红景天和麝香保心丸对兔动脉粥样斑块稳定性的影响及机制研究

【作者】 李天奇；

【导师】 范维琥； 李勇； 罗心平；

【作者基本信息】 复旦大学 ， 内科学， 2006， 博士

【摘要】 目的：1、通过高脂饲料喂养和球囊扩张术建立兔股动脉粥样硬化模型，了解以此建立模型的大体形态，以及显微镜下的细胞组成情况。2、研究具有促进血管生成作用的中药麝香保心丸、红景天和调脂药物辛伐他汀（作为阳性对照药物）干预对于兔股动脉粥样硬化斑块内新生血管、细胞外基质代谢、炎症反应以及血脂水平的影响。3、比较红景天水煎剂和红景天苷悬浊液在相同红景天苷浓度下对兔股动脉粥样斑块内新生血管、细胞外基质代谢、炎症反应以及血脂水平的影响。通过研究两者的药效在本模型上的差异，为中草药红景天应用的定量化和标准化探索途径。 方法：1、动物造模：50只健康雄性新西兰兔以高脂饲料喂养2周后，行球囊扩张术损伤股动脉内膜建立动脉粥样硬化模型。2、实验分组和给药方法：将动物随机分为对照组（10只）、红景天组（10只）、红景天苷组（10只）、麝香保心丸组（10只）和辛伐他汀组（10只）。继续喂养高脂饲料4周。干预组同时给予药物灌胃4周。红景天制成水煎剂（浓度为0.5g／ml），灌胃剂量为1ml／kg每天；测定红景天水煎剂中红景天苷含量为0.173mg／g，将1％的红景天苷干粉与蒸馏水配成红景天苷浓度同样为0.173mg／g的悬浊液，灌胃剂量也是1ml／kg每天；麝香保心丸给药剂量为一粒（22.5mg）每只每天；辛伐他汀碾碎后配成悬浊液，灌胃剂量是2.5mg／kg每天。3、血脂测定，分别于实验第1周、第3周以及第7周处死前采血。采血前禁食：选择耳缘静脉采血。测定血清总胆固醇、甘油三酯、低密度脂蛋白和高密度脂蛋白。4、实验第7周采用酶联免疫吸附法（ELISA）测定血清基质金属蛋白酶-3（MMP-3）、血清白介素6（IL-6）和血清超敏C反应蛋白（hsCRP）。5、用病理学染色和免疫组化染色定量兔股动脉粥样硬化斑块中CD34、血管内皮生长因子（VEGF）、MMP-3、胶原、增殖细胞核抗原（PCNA）、单核细胞趋化蛋白-1（MCP-1）、糜酶（chymase）和α平滑肌肌动蛋白（αSMA）的阳性面积。6、以实时荧光PCR检测各组粥样斑块中的VEGFmRNA含量。7、检测结果用（?）±S表示，各组间差异比较用单因素方差分析中的多重比较q检验。SPSS10.0软件进行统计分析。 结果：1 一般情况和粥样斑块模型的大体观察 整个实验过程中共死亡兔子10只。实验结束时动物数目为：对照组8只，麝香保心丸组8只，红景天组6只，红景天苷组9只，辛伐他汀组9只。可见股动脉球囊扩张处有较大的灰白色斑块形成；光镜下看到斑块包含泡沫细胞、胆固醇结晶、平滑肌细胞和胶原组织等人类粥样斑块主要成分。2 斑块内新生血管情况 所有药物干预组的新生血管面积（用CD34阳性染色面积表示）都比对照组显著减少（辛伐他汀组vs对照组：15.24±3.34％ vs 20.39±5.96％，P＜0.01；红景天组vs对照组：10.99±3.59％ vs 20.39±5.96％，P＜0.01；麝香保心丸组vs对照组：13.85±2.97％ vs 20.39±5.96％，P＜0.01）；红景天苷也有类似的作用（13.28±4.01％ vs 20.39±5.96％，P＜0.01），与红景天组比较无显著性差异。同样药物干预组的VEGF阳性面积比对照组明显减少（辛伐他汀组vs对照组：13.35±7.27％ vs 20.14±6.15％，P＜0.01；红景天组vs对照组：11.43±3.41％ vs 20.14±6.15％，P＜0.01；麝香保心丸组vs对照组：15.23±2.93％ vs 20.14±6.15％，P＜0.01），并以红景天组减少最为显著。红景天苷也相仿（13.59±4.74％ vs 20.14±6.15％，P＜0.01），与红景天组比较没有显著差别。两个中药干预组斑块内VEGF的mRNA拷贝数不比对照组减少(红景天组vs对照组：1.38×10^-2 vs 6.07×10^-3；麝香保心丸组vs对照组：2.00×10^-2 vs 6.07×10^-3)。3 斑块内细胞外基质代谢情况 药物干预组的胶原面积都比对照组显著减少（辛伐他汀组vs对照组：12.54±2.82％ vs 20.02±6.11％，P＜0.01；红景天组vs对照组：16.99±6.00％ vs 20.02±6.11％，P＜0.05；麝香保心丸组vs对照组：11.35±1.87％ vs 20.02±6.11％，P＜0.01），其中又以麝香保心丸组下降最为明显；红景天苷也有降低胶原作用（15.65±1.97％ vs 20.02±6.11，P＜0.01），与红景天组差别无显著性。辛伐他汀组和麝香保心丸组粥样斑块的α平滑肌肌动蛋白抗体阳性面积都与对照组相仿；而红景天组则显著减少（红景天组vs对照组：10.34±1.62％ vs 14.44±2.76％，P＜0.01）；红景天苷也有降低作用（11.94±2.84％ vs 14.44±2.76％，P＜0.01）；但该组降低程度不如红景天组，这种差别具有显著性（P＜0.05）。辛伐他汀组和红景天苷组的斑块内的PCNA阳性面积比对照组明显减少（辛伐他汀组vs对照组：8.86±5.60％ vs 15.07±5.66％，P＜0.01；红景天苷组9.42±4.16％ vs 15.07±5.66％，P＜0.01）；红景天苷组和红景天组比较有显著性差异（P＜0.05）。各药物干预组斑块内的MMP-3阳性面积都比对照组明显减少（辛伐他汀组vs对照组：11.24±2.81％ vs 16.30±5.96％，P＜0.01；红景天组vs对照组：11.89±4.29％ vs 16.30±5.96％，P＜0.01；麝香保心丸组vs对照组：11.52±4.97％ vs 16.30±5.96％，P＜0.01；红景天苷组vs对照组10.51±2.34％ vs 16.30±5.96％，P＜0.01）；红景天组和红景天苷组比较没有显著性差别。有两种药物能显著下降血清MMP-3水平（辛伐他汀组vs对照组：8.38±19.44mg／L vs 21.01±17.22mg／L，P＜0.01；红景天组vs对照组：12.43±9.32mg／L vs 21.01±17.22mg／L，P＜0.05）。红景天苷组和红景天组比较有显著性差别（P＜0.05）。4 与斑块炎症相关的指标 除了红景天苷（8.05±4.14％ vs 12.21±2.59％，P＜0.01）外，其他药物降低斑块内MCP-1的作用都不显著；红景天苷与红景天组比较有显著性差异（MCP-1，P＜0.01）。所有药物都能减少斑块内的chymase阳性面积（辛伐他汀组vs对照组：9.74±2.11％ vs 12.53±2.78％，P＜0.01；红景天组vs对照组：9.13±1.95％ vs 12.53±2.78％，P＜0.01；麝香保心丸vs对照组：6.23±3.71％ vs 12.53±2.78％，P＜0.01；红景天苷组vs对照组：5.57±2.13％ vs 12.53±2.78％，P＜0.01）；红景天苷组与红景天组比较差别达到显著性（P＜0.01）。所有药物都没有显著降低血IL—6和CRP的水平。5 血脂 除辛伐他汀能显著降低血清总胆固醇、LDL-C和甘油三酯外，麝香保心丸和红景天都能显著降低血清LDL-C水平（麝香保心丸组vs对照组：6.19±14.34 mmol／L vs 12.88±9.82 mmol／L，P＜0.01；红景天组vs对照组8.87±4.00 mmol／L vs 12.88±9.82 mmol／L，P＜0.05），红景天还可以显著降低血清总胆固醇（10.11±3.59 mmol／L vs 20.32±6.33mmol／L，P＜0.01）；两种中药对甘油三酯都无降低作用。红景天苷对血脂的影响没有达到显著性，该组的血清总胆固醇水平与红景天组比较有显著性差异（19.34±11.44 vs 10.11±3.59 mmol／L，P＜0.01）。 结论： 1、采用高脂饲料喂养结合球囊扩张术建立的动脉粥样硬化模型，包含人类粥样斑块的基本组成。 2、红景天和麝香保心丸都能减少粥样斑块内血管新生，这种作用可能和促进斑块内VEGF的分解有关；抑制血管新生的部分直接机制是抑制肥大细胞的活性，部分间接机制是对斑块内基质的影响如减少斑块内MMP-3和抑制平滑肌增殖和转化。两种中药减少斑块内MMP-3的作用有利于斑块的稳定，机制包括抑制平滑肌增殖以及转化、抑制chymase活性和降低血清LDL-C等。两种中药对粥样斑块内胶原和MMP-3含量的净效应与降低血清LDL-C有关；对于斑块内平滑肌细胞的增殖和转化，MCP-1含量以及肥大细胞活性的影响似乎有降低LDL-C以外的其他机制参与。 3、红景天主要通过红景天苷对粥样斑块发生的作用有：减少粥样斑块内新生血管和VEGF含量，抑制斑块内胶原生成和MMP-3活性，以及下降血清LDL-C。两种药物有差异的作用包括：对平滑肌增殖和转化的影响，对MCP-1的作用，对肥大细胞以及chymase的作用，还有下降血清VLDL-C的作用。产生这些差异的原因，考虑一是红景天含有其他与红景天苷药理作用不同的组成（尤其是显著下降血清VLDL-C的成分）；二是红景天所含的其他成分直接拮抗红景天苷。更多还原

【Abstract】 Objective: 1. To get acquainted with establishment of rabbit arteriosclerotic model through high-fat diet and femoral artery angioplasty operation. To observe morphology of arteriosclerotic plaque so as to analyze its component such as foam cells,extracellular cholesterol crystal,vascular smooth muscle cells,and collagen.2. To investigate effects of simvastatin, Rhodiola rosea, She Xiang Bao Xin Wan（SXBXW） ,and Salidroside（all as angiogenesis inducing agents） on angiogenesis, extracellular matrix,and inflammation response in the arteriosclerotic plaque . Blood lipid changes are also examined.3. To compare effects of Rhodiola rosea and Salidroside on angiogenesis ,extracellular matrix,and inflammation response in the arteriosclerotic plaque and blood lipid changes. To try finding a way of standardizition and quantification of usage of Rhodiola rosea.Methods: 1. Animal models: 50 Newsealand Rabbits were feed with high-fat diet for 2 weeks. Then femoral artery angioplasty operation were performed. 2. Grouping and medicine administration: All rabbits were divided into: control group ,simvastatin group, Rhodiola rosea group, SXBXW group ,and Salidroside group.All rabbits were fed high-fat diet for another 4 weeks. Rabbits of intervention groups were fed together with medicines for 4 weeks. The dose of Rhodiola rosea （decoction with concentration of 0.5g/ml） intragastric administration was 1 ml/kg everyday; The intragastric dose of Salidroside suspension was also 1 ml/kg everyday（by mixing 1% salidroside powder with distilled water with the same concentration of salidroside as in the Rhodiola rosea decoction as 0.173mg/g） ; rabbits of SXBXW group were given 1 pills everyday;simvastatin pills were scrunched and made suspension with the administration dose of 2.5mg/kg everyday.3. Blood lipid examination:Blood total cholesterol,LDL-C,HDL,and triglyceride were determined in 1^st week,3^rd week,and 7^th week.4. Serum MMP-3,IL-6,and hsCRP were determined in 7^th week by ELISA.5. CD34,VEGF,MMP-3 ,collagen ,PCNA,MCP-1,chymase,and α SMA positive area in the arteriosclerotic plaque were analyzed and quantification by means of immunohistochemistry techniques. 6.VEGF mRNA in arterosclerotic plaque were examed by realtime PCR.7. The results were express with X|- ± S, and the difference among the groups was tested by q test （multiple comparisons of one-factor analysis of variance）. SPSS10.0 was used in the data processing.Results: 1 General obersvation and description of arteriosclerotic plaque model: 10 rabbits died during the 7 weeks experiment. At the end of the experiment,the number of rabbits of each group was: control group-8 ,simvastatin group-9, Rhodiola rosea group-6, SXBXW group-8 ,and Salidroside group-9. Big grey plaques could be seen in the femoral artery and foam cells,extracellular cholesterol crystal,vascular smooth muscle cells,and collagen could be found under microscope.2 Angiogenesis in the arteriosclerotic plaque: CD34 positive areas in arteriosclerotic plaques of All medicine intervention groups were decreased（simvastatin vs cotrol:15.24±3.34% vs 20.39 ± 5.96%, P<0.01; Rhodiola rosea vs cotrol: 10.99 ± 3.59% vs 20.39 ± 5.96%, P<0.01; SXBXW vs cotrol: 13.85 ± 2.97% vs 20.39±5.96%, P<0.01; Salidroside vs control: 13.28 ± 4.01% vs 20.39 ± 5.96%, P<0.01） . VEGF positive areas in arteriosclerotic plaques of all medicine intervention groups were also decreased （simvastatin vs cotrol: 13.35 ± 7.27% vs 20.14 ± 6.15%, P<0.01; Rhodiola rosea vs cotrol: 11.43± 3.41% vs 20.14 ± 6.15%, P<0.01; SXBXW vs cotrol: 15.23 ± 2.93% vs 20.14 ± 6.15%, P<0.01; Salidroside vs control 13.59 ± 4.74% vs 20.14 ± 6.15%, P<0.01） . Rhodiola rosea and Salidroside group didn’t have significant difference on those two results.3. Changes of extracellular matrix in the arteriosclerotic plaque: Collagen positive areas in arteriosclerotic plaques of All medicine intervention groups were decreased （simvastatin vs cotrol: 12.54 ± 2.82% vs 20.02 ± 6.11%, P<0.01; Rhodiola rosea vs cotrol: 16.99 ± 6.00% vs 20.02 ± 6.11%, P<0.05; SXBXW vs cotrol: 11.35 ± 1.87% vs 20.02 ± 6.11%, P<0.01; Salidroside vs control 15.65 ± 1.97% vs 20.02 ± 6.11, P<0.01） . MMP-3 positive areas in arteriosclerotic plaques of all medicine intervention groups were also decreased（simvastatin vs cotrol: 11.24 ± 2.81% vs 16.30 ± 5.96%, P<0.01; Rhodiola rosea vs cotrol: 11.89 ± 4.29% vs 16.30 ± 5.96%, P<0.01; SXBXW vs cotrol: 11.52 ± 4.97% vs 16.30 ± 5.96%, P<0.01; Salidroside vs control: 10.51 ± 2.34% vs 16.30 ± 5.96%, P<0.01） . Rhodiola rosea and Salidroside group didn’t have significant difference on those two results, α SMA positive areas in arteriosclerotic plaque of Simvastatin group and SXBXW group were similar with that of control group. However, Rhodiola rosea and Salidroside could both decease α SMA positive areas （Rhodiola rosea vs cotrol: 10.34±1.62% vs 14.44 ± 2.76%, P<0.01; Salidroside vs control: 11.94±2.84% vs 14.44 ± 2.76%, P<0.01） ,and they had significant difference（P<0.05） .Simvastatin and Salidroside could decrease PCNA positive area in the arteriosclerotic plaque （simvastatin vs cotrol: 8.86 ± 5.60% vs15.07 ± 5.66%, P<0.01; Salidroside vs control 9.42±4.16% vs 15.07 ± 5.66%, P<0.01） ,but Rhodiola rosea and SXBXW could not. The difference between Rhodiola rosea group and Salidroside group was signifibcant （P<0.05） . Two medicines could reduce serum MMP-3 level（simvastatin vs cotrol: 8.38 ± 19.44 mg/L vs 21.01 ± 17.22 mg/L, P<0.01; Rhodiola rosea vs control: 12.43 ± 9.32 mg/L vs 21.01 ± 17.22 mg/L, P<0.05 ） . The difference between Rhodiola rosea group and Salidroside group was signifibcant （P<0.05） .4. Inflammation response in the arteriosclerotic plaque: All medicines could decrease chymase positive areas in the arteriosclerotic plaque （simvastatin vs cotrol: 9.74±2.11% vs 12.53 ± 2.78%, P<0.01; Rhodiola rosea vs control: 9.13 ± 1.95% vs 12.53 ± 2.78%, P<0.01; SXBXW vs control: 6.23 ± 3.71% vs 12.53±2.78%, P<0.01; Salidroside vs control: 5.57±2.13% vs 12.53±2.78%, P<0.01） . Salidroside could decrease MCP-1 positive areas （8.05±4.14% vs 12.21 ±2.59%, P<0.01）, but the other medicines could not. P<0.01) .Salidroside and Rhodiola rosea group had significant difference on those two results （P<0.01） . All medicines couldn’t reduce serum IL-6 and CRP signicantly.5. Blood lipid: Simvastatin could reduce serum total cholesterol ,LDL-C,HDL and triglyceride significantly. Rhodiola rosea and SXBXW could reduce LDL-C（SXBXW vs control: 6.19±14.34 mmol/L vs 12.88 ± 9.82 mmol/L, P<0.01; Rhodiola rosea vs control :8.87±4.00 mmol/L vs 12.88 ± 9.82 mmol/L, P<0.05 ）),and Rhodiola rosea could further reduce total cholesterol （10.11±3.59 mmol/L vs 20.32 ±6.33 mmol/L, P<0.01） . Salidroside had little effect on blood lipid and had significant difference on serum total cholesterol with Rhodiola rosea （19.34±11.44 vs 10.11±3.59 mmol/L, P<0.01） .Conclusions: 1. Rabbit arteriosclerotic plaque model establishe with high-fat diet and femoral artery angioplasty operation contained main cells and matrix components of human lesions.2. Rhodiola rosea and SXBXW could both decrease VEGF,MMP-3, and chymase in the arteriosclerotic plaque,inhibit angiogenesis in the plaque,and reduce serum LDL-C. Those mechanisms might benefit the stability of plaque.3. The effects of inhibition of angiogenesis and production of collagen,decrease of MMP-3 and VEGF in the arteriosclerotic plaque,and reduction of serum LDL-C by Rhodiola rosea may be attributed to salidroside . Rhodiola rosea might have other important components with functions such as increasing MCP-1 and chymase in the arteriosclerotic plaque, affecting proliferation of VSMCs andreducing serum VLDL-C.更多还原