舒脉胶囊干预动脉粥样硬化斑块内血管新生的作用及机理研究
|
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。
Background
Atherosclerosis (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 events
Recent 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.
Aim
To 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.
Methods
Mail50apolipoprotein 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 sacrificed
Detection 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.
Results
1. Comparison of serum lipid concentration
The 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 analysis
Hematoxylin 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).
Conclusion
1. 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.
Background
The 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,
Aim
To 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.
Methods
Mail50apolipoprotein 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 sacrificed
Detection 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.
Results
1. Immunohistochemical analysis
There 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).
Conclusion
Shumai 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.
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。
Background
Atherosclerosis (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 events
Recent 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.
Aim
To 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.
Methods
Mail50apolipoprotein 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 sacrificed
Detection 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.
Results
1. Comparison of serum lipid concentration
The 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 analysis
Hematoxylin 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).
Conclusion
1. 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.
Background
The 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,
Aim
To 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.
Methods
Mail50apolipoprotein 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 sacrificed
Detection 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.
Results
1. Immunohistochemical analysis
There 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).
Conclusion
Shumai 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.
引文
1. Naghavi M, Libby P, Falk E, et al. From vulnerable plaque to vulnerable patient: Acall for new definitions and risk assessment strategies:Part Ⅰ [J].Circulation,2003,108(4):1664-1672.
2. Renu Virmani, Frank D. Kolodgie, et al. Atherosclerotic Plaque Progression and Vulnerability to Rupture—Angiogenesis as a Source of Intraplaque Hemorrhage[J]. Arterioscler Thromb Vasc Biol,2005,25:2054-2061.
3. Moreno PR, Purushothaman KR, Sirol M, et al. Neovascularization in human atherosclerosis[J]. Criculation,2006,113(18):2245-2252.
4. Chen YX, Nakashima Y, Tanaka K, et al. Immunohistochemical expression of vascular endothelial growth factor/vascular permeability factor in atherosclerotic intimas of human coronary arteries[J]. Arterioscler Thromb Vasc Biol,1999,19(1):131-9.
5. Pandya NM, Dhalla NS, Santani DD. Angiogeneses——a new target for future therapy[J].Vascular Pharmacology,2006,44(5):265-274.
6. Karagiannis ED, Popel AS. Identification of novel short peptides derived from the a4,a5,and a6 fibrils of type Ⅳ collagen with anti-angiogenic properties [J]. Biochemical and Biophysical Research Communication,2007,354(2):434-439.
7. Rogers MS, D'Amato RI. The effect of genetic diversity on angiogenesis [J]. Experimental Cell Reseach,2006,312(5):561-574.
8.沈伟,范维琥,施海明,等.红景天对兔动脉粥样硬化斑块内血管内皮细胞生长因子表达及血管新生的影响[J].中国中西医结合杂,2008；11(28)：1022-1025.
9.李天奇,范维琥,李勇,等.麝香保心丸减少兔股动脉斑块内血管新生机制探讨[J].中西医结合心脑血管病杂志,2009,7(6)：680-682.
10.张路,吴宗贵,廖德宁,等.通心络对实验性家兔主动脉粥样斑块内血管内皮生长因子表达的影响[J].中国动脉硬化杂志,2004,12(2)：177-182.
11.杨涛,袁肇凯,胡志希,等.养心通脉片对动脉粥样硬化白兔VEGF的表达 和新生血管数的影响[J].中西医结合心脑血管病杂志,2006,4(5)：407-408.
12.尹慧秋,张继东,林海青,等.舒脉胶囊对大鼠缺血心肌促血管新生的实验研究[J].中国中西医结合杂志,2007,27(11)：1020-1023.
13. Ignatowski AC. Influence of animal food on the organism of rabbits[J]. S Peterb Izviest ImpVoyenno-Med Akad,1908,16:154-173.
14. Piedrahita JA, Zhang SH, Hagaman JR, et al. Generation of mice carrying a mutant apolipoprotein E gene inactivated by gene targeting in embryonic stem cells[J]. PNAS,1992,89:4471-4475.
15. Plump AS, Smith JD, Hayek T, et al. Severe hypercholesterolemia and atherosclerosis in apolipoprotein E-deficient mice created by homologous recombination in ES cells[J]. Cell,1992,71:343-353.
16. Cha J, Ivanov V, Ivanova S, et al. Evolution of angiotensin II-mediated atherosclerosis in ApoE KO mice[J]. Mol Med Report,2010,3:565-70.
17. Menu P, Pellegrin M, Aubert JF, et al. Atherosclerosis in ApoE-deficient mice progresses independently of the NLRP3 inflammasome [J]. Cell Death Dis 2011,2:e137.
18. Zhang SH, Reddick RL, Piedrahita JA, et al. Spontaneous hypercholesterolemia and arterial lesions in mice lacking apolipoprotein E[J]. Science,1992,258: 468-471.
19. Coleman R, Hayek T, Keidar S, et al. Mouse model for human atherosclerosis: long-term histopathological study of lesion development in the aortic arch of apolipoprotein E-deficient(EO)mice[J]. Acta Histochem,2006,108(6):415-424.
20. Nakashima Y, Plump AS, Raines EW, et al. ApoE-deficient mice develop lesions of all phases of atherosclerosis throughout the arterial tree[J]. Arterioscler Thromb,1994,14:133-140.
21. Jawien J, Nastalek P, Korbut R. Mouse models of experimental atherosclerosis[J]. Physiol Pharmacol,2004,55(3):503-517.
22. Dawber TR, Kannel WB, Revotskie N, et al. The epidemiology of coronary heart disease--the Framingham enquiry[J]. Proc R Soc Med,1962,55:265-71.
23. Folsom AR, Wu KK, Rosamond WD, et al. Prospective study of hemostatic factors and incidence of coronary heart disease:the Atherosclerosis Risk in Communities (ARIC) Study[J]. Circulation,1997,96(4):1102-8.
24.刘静,赵冬,晃兆苏,等.低密度脂蛋白胆固醇与心血管病发病关系的前瞻性研究[J].中华心血管病杂志,2001,29(9)：561-565.
25.李莹,陈志红,周北凡,等.血脂和脂蛋白水平对我国中年人群缺血性心血管病事件的预测作用[J].中华心血管病杂志,2004,32(7)：643-647.
26. Kher N, Marsh JD. Pathobiology of atherosclerosis—a brief review[J]. Semin Thromb Hemost,2004,30:665-672.
27. Sugano R, Matsuoka H, Haramaki N, et al. Polymorphonuclear leukocytes may impair endothelial function:results of crossover randomized study of lipid-lowering therapies[J]. Arterioscler Thromb Vasc Biol,2005,25(6):1262-7.
28. Rodriguez JA, Nespereira B, Perez-Ilzarbe M, et al. Vitamins C and E prevent endothelial VEGF and VEGFR2 overexpression induced by porcine hypercholesterolemic LDL[J]. Cardiovasc Res,2005,65(3):665-73.
29. Petnehazy T, Stokes KY, Russell JM, et al. Angiotensin II type-1 receptor antagonism attenuates the inflammatory and thrombogenic responses to hypercholesterolemia in venules[J]. Hypertension,2005,45(2):209-15. Epub 2005 Jan 17.
30. Steinberg D. Thematic review series:the pathogenesis of atherosclerosis. An interpretive history of the cholesterol controversy, part Ⅴ:the discovery of the statins and the end of the controversy [J]. J Lipid Res,2006,47(7):1339-51.
31. Han SN, Leka LS, Lichtenstein AH, et al. Effect of hydrogenated and saturated, relative to polyunsaturated, fat on immune and inflammatory responses of adults with moderate hypercholesterolemia[J]. J Lipid Res,2002, 43:445-452.
32. Lusis AJ. Atherosclerosis. Nature,2000,407:233-241.
33. Nissen SE, Tuzcu EM, Schoenhagen P, et al. Effect of intensive compared with moderate lipid-lowering therapy on progression of coronary atherosclerosis:a randomized controlled trial[J]. JAMA,2004,291(9):1071-80.
34. Wissler RW. Update on the pathogenesis of atherosclerosis [J]. Am J Med,1991, 91:3S-9S.
35. Naghavi M, Libby P, Falk E, et al. From vulnerable plaque to vulnerable patient: A call for new definitions and risk assessment strategies:Part Ⅰ[J].Circulation, 2003,108(4):1664-1672.
36. Virmani R, Kolodgie FD, Burke AP, et al. Lessons from sudden coronary death: a comprehensive morphological classification scheme for atherosclerotic lesions[J]. Arterioscler Thromb Vasc Biol,2000,20:1262-1275.
37.吴平生,张远慧,许乙凯,等.易损斑块及易损患者的新定义及危险分层[J].中华心血管病杂志,2004,32(3)：283-285.
38. Schaar JA, Muller JE, Falk E, et al. Terminology for high-risk and vulnerable coronary artery plaques. Report of a meeting on the vulnerable plaque [J]. Eur Heart J 2004,25:1077-1082.
39.陈在嘉,高润霖.主编.冠心病.北京；人民卫生出版社,2002：72.
40. Ueda Y, Ogasawara N, Matsuo K, et al.Acute coronary syndrome:insight from angioscopy[J]. Circ J,2010,74(3):411-7.
41.金惠铭,李先涛.血管新生的调控[J].中国微循环,2001,5(2)：85-88.
42. Schaper W, Ito WD. Molecular mechanisms of coronary collateral vessel growth[J].Circ Res,1996,79(5):911-919.
43. Gossl M, Versari D, Hildebrandt HA, et al. Segmental heterogeneity of vasa vasorum neovascularization in human coronary atherosclerosis[J]. JACC Cardiovasc Imaging,2010,3(1):32-40.
44.孙璐,韦立新,石怀银,等.冠状动脉粥样硬化斑块内血管新生与斑块稳定的关系[J].中华病理学杂志,2003,32(5)：
45. Moreno PR, Purushothaman KR, Fuster V, et al. Plaque neovascularization is increased in ruptured atherosclerotic lesions of human aorta:implications for plaque vulnerability [J]. Circulation,2004,110(14):2032-8.
46. Karen S. Moulton, Eric Heller, Moritz A. Konerding, Evelyn Flynn, Wulf Palinski,et al. Angiogenesis Inhibitors Endostatin or TNP-470 Reduce Intimal Neovascularization and Plaque Growth in Apolipoprotein E-Deficient Mice[J]. Circulation,1999,99:1726-1732.
47. Teo NB, Shoker BS, Martin L, et al. Angiogenesis in pre-invasive cancers[J]. Anticancer Res,2002,22:2061.
48. Hannen EJ, Riediger D. The quantification of angiogenesis in relation to metastasis in oral cancer:a review[J]. Int J Oral Maxillofac Surg,2004,33:2.
49. Krause DS, Fackler MJ, Civin CI, et al. CD34:structure, biology, and clinical utility[J]. Blood,1996,87:1-13.
50. Poncelet C, Madelenat P, Feldmann G, et al. Expression of vonWillebrand's factor, CD34, CD31, and vascular endothelial growth factor in uterine leiomyomas[J]. Fertil Steril,2002,78:581.
51.王智,龚艳君,洪涛.他汀与动脉粥样硬化斑块逆转[J].临床荟萃,2013,28(7)：831-834.
52. FDA.org—The Center For Health and Wellness. Accessed April 25,2012.
53.吕越,张丽萍.黄芪煎剂降脂作用实验研究[J].时珍国医国药,2007,18(11)：2613.
54.童红莉,田亚平,汪德清,等.黄芪多糖对高脂血症大鼠血脂的调节[J].中国组织工程学研究与临床康复,2006,10(11)：
55.潘娜.丹参注射液分疗程治疗的降脂疗效研究[J].临床合理用药杂志,2011,04(18)：30.
56.陈礼波,李晓辉,张海港,等.三七总皂苷对兔腹主动脉粥样硬化防治作用的超声学评价[J].现代生物医学进展,2008,8(6)：1054-1056.
57. Liu G, Wang B, Zhang J, et al. Total panax notoginsenosides prevent atherosclerosis in apolipoprotein E-knockout mice:Role of downregulation of CD40 and MMP-9 expression[J]. J Ethnopharmacol,2009,126:350-354.
58.贺立勃.瓜蒌、薤白降脂作用的析因研究[J].中医药导报,2002,8(4)：205-207.
59.王冬梅,代世元,芦丽莉,等.瓜蒌皮提取物对大鼠动脉粥样硬化保护作用的实验研究[J].北华大学学报自然科学版,2008,9(2)：128-131.
60.李宁,赵霞,张文高.水蛭微粉治疗高脂血症疗效观察[J].中国误诊学杂志,2008,8(4)：802-803.
61.于燕,刘继兰,王菊英,等.土鳖虫水提液对实验性高脂血症大鼠血管内皮 细胞的保护作用[J].山东大学学报医学版,2002,40(5)：398-400.
62.李海涛,朱雪晶,吴其标,等.人工麝香对大鼠高脂血症影响的实验研究[J].中国临床药理学与治疗学,2010,15(9)：11-13.
1. Inoue M, Itoh H, Ueda M, et al. Vascular endothelial growth factor (VEGF) expression in human coronary atherosclerotic lesions:possible pathophysiological significance of VEGF in progression of atherosclerosis[J]. Circulation,1998,98: 2108-2116.
2. Belgore F, Blann A, Neil D, et al. Localisation of members of the vascular endothelial growth factor (VEGF) family and their receptors in human atherosclerotic arteries[J]. J Clin Pathol,2004,57:266-272.
3. Herrmann J, Lerman LO, Mukhopadhyay D, et al. Angiogenesis in atherogenesis[J]. Arterioscler Thromb Vasc Biol,2006,26:1948-1957.
4. Ferrara N, Gerber HP, LeCouter J. The biology of VEGF and its receptors[J]. Nat Med,2003,9:669-676.
5. Kim I, Moon SO, Park SK, et al. Angiopoietin-1 reduces VEGF-stimulated leukocyte adhesion to endothelial cells by reducing ICAM-1, VCAM-1, and E-selectin expression[J]. Circ Res,2001,89:477-479.
6. Bjornheden T, Levin MM, Evaldsson, et al. Evidence of hypoxic area within the arterial wall in vivo[J]. Arterioscler Thromb Vasc Biol,1999,19:870-876.
7. Losso JN, Bawadi HAJ. Hypoxia inducible factor pathways as targets for functional foods[J]. Agric Food Chem,2005,53 (10):3751-68.
8. Kelly BD, Hackett SF, Hirota K, et al. Cell type-specific regulation of angiogenic growth factor gene expression and induction of angiogenesis in nonischemic tissue by a constitutively active form of hypoxia-inducible factor 1 [J]. Circ Res,2003,93:1074-1081
9. Ushio-Fukai M. Redox signaling in angiogenesis:role of NADPH oxidase[J]. Cardiovasc Res,2006,71:226-235.
10. Nespereira B, Perez-Ilzarbe M, Fernandez P, et al. Vitamins C and E downregulate vascular VEGF and VEGFR2 expression in apolipoprotein-E-deficient mice[J]. Atherosclerosis,2003,171:67-73.
11. Masuko Ushio-Fukai, Norifumi Urao. Novel Role of NADPH Oxidase in Angiogenesis and Stem/Progenitor Cell Function[J]. Antioxid Redox Signal, 2009,11:2517-2533.
12. Ago T, Kitazono T, Ooboshi H, et al. Nox4 as the major catalytic component of an endothelial NAD(P)H oxidase[J]. Circulation,2004,109:227-233.
13. Meng D, Mei A, Liu J, et al. NADPH oxidase 4 mediates insulin-stimulated HIF-1α and VEGF expression, and angiogenesis in vitro[J]. PLoS One,2012, 7:e48393.
14. Livak KJ, Schmittqen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C (T)) Method [J]. Methods, 2001,25(4):402-408.
15. Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR [J]. Nucleic Acids Res,2001,29(9):e45.
16. Senger DR, Galli SJ, Dvorak AM, et al. Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid[J]. Science,1983, 219:983-985.
17. Ferrara N, Henzel WJ. Pituitary follicular cells secrete a novel heparin-binding growth factor specific for vascular endothelial cells[J]. Biochem Biophys Res Commun,1989,161:851-858.
18. Carmeliet P, Ferreira V, Breier G, et al. Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele[J]. Nature,1996,380:435-439.
19. Ferrara N, Carver-Moore K, Chen H, et al. Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene[J]. Nature,1996, 380:439-442.
20. Benjamin LE, Hemo I, Keshet E. A plasticity window for blood vessel remodelling is defined by pericyte coverage of the preformed endothelial network and is regulated by PDGF-B and VEGF[J]. Development,1998,125(9):1591-8.
21. Ku DD, Zaleski JK, Liu S, et al. Vascular endothelial growth factor induces EDRF-dependent relaxation in coronary arteries[J]. Am J Physiol,1993,265(2 Pt 2):H586-92.
22. Ferrara N, Gerber HP, LeCouter J. The biology of VEGF and its receptors[J]. Nat Med,2003,9:669-676.
23. Kim I, Moon SO, Park SK, et al. Angiopoietin-1 reduces VEGF-stimulated leukocyte adhesion to endothelial cells by reducing ICAM-1, VCAM-1, and E-selectin expression[J]. Circ Res,2001,89:477-479.
24. Shalaby F, Rossant J, Yamaguchi TP, et al. Failure of blood-island formation and vasculogenesis in Flk-1-deficient mice[J]. Nature,1995,376:62-66.
25. Benjamin LE, Hemo I, Keshet E. A plasticity window for blood vessel remodelling is defined by pericyte coverage of the preformed endothelial network and is regulated by PDGF-B and VEGF[J]. Development,1998,125(9):1591-8.
26. Gerber HP, McMurtrey A, Kowalski J, et al. Vascular endothelial growth factor regulates endothelial cell survival through the phosphatidylinositol 3'-kinase/Akt signal transduction pathway. Requirement for Flk-1/KDR activation[J].J Biol Chem,1998,273(46):30336-43.
27. Gerber HP, Dixit V, Ferrara N. Vascular endothelial growth factor induces expression of the antiapoptotic proteins Bcl-2 and A1 in vascular endothelial cells[J].J Biol Chem,1998,273(21):13313-6.
28.李树合,周定标,孙同柱,等.颈动脉粥样硬化斑块中VEGF受体Flk-1和Flt-1表达的改变及其意义[J].感染、炎症、修复,2005,1(6)：204-05.
29. Takahashi H, Shibuya M. The vascular endothelial growth factor(VEGF)/VEGF receptor systerm and its role under physiological and pathological conditions[J]. Clin Sci(Lond),2005,109(3):227-241.
30. Li J, Perel la MA, Tsai TC. Induct ion of vascular endothel ial growth factor gene expression by interleukin-13 in rat aortic smooth muscle cell[J]. J Biol Chem, 1995,270(1):308.
31. Lemstrem AD, Krebs R, Nyknen AI, et al. Vascular Endothel ial Growth Factor Enhances Cardiac Al lograft Arteriosclerosis[J]. Circulation,2002,105:45 2524-2530.
32. WilliamsB, BakerAO, GallacherB, et al. AngiogensinⅡ increases vascular permeability factor gene expression by human vascular smooth muscle cells[J]. Hypertension,1995,25(5):913.
33. Inoue M, Itoh H, Ueda M et al. Vascular endothelial growth factor (VEGF) expression in human coronary atherosclerotic lesions:possible pathophysiological significance of VEGF in progression of atherosclerosis[J]. Circulation,1998,98: 2108-2116.
34. Belgore F, Blann A, Neil D, et al. Localisation of members of the vascular endothelial growth factor (VEGF) family and their receptors in human atherosclerotic arteries[J]. J Clin Pathol,2004,57:266-272.
35. Herrmann J, Lerman LO, Mukhopadhyay D, et al. Angiogenesis in atherogenesis[J]. Arterioscler Thromb Vasc Biol,2006,26:1948-1957.
36. Celletti FL, Hilfiker PR, Ghafouri P, et al. Effect of human recombinant vascular endothelial growth factor165 on progression of atherosclerotic plaque[J]. J Am Coll Cardiol,2001,37:2126-2130.
37. Moulton KS, Vakili K, Zurakowski D, et al. Inhibition of plaque neovascularization reduces macrophage accumulation and progression of advanced atherosclerosis[J].Proc Natl Acad Sci,2003,100(8):4736-41.
38. Bjornheden T, Levin MM, Evaldsson, et al. Evidence of hypoxic area within the arterial wall in vivo[J]. Arterioscler Thromb Vasc Biol,1999,19:870-876.
39. Losso JN, Bawadi HAJ. Hypoxia inducible factor pathways as targets for functional foods[J]. Agric Food Chem,2005,53 (10):3751-68.
40. Forsythe JA. Jiang BH, Iyer NV, et al. Activation of vascular endothelial growth factor gene transcription by hypoxia—inducible factor 1[J]. Mol Cell Biol,1996,16:4604-4613.
41. Enholm B,Paavonen K, Ristimaki A, et al. Comparison of VEGF, VEGF-B, VEGF-C and Ang-1 mRNA regulation by serum, growth factors, oncoproteins and hypoxia[J]. Oncogene,1997,14(20):2475-83.
42. Wenger RH. Cellular adaptation to hypoxia; O2-sensing protein hydroxylases, hypoxia—inducible transcription factors, and O2-regulated gene expression[J]. FASEB J,2002,16:1151-1162.
43.Shweiki D,Itin A,Soffer D,et al.Vascular endotheli—al,growth factor inducedby hypoxia may mediae hypoxia—initia—ted angiogenesis[J]. Nature,1992, 359:843-846.
44. Steinberg D. Lipoproteins and atherosclerosis. A look back and a look ahead[J]. Arteriosclerosis,1983,3:283-301.
45. Cho M, Hunt T K, Hussain M Z. Hydrogen peroxide stimulates macrophage vascular endothelial growth factor release[J]. Physiol Heart Circ Physiol,2001, 280:2357-2363.
46. Kuroki M, Voest E E, Amano S, et al. Reative oxygen intermediates increase vascular endothelial growth facter expression in vitro and vivo[J]. Clin Invest, 1996,98:1667-1675.
47. Yasuda M, Ohzeki Y, Shimizu S, et al. Stimulation of in vitro angiogenesis by hydrogen peroxide and the relation with ETS-1 in endothelial cells[J]. Life Sci, 1999,64(4):249-258.
48. Masuko Ushio-Fukai, Norifumi Urao. Novel Role of NADPH Oxidase in Angiogenesis and Stem/Progenitor Cell Function[J]. Antioxid Redox Signal, 2009,11:2517-2533.
49. Chen JX, Zeng H, Lawrence ML, et al. Angiopoietin-1-induced angiogenesis is modulated by endothelial NADPH oxidase[J]. Am J Physiol Heart Circ Physiol, 2006,291:H1563-1572.
50. Ago T, Kitazono T, Ooboshi H, et al. Nox4 as the major catalytic component of an endothelial NAD(P)H oxidase[J]. Circulation,2004,109:227-233.
51. Park HS, Chun JN, Jung HY, et al. Role of NADPH oxidase 4 in lipopolysaccharide-induced proinflammatroy responses by human aortic endothelial cells[J]. Cardiovase Res,2006,72:447-455.
52.张路,吴宗贵,廖德宁,等.通心络对实验性家兔主动脉粥样斑块内血管内皮生长因子表达的影响[J].中国动脉硬化杂志,2004,12(2)：177-182.
53.杨涛,袁肇凯,胡志希,等.养心通脉片对动脉粥样硬化白兔VEGF的表达和新生血管数的影响[J].中西医结合心脑血管病杂志,2006,4(5)：407-408.
54.李天奇,范维琥,李勇,等.麝香保心丸减少兔股动脉斑块内血管新生机制探讨[J].中西医结合心脑血管病杂志,2009,7(6)：680-682.
55.崔伊梅.黄芪及其有效成分黄芪苷Ⅳ在心血管药理作用的研究进展[J].天津医科大学学报,2005,11(4)：656-659.
56.杨五彪,陈群立,马灵筠,等.黄芪多糖对兔动脉粥样硬化血管内皮细胞功能的影响[J].陕西医学杂志,2005,34(8)：914-918.
57.曾国安,陈玉燕,李博,等.黄芪多糖对兔动脉粥样硬化血管内皮细胞功能的影响[J].中西医结合心脑血管病杂志,2010,8(10)：1206-1208.
58.杨志红,赵晓龙,陈凤玲,等.黄芪多糖对THP-1.巨噬细胞源性泡沫细胞功能的影响[J].中国分子心脏病学杂志,2007,7(3)：138-142.
59.郭静,王庸晋,燕李晨,等.黄芪多糖对RAW264.7巨噬细胞源性泡沫细胞内胆固醇含量的影响[J].中国心血管病研究,2013,11(3)：216-219.
60.高建,徐先样,徐先俊,等.黄芪总皂甙抗血栓形成作用实验研究[J].中成药,2002,24(2)：116-118.
61.刘宽芝,李静波,吕海莉,等.黄芪、三七总皂苷对2型糖尿病大血管病变患者MMP-9的影响[J].中国中药杂志,2004,29(3)：264-266.
62.游洋,段岩,张效林,等.黄芪水提取物对载脂蛋白E基因敲除小鼠动脉粥样硬化斑块部位基质金属蛋白酶-9表达及斑块形成的影响[J].中华心血管病杂志,2012,40(6)：522-526.
63.李家实.中药鉴定学[M].上海：上海科学技术出版社.1998；172.
64.高学敏,张廷模,张俊荣,等.中药学[M].中国中医药出版社,2002,9(1)：51-500.
65. Fang ZY, Lin R, Yuan BX, et al. Tanshinone IIA downregulates the CD40 expression and decreases MMP-2 activity on atherosclerosis induced by high fatty diet in rabbit[J]. J Ethnopharmacol,2008,115(2):217-22.
66.张知新,高福云,李克明,等.丹参酚酸B镁对自由基损伤人主动脉内皮细胞的影响[J].中国中西医结合杂志,2004,24-521-4.
67. Kang BY, Chung SW, Kim SH,et al. Inhibition of interleukin-12 and interferon-gamma production in immune cells by tanshinones from Sal via miltiorrhiza[J].Immunopharmacology,2000,49(3):355-61.
68.高玉琪,罗南萍,梁春,等.丹参多酚酸盐对ApoE-/-小鼠血清IL-6的影响[J].放射免疫学杂志,2011,24(1)：15-17.
69. Fang ZY, Lin R, Yuan BX, et al. Tanshinone ⅡA downregulates the CD40 expression and decreases MMP-2 activity on atherosclerosis induced by high fatty diet in rabbit[J]. J Ethnopharmacol,2008,115(2):217-22.
70.许涛,喻莉,郑智,等.丹参对实验性动脉粥样硬化形成的预防[J].临床心血管病杂志,2005,21(1)：54-55.
71.姜开余,顾振纶,阮长耿.丹参素对CD11b、P-Selectin、ICAM-1、VCAM-1、 E-Selectin表达的影响[J].中国药理学通报,2000,16(6)：682-685.
72. Huang KJ, Wang H, Xie WZ. Investigation of the effect of tanshinone ⅡA on nitric oxide production in human vascularendothelial cells by fluorescence imaging[J]. Spectrochimica Acta Part A,2007,68:1180.
73.李永胜,王进,王照华,等.丹参酮ⅡA对主动脉内皮细胞功能损伤的保护机制[J].中国急救医学,2007,27(8)：720.
74.成之福,王毅华,王家安,等.复方丹参注射液对冠心病患者一氧化氮、内皮素-1的影响[J].中国医院药学杂志,2006,26(10)：1275.
75.袁恒杰.丹参素药理作用研究新进屉[J].中国医院药学杂志,2006,26(5)：604-606.
76.刘虹彬,温进坤.丹参对血管平滑肌细胞基质金属蛋白酶和骨桥蛋白基因表达及细胞增殖的影响[J].中国中西医结合杂志,2002,22(10)：764-766.
77.扬鹏远,芮耀城,张黎,等.U937泡沫细胞中血管内皮生长因子的表达及药物的抑制作用[J].药学学报,2002,37(2)：86-89.
78.甘烦远,郑光植.三七化学成分研究概况[J].中国药学杂志,1992,27：138.
79.何雪峰.李晓辉,李淑惠,等.三七总皂苷对家兔实验性动脉粥样硬化的预防作用[J].中国药房,2007,18(6)：408-409.
80.李韬,曲德英,雷菠,等.三七粉对家兔实验性动脉粥样硬化的影响[J].中医研究,2006,19(1)：17-19.
81.文川,徐浩,黄启福,等.活血中药对ApoE基因缺陷小鼠血脂及动脉粥样硬化斑块炎症反应的影响[J].中国中西医结合杂志,2005,25(4)：345-349.
82. Ng T B, Liu F. Wang H X. The antioxidant effects of aqueous and organic extracts of Panax quinquefolium, Panaxnotoginseng, Codonopsis pilosula, Pseudostellaria heterophy—lla and Glehnia littoralis[J]. J Ethnopharmacol, 2004,93(2-3):285-288.
83. Rhule A, Navarro S, Smith J R, et al. Panax notoginseng attenuates LPS-induced pro-inflammatory mediators in RAW264.7 cells. J Ethnopharmacol,2006,106(1):121-128.
84. Liu G, Wang B, Zhang J, et al. Total panax notoginsenosides prevent atherosclerosis in apolipoprotein E-knockout mice:Role of downregulation of CD40 and MMP-9 expression. J Ethnopharmacol,2009,126:350-354.
85.何翠瑶.李晓辉,何雪峰.三七皂苷单体不同配伍对单核一内皮细胞黏附作用的影响[J].中医杂志,2007,48(4)：354-361.
86.胡俊,沈中晨,沈芸,等.三七毛根皂苷对动脉粥样硬化大鼠血脂及血管细胞黏附分子表达影响.上海大学学报(自然科学版),2009,15(3)：320-325.
87.陈剑鸿。王碧江,刘松青,等.三七总皂苷对内毒紊损伤血管内皮细胞炎症特性的影响[J].中国医院药学杂志,2004,24(3)：140-141.
88.吕志军,梁绪国.三七总皂苷对血管内皮细胞凋亡及凋亡调控基因的影响[J].中西医结合心脑血管病杂志,2005,3(11)：975-977.
89.闫彦芳,张壮.孙甥伦.等.三七总皂苷及其主要成分对血管内皮细胞缺氧损伤的保护作用[J].中国实验方剂学杂志,2002,8(1)：34-37.
90.曾桂凤,刘建勋,李澎,等.丹参三七不同配比对缺氧复氧损伤人脐静脉内皮细胞的保护作用[J].精细化工,2006,23(2)：126-129.
91.李晓宇,孙建国,郑媛婷.等.三七总皂苷对抗H202所致大鼠脑微血管内皮细胞损伤的物质基础研究[J].中国药理学通报,2007,23(8)t 1030-1034.
92.袁志兵,李晓辉,李淑慧,等.三七总皂苷对动脉粥样硬化斑块稳定性的影响[J].中国天然药物,2006,4(1)：62-65.
93.潘贺,刚宏林,苏云明.中药水蛭的活性成分及药理作用研究概况[J].中医药信息,2006,23(1)：20-22.
94.李秀珍.开发中的抗栓剂-水蛭素及其12肽[J].军事医学科学院院报,1996,20(1)：73-75.
95.冉春风,白淑杰,杨静.水蛭注射液抗血栓作用的实验研究[J].现代康复,2001,5：73-4
96. Salzet M. Theromin, a novel leech thrombin inhibitor [J]. J Niological Chemistry, 2000,275:774.
97.胡志坚.中药临床应用[M].内蒙古人民出版社,1980,414.
98.李宁,赵霞,张文高.水蛭微粉对不稳定性心绞痛血管内皮保护作用的观察[J].中国误诊学杂志,2009,9(7)：1539.
99.梁桂文,姜敏辉.水蛭素对动脉内皮保护作用的实验研究[J].天津医药,2012,40(12)：1234-1236.
100.李凤文,刘晓颖,张立石,等.水蛭及其复方对实验性动脉粥样硬化家兔主动脉内皮素基因表达的影响[J].中医杂志,1999,40(1)：50-51
101.陈国伟,潘阳,商亮,等.水蛭对载脂蛋白E基因敲除鼠动脉粥样硬化斑块的影响[J].武汉大学学报(医学版),2013,34(3)：11-13.
102.周春凤,蔡萌.土鳖虫抗凝血作用研究[J].长春中医学院学报,1999,15：47.
103.杨红莲,刘梅.土鳖虫的化学成分及药理研究[J].陕西中医学学报,2005,282：48-50.
104.郭庆民,刘继兰,王菊英,等.土鳖虫对红细胞变形性和膜成分的影响[J].中国生化药物杂志,2000,21：235-7.
105.于燕,刘继兰,王菊英,等.土鳖虫水提物对实验性高脂血症大鼠血管内皮和内皮素的影响[J].中国生化药物杂志,2003,24(1)：15-17.
106.于燕,刘继兰,王菊英,等.土鳖虫水提液对实验性高脂血症大鼠血管内皮细胞的保护作用[J].山东大学学报(医学版),2002,40(5)：398-400.
107.屠婕红,余菁,陈伟光.瓜蒌的化学成分和药理作用研究概况[J].中国药师,2004,7(7)：562-563.
108.贝伟剑.大子栝楼和栝楼的药理作用比较[J].湖南中医药导报,1996,2：37-9.
109.贺立勃.瓜萎、薤白降脂作用的析因研究[J].中医药导报,2002,8(4)：205-207.
110.蒋文跃,杨宇.化痰药半夏、瓜蒌、浙贝母、石菖蒲对大鼠血液流变性的 影响[J].中医杂志,2002；43(3)：215-216.
111.凌宏,鲁翔,董传仁,等.瓜蒌、心得安对血小板体外聚集和TXA2合成的影响[J].湖北医学院学报,1988,9(2)：138.
112.王冬梅,代世元,芦丽莉,等.瓜蒌皮提取物对大鼠动脉粥样硬化保护作用的实验研究[J].北华大学学报自然科学版,2008,9(2)：128-131.
113.卢丽莉,王冬梅.瓜蒌皮提取液对实验性高脂血症大鼠血清NO、SOD、MDA的影响[J].北华大学学报(自然科学版),2008,9(5)：423-425.
114.上海瓜蒌协作组,瓜萎注射液鉴定书,上海,1976.
115. Kimura M.麝香中新的蛋白激酶活化剂及对豚鼠心肌强心作用的研究[J].国外医学·植物药分册,1992,14(1)：57.
116.黄正良.麝香的药理作用及临床应用研究[J].中成药研究,1987,(5)：23-25.
117.胡秉龙等.麝香研究新进展[J].中医药信息,1997,2：11.
118.尹士敏,王士贤.麝香的药理作用及临床研究近况[J].天津药学,2002,14(3)：42-44.
119.朱秀媛.人工麝香的药用潜力[J].百科知识,1995,(7)：10-11.
120.周元风,胡永兵,洪浪.麝香保心丸对动脉粥样硬化兔血脂及hs-CRP的影响[J].实用临床医学,2013,14(4)：10-11.
121.李天奇,李勇,范维琥,等.麝香保心丸和辛伐他汀对兔股动脉粥样硬化斑块稳定性的影响[J].中华老年心血管病杂志,2006,8(5)：296-299.
122.李天奇,范维琥,李勇,等.麝香保心丸减少兔股动脉斑块内血管新生机制探讨[J].中西医结合心脑血管病杂志,2009,7(6)：680-682.
2. Renu Virmani, Frank D. Kolodgie, et al. Atherosclerotic Plaque Progression and Vulnerability to Rupture—Angiogenesis as a Source of Intraplaque Hemorrhage[J]. Arterioscler Thromb Vasc Biol,2005,25:2054-2061.
3. Moreno PR, Purushothaman KR, Sirol M, et al. Neovascularization in human atherosclerosis[J]. Criculation,2006,113(18):2245-2252.
4. Chen YX, Nakashima Y, Tanaka K, et al. Immunohistochemical expression of vascular endothelial growth factor/vascular permeability factor in atherosclerotic intimas of human coronary arteries[J]. Arterioscler Thromb Vasc Biol,1999,19(1):131-9.
5. Pandya NM, Dhalla NS, Santani DD. Angiogeneses——a new target for future therapy[J].Vascular Pharmacology,2006,44(5):265-274.
6. Karagiannis ED, Popel AS. Identification of novel short peptides derived from the a4,a5,and a6 fibrils of type Ⅳ collagen with anti-angiogenic properties [J]. Biochemical and Biophysical Research Communication,2007,354(2):434-439.
7. Rogers MS, D'Amato RI. The effect of genetic diversity on angiogenesis [J]. Experimental Cell Reseach,2006,312(5):561-574.
8.沈伟,范维琥,施海明,等.红景天对兔动脉粥样硬化斑块内血管内皮细胞生长因子表达及血管新生的影响[J].中国中西医结合杂,2008；11(28)：1022-1025.
9.李天奇,范维琥,李勇,等.麝香保心丸减少兔股动脉斑块内血管新生机制探讨[J].中西医结合心脑血管病杂志,2009,7(6)：680-682.
10.张路,吴宗贵,廖德宁,等.通心络对实验性家兔主动脉粥样斑块内血管内皮生长因子表达的影响[J].中国动脉硬化杂志,2004,12(2)：177-182.
11.杨涛,袁肇凯,胡志希,等.养心通脉片对动脉粥样硬化白兔VEGF的表达 和新生血管数的影响[J].中西医结合心脑血管病杂志,2006,4(5)：407-408.
12.尹慧秋,张继东,林海青,等.舒脉胶囊对大鼠缺血心肌促血管新生的实验研究[J].中国中西医结合杂志,2007,27(11)：1020-1023.
13. Ignatowski AC. Influence of animal food on the organism of rabbits[J]. S Peterb Izviest ImpVoyenno-Med Akad,1908,16:154-173.
14. Piedrahita JA, Zhang SH, Hagaman JR, et al. Generation of mice carrying a mutant apolipoprotein E gene inactivated by gene targeting in embryonic stem cells[J]. PNAS,1992,89:4471-4475.
15. Plump AS, Smith JD, Hayek T, et al. Severe hypercholesterolemia and atherosclerosis in apolipoprotein E-deficient mice created by homologous recombination in ES cells[J]. Cell,1992,71:343-353.
16. Cha J, Ivanov V, Ivanova S, et al. Evolution of angiotensin II-mediated atherosclerosis in ApoE KO mice[J]. Mol Med Report,2010,3:565-70.
17. Menu P, Pellegrin M, Aubert JF, et al. Atherosclerosis in ApoE-deficient mice progresses independently of the NLRP3 inflammasome [J]. Cell Death Dis 2011,2:e137.
18. Zhang SH, Reddick RL, Piedrahita JA, et al. Spontaneous hypercholesterolemia and arterial lesions in mice lacking apolipoprotein E[J]. Science,1992,258: 468-471.
19. Coleman R, Hayek T, Keidar S, et al. Mouse model for human atherosclerosis: long-term histopathological study of lesion development in the aortic arch of apolipoprotein E-deficient(EO)mice[J]. Acta Histochem,2006,108(6):415-424.
20. Nakashima Y, Plump AS, Raines EW, et al. ApoE-deficient mice develop lesions of all phases of atherosclerosis throughout the arterial tree[J]. Arterioscler Thromb,1994,14:133-140.
21. Jawien J, Nastalek P, Korbut R. Mouse models of experimental atherosclerosis[J]. Physiol Pharmacol,2004,55(3):503-517.
22. Dawber TR, Kannel WB, Revotskie N, et al. The epidemiology of coronary heart disease--the Framingham enquiry[J]. Proc R Soc Med,1962,55:265-71.
23. Folsom AR, Wu KK, Rosamond WD, et al. Prospective study of hemostatic factors and incidence of coronary heart disease:the Atherosclerosis Risk in Communities (ARIC) Study[J]. Circulation,1997,96(4):1102-8.
24.刘静,赵冬,晃兆苏,等.低密度脂蛋白胆固醇与心血管病发病关系的前瞻性研究[J].中华心血管病杂志,2001,29(9)：561-565.
25.李莹,陈志红,周北凡,等.血脂和脂蛋白水平对我国中年人群缺血性心血管病事件的预测作用[J].中华心血管病杂志,2004,32(7)：643-647.
26. Kher N, Marsh JD. Pathobiology of atherosclerosis—a brief review[J]. Semin Thromb Hemost,2004,30:665-672.
27. Sugano R, Matsuoka H, Haramaki N, et al. Polymorphonuclear leukocytes may impair endothelial function:results of crossover randomized study of lipid-lowering therapies[J]. Arterioscler Thromb Vasc Biol,2005,25(6):1262-7.
28. Rodriguez JA, Nespereira B, Perez-Ilzarbe M, et al. Vitamins C and E prevent endothelial VEGF and VEGFR2 overexpression induced by porcine hypercholesterolemic LDL[J]. Cardiovasc Res,2005,65(3):665-73.
29. Petnehazy T, Stokes KY, Russell JM, et al. Angiotensin II type-1 receptor antagonism attenuates the inflammatory and thrombogenic responses to hypercholesterolemia in venules[J]. Hypertension,2005,45(2):209-15. Epub 2005 Jan 17.
30. Steinberg D. Thematic review series:the pathogenesis of atherosclerosis. An interpretive history of the cholesterol controversy, part Ⅴ:the discovery of the statins and the end of the controversy [J]. J Lipid Res,2006,47(7):1339-51.
31. Han SN, Leka LS, Lichtenstein AH, et al. Effect of hydrogenated and saturated, relative to polyunsaturated, fat on immune and inflammatory responses of adults with moderate hypercholesterolemia[J]. J Lipid Res,2002, 43:445-452.
32. Lusis AJ. Atherosclerosis. Nature,2000,407:233-241.
33. Nissen SE, Tuzcu EM, Schoenhagen P, et al. Effect of intensive compared with moderate lipid-lowering therapy on progression of coronary atherosclerosis:a randomized controlled trial[J]. JAMA,2004,291(9):1071-80.
34. Wissler RW. Update on the pathogenesis of atherosclerosis [J]. Am J Med,1991, 91:3S-9S.
35. Naghavi M, Libby P, Falk E, et al. From vulnerable plaque to vulnerable patient: A call for new definitions and risk assessment strategies:Part Ⅰ[J].Circulation, 2003,108(4):1664-1672.
36. Virmani R, Kolodgie FD, Burke AP, et al. Lessons from sudden coronary death: a comprehensive morphological classification scheme for atherosclerotic lesions[J]. Arterioscler Thromb Vasc Biol,2000,20:1262-1275.
37.吴平生,张远慧,许乙凯,等.易损斑块及易损患者的新定义及危险分层[J].中华心血管病杂志,2004,32(3)：283-285.
38. Schaar JA, Muller JE, Falk E, et al. Terminology for high-risk and vulnerable coronary artery plaques. Report of a meeting on the vulnerable plaque [J]. Eur Heart J 2004,25:1077-1082.
39.陈在嘉,高润霖.主编.冠心病.北京；人民卫生出版社,2002：72.
40. Ueda Y, Ogasawara N, Matsuo K, et al.Acute coronary syndrome:insight from angioscopy[J]. Circ J,2010,74(3):411-7.
41.金惠铭,李先涛.血管新生的调控[J].中国微循环,2001,5(2)：85-88.
42. Schaper W, Ito WD. Molecular mechanisms of coronary collateral vessel growth[J].Circ Res,1996,79(5):911-919.
43. Gossl M, Versari D, Hildebrandt HA, et al. Segmental heterogeneity of vasa vasorum neovascularization in human coronary atherosclerosis[J]. JACC Cardiovasc Imaging,2010,3(1):32-40.
44.孙璐,韦立新,石怀银,等.冠状动脉粥样硬化斑块内血管新生与斑块稳定的关系[J].中华病理学杂志,2003,32(5)：
45. Moreno PR, Purushothaman KR, Fuster V, et al. Plaque neovascularization is increased in ruptured atherosclerotic lesions of human aorta:implications for plaque vulnerability [J]. Circulation,2004,110(14):2032-8.
46. Karen S. Moulton, Eric Heller, Moritz A. Konerding, Evelyn Flynn, Wulf Palinski,et al. Angiogenesis Inhibitors Endostatin or TNP-470 Reduce Intimal Neovascularization and Plaque Growth in Apolipoprotein E-Deficient Mice[J]. Circulation,1999,99:1726-1732.
47. Teo NB, Shoker BS, Martin L, et al. Angiogenesis in pre-invasive cancers[J]. Anticancer Res,2002,22:2061.
48. Hannen EJ, Riediger D. The quantification of angiogenesis in relation to metastasis in oral cancer:a review[J]. Int J Oral Maxillofac Surg,2004,33:2.
49. Krause DS, Fackler MJ, Civin CI, et al. CD34:structure, biology, and clinical utility[J]. Blood,1996,87:1-13.
50. Poncelet C, Madelenat P, Feldmann G, et al. Expression of vonWillebrand's factor, CD34, CD31, and vascular endothelial growth factor in uterine leiomyomas[J]. Fertil Steril,2002,78:581.
51.王智,龚艳君,洪涛.他汀与动脉粥样硬化斑块逆转[J].临床荟萃,2013,28(7)：831-834.
52. FDA.org—The Center For Health and Wellness. Accessed April 25,2012.
53.吕越,张丽萍.黄芪煎剂降脂作用实验研究[J].时珍国医国药,2007,18(11)：2613.
54.童红莉,田亚平,汪德清,等.黄芪多糖对高脂血症大鼠血脂的调节[J].中国组织工程学研究与临床康复,2006,10(11)：
55.潘娜.丹参注射液分疗程治疗的降脂疗效研究[J].临床合理用药杂志,2011,04(18)：30.
56.陈礼波,李晓辉,张海港,等.三七总皂苷对兔腹主动脉粥样硬化防治作用的超声学评价[J].现代生物医学进展,2008,8(6)：1054-1056.
57. Liu G, Wang B, Zhang J, et al. Total panax notoginsenosides prevent atherosclerosis in apolipoprotein E-knockout mice:Role of downregulation of CD40 and MMP-9 expression[J]. J Ethnopharmacol,2009,126:350-354.
58.贺立勃.瓜蒌、薤白降脂作用的析因研究[J].中医药导报,2002,8(4)：205-207.
59.王冬梅,代世元,芦丽莉,等.瓜蒌皮提取物对大鼠动脉粥样硬化保护作用的实验研究[J].北华大学学报自然科学版,2008,9(2)：128-131.
60.李宁,赵霞,张文高.水蛭微粉治疗高脂血症疗效观察[J].中国误诊学杂志,2008,8(4)：802-803.
61.于燕,刘继兰,王菊英,等.土鳖虫水提液对实验性高脂血症大鼠血管内皮 细胞的保护作用[J].山东大学学报医学版,2002,40(5)：398-400.
62.李海涛,朱雪晶,吴其标,等.人工麝香对大鼠高脂血症影响的实验研究[J].中国临床药理学与治疗学,2010,15(9)：11-13.
1. Inoue M, Itoh H, Ueda M, et al. Vascular endothelial growth factor (VEGF) expression in human coronary atherosclerotic lesions:possible pathophysiological significance of VEGF in progression of atherosclerosis[J]. Circulation,1998,98: 2108-2116.
2. Belgore F, Blann A, Neil D, et al. Localisation of members of the vascular endothelial growth factor (VEGF) family and their receptors in human atherosclerotic arteries[J]. J Clin Pathol,2004,57:266-272.
3. Herrmann J, Lerman LO, Mukhopadhyay D, et al. Angiogenesis in atherogenesis[J]. Arterioscler Thromb Vasc Biol,2006,26:1948-1957.
4. Ferrara N, Gerber HP, LeCouter J. The biology of VEGF and its receptors[J]. Nat Med,2003,9:669-676.
5. Kim I, Moon SO, Park SK, et al. Angiopoietin-1 reduces VEGF-stimulated leukocyte adhesion to endothelial cells by reducing ICAM-1, VCAM-1, and E-selectin expression[J]. Circ Res,2001,89:477-479.
6. Bjornheden T, Levin MM, Evaldsson, et al. Evidence of hypoxic area within the arterial wall in vivo[J]. Arterioscler Thromb Vasc Biol,1999,19:870-876.
7. Losso JN, Bawadi HAJ. Hypoxia inducible factor pathways as targets for functional foods[J]. Agric Food Chem,2005,53 (10):3751-68.
8. Kelly BD, Hackett SF, Hirota K, et al. Cell type-specific regulation of angiogenic growth factor gene expression and induction of angiogenesis in nonischemic tissue by a constitutively active form of hypoxia-inducible factor 1 [J]. Circ Res,2003,93:1074-1081
9. Ushio-Fukai M. Redox signaling in angiogenesis:role of NADPH oxidase[J]. Cardiovasc Res,2006,71:226-235.
10. Nespereira B, Perez-Ilzarbe M, Fernandez P, et al. Vitamins C and E downregulate vascular VEGF and VEGFR2 expression in apolipoprotein-E-deficient mice[J]. Atherosclerosis,2003,171:67-73.
11. Masuko Ushio-Fukai, Norifumi Urao. Novel Role of NADPH Oxidase in Angiogenesis and Stem/Progenitor Cell Function[J]. Antioxid Redox Signal, 2009,11:2517-2533.
12. Ago T, Kitazono T, Ooboshi H, et al. Nox4 as the major catalytic component of an endothelial NAD(P)H oxidase[J]. Circulation,2004,109:227-233.
13. Meng D, Mei A, Liu J, et al. NADPH oxidase 4 mediates insulin-stimulated HIF-1α and VEGF expression, and angiogenesis in vitro[J]. PLoS One,2012, 7:e48393.
14. Livak KJ, Schmittqen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C (T)) Method [J]. Methods, 2001,25(4):402-408.
15. Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR [J]. Nucleic Acids Res,2001,29(9):e45.
16. Senger DR, Galli SJ, Dvorak AM, et al. Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid[J]. Science,1983, 219:983-985.
17. Ferrara N, Henzel WJ. Pituitary follicular cells secrete a novel heparin-binding growth factor specific for vascular endothelial cells[J]. Biochem Biophys Res Commun,1989,161:851-858.
18. Carmeliet P, Ferreira V, Breier G, et al. Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele[J]. Nature,1996,380:435-439.
19. Ferrara N, Carver-Moore K, Chen H, et al. Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene[J]. Nature,1996, 380:439-442.
20. Benjamin LE, Hemo I, Keshet E. A plasticity window for blood vessel remodelling is defined by pericyte coverage of the preformed endothelial network and is regulated by PDGF-B and VEGF[J]. Development,1998,125(9):1591-8.
21. Ku DD, Zaleski JK, Liu S, et al. Vascular endothelial growth factor induces EDRF-dependent relaxation in coronary arteries[J]. Am J Physiol,1993,265(2 Pt 2):H586-92.
22. Ferrara N, Gerber HP, LeCouter J. The biology of VEGF and its receptors[J]. Nat Med,2003,9:669-676.
23. Kim I, Moon SO, Park SK, et al. Angiopoietin-1 reduces VEGF-stimulated leukocyte adhesion to endothelial cells by reducing ICAM-1, VCAM-1, and E-selectin expression[J]. Circ Res,2001,89:477-479.
24. Shalaby F, Rossant J, Yamaguchi TP, et al. Failure of blood-island formation and vasculogenesis in Flk-1-deficient mice[J]. Nature,1995,376:62-66.
25. Benjamin LE, Hemo I, Keshet E. A plasticity window for blood vessel remodelling is defined by pericyte coverage of the preformed endothelial network and is regulated by PDGF-B and VEGF[J]. Development,1998,125(9):1591-8.
26. Gerber HP, McMurtrey A, Kowalski J, et al. Vascular endothelial growth factor regulates endothelial cell survival through the phosphatidylinositol 3'-kinase/Akt signal transduction pathway. Requirement for Flk-1/KDR activation[J].J Biol Chem,1998,273(46):30336-43.
27. Gerber HP, Dixit V, Ferrara N. Vascular endothelial growth factor induces expression of the antiapoptotic proteins Bcl-2 and A1 in vascular endothelial cells[J].J Biol Chem,1998,273(21):13313-6.
28.李树合,周定标,孙同柱,等.颈动脉粥样硬化斑块中VEGF受体Flk-1和Flt-1表达的改变及其意义[J].感染、炎症、修复,2005,1(6)：204-05.
29. Takahashi H, Shibuya M. The vascular endothelial growth factor(VEGF)/VEGF receptor systerm and its role under physiological and pathological conditions[J]. Clin Sci(Lond),2005,109(3):227-241.
30. Li J, Perel la MA, Tsai TC. Induct ion of vascular endothel ial growth factor gene expression by interleukin-13 in rat aortic smooth muscle cell[J]. J Biol Chem, 1995,270(1):308.
31. Lemstrem AD, Krebs R, Nyknen AI, et al. Vascular Endothel ial Growth Factor Enhances Cardiac Al lograft Arteriosclerosis[J]. Circulation,2002,105:45 2524-2530.
32. WilliamsB, BakerAO, GallacherB, et al. AngiogensinⅡ increases vascular permeability factor gene expression by human vascular smooth muscle cells[J]. Hypertension,1995,25(5):913.
33. Inoue M, Itoh H, Ueda M et al. Vascular endothelial growth factor (VEGF) expression in human coronary atherosclerotic lesions:possible pathophysiological significance of VEGF in progression of atherosclerosis[J]. Circulation,1998,98: 2108-2116.
34. Belgore F, Blann A, Neil D, et al. Localisation of members of the vascular endothelial growth factor (VEGF) family and their receptors in human atherosclerotic arteries[J]. J Clin Pathol,2004,57:266-272.
35. Herrmann J, Lerman LO, Mukhopadhyay D, et al. Angiogenesis in atherogenesis[J]. Arterioscler Thromb Vasc Biol,2006,26:1948-1957.
36. Celletti FL, Hilfiker PR, Ghafouri P, et al. Effect of human recombinant vascular endothelial growth factor165 on progression of atherosclerotic plaque[J]. J Am Coll Cardiol,2001,37:2126-2130.
37. Moulton KS, Vakili K, Zurakowski D, et al. Inhibition of plaque neovascularization reduces macrophage accumulation and progression of advanced atherosclerosis[J].Proc Natl Acad Sci,2003,100(8):4736-41.
38. Bjornheden T, Levin MM, Evaldsson, et al. Evidence of hypoxic area within the arterial wall in vivo[J]. Arterioscler Thromb Vasc Biol,1999,19:870-876.
39. Losso JN, Bawadi HAJ. Hypoxia inducible factor pathways as targets for functional foods[J]. Agric Food Chem,2005,53 (10):3751-68.
40. Forsythe JA. Jiang BH, Iyer NV, et al. Activation of vascular endothelial growth factor gene transcription by hypoxia—inducible factor 1[J]. Mol Cell Biol,1996,16:4604-4613.
41. Enholm B,Paavonen K, Ristimaki A, et al. Comparison of VEGF, VEGF-B, VEGF-C and Ang-1 mRNA regulation by serum, growth factors, oncoproteins and hypoxia[J]. Oncogene,1997,14(20):2475-83.
42. Wenger RH. Cellular adaptation to hypoxia; O2-sensing protein hydroxylases, hypoxia—inducible transcription factors, and O2-regulated gene expression[J]. FASEB J,2002,16:1151-1162.
43.Shweiki D,Itin A,Soffer D,et al.Vascular endotheli—al,growth factor inducedby hypoxia may mediae hypoxia—initia—ted angiogenesis[J]. Nature,1992, 359:843-846.
44. Steinberg D. Lipoproteins and atherosclerosis. A look back and a look ahead[J]. Arteriosclerosis,1983,3:283-301.
45. Cho M, Hunt T K, Hussain M Z. Hydrogen peroxide stimulates macrophage vascular endothelial growth factor release[J]. Physiol Heart Circ Physiol,2001, 280:2357-2363.
46. Kuroki M, Voest E E, Amano S, et al. Reative oxygen intermediates increase vascular endothelial growth facter expression in vitro and vivo[J]. Clin Invest, 1996,98:1667-1675.
47. Yasuda M, Ohzeki Y, Shimizu S, et al. Stimulation of in vitro angiogenesis by hydrogen peroxide and the relation with ETS-1 in endothelial cells[J]. Life Sci, 1999,64(4):249-258.
48. Masuko Ushio-Fukai, Norifumi Urao. Novel Role of NADPH Oxidase in Angiogenesis and Stem/Progenitor Cell Function[J]. Antioxid Redox Signal, 2009,11:2517-2533.
49. Chen JX, Zeng H, Lawrence ML, et al. Angiopoietin-1-induced angiogenesis is modulated by endothelial NADPH oxidase[J]. Am J Physiol Heart Circ Physiol, 2006,291:H1563-1572.
50. Ago T, Kitazono T, Ooboshi H, et al. Nox4 as the major catalytic component of an endothelial NAD(P)H oxidase[J]. Circulation,2004,109:227-233.
51. Park HS, Chun JN, Jung HY, et al. Role of NADPH oxidase 4 in lipopolysaccharide-induced proinflammatroy responses by human aortic endothelial cells[J]. Cardiovase Res,2006,72:447-455.
52.张路,吴宗贵,廖德宁,等.通心络对实验性家兔主动脉粥样斑块内血管内皮生长因子表达的影响[J].中国动脉硬化杂志,2004,12(2)：177-182.
53.杨涛,袁肇凯,胡志希,等.养心通脉片对动脉粥样硬化白兔VEGF的表达和新生血管数的影响[J].中西医结合心脑血管病杂志,2006,4(5)：407-408.
54.李天奇,范维琥,李勇,等.麝香保心丸减少兔股动脉斑块内血管新生机制探讨[J].中西医结合心脑血管病杂志,2009,7(6)：680-682.
55.崔伊梅.黄芪及其有效成分黄芪苷Ⅳ在心血管药理作用的研究进展[J].天津医科大学学报,2005,11(4)：656-659.
56.杨五彪,陈群立,马灵筠,等.黄芪多糖对兔动脉粥样硬化血管内皮细胞功能的影响[J].陕西医学杂志,2005,34(8)：914-918.
57.曾国安,陈玉燕,李博,等.黄芪多糖对兔动脉粥样硬化血管内皮细胞功能的影响[J].中西医结合心脑血管病杂志,2010,8(10)：1206-1208.
58.杨志红,赵晓龙,陈凤玲,等.黄芪多糖对THP-1.巨噬细胞源性泡沫细胞功能的影响[J].中国分子心脏病学杂志,2007,7(3)：138-142.
59.郭静,王庸晋,燕李晨,等.黄芪多糖对RAW264.7巨噬细胞源性泡沫细胞内胆固醇含量的影响[J].中国心血管病研究,2013,11(3)：216-219.
60.高建,徐先样,徐先俊,等.黄芪总皂甙抗血栓形成作用实验研究[J].中成药,2002,24(2)：116-118.
61.刘宽芝,李静波,吕海莉,等.黄芪、三七总皂苷对2型糖尿病大血管病变患者MMP-9的影响[J].中国中药杂志,2004,29(3)：264-266.
62.游洋,段岩,张效林,等.黄芪水提取物对载脂蛋白E基因敲除小鼠动脉粥样硬化斑块部位基质金属蛋白酶-9表达及斑块形成的影响[J].中华心血管病杂志,2012,40(6)：522-526.
63.李家实.中药鉴定学[M].上海：上海科学技术出版社.1998；172.
64.高学敏,张廷模,张俊荣,等.中药学[M].中国中医药出版社,2002,9(1)：51-500.
65. Fang ZY, Lin R, Yuan BX, et al. Tanshinone IIA downregulates the CD40 expression and decreases MMP-2 activity on atherosclerosis induced by high fatty diet in rabbit[J]. J Ethnopharmacol,2008,115(2):217-22.
66.张知新,高福云,李克明,等.丹参酚酸B镁对自由基损伤人主动脉内皮细胞的影响[J].中国中西医结合杂志,2004,24-521-4.
67. Kang BY, Chung SW, Kim SH,et al. Inhibition of interleukin-12 and interferon-gamma production in immune cells by tanshinones from Sal via miltiorrhiza[J].Immunopharmacology,2000,49(3):355-61.
68.高玉琪,罗南萍,梁春,等.丹参多酚酸盐对ApoE-/-小鼠血清IL-6的影响[J].放射免疫学杂志,2011,24(1)：15-17.
69. Fang ZY, Lin R, Yuan BX, et al. Tanshinone ⅡA downregulates the CD40 expression and decreases MMP-2 activity on atherosclerosis induced by high fatty diet in rabbit[J]. J Ethnopharmacol,2008,115(2):217-22.
70.许涛,喻莉,郑智,等.丹参对实验性动脉粥样硬化形成的预防[J].临床心血管病杂志,2005,21(1)：54-55.
71.姜开余,顾振纶,阮长耿.丹参素对CD11b、P-Selectin、ICAM-1、VCAM-1、 E-Selectin表达的影响[J].中国药理学通报,2000,16(6)：682-685.
72. Huang KJ, Wang H, Xie WZ. Investigation of the effect of tanshinone ⅡA on nitric oxide production in human vascularendothelial cells by fluorescence imaging[J]. Spectrochimica Acta Part A,2007,68:1180.
73.李永胜,王进,王照华,等.丹参酮ⅡA对主动脉内皮细胞功能损伤的保护机制[J].中国急救医学,2007,27(8)：720.
74.成之福,王毅华,王家安,等.复方丹参注射液对冠心病患者一氧化氮、内皮素-1的影响[J].中国医院药学杂志,2006,26(10)：1275.
75.袁恒杰.丹参素药理作用研究新进屉[J].中国医院药学杂志,2006,26(5)：604-606.
76.刘虹彬,温进坤.丹参对血管平滑肌细胞基质金属蛋白酶和骨桥蛋白基因表达及细胞增殖的影响[J].中国中西医结合杂志,2002,22(10)：764-766.
77.扬鹏远,芮耀城,张黎,等.U937泡沫细胞中血管内皮生长因子的表达及药物的抑制作用[J].药学学报,2002,37(2)：86-89.
78.甘烦远,郑光植.三七化学成分研究概况[J].中国药学杂志,1992,27：138.
79.何雪峰.李晓辉,李淑惠,等.三七总皂苷对家兔实验性动脉粥样硬化的预防作用[J].中国药房,2007,18(6)：408-409.
80.李韬,曲德英,雷菠,等.三七粉对家兔实验性动脉粥样硬化的影响[J].中医研究,2006,19(1)：17-19.
81.文川,徐浩,黄启福,等.活血中药对ApoE基因缺陷小鼠血脂及动脉粥样硬化斑块炎症反应的影响[J].中国中西医结合杂志,2005,25(4)：345-349.
82. Ng T B, Liu F. Wang H X. The antioxidant effects of aqueous and organic extracts of Panax quinquefolium, Panaxnotoginseng, Codonopsis pilosula, Pseudostellaria heterophy—lla and Glehnia littoralis[J]. J Ethnopharmacol, 2004,93(2-3):285-288.
83. Rhule A, Navarro S, Smith J R, et al. Panax notoginseng attenuates LPS-induced pro-inflammatory mediators in RAW264.7 cells. J Ethnopharmacol,2006,106(1):121-128.
84. Liu G, Wang B, Zhang J, et al. Total panax notoginsenosides prevent atherosclerosis in apolipoprotein E-knockout mice:Role of downregulation of CD40 and MMP-9 expression. J Ethnopharmacol,2009,126:350-354.
85.何翠瑶.李晓辉,何雪峰.三七皂苷单体不同配伍对单核一内皮细胞黏附作用的影响[J].中医杂志,2007,48(4)：354-361.
86.胡俊,沈中晨,沈芸,等.三七毛根皂苷对动脉粥样硬化大鼠血脂及血管细胞黏附分子表达影响.上海大学学报(自然科学版),2009,15(3)：320-325.
87.陈剑鸿。王碧江,刘松青,等.三七总皂苷对内毒紊损伤血管内皮细胞炎症特性的影响[J].中国医院药学杂志,2004,24(3)：140-141.
88.吕志军,梁绪国.三七总皂苷对血管内皮细胞凋亡及凋亡调控基因的影响[J].中西医结合心脑血管病杂志,2005,3(11)：975-977.
89.闫彦芳,张壮.孙甥伦.等.三七总皂苷及其主要成分对血管内皮细胞缺氧损伤的保护作用[J].中国实验方剂学杂志,2002,8(1)：34-37.
90.曾桂凤,刘建勋,李澎,等.丹参三七不同配比对缺氧复氧损伤人脐静脉内皮细胞的保护作用[J].精细化工,2006,23(2)：126-129.
91.李晓宇,孙建国,郑媛婷.等.三七总皂苷对抗H202所致大鼠脑微血管内皮细胞损伤的物质基础研究[J].中国药理学通报,2007,23(8)t 1030-1034.
92.袁志兵,李晓辉,李淑慧,等.三七总皂苷对动脉粥样硬化斑块稳定性的影响[J].中国天然药物,2006,4(1)：62-65.
93.潘贺,刚宏林,苏云明.中药水蛭的活性成分及药理作用研究概况[J].中医药信息,2006,23(1)：20-22.
94.李秀珍.开发中的抗栓剂-水蛭素及其12肽[J].军事医学科学院院报,1996,20(1)：73-75.
95.冉春风,白淑杰,杨静.水蛭注射液抗血栓作用的实验研究[J].现代康复,2001,5：73-4
96. Salzet M. Theromin, a novel leech thrombin inhibitor [J]. J Niological Chemistry, 2000,275:774.
97.胡志坚.中药临床应用[M].内蒙古人民出版社,1980,414.
98.李宁,赵霞,张文高.水蛭微粉对不稳定性心绞痛血管内皮保护作用的观察[J].中国误诊学杂志,2009,9(7)：1539.
99.梁桂文,姜敏辉.水蛭素对动脉内皮保护作用的实验研究[J].天津医药,2012,40(12)：1234-1236.
100.李凤文,刘晓颖,张立石,等.水蛭及其复方对实验性动脉粥样硬化家兔主动脉内皮素基因表达的影响[J].中医杂志,1999,40(1)：50-51
101.陈国伟,潘阳,商亮,等.水蛭对载脂蛋白E基因敲除鼠动脉粥样硬化斑块的影响[J].武汉大学学报(医学版),2013,34(3)：11-13.
102.周春凤,蔡萌.土鳖虫抗凝血作用研究[J].长春中医学院学报,1999,15：47.
103.杨红莲,刘梅.土鳖虫的化学成分及药理研究[J].陕西中医学学报,2005,282：48-50.
104.郭庆民,刘继兰,王菊英,等.土鳖虫对红细胞变形性和膜成分的影响[J].中国生化药物杂志,2000,21：235-7.
105.于燕,刘继兰,王菊英,等.土鳖虫水提物对实验性高脂血症大鼠血管内皮和内皮素的影响[J].中国生化药物杂志,2003,24(1)：15-17.
106.于燕,刘继兰,王菊英,等.土鳖虫水提液对实验性高脂血症大鼠血管内皮细胞的保护作用[J].山东大学学报(医学版),2002,40(5)：398-400.
107.屠婕红,余菁,陈伟光.瓜蒌的化学成分和药理作用研究概况[J].中国药师,2004,7(7)：562-563.
108.贝伟剑.大子栝楼和栝楼的药理作用比较[J].湖南中医药导报,1996,2：37-9.
109.贺立勃.瓜萎、薤白降脂作用的析因研究[J].中医药导报,2002,8(4)：205-207.
110.蒋文跃,杨宇.化痰药半夏、瓜蒌、浙贝母、石菖蒲对大鼠血液流变性的 影响[J].中医杂志,2002；43(3)：215-216.
111.凌宏,鲁翔,董传仁,等.瓜蒌、心得安对血小板体外聚集和TXA2合成的影响[J].湖北医学院学报,1988,9(2)：138.
112.王冬梅,代世元,芦丽莉,等.瓜蒌皮提取物对大鼠动脉粥样硬化保护作用的实验研究[J].北华大学学报自然科学版,2008,9(2)：128-131.
113.卢丽莉,王冬梅.瓜蒌皮提取液对实验性高脂血症大鼠血清NO、SOD、MDA的影响[J].北华大学学报(自然科学版),2008,9(5)：423-425.
114.上海瓜蒌协作组,瓜萎注射液鉴定书,上海,1976.
115. Kimura M.麝香中新的蛋白激酶活化剂及对豚鼠心肌强心作用的研究[J].国外医学·植物药分册,1992,14(1)：57.
116.黄正良.麝香的药理作用及临床应用研究[J].中成药研究,1987,(5)：23-25.
117.胡秉龙等.麝香研究新进展[J].中医药信息,1997,2：11.
118.尹士敏,王士贤.麝香的药理作用及临床研究近况[J].天津药学,2002,14(3)：42-44.
119.朱秀媛.人工麝香的药用潜力[J].百科知识,1995,(7)：10-11.
120.周元风,胡永兵,洪浪.麝香保心丸对动脉粥样硬化兔血脂及hs-CRP的影响[J].实用临床医学,2013,14(4)：10-11.
121.李天奇,李勇,范维琥,等.麝香保心丸和辛伐他汀对兔股动脉粥样硬化斑块稳定性的影响[J].中华老年心血管病杂志,2006,8(5)：296-299.
122.李天奇,范维琥,李勇,等.麝香保心丸减少兔股动脉斑块内血管新生机制探讨[J].中西医结合心脑血管病杂志,2009,7(6)：680-682.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |