【作者】 乔云；

【导师】 张继东；

【作者基本信息】 山东大学 ， 中西医结合临床， 2014， 博士

【摘要】 研究背景动脉粥样硬化(Atherosclerosis, AS)严重危害人类的健康,其所引发的心脑血管疾病发病率逐年上升,且发病呈年轻化趋势。研究发现,具有脂质中心大、纤维帽薄、平滑肌细胞和胶原含量少、巨噬细胞浸润多、炎症细胞和炎症介质水平较高的不稳定性斑块和急性心血管事件发生关系密切。近年研究证明,斑块内血管新生成为衡量斑块稳定性的一项新指标。在动脉粥样斑块,来自正常血管外膜与中膜外层的滋养血管更加丰富,延伸到动脉粥样硬化的内膜,形成斑块的新生血管。这些新生血管可促进炎症细胞聚集,并能诱发斑块出血和斑块破裂。减少斑块内新生血管亦成为治疗动脉粥样硬化斑块的一个备受关注的治疗靶点。中医防治动脉粥样硬化取得了长足的进展,近年来,在中药对动脉粥样硬化斑块内血管新生的研究方面亦显示出有效的抑制作用。前期工作证实,中药复方制剂舒脉胶囊可促进缺血心肌血管新生,课题组将进一步通过在体实验观察舒脉胶囊对载脂蛋白E基因敲除小鼠斑块面积、斑块内胶原、脂质、平滑肌细胞及巨噬细胞含量和斑块内新生血管标志物CD34的影响,从而观察舒脉胶囊对斑块内部成分及斑块内新生血管的影响。目的观察舒脉胶囊舒脉胶囊对载脂蛋白E基因敲除(apoE-/-)小鼠血脂水平、斑块内部成分(脂质、胶原纤维、平滑肌细胞、巨噬细胞等含量)、斑块内新生血管的影响,探讨舒脉胶囊对斑块及斑块内血管新生的疗效。方法1.研究对象8周龄雄性apoE-/-小鼠50只,同种属C57BL/6J小鼠10只。apoE-/-小鼠给予高脂饮食喂养(15%脂肪,0.25%胆固醇,84.75%基础饲料),C57BL/6J小鼠给予普通饲料喂养,于高脂喂养12周末,将apoE-/-小鼠随机分为模型组(模型组)、舒脉胶囊小剂量组(小剂量组)、舒脉胶囊大剂量组(大剂量组)、辛伐他汀对照组(辛伐他汀组)和三七总皂苷对照组(三七总皂苷组),每组10只。C57BL/6J小鼠为正常对照组(正常组),共6组。小剂量组和大剂量组分别给予舒脉胶囊700mg/kg,3500mg/kg灌胃,1次/d；辛伐他汀组给予辛伐他汀3mg/kg灌胃,1次/d；三七总皂苷组给予三七总皂苷60mg/kg灌胃,1次/d；灌胃时间为12周,模型组和正常组给予灭菌蒸馏水灌胃0.2ml/只,1次/d,时间12周。于试验24周末,处死各组实验鼠。2.研究指标(1)血脂检测：氧化酶法测定各实验组动物血清总胆固醇(Total cholesterol,TC)、低密度脂蛋白胆固醇(Low density lipoprotein cholesterol, LDL-C)、甘油三酯(Triglyceride, TG)浓度。(2)主动脉组织切片病理学检测：HE染色及斑块面积/管腔面积测定；油红O染色检测斑块内脂质含量；天狼猩红染色检测斑块内胶原含量；(3)免疫组织化学方法检测：斑块内巨噬细胞(MOMA-2)、平滑肌肌动蛋白(α-S4A)含量表达；斑块内CD34含量表达。结果1.小鼠血脂测定与正常对照组相比,模型组小鼠血清TC、TG、LDL-C浓度明显升高(P均<0.001)；与模型组比较,大小剂量组、辛伐他汀组、三七总皂苷组均可明显降低血清TC、TG、LDL-C浓度(P<0.05)；其中,大剂量组TG、LDL-C浓度下降最为显著,辛伐他汀组降低TC最为显著,大剂量组和小剂量组比较有显著性差异(P<0.05)。2.病理学检测HE染色：正常对照组小鼠动脉管腔未见异常改变,内膜、中膜、外膜三层结构明显,无AS斑块形成。模型组小鼠主动脉壁有明显AS斑块形成,管腔狭窄较重,内膜表面不光滑,内皮细胞缺失或不连续。各组斑块面积/管腔面积分别是54.64±9.31%(模型组),43.21±6.69%(小剂量组),24.16±7.88%(大剂量组),25.85±6.66%(辛伐他汀组),31.90±9.79%(三七总皂苷组)。与模型组相比,各用药组斑块面积／管腔面积比值有不同程度降低(P<0.01或P<0.001),其中大剂量组,辛伐他汀组,三七总皂苷组与小剂量组相比差异有统计学意义(P<0.05或P<0.01)。油红O染色：对照组小鼠动脉壁较薄,无脂质沉积,模型组小鼠斑块内见大量红染的脂质沉积。各组斑块内脂质含量分别是25.85±3.17%(模型组)、20.95±3.24%(小剂量组)、12.33±3..13%(大剂量组)、12.67±5.52%(三七总皂苷组)、13.80±3.92%(辛伐他汀组)。各药物治疗组斑块内脂质含量低于模型组(P<0.05或P<0.001),大剂量组与小剂量组相比,差异有显著性(P<0.01)。大剂量组与辛伐他汀组、三七总皂苷组之间差异无显著性(P均>0.05)。天狼猩红染色：偏振光显微镜下显示红色或棕黄色及绿色表示斑块中的Ⅰ型和Ⅲ型胶原。各组胶原含量分别为模型组7.80±2.08%、大剂量组13.86±2.98%、小剂量组10.04±2.38%、辛伐他汀组12.88±2.95%、三七总皂苷组11.68±4.02%；与模型组比较,各药物治疗组均增加了斑块中的胶原含量(P<0.05),大剂量组胶原含量和辛伐他汀组比较无显著性差异(P>0.05),大剂量组和小剂量组比较差异有显著性(P<0.05)。3.免疫组化染色免疫组化染色显示斑块内均有MOMA-2(巨噬细胞)和α-SMA(平滑肌细胞)的局部表达。与模型组比较,大剂量组、辛伐他汀组、三七总皂苷组巨噬细胞含量明显低于模型组(P<0.001或P<0.01),而小剂量组和模型组比较巨噬细胞含量无显著性差异(P>0.05),大剂量组与辛伐他汀组、三七总皂苷组之间差异无显著性(P>0.05)；与模型组比较,大剂量组、辛伐他汀组、三七总皂苷组平滑肌细胞含量明显高于模型组(P<0.01或P<0.05),而小剂量组和模型组比较平滑肌细胞含量无显著性差异(P>0.05)；组间两两比较,大剂量组与辛伐他汀组、三七总皂苷组之间差异无显著性(P>0.05)。各药物治疗组CD34阳性染色的新生血管含量均较模型组减少(P<0.01或P<0.05),大剂量组、辛伐他汀组和三七总皂苷组组间两两比较差异无显著性(P>0.05),大剂量组、辛伐他汀组和三七总皂甙组与小剂量组比较有显著性差异(P<0.05)。结论1.舒脉胶囊能够降低apoE-/-小鼠血脂水平,对TG和LDL-C降低作用明显。2.舒脉胶囊能够有效抑制动脉粥样硬化斑块形成,表现为减少动脉粥样硬化斑块面积,影响斑块内部成分,降低斑块内巨噬细胞和脂质的含量,增加斑块内平滑肌细胞和胶原的含量,从而增加斑块的稳定性。3.舒脉胶囊能够减少斑块内新生血管,且疗效具有剂量依赖性。研究背景动脉粥样硬化(Atherosclerosis, AS)疾病的发生发展对人类的健康产生了严重的危害,是临床心血管事件重要的病理学基础。近年有大量研究证实,血管新生和动脉粥样硬化斑块进展密切相关。血管新生的发生机制非常复杂,血管内皮生长因子(vascular endothelial growth factor, VEGF)是目前所知的最关键的血管生成促进因子,能特异性作用于血管内皮细胞,诱导内皮细胞的增生、迁移,增加血管通透性,在生理性和病理性血管新生过程中发挥着重要作用。VEGF通过其受体发挥作用,VEGF受体目前发现有三种：VEGFR-1、VEGFR2和VEGFR-3,新生血管内皮细胞的增殖及VEGF所诱导的粘附分子表达和血管通透性的增加主要通过VEGFR2这一重要介质实现。缺氧是引起斑块内血管新生的基本原因。缺氧环境中,缺氧诱导因子-1α(HIF-1α)是对VEGF起主要调控作用的转录因子,其可以上调下游促血管形成物质VEGF的表达。活性氧(Reactive oxygen species, ROS)在血管生成过程中发挥了重要作用,过量的活性氧参与了内皮细胞及干细胞的衰老和凋亡,从而导致了不成熟的新生血管的产生。尼克酰胺腺嘌呤二核苷酸氧化酶(NADPH氧化酶)是血管内皮细胞生成活性氧的主要酶体,NADPH氧化酶来源的ROS在内皮细胞的血管生成中起着重要作用。血管内皮细胞主要表达尼克酰胺腺嘌呤二核苷酸氧化酶-4(NOX4),研究发现NOX4来源的ROS在激活VEGF表达中起着重要作用。中医防治动脉粥样硬化的研究不断深入。前期工作证实,中药复方制剂舒脉胶囊可有效减少AS斑块内血管新生。本研究通过实时定量RT-PCR技术检测斑块内VEGF、VEGFR2、HIF-1α、NOX4mRNA的表达,Western blot技术检测VEGF、VEGFR2、HIF-1α、NOX4蛋白的表达,更加深入研究舒脉胶囊干预斑块内血管新生的作用机制。目的观察舒脉胶囊对载脂蛋白E基因敲除(apoE以)小鼠主动脉VEGF、VEGF-R2、 HIF-1α、NOX4mRNA及蛋白表达的影响,探讨舒脉胶囊干预动脉粥样硬化斑块内血管新生的机理。方法1.研究对象8周龄雄性apoE-/-小鼠50只,同种属C57BL/6J小鼠10只。apoE-/-小鼠给予高脂饮食喂养(15%脂肪,0.25%胆固醇,84.75%基础饲料),C57BL/6J小鼠给予普通饲料喂养,于高脂喂养12周末,将apoE-/-小鼠随机分为模型组(模型组)、舒脉胶囊小剂量组(小剂量组)、舒脉胶囊大剂量组(大剂量组)、辛伐他汀对照组(辛伐他汀组)和三七总皂苷对照组(三七总皂苷组),每组10只。C57BL/6J小鼠为正常组(正常组),共6组。小剂量组和大剂量组分别给予舒脉胶囊700mg/kg,3500mg/kg灌胃,1次/d；辛伐他汀组给予辛伐他汀3mg/kg灌胃,1次/d；三七总皂苷组给予三七总皂苷60mg/kg灌胃,1次/d；灌胃时间为12周,模型组和正常组给予灭菌蒸馏水灌胃0.2ml/只,1次/d,时间12周。于试验24周末,处死各组实验鼠。2.研究指标(1)免疫组织化学方法检测：斑块内VEGF含量表达。(2)实时定量RT-PCR(Real-time Quantitative RT-PCR)技术检测：主动脉VEGF、VEGFR2、HIF-1α、 NOX4mRNA的表达。(3)免疫印记(Western blot)检测：主动脉VEGF、VEGF-R2、 HIF-1α、NOX4蛋白的表达。结果1.免疫组化染色免疫组化染色显示模型组和正常对照组比较VEGF表达显著升高(P<0.001),各药物治疗组斑块内VEGF阳性面积均较模型组减少(P<0.01或P<0.05),其中,以大剂量组和辛伐他汀组效果最为显著,和小剂量组比较有显著性差异(P<0.01)。2.主动脉VEGF、VEGFR2、HIF-1α、NOX4mRNA表达水平比较(1)VEGF mRNA表达比较：与正常对照组相比,模型组小鼠血管壁VEGF mRNA表达显著升高(P<0.001)；各药物处理组VEGF mRNA表达较模型组均有显著下降(P<0.01),其中辛伐他汀组下降最为显著(P<0.001),小剂量组与大剂量组比较有显著差异(P<0.01),辛伐他汀组与三七总皂苷组比较有显著差异(P<0.01)(2) VEGFR2mRNA表达比较：与正常对照组相比,模型组小鼠血管壁VEGFR2mRNA表达显著升高(P<0.001)；与模型组比较,各药物处理组VEGFR2mRN表达均显著下降(P<0.01),大剂量组和小剂量组比较有显著性差异(P<0.01)。(3) HIF-1α mRNA表达比较：与正常对照组相比,模型组小鼠血管壁HIF-1αmRNA表达显著升高(P<0.001)；各药物处理组HIF-1αmRNA表达较模型组均有不同程度下降(P<0.01),各药物处理组之间差异无显著性(P>0.05)。(4)NOX4mRNA表达比较：与正常对照组相比,模型组小鼠血管壁NOX4mRNA表达显著升高(P<0.001)；与模型组比较,各药物处理组NOX4mRNA表达均显著下降(P<0.01),其中三七总皂甙组下降最为显著,其次为大剂量组；两组和辛伐他汀组及小剂量组比较均有显著性差异(P均<0.01)。3.主动脉VEGF、VEGFR2、HIF-1α、NOX4蛋白表达水平比较(1) VEGF蛋白表达比较：与正常组比较,模型组小鼠血管壁VEGF表达明显升高(P<0.001)；各药物干预组VEGF蛋白表达与模型组相比,有非常显著性差异(P<0.01)；其中,大剂量组和辛伐他汀组比较无显著性差异(P>0.05),两组和小剂量组及三七总皂苷组比较均有显著性差异(P均<0.05)(2) VEGFR2蛋白表达比较：与正常对照组相比,模型组小鼠血管壁VEGFR2蛋白表达显著升高(P<0.001)；与模型组相比,各药物处理组VEGFR2蛋白表达均明显下降(P<0.01或P<0.05)；大剂量组、辛伐他汀组和三七总皂苷组和小剂量组比较下降有显著性差异(P均<0.01)。(3) HIF-1α蛋白表达比较：与正常对照组相比,模型组小鼠血管壁HIF-1α蛋白表达显著升高(P<0.001)；与模型组相比,各药物治疗组HIF-1α蛋白表达均明显下降(P<0.01)；辛伐他汀组下降最为显著,和其余各治疗组比较差异有显著性(P<0.01)。(4)NOX4蛋白表达比较：与正常对照组相比,模型组小鼠血管壁NOX4蛋白表达显著升高(P<0.001)；与模型组相比,各药物治疗组NOX4蛋白表达均明显下降(P<0.01)；其中三七总皂苷组下降最为显著(P<0.01),大剂量组和辛伐他汀组及小剂量组比较有显著性差异(P均<0.01)。结论舒脉胶囊抑制斑块内血管新生,稳定斑块的机制和影响HIF-1α、VEGF、VEGFR2、NOX4mRNA及蛋白水平的表达有关。更多还原

【Abstract】 BackgroundAtherosclerosis (Atherosclerosis, AS) serious harm to human health, it raised the incidence of cardiovascular and cerebrovascular diseases increased year by year, and the incidence was getting younger and younger. The study, found that the instable atherosclerotic plaque which has a big lipid, a thin fibrous cap, fewer smooth muscle cells and collagen content, more macrophages, higher levels of inflammatory cells and inflammatory mediators, are closely related to acute cardiovascular eventsRecent studies have demonstrated that plaque angiogenesis have became a new measurement of plaque stability. In atherosclerotic plaque, the blood vessels from adventitia and outer middle-membrane of the normal blood vessels get richer, extend into the intima of atherosclerosis, thus form plaque neovascularization. These new blood vessels can promote accumulation of inflammatory cells, and can induce plaque hemorrhage and plaque rupture. Reduce plaque neovascularization has become the treatment of atherosclerotic plaque concern a therapeutic target.Traditional Chinese Medicine has made considerable progress in Prevention and treatment of atherosclerosis. In recent years, the study of traditional Chinese medicine on the atherosclerotic plaque angiogenesis also shows the effective inhibition. Previous work demonstrated that traditional Chinese medicine Shumai capsule can promote angiogenesis in ischemic myocardium. Our team will further observe the effect of Shumai Capsules on collagen, lipids, smooth muscle cells, macrophages and angiogenesis marker CD34in atherosclerotic plaque, to investigate the impact on the internal components of plaque and plaque neovascularization.AimTo observe the effect of Shumai Capsules on collagen, lipids, smooth muscle cells, macrophages and CD34in atherosclerotic plaque, to investigate the impact on the internal components of plaque and plaque neovascularization.MethodsMail50apolipoprotein E-knockout (apoE-/-) mice at8weeks of age were fed a high fat high cholesterol diet including15%fat and0.25%cholesterol. Mail10C57BL/6J mice at8weeks of age were fed a normal chow diet. At20weeks of age, apoE-/-mice were randomly divided into five groups, the ApoE-KO group(Model group), the apoE-/-+SMCH group (SMCH group), the ApoE-/-+SMCL group (SMCL group), the apoE-/-+total panax notoginsenoside group (PNS group) and the apoE-/-+Simvastatin group (Simvastatin group)(n=10for each group). C57BL/6J mice were the normal control group (Control group). Mice in SMCH and SMCL group were given SMC at3500mg/kg,700mg/kg, respectively. Mice in PNS group were given PNS at60mg/kg. And mice in Simvastatin group were given Simvastatin at3mg/kg. All drugs were given orally, once a day for12weeks. Mice in model and control groups were given0.2ml sterile distilled water, once a day for12weeks. After administered for12weeks, all mice were sacrificedDetection contents:(1)Serum levels of total cholesterol (TC), low density lipoprotein cholesterol (LDL-C) and trigliyceride (TG) was detected by oxidation enzyme method.(2)Histopathological analysis:Sections were stained with hematoxylin and eosin, oil red0staining and Sirius red staining.(3) Immunohistochemical analysis:Immunohistochemical staining were performed and the expressions of MOMA-2, a-SMA,CD34were detected.Results1. Comparison of serum lipid concentrationThe serum TC, TG and LDL-C concentration was significantly higher in the Model group than that in Control group (P＜0.001). Lipid levels in SMCH, SMCL, Simvastatin and PNS group were significantly lower compared with that in Model group (P＜0.05). The levels of TG and LDL-C in SMCH group decreased the most significant, while Simvastatin group significantly reduce the level of TC than other groups. There was a significant difference between SMCH group and SMCL group (P＜0.05).2. Histological and Morphology analysisHematoxylin and eosin staining:A normal aorta wall was observed in Control group mice. Atherosclerotic lesions were observed in all apoE-/-mice and the lumens were narrowed in a different degree. The ratio of plaque area to lumen area was54.64±9.31%(Model group),43.21±6.69%(SMCL group),24.16±7.88%(SMCH group),25.85±6.66%(Simvastatin group) and31.90±9.79%(PNS group). The ratio of plaque area to lumen area reduced to varying significant degrees in each treatment group compare with that in Model group (P＜0.01or P＜0.001). SMCH, Simvastatin and PNS group differ statistically significant compared to SMCL group(P<0.05or P＜0.01).Oil red0staining:There was no lipid deposition in normal aortic wall. There were a lot of red dye plaque lipid deposition in model group. The lipid content within plaque was25.85±3.17%(model group),20.95±3.24%(SMCL group),12.33±3..13%(SMCH group),13.80±3.92%(Simvastatin group) and12.67±5.52%(PNS group). Compared with model group, lipid infiltration in plaques were lower in each treatment group(P＜0.05or P＜0.001). There was a significant difference between SMCH group and SMCL group(P＜0.01). There was no significant difference between SMCH, Simvastatin and PNS group(P＞0.05).Sirius red staining:Under polarized light microscopy AS plaque shows red or brown and green patches of collagen type Ⅰ and Ⅲ. Compared with the Model group, the treatment group had increased the collagen content of the plaque (P＜0.05). There was no significant difference in collagen content between SMCH group and Simvastatin group (P＞0.05). There was a significant difference between SMCH group and SMCL group(P＜0.05).3. Immunohistochemical analysis Immunohistochemical staining showed that the expression of both local MOMA-2(macrophages) and a-SMA (smooth muscle cells) within the plaque. Compared with the Model group, the SMCH, Simvastatin and PNS groups were significantly reduced macrophage expression(P＜0.01). But there was no difference between SMCL and model group in reducing macrophage expression(P＞0.05). Meanwhile, the expressions of smooth muscle cells in SMCH, Simvastatin and PNS group treatment groups were significantly higher than that in Model group(P＜0.05). But there was no difference between SMCL and model group in increasing smooth muscle cells expression(P＞0.05). Comparison between the two groups, There was no significant difference between SMCH, Simvastatin and PNS group(P＞0.05).CD34-positive staining neovascularization in each treatment group significantly decreased compared with that in model group(P＜0.05). There was no difference between SMCH, Simvastatin and PNS group(P＞0.05). CD34-positive staining areas were lower in SMCH, Simvastatin and PNS groups than those in SMHL group(P＜0.05).Conclusion1. Shumai capsule could decrease blood lipid level in apoE-/-mice. It had an obvious effect in decreasing TG and LDL-C levels.2. Shumai capsule could effectively inhibit the formation of atherosclerotic plaques. SMC could reduce atherosclerotic plaque area, affect the internal components of the plaque, reduce the amount of plaque macrophages and lipids, increase expression of smooth muscle cells and collagen in plaque. Thereby increased the stability of the plaque.3. Shumai capsule could reduce neovascularization within atherosclerotic plaques. And the affection was dose-dependent. BackgroundThe development of atherosclerosis (Atherosclerosis, AS) disease on human health have had a serious harm, is an important clinical cardiovascular events pathological basis. In recent years, numerous studies have demonstrated that angiogenesis and atherosclerotic plaque progression are closely related.The mechanism is very complex of angiogenesis. Vascular endothelial growth factor (vascular endothelial growth factor, VEGF) is the most critical for angiogenesis promoting factors currently known. It can specifically acts on endothelial cells and induce endothelial cell proliferation, migration, increased vascular permeability, plays an important role in physiological and pathological angiogenesis process. VEGF play a role via its receptor. Currently there are three VEGF receptors found: VEGFR-1, VEGFR2and VEGFR3. And increased vascular permeability neovascular endothelial cell proliferation and VEGF induced expression of adhesion molecules, mainly through VEGFR2to achieve this important medium.Hypoxia is the basic cause of plaque angiogenesis. In Hypoxic conditions, hypoxia-inducible factor-1α (HIF-la), as a important transcription factor, plays a major role in the regulation VEGF. It can increase the expression VEGF, the downstream of pro-angiogenic substances. Reactive oxygen species (Reactive oxygen species, ROS) play an important role in the angiogenic process. The excess of reactive oxygen species involved senescence and apoptosis of endothelial cells and stem cells, resulting in the generation of new blood vessels immature.Nicotinamide adenine dinucleotide phosphate oxidase (NADPH Oxidase) is the main body of the enzyme to produce ROS in vascular endothelial cells. NADPH oxidase-derived ROS plays an important role in the angiogenic endothelial cells NOX4mainly expressed in vascular endothelial cells. The study found that ROS sources from NOX4activation plays an important role in the expression of VEGF.Traditional Chinese Medicine has made considerable progress in Prevention and treatment of atherosclerosis. Previous work had demonstrated that traditional Chinese medicine Shumai capsule can effectively reduce AS plaque angiogenesis. Our research will further investigate the mechanism of Shumai capsule on atherosclerotic plaque. Real-time quantitative RT-PCR technique to detect the plaque VEGF, VEGFR2, HIF-la and NOX4mRNA expression. Western blot technique to detect VEGF, VEGFR2,HIF-la and NOX4protein expression,AimTo detect VEGF, VEGFR2, HIF-la and NOX4mRNA expression by Real-time quantitative RT-PCR technique. To detect VEGF, VEGFR2, HIF-la and NOX4protein expression by Western blot technique. To investigate the mechanism of Shumai capsule on atherosclerotic plaque angiogenesis.MethodsMail50apolipoprotein E-knockout (apoE-/-) mice at8weeks of age were fed a high fat high cholesterol diet including15%fat and0.25%cholesterol. Mail10C57BL/6J mice at8weeks of age were fed a normal chow diet. At20weeks of age, apoE-/-mice were randomly divided into five groups, the ApoE-KO group(Model group), the apoE-/-+SMCH group (SMCH group), the ApoE-/-+SMCL group (SMCL group), the apoE-/-+total panax notoginsenoside group (PNS group) and the apoE-/-+Simvastatin group (Simvastatin group)(n=10for each group). C57BL/6J mice were the normal control group (Control group). Mice in SMCH and SMCL group were given SMC at3500mg/kg,700mg/kg, respectively. Mice in PNS group were given PNS at60mg/kg. And mice in Simvastatin group were given Simvastatin at3mg/kg. All drugs were given orally, once a day for12weeks. Mice in model and control groups were given0.2ml sterile distilled water, once a day for12weeks. After administered for12weeks, all mice were sacrificedDetection contents:(1) Immunohistochemical analysis:Immunohistochemical staining were performed and the expressions of VEGF were detected.(2) RT-PCR: The mRNA expressions of VEGF, VEGF-R2, HIF-1α and NOX4in the aortic tissue were analyzed by using RT-PCR technique.(3) Western blot:The protein expressions of VEGF, VEGF-R2, HIF-la and NOX4in the aortic tissue were analyzed with Western blot technique.Results1. Immunohistochemical analysisThere was a significant difference between Model group and Control group in reducing VEGF expression(P＜0.001). VEGF positive staining area in each treatment group significantly decreased compared with that in Model group(P＜0.01or P＜0.05). SMCH and Simvastatin group exerted the most effective action, and there was a significant difference compared with SMCL group(P＜0.01).2. RT-PCR analysis(1) Comparison of expression of VEGF mRNA:The expression of VEGF mRNA in the model group was higher than that in the normal group (P＜0.001). After treatment with SMC, Simvastatin and PNS, VEGF mRNA was decreased in a different degree compared with the model group(P＜0.01), and among those treatment groups, the Simvastatin group had the lowest VEGF mRNA expression (P＜0.001). There was a significant difference between SMCH group and SMCL group(P＜0.01). There was also a significant difference between Simvastatin and PNS group(P＜0.01).(2) Comparison of expression of VEGFR2mRNA:The expression of VEGFR2mRNA in the model group was higher than that in the normal group (P＜0.001). VEGFR2mRNA was decreased in a different degree in drugs treatment groups compared with the model group (P＜0.01). There was a significant difference between SMCH group and SMCL group (P＜0.01).(3) Comparison of expression of HIF-1α mRNA:The expression of NOX4mRNA in the model group was higher than that in the normal group (P＜0.001). HIF-1α mRNA was decreased in a different degree in drugs treatment groups compared with the model group (P＜0.01). There was no difference between all drugs treatment groups (P＞0.05).(4) Comparison of expression of NOX4mRNA:The expression of NOX4mRNA in the model group was higher than that in the normal group (P＜0.001), After treatment with SMC, Simvastatin and PNS, NOX4mRNA was decreased in a different degree compared with the model group(P＜0.01). Among those treatment groups, PNS group and SMCH group decreased the most significant, and there was a significant difference of those two groups compared with Simvastatin and SMCL group (P＜0.01).3. Western blot analysis(1) Comparison of expression of VEGF protein:The expression of VEGF protein in Model group was higher than that in Control group (P＜0.001). The expression of VEGF protein was significantly decreased after treatment with SMC, Simvastain and PNS (P＜0.01). There was no significant difference between SMCH and Simvastatin group (P＞0.05). SMCH and Simvastatin group had significant differences compared with PNS group and SMCL group(P＜0.05).(2) Comparison of expression of VEGFR2protein:The expression of VEGFR2protein in Model group was higher than that in Control group (P＜0.001). The expression of VEGFR2protein was significantly decreased after treatment with SMC, Simvastain and PNS (P＜0.01or P＜0.05). The expression of VEGFR2protein was lower in SMCH, Simvastatin and PNS groups than that in SMHL group (P＜0.01).(3) Comparison of expression of HIF-la protein:The expression of HIF-la protein in Model group was higher than that in Control group (P＜0.001). The expression of HIF-la protein was significantly decreased after treatment with SMC, Simvastain and PNS (P＜0.01). Simvastatin group had the lowest HIF-1α protein expression(P＜0.01).(4) Comparison of expression of NOX4protein:The expression of NOX4protein in Model group was higher than that in Control group (P＜0.001). After treatment with SMC, Simvastatin and PNS, NOX4protein expression was decreased in a different degree compared with the Model group(P＜0.01). PNS group had the lowest NOX4protein expression(P＜0.01). SMCH group decreased NOX4protein expression in a different degree compared with Simvastatin and SMCL group (P＜0.01).ConclusionShumai capsule could inhibit plaque angiogenesis and promote plaque stabilization. The mechanisms were possibly associated with the suppressive effect of VEGF, VEGFR2, HIF-la and NOX4expression in aortas of apoE-/-mice.更多还原