation.
The expression and activity of eNOS directly influence NO production and affect endothelial function,eNOS activity is regulated by the phosphorylation of eNOS at many sites,among these sites,the phosphorylation at Ser1177 and Thr495 were most important.Phosphorylation on Ser1177 activates eNOS and increases its sensitivity to Ca~(2+)/calmodulin,leading to enhanced NO production in endothelial cells.In contrast to Ser1177,Thr495 acts on eNOS as a negative regulatory site,i.e.,phosphorylation of this site is associated with a decrease in eNOS activity.In physiology,NO synthesized by eNOS plays a crucial role in the maintenance of vascular homeostasis by relaxation of the vascular smooth muscle,protection of the intima from platelet aggregation and leukocyte adhesion and inhibition of vascular smooth muscle cell proliferation.Endothelial dysfunction led to the decrease of NO production and release,and activate the inflammatory cells by combining with various kinds of leukocyte through adhesion molecules expressed on the surface of endothelial cells, and the active inflammatory cells ingress the artery wall and release hydrolytic enzyme,cytokines,chemokines,growth factor,and so on.These changes induced the plaque formation.So eNOS dysfunction plays an important role in the formation and progression of AS.
The improvement of endothelial function and the prevention of endothelial dysfunction are new development tendency in the area of coronary heart disease treatment.Since coronary heart disease is associated with a decreased bioavailability of NO,enhancement of eNOS activity and upregulation of functional eNOS in the long-run may protect the cardiovascular system.Our previous study has proved that QiShenYiQi drop pills(QSYQ drop pills)could regulate the blood lipid and has anti-inflammatory effect.Radix Astragali,the principal drug in this prescription,can protect endothelial function,and the effect was associated with inhibiting apoptosis, changing permeability,anti-inflammatory effect and regulating vasomotion. Astragalus polysaccharides(APS),the major component of radix astragali,can improve endothelial cell function.However,it has not been fully elucidated whether the molecular mechanisms responsible for these effects are associated with the expression and activity of eNOS.Previous researches paid more attention to the role of iNOS on endothelial dysfunction,eNOS and Phospho-eNOS were less mentioned. In addition,intravenous endothelial cells or bovine aortic endothelial cells were adopted usually in these researches,moreover,these studies focus on iNOS,thus,in the present study,in order to elucidate the prevention therapeutic effect on the endothelial cell damage induced by LPS,human aortic endothelial cells(HAECs) were cultured in vitro,and we observed the effect of LPS on the expression of eNOS and Phospho-eNOS and the protective effect of APS on the endothelial cells damage induced by LPS.
Objective
(1)To establish the model of human aortic endothelial cells damage;
(2)To investigate whether APS attenuates endothelial dysfunction in LPS-activated HAECs and approach the possible mechanism.
Methods
To establish a model of endothelial cell damage,we observe the effect of LPS with different concentration and different time on the expression of eNOS and Phospho-eNOS.
The cultured HAECs were pretreated with or without APS(0.025mg/ml、0.25mg/ml、2.5mg/ml)for 12h and were observed under inverted microscope.The effects of APS with different concentration on eNOS mRNA expression were investigated by real-time PCR and the effects of APS on the protein expression of eNOS and Phospho-eNOS 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 LPS damage group,and mainly normal structure of endothelial cells in APS groups.
Based on the aim of the present study and the effect of LPS with different concentration and different time on the expression of eNOS and phosphated eNOS, 100ng/ml LPS for 24h is the optimized stimulus for the present study.
In cultured HAECs,100ng/ml LPS significantly decreased eNOS expressions at protein and mRNA levels.In addition,100ng/ml LPS significantly decreased the phosphorylation of eNOS on Ser1177 and enhanced the phosphorylation of eNOS on Thr495.
Cultured HAECs pretreated with APS for 12h,0.25mg/ml and 2.5mg/ml APS significantly unregulated eNOS expressions at protein and mRNA levels,and enhanced phosphorylation of eNOS on Ser1177,but no effect on Thr495.
Conclusion
(1)100ng/ml LPS for 24h not only decreased the eNOS expression but also inhibited the eNOS activity by decreasing the phosphorylation of eNOS on Ser1177 and enhancing the phosphorylation of eNOS on Thr495.So 100ng/ml for 24h is the optimized stimulus for the present study.
(2)0.25mg/ml and 2.5mg/ml APS significantly upregulated eNOS expressions at protein and mRNA levels,and enhanced the increased phosphorylation of eNOS on Ser1177.APS enhanced the expression and activity of eNOS to improve the endothelial function.
心血管疾病是严重威胁人类生命健康的常见病和多发病,全球每年大约有2000万人死于急性心血管事件,其中,半数以上死于急性心肌梗死(acutemyocardial infarction,AMI)。随着人口老龄化的发展,我国急性心肌梗死的发病率呈明显上升趋势,已接近国际平均水平。近年来,由于监控和治疗水平的提高,心肌梗死的死亡率明显降低,但存活患者再次发生心肌梗死、充血性心力衰竭和猝死等急性心血管事件的危险性仍很高。因此,除在急性期应积极治疗外,还应加强心肌梗死后的二级预防。
心肌梗死的二级预防是指心肌梗死发生后,预防再梗死和猝死,大量研究业已证明,抗血小板制剂、β-受体阻滞剂、他汀类调脂药物及血管紧张素转换酶抑制剂等对心肌梗死后的长期二级预防具有积极而肯定的疗效,并且其疗效不受患者其他情况(如年龄、性别等)的影响。自从第一个标志性试验显示他汀类药物能够降低心肌梗死后患者冠心病危险事件的发生率和总病死率以来,该类药物已经成为预防治疗的基础。调脂治疗虽只能使冠状动脉狭窄的病变减轻1%～4%,但可显著降低心肌梗死的再发率和病死率,其原因在于心肌梗死多发生于轻或中度狭窄的冠状动脉,导致心肌坏死的主要原因为粥样斑块的破裂、出血及继发形成的血栓与血管腔闭塞,而非冠状动脉的固定性狭窄。调脂治疗在防止斑块破裂和出血方面的功效,正是其用于心肌梗死二级预防的基础和依据。
冠状动脉粥样硬化斑块不稳定或易于破裂者及血栓形成倾向严重者易发生再梗死,因此,心肌梗死后急性心血管事件的发生与否取决于斑块的易损性,与防治血栓相比,稳定易损斑块具有更大的临床价值,是心肌梗死二级预防的最终目标。近年来的研究发现,急性冠脉综合征(acute coronary syndrome,ACS)患者常具有多个易损斑块,因此,全身性的药物治疗成为稳定易损斑块的首选方法。虽然基础研究证明,他汀类药物可以通过改善血脂、抑制斑块炎症及增加纤维帽厚度等作用稳定易损斑块。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。
炎症反应在不稳定斑块形成及破裂中起着重要的作用,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、抑制炎症反应和抑制胶原纤维降解等多个角度稳定动脉粥样硬化斑块,在稳定易损斑块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。近年来,中医对心肌梗死的研究很多,进展很快,但有关心肌梗死二级预防与斑块稳定性的相关研究甚少。芪参益气滴丸是天士力集团研制的治疗心血管疾病的现代中药,具有益气活血、通络止痛的功效,在冠心病防治中疗效肯定,但对于不稳定斑块的干预作用如何,尚未见系统研究报道。近年来,血管生物学和分子生物学技术的发展为深入研究中医药稳定动脉粥样斑块的机制提供了新的手段。大量循证医学研究证明,他汀类降脂药物具有一定稳定斑块的作用,可显著降低急性心血管事件的发生率和死亡率,因此,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易损斑块动物模型,运用分子生物学及影像学等技术,观察芪参益气滴丸消退和稳定动脉粥样硬化斑块的疗效,并阐明其分子生物学机制。
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易损斑块动物模型;
(2)应用血管内超声显像技术观察易损斑块影像学特征,评价其动态观察动脉粥样硬化斑块变化的价值;
(3)对比芪参益气滴丸和辛伐他汀稳定易损斑块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70只,随机分为正常对照组和实验组。正常对照组10只,给予普通颗粒饲料;实验组60只,在适应性饲养1周后,球囊损伤腹主动脉+高胆固醇饲料喂养,造模期为12周。
2.实验分组及药物干预
球囊损伤手术中2只实验兔麻醉意外死亡,造模过程中1只实验兔死于腹泻,8只作为AS验证组,49只动脉粥样硬化模型兔停止高胆固醇饲料饲养,随机分为3组。对照组15只,喂以普通饲料;中药组(芪参益气滴丸组)17只,喂以普通饲料加芪参益气滴丸1.5g/kg/d;西药组(辛伐他汀组)17只,喂以普通饲料加辛伐他汀5mg/kg/d,持续12周。
3.斑块局部的p53基因转染
24周末对3组实验兔进行斑块局部的基因转染,向转染部位的管腔中注入10μl携带人野生型p53基因的复制缺陷型重组腺病毒载体(Ad5-p53,滴度为1.5×10~(10)pfu/ml)。所有实验兔继续普通颗粒饲料喂养2周。
4.药物触发
26周末,对实验兔进行药物触发:中国斑点蝰蛇毒0.15mg/kg腹膜下注射,30min后耳缘静脉注射组胺0.02mg/kg,于处死动物前24h、48h给予两次药物触发。
5.称量体重
实验前、12周造模后及26周末药物干预后分别对所有实验兔称量体重。
6.血脂检测
分别于实验开始、12周末及24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、12周末及26周末采血,采用速率散射光比浊法测定血浆纤维蛋白原(fibrinogen,Fib)水平;应用酶联免疫吸附法(enzyme-linkedimmunosorbent assay,ELISA)检测触发前后血清高敏C反应蛋白(highsensitivity C-reactive protein,hs-CRP)、可溶性血管间细胞黏附分子-1(solublevascular cell adhesion molecule-1,sVCAM-1)和氧化低密度脂蛋白(oxidizedlow-density lipoprotein,ox-LDL)的水平。
8.高频体表超声检查
分别于实验开始、12周末、24周末行腹主动脉高频体表超声检查。测量腹主动脉舒张末期内径(end-diastolic luminal diameter,Dd)、收缩末期内径(endsystolicluminal diameter,Ds)、腹主动脉后壁内膜-中层厚度(intima-mediathickness,IMT)、腹主动脉收缩期血流峰值速度(peak velocity,Vp)、收缩期平均速度(mean velocity,Vm)和收缩期流速积分(velocity integral,VTI)。将内-中膜平均回声强度AⅡ-Ⅰ与外膜平均回声强度AⅡ-A的比值作为内-中膜校正的AD值(AⅡc%),AⅡc%=AⅡ-Ⅰ/(AⅡ-A)×100%。
9.高频血管内超声显像检查
分别于实验开始、12周末及26周末处死动物前行腹主动脉血管内超声(IVUS)检查。测量血管外弹力膜面积(external elastic membrane area,EEMA)、管腔面积(lumen area,LA)、斑块面积(plaque area,PA)、血管内径(vesseldiameter,VD)、管腔内径(lumen diameter,LD),并计算偏心指数(eccentricityindex,EI)和血管重构指数(remodeling index,RI)。
10.病理染色
留取腹主动脉标本,苏木素—伊红(hematoxylin-eosin,HE)染色、Masson三色染色、油红O染色和Movat五色套染法染色。
11.免疫组织化学染色
行p53、RAM11、a-actin、VCAM-1、MCP-1、MMP-9及TIMP-1免疫组织化学染色。
12.电镜检查
留取标本进行扫描电子显微镜和透射电子显微镜检查,观察血管超微结构的变化。
13.Real-time PCR
检测血管组织中p53、VCAM-1、MCP-1、MMP-9及TIMP-1的mRNA表达水平,并以管家基因GAPDH作为参照。
14.Western blot
检测血管组织中p53、VCAM-1、MCP-1、MMP-9及TIMP-1的蛋白表达水平。
15.统计学分析
连续性数据用(?)±SD表示,离散数据用例数和百分数表示。三组间斑块破裂率的比较应用Pearson Chi-Square检验,其他指标应用单因素方差分析。应用SPSS统计软件进行统计学处理(version 13.0;SPSS Inc),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49只实验兔中,中药组和西药组各有1只实验兔死亡,分别死于腹泻和呼吸道感染。最终完成药物干预的实验兔共47只,其中,对照组15只,中药组和西药组各16只。本文对完成药物干预的47只实验兔的资料进行分析。
2.实验动物体重的变化
基础状态和12周末各组实验兔体重的比较均无显著性差异(P均＞0.05),26周末中药组实验兔体重较对照组显著降低(P＜0.05),而西药组和对照组之间无显著差异。
3.斑块破裂率的比较
大体病理形态学特征结合病理学及血管内超声显像证实对照组、中药组和西药组分别有12只,6只和7只实验兔出现斑块破裂、血栓形成。破裂率分别为80.00%,37.50%和43.75%。与对照组比较,中药组和西药组斑块破裂率均显著降低(P均＜0.05),而两药物组间斑块破裂率无显著差异。
4.血脂检测
与对照组相比,中药组和西药组LDL-C水平均显著降低(P均＜0.01),且西药组水平显著低于中药组(P＜0.01);
与对照组相比,中药组的TG水平显著降低(P＜0.01),西药组虽有所降低,但未达统计学意义;
与对照组相比,西药组的TC水平显著降低(P＜0.01),中药组虽有所降低,但未达统计学意义,且西药组水平显著低于中药组(P＜0.01);
三组间HDL-C水平无显著差异。
5.血清炎性因子的检测
与对照组相比,中药组和西药组hs-CRP水平均显著降低(P均＜0.01),且西药组显著低于中药组(P＜0.05);
与对照组相比,中药组和西药组纤维蛋白原水平均显著降低(P＜0.05和P＜0.01),且西药组显著低于中药组(P＜0.01);
与对照组相比,中药组和西药组ox-LDL水平均显著降低(P＜0.01和P＜0.05),且中药组显著低于西药组(P＜0.01);
与对照组相比,中药组sVCAM-1显著降低(P＜0.05),西药组虽有所降低,但未达统计学意义。
6.高频体表超声
与对照组相比,中药组和西药组IMT均显著降低(P均＜0.01),且西药组显著低于中药组(P＜0.05);
与对照组相比,中药组和西药组AⅡc%均显著升高(P均＜0.01),而两药物组之间无显著差异;
各组间血流速度测值无显著差异(P均＞0.05)。
7.血管内超声显像检查
与对照组相比,中药组和西药组PA均显著减小(P均＜0.01),且西药组显著低于中药组(P＜0.05);
与对照组相比,西药组LAS%和EEMA均显著降低(P＜0.05和P＜0.01),中药组虽有所降低,但未达统计学意义;
三组间其他测值无显著性差异(P均＞0.05)。
8.病理染色
病理染色显示,对照组斑块内脂质含量较高,多数斑块破裂,且有血栓形成,斑块破裂处及血栓内可见大量炎性细胞浸润,血栓与血管壁附着紧密,中膜明显萎缩。中药组和西药组泡沫细胞减少,纤维组织增生明显。
9.免疫组织化学染色
免疫组化染色显示转染Ad5-p53兔腹主动脉p53表达明显较之未转染兔明显增强。三组斑块内均有a-actin(平滑肌细胞)、RAM11(巨噬细胞)、VCAM-1、MCP-1、MMP-9及TIMP-1的局部表达,且与对照组相比,两药物组a-actin的表达明显增强,其余炎症因子的表达明显减弱。
10.电镜检查
对照组大部分实验兔发生斑块破裂、血栓形成,内皮细胞不完整,平滑肌细胞核固缩,胞质浓染,胶原纤维排列紊乱,线粒体空泡状变性。中药组和西药组泡沫细胞减少,平滑肌形态基本正常,细胞间连接较紧密,胶原纤维排列稍紊乱,异染色质轻微边聚。
11.Real-time PCR
与对照组比,中药组和西药组腹主动脉VCAM-1、MCP-1、MMP-9及TIMP-1mRNA水平均显著降低(P＜0.01或P＜0.05),其中,中药组VCAM-1 mRNA表达水平显著低于西药组(P＜0.01),而西药组MMP-9 mRNA表达显著低于中药组(P＜0.05),两药物组间其余炎症因子mRNA表达水平无显著差异;
三组间p53 mRNA表达水平无显著差异。
12.Western blot
与对照组比,中药组和西药组腹主动脉MCP-1、MMP-9和TIMP-1蛋白表达均显著降低(P均＜0.01),而两药物组间无显著差异;
与对照组比,中药组和西药组腹主动脉VCAM-1蛋白表达水平均显著降低(P均＜0.01),且中药组水平显著低于西药组(P＜0.01);
三组间p53蛋白表达水平无显著差异。
结论
(1)应用内皮损伤、高脂饲养、斑块局部转染p53基因和药物触发的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易损斑块的动物模型;
(2)血管内超声成像技术是识别易损斑块的可靠影像学技术,可用于动态观察AS斑块变化及判断药物疗效;
(3)芪参益气滴丸和辛伐他汀均能通过降低血脂和血清炎症因子水平、抗脂质过氧化、减少斑块局部的脂质含量及抑制斑块局部炎性反应等作用稳定易损斑块,辛伐他汀的降血脂及抗炎效果明显优于芪参益气滴丸,而芪参益气滴丸的抗脂质过氧化作用强于辛伐他汀,两药合用可能在心肌梗死二级预防方面起到更好的疗效。
背景
内皮功能障碍与动脉粥样硬化(atherosclerosis,AS)等心血管疾病有密切关系,血管内皮细胞损伤被认为是AS发展的一个早期关键性环节,持续、轻度的内皮细胞损伤可使内皮细胞的调节功能出现异常,且急性冠脉综合征(acutecoronary syndrome,ACS)患者体内亦有不同程度的血管内皮细胞功能紊乱和损伤。一氧化氮(Nitric oxide,NO)是影响内皮细胞功能的一种重要生物活性物质,由L-精氨酸在一氧化氮合酶(Nitric oxide synthase,NOS)作用下氧化生成,具有维持血管的舒张状态、抑制血小板和单核巨噬细胞的粘附和聚集、抑制血管平滑肌细胞分裂增殖等作用。NO的减少可增加血管紧张度、使血管易于发生炎性改变、促使斑块不稳定及诱发血栓形成。由于内皮细胞直接和血液接触,因此,很易受到血液中有害物质如细菌内毒素脂多糖(lipopolysaccharide,LPS)、炎症介质和自由基等的刺激而受损。其中,LPS由于其广泛的生物学作用,越来越引起人们的关注。大量研究表明,内毒素参与AS的形成过程,特别是AS血管炎症的初始过程。
内皮型一氧化氮合酶(Endothelial nitric oxide synthase,eNOS)的表达或活性改变均可直接影响内皮NO生成,从而影响内皮细胞功能,eNOS磷酸化/去磷酸化对于机体的eNOS活化具有重要作用。在eNOS众多的磷酸化位点中,丝氨酸-1177(Ser1177)和苏氨酸-495(Thr495)是2个最重要的磷酸化位点,前者磷酸化可以增强其活性,而后者磷酸化可以抑制其活性。在生理状态下,内皮释放的NO可减少血小板聚集、抑制单核细胞黏附和平滑肌细胞增殖;假如内皮功能障碍,NO合成和释放减少,未受伤但功能失调的内皮细胞通过在其表面表达的黏附分子与各种类型的白细胞结合,激活炎症细胞进入动脉壁,释放水解酶、细胞因子、化学因子和生长因子等,引起斑块的生长,启动AS的发生。因此,eNOS功能障碍在AS发生和发展中发挥着重要作用。
改善内皮细胞功能、加强对内皮功能障碍的预防性治疗是冠心病治疗领域的一个新的发展趋势。冠心病发病与NO的生物利用度降低有关,因此,增强eNOS活性和表达可能会对心血管系统有保护作用。芪参益气滴丸经前期体内实验已证实具有调节血脂及抗炎作用,方中主要代表药物黄芪的现代药理研究表明,该药物对血管内皮细胞具有保护作用,可能与其防止细胞凋亡、改变血管通透性、抗氧化、调节血管舒缩等作用有关。黄芪多糖(Astragalus polysaccharides,APS)是黄芪的主要有效成分之一,具有保护血管内皮细胞的功能,但这种作用与上调eNOS的表达与活性是否有关还不甚清楚。以往研究多采用静脉内皮细胞或牛动脉内皮细胞进行培养,且研究多关注LPS对iNOS的影响,为更好地模拟人动脉内皮细胞的功能,本实验采用原代人主动脉内皮细胞(human aortic endothelial cells,HAECs),以LPS作为形成内皮细胞损伤的诱导因子,观察不同浓度及不同时间点LPS对于体外培养人主动脉内皮细胞eNOS及Phospho-eNOS蛋白表达的影响,并观察黄芪多糖对LPS诱导的内皮细胞损伤的防治疗效。
目的
(1)建立人主动脉内皮细胞损伤的模型;
(2)观察不同浓度黄芪多糖对脂多糖所致的内皮细胞损伤的保护作用及其机制。
方法
为建立内皮细胞损伤模型,确定LPS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LPS刺激后,人主动脉内皮细胞eNOS及Phospho-eNOS表达变化,选定LPS的最佳刺激条件。
不同浓度的黄芪多糖(0.025mg/ml、0.25mg/ml、2.5mg/ml)预孵育12h后再加LPS,倒置显微镜下观察细胞形态的变化,并且用RT-PCR方法检测各组eNOS的mRNA表达水平,western blot方法测定各组eNOS及Phospho-eNOS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。LPS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;黄芪多糖2.5mg/ml治疗组细胞形态与正常组差别不明显。
根据不同浓度及不同时间LPS刺激后人主动脉内皮细胞eNOS及Phospho-eNOS变化情况及本实验的研究目的,我们选定100ng/ml的LPS培养液培养细胞24h为最佳刺激条件。
100ng/ml LPS培养细胞24h,eNOS的mRNA及蛋白表达水平均显著下降,Phospho-eNOS(Ser1177)蛋白表达水平显著下降,而Phospho-eNOS(Thr495)蛋白表达水平显著上升。
0.25mg/ml和2.5mg/ml黄芪多糖可以显著缓解LPS对eNOS mRNA表达、eNOS和Phospho-eNOS(Ser1177)蛋白表达的抑制作用,但对于Phospho-eNOS(Thr495)蛋白表达无明显影响。
结论
(1)100ng/ml LPS培养人主动脉内皮细胞24h不仅可以降低eNOS表达水平,还可以通过降低Phospho-eNOS(Ser1177)表达水平及升高Phospho-eNOS(Thr495)表达水平,使eNOS活性降低,为适合本实验的最佳刺激条件;
(2)0.25mg/ml和2.5mg/ml黄芪多糖可以通过上调eNOS表达水平及Phospho-eNOS(Ser1177)蛋白表达水平,从而改善内皮细胞功能;
(3)黄芪多糖对Phospho-eNOS(Thr495)蛋白表达无明显影响。
Background
Acute cardiovascular diseases are common and multi-onsct diseases,which have serious effects on the hcalth of human beings.It has been reported that 20 million patients died of acute cardiovascular events,and over ten thousands patients occurred acute myocardial infarction(AMI).With the development of aging,the incidence of AMI in our country was at the increased tendency and got close to thc international average level.In recent years,although the advances in monitoring and therapy have improved the survival of patients who cxpcrience myocardial infarction,patients who survive an MI are still at increased risk of subsequent re-infarction,congestive heart failure,and sudden death.So much more attention should be paid to the secondary treatment of MI.
The secondary treatment of MI is thc prevention of re-infarction and sudden death after MI.Several pharmacological agents arc known to improve prognosis in these patients,i.e.beta-blockers,antiplatelet agents,statins,and angiotensin converting enzyme inhibitors(ACEI).Statins is the basis of preventive treatment since the first trial which demonstrated that statins could decrease the risk and the total mortality of coronary incidences of the patients who survived from MI. Improving the blood lipid could decrease the rate of recurrence and mortality of MI despite it only reduced the coronary stenosis by 1%-4%because the most AMI occurred at slight or moderate stenotic coronary artery,and the lesion which induced myocardiolysis is the rupture and hemorrhage of plaques and subsequent thrombus formation and occlusive blood vessel not the fixed stenosis.The secondary treatment of MI for statins is based on the prevention of plaques ruptures and hemorrhage.
The plaques unstable or prone to rupture and thrombosis were at increased risk of reinfarction,so the stablization of vulnerable plaques has more clinical values compared with the prevention of thrombis,and it is the final target of secondary treatment of MI.Recently,many studies have discovered that there are multiple vulnerable piques in patients with ACS.So the general drug therapy is the basis of stabilizing vulnerable plques.Many basic researchs have demonstrated statins could significantly stabilize the vulnerable plaques by improving the blood lipid,inhibiting the inflammatory reaction in plaques and thickening the fibrous caps of plaques. However,the Prove-It Trial showed that 22.4%of patients experienced acute coronary event in 2 years despite an intensive statin therapy.Moreover,some patients withdraw from statin treatment because of liver dysfunction.In addition,the drug combination could have better effect compared with one kind of medicine,so the "polypill" was prefered in secondary prevent treatment by two British scholars in 2004.Although the manuscription met with the doubt,the change of mode has important meaning.
The inflammatory reaction plays an important role in the formation and rupture M of unstable plaque.Chinese herb medicine could protect vascular endothelial cells, improve the lipid metabolism,inhibit the inflammatory reaction to stabilize the vulnerable plaques and might have potential therapeutic effect on stabilization of vulnerable plaques.In addition,Chinese drugs pharmaceutics is moderate and have less toxicant and more effect when compatibility with other Chinese drugs,so it could be used for a long time in secondery treatment of MI.In recent years,the researchs on MI were much and made rapid advancement,however,there were very few studies in the relationship between the secondary treatment of MI and plaque stabilization. QiShenYiQi drop pills(QSYQ drop pills)made by TianShiLi Group has the effects of benefiting vital energy and promoting blood flow and has been used in cardiovascular system diseases with positive effects,however the effects on stabilizing unstable plaques is unknown.The present study was aimed to observe the therapeutic effects QSYQ drop pills on vulnerable plaques through the molecular biology and imageology technology and to elucidate the possible mechanism.
Objective
(1)To establish an animal model of vulnerable plaques that is mimic to human pathological changes and convenient for intervention;
(2)To observe the imaging feature of vulnerable plaques using intravascular ultrasound technique and to assess the value of this technique in evaluating the development of atherosclerotic plaques;
(3)To compare the therapeutic effects of QSYQ drop pills and Simvastatin on stabilizing vulnerable plaques and approach the possible mechanism.
Methods
1.Establishing the animal model:70 rabbits were randomly divided into the normal group and the experiment group.10 rabbits in the normal group were fed normal diet,60 rabbits in the experiment group were fed on an atherogenic diet containing 1%cholesterol for 12 weeks after abdominal aortic wall injuries were induced using an intravascular balloon.
2.Intervention methods:Two rabbits died from the anesthetic accident in the operation of abdominal aortic wall injuries induced by an intravascular balloon and one rabbit died of diarrhea.Eight rabbit were used to prove the establishment of the animal model.At the end of 12~(th)week,49 rabbits were randomly divided into three groups and given drugs for another 12 weeks.15 rabbits in the control group were fed with a normal diet plus 20ml distilled water.17 rabbits in QSYQ Drop pills group were given the same diet as in the first group plus QSYQ Drop pills(1.5g/kg/d).17 rabbits in Simvastatin group of rabbits were fed the same diet as the other two groups but supplemented with Simvastatin(5mg/kg/d).
3.Gene transfection:At the end of the 24~(th)week,laparotomy was conducted in all rabbits after having been anesthetized with an intravenous injection of sodium pentobarbital(30mg/kg)and an adenoviral vector containing recombinant p53 was transfected into the location of plaques of these rabbits.These rabbits were maintained on a normal diet for additional 2 weeks.
4.Pharmacological triggering:At the end of the 26~(th)week 26,plaque rupture was induced by pharmacological triggering using Chinese Russell's Viper Venom and histamine.Rabbits were euthanized 24-48 hours after the above procedure.
5.Body weight:Body weights of all rabbits were measured at baseline,the end of the 12~(th)and 26~(th)week.
6.Lipid measurement:blood samples were collected from all rabbits at the baseline,the end of the 12~(th)and 26~(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.Biochemical studies:The blood samples were collected from all rabbits at the baseline,the end of the 12~(th)and 26~(th)week.Plasma fibrinogen was measured by rate-nephelometry.Serum high sensitivity C-reactive protein(hsCRP),soluble vascular cell adhesion molecule-1(sVCAM-1)and oxidized low-density lipoprotein (ox-LDL)were assayed using highly sensitive enzyme-linked immunosorbent assay (ELISA)kits.
8.High frequency ultrasound:At the baseline,end of the 12~(th)and 24~(th)week,the abdominal aorta was scanned using a high frequency duplex ultrasonographic system. The intima-media thickness(IMT),the end-diastolic luminal diameters(Dd),the end-systolic luminal diameter(Ds),the mean velocity(Vm),peak velocity(Vp), velocity integral(VTI)and the average image intensity(AⅡ)of the abdominal aorta were measured.
9.Intravascular ultrasound(IVUS):IVUS studies were performed at the baseline, the end of the 12~(th)and 26~(th)week.The external elastic membrane area(EEMA),lumen area(LA),plaque area(PA)and vessel diameter(VD)were measured.The EI and RI were calculated.
10.Histopathological analysis:the abdominal aorta was processed and examined by hematoxylin and eosin,Masson and oil red O staining.
11.Immunohistochemical staining:The expression of RAM11,a-actin,p53, VCAM-1,MCP-1,MMP-9 and TIMP-1 were determined.
12.Electron microscope:Scanning electron microscope and transmission electron microscope were used to observe the ultrastructure of the abdominal aorta.
13.Real-time PCR:The mRNA expressions of p53,VCAM-1,MCP-1,MMP-9 and TIMP-1 in the abdominal aorta tissue were analyzed.
14.Western blot:The protein expressions of p53,VCAM-1,MCP-1,MMP-9 and TIMP-1 in the abdominal aorta tissue were analyzed.
15.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:15 rabbits in the control group,16 rabbits in QSYQ Drop pills group and Simvastatin group completed the study.
2.Body weight:Body weights in the three groups were essentially equal. However,body weight in QSYQ Drop pills group was significantly lower than that in the control group(P＜0.05).But there was no difference between control group and Simvastatin group.
3.Incidence of Plaque Rupture:12 rabbits in the control group(80.00%),6 in QSYQ Drop pills group(37.50%)and 7 in Simvastatin group(43.75%)had ruptured atherosclerotic plaques in the abdominal aorta.The incidence of rupture in QSYQ Drop pills group and Simvastatin group was significantly lower than that in the control group(both P＜0.05),while there were no differences in the plaque rupture rates between QSYQ Drop pills group and Simvastatin group.
4.Lipid measurement:Compared with the control group,the levels of LDL-C decreased significantly in QSYQ Drop pills group and Simvastatin group(both P＜0.01),and the LDL-C levels were lower in Simvastatin group than those in QSYQ Drop pills group.In addition,Simvastatin decreased the levels of TC(P＜0.01)and QSYQ Drop pills decreased TG levels significantly(P＜0.01).
5.Inflammatory markers:Compared with the control group,the serum hs-CRP levels decreased significantly in QSYQ Drop pills group and Simvastatin group at the end of the 26~(th)week(both P＜0.01),and the hs-CRP levels in Simvastatin group were significantly lower than those in QSYQ Drop pills group(P＜0.05).The plasma fibrinogen increased in QSYQ Drop pills group and Simvastatin group compared with the control group(P＜0.05 and P＜0.01),and the fibrinogen levels in Simvastatin group were significantly lower than those in QSYQ Drop pills group(P＜0.01).The serum sVCAM-1 levels in QSYQ Drop pills group were significantly lower than those in the control group(P＜0.05).
6.High frequency ultrasound measurement:The maximum IMT of the abdominal aorta in QSYQ Drop pills group and Simvastatin group were significantly lower than that in the control group(both P＜0.01),and the value of the maximum IMT in Simvastatin group was lower than that in QSYQ Drop pills group(P＜0.05).In addition,AIIc%values were increased in QSYQ Drop pills group and Simvastatin group compared with the control group(both P＜0.01).
7.IVUS measurement:The values of PA in QSYQ Drop pills group and Simvastatin group were significantly lower than those in the control group(both P＜0.01),and the values of PA in Simvastatin group were lower than those in QSYQ Drop pills group.The LAS%and EEMA values decreased in Simvastatin group compared with the control group(P＜0.05 and P＜0.01).
8.Pathologic staining:Most rabbits in the control group developed plaque rupture and intravascular thrombosis with a dense infiltration of inflammatory cells. In contrast,the atherosclerotic lesions in QSYQ Drop pills group and Simvastatin group were similar in that plaque thickness was diminished,foam cells decreased and collagen proliferation present.
9.Inununohistochemical staining:The expressions of RAM11,VCAM-1, MCP-1,MMP-9 and TIMP-1 in plaques were lower in the two drug group than those in the control group.The expressions of a-actin in control group were lower than those in the two drug groups.
10.Electron microscopy examination:Most rabbits in the control group developed plaque rupture and intravascular thrombosis.Transmission electron microscopy showed karyopyknosis,heavy dyed-cytochylema,vacuolar degeneration chondriosome,and chaoticly alined muscle fiber in the control group and less disorderly-alined SMC,mainly normal structure of SMC and collagenous fibers in QSYQ Drop pills group and simvastatin group.
11.Real-time PCR:The mRNA expression levels of VCAM-1,MCP-1,MMP-9 and TIMP-1 in the atherosclerostic lesions of the abdominal aorta in the two drug groups were significantly lower than those in the control group(P＜0.01 or P＜0.05). The expression levels of VCAM-1 in QSYQ Drop pills group were lower than those in Simvastatin group(P＜0.01),and the expression levels of MMP-9 in Simvastatin group is lower than those in QSYQ Drop pills group(P＜0.05).
12.Western blot:The protein expression levels of MCP-1,MMP-9 and TIMP-1 in the atherosclerostic lesions of the abdominal aorta in the two drug groups were significantly lower than those in the control group(both P＜0.01).Compared with the control group,the protein expression levels of VCAM-1 in the atherosclerostic lesions of the abdominal aorta in the two drug groups decreased(P＜0.01 or P＜0.05),and the expression levels of VCAM-1 in QSYQ Drop pills group than those in Simvastatin group(P＜0.01).
Conclusions
(1)The rabbit vulnerable plaque model,which is established with balloon-induced abdominal aortic wall injury together with a cholesterol-rich diet,gene transfection and drug triggering,is mimic to human disease and suitable for the observation of drug therapeutic effects.
(2)Intravascular unltrasound technique is a realiable technique which can be used evaluate vulnerable plaques quantificationally to monitor the atherosclerotic plaque progression and evaluate the therapeutic effects.
(3)QSYQ Drop pills and Simvastatin could stabilize the vulnerable plaque by improving the lipid metabolism,decreasing the levels of blood inflammatory factors, inhibiting the oxidation of lipid,decreasing the lipid contents in plaques and inhibiting the inflammatory reaction in plaques.The effects of Simvastatin about improving the lipid metabolism and anti-inflammation are better,and the anti-oxidation of QSYQ Drop pills is powerful.The combination of the two drugs might have better therapeutic effects on the secondary prevention of MI.
Background
Endothelial dysfunction was associated with cardiovascular disease such as atherosclerosis(AS).The vascular endothelial cell damage is the critical stage of AS, and the continuous endothelial cell injury led to the regulation dysfunction.In addition,the endothelial functional disorder was also in patients with acute coronary syndrome(ACS).Endothelium-derived nitric oxide(NO)is synthesized from L-arginine by endothelial nitric oxide synthase(eNOS)and plays a critical role in the regulation of endothelial cell function.NO possesses complex cardiovascular actions such as regulating vascular tone,protecting the intima from platelet aggregation and mononuclear macrophage adhesion,and inhibiting vascular smooth muscle cell proliferation.A relative deficiency in vascular NO influences the vascular tone, increases vascular wall inflammation,plaque instability and thrombosis.Endothelial cells line the inner wall of blood vessels,so it is sensitive to injury by the stimulus in the blood such as endotoxin(i.e.lipopolysaccharide,LPS),mediators of inflammation, free radical.LPS was paid close attention for its extensive biological effect.Many investigations have demonstrated that the endotoxin was involved in the AS formation especially the initial procedure of the vascular inflammation.
The expression and activity of eNOS directly influence NO production and affect endothelial function,eNOS activity is regulated by the phosphorylation of eNOS at many sites,among these sites,the phosphorylation at Ser1177 and Thr495 were most important.Phosphorylation on Ser1177 activates eNOS and increases its sensitivity to Ca~(2+)/calmodulin,leading to enhanced NO production in endothelial cells.In contrast to Ser1177,Thr495 acts on eNOS as a negative regulatory site,i.e.,phosphorylation of this site is associated with a decrease in eNOS activity.In physiology,NO synthesized by eNOS plays a crucial role in the maintenance of vascular homeostasis by relaxation of the vascular smooth muscle,protection of the intima from platelet aggregation and leukocyte adhesion and inhibition of vascular smooth muscle cell proliferation.Endothelial dysfunction led to the decrease of NO production and release,and activate the inflammatory cells by combining with various kinds of leukocyte through adhesion molecules expressed on the surface of endothelial cells, and the active inflammatory cells ingress the artery wall and release hydrolytic enzyme,cytokines,chemokines,growth factor,and so on.These changes induced the plaque formation.So eNOS dysfunction plays an important role in the formation and progression of AS.
The improvement of endothelial function and the prevention of endothelial dysfunction are new development tendency in the area of coronary heart disease treatment.Since coronary heart disease is associated with a decreased bioavailability of NO,enhancement of eNOS activity and upregulation of functional eNOS in the long-run may protect the cardiovascular system.Our previous study has proved that QiShenYiQi drop pills(QSYQ drop pills)could regulate the blood lipid and has anti-inflammatory effect.Radix Astragali,the principal drug in this prescription,can protect endothelial function,and the effect was associated with inhibiting apoptosis, changing permeability,anti-inflammatory effect and regulating vasomotion. Astragalus polysaccharides(APS),the major component of radix astragali,can improve endothelial cell function.However,it has not been fully elucidated whether the molecular mechanisms responsible for these effects are associated with the expression and activity of eNOS.Previous researches paid more attention to the role of iNOS on endothelial dysfunction,eNOS and Phospho-eNOS were less mentioned. In addition,intravenous endothelial cells or bovine aortic endothelial cells were adopted usually in these researches,moreover,these studies focus on iNOS,thus,in the present study,in order to elucidate the prevention therapeutic effect on the endothelial cell damage induced by LPS,human aortic endothelial cells(HAECs) were cultured in vitro,and we observed the effect of LPS on the expression of eNOS and Phospho-eNOS and the protective effect of APS on the endothelial cells damage induced by LPS.
Objective
(1)To establish the model of human aortic endothelial cells damage;
(2)To investigate whether APS attenuates endothelial dysfunction in LPS-activated HAECs and approach the possible mechanism.
Methods
To establish a model of endothelial cell damage,we observe the effect of LPS with different concentration and different time on the expression of eNOS and Phospho-eNOS.
The cultured HAECs were pretreated with or without APS(0.025mg/ml、0.25mg/ml、2.5mg/ml)for 12h and were observed under inverted microscope.The effects of APS with different concentration on eNOS mRNA expression were investigated by real-time PCR and the effects of APS on the protein expression of eNOS and Phospho-eNOS 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 LPS damage group,and mainly normal structure of endothelial cells in APS groups.
Based on the aim of the present study and the effect of LPS with different concentration and different time on the expression of eNOS and phosphated eNOS, 100ng/ml LPS for 24h is the optimized stimulus for the present study.
In cultured HAECs,100ng/ml LPS significantly decreased eNOS expressions at protein and mRNA levels.In addition,100ng/ml LPS significantly decreased the phosphorylation of eNOS on Ser1177 and enhanced the phosphorylation of eNOS on Thr495.
Cultured HAECs pretreated with APS for 12h,0.25mg/ml and 2.5mg/ml APS significantly unregulated eNOS expressions at protein and mRNA levels,and enhanced phosphorylation of eNOS on Ser1177,but no effect on Thr495.
Conclusion
(1)100ng/ml LPS for 24h not only decreased the eNOS expression but also inhibited the eNOS activity by decreasing the phosphorylation of eNOS on Ser1177 and enhancing the phosphorylation of eNOS on Thr495.So 100ng/ml for 24h is the optimized stimulus for the present study.
(2)0.25mg/ml and 2.5mg/ml APS significantly upregulated eNOS expressions at protein and mRNA levels,and enhanced the increased phosphorylation of eNOS on Ser1177.APS enhanced the expression and activity of eNOS to improve the endothelial function.
引文
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