【作者】 陈博；

【导师】 刘玲；

【作者基本信息】 湖北中医药大学 ， 中医内科学， 2014， 博士

【摘要】 研究目的：探讨软坚散结配方干预颈动脉粥样硬化斑块作用的机制。①探讨软坚散结配方对颈动脉粥样硬化（CAS）的颈动脉处粥样硬化斑块病理形态学的影响以及粥样硬化的颈动脉处的内膜、中膜厚度的影响。②探讨软坚散结配方对CAS兔血清内总胆固醇（Total cholesterol,TC）、甘油三酯（Triglyceride,TG）、低密度脂蛋白胆固醇（Low densitylipoprotein,LDL-C）、高密度脂蛋白胆固醇（High-densitylipoprotein,HDL-C）的干预作用影响。③探讨软坚散结配方对CAS的斑块细胞间粘附分子-1（intercellularadhesion molecule-1，ICAM-1）、基质金属蛋白酶-2（matrix metalloproteinase-2，MMP-2）、金属蛋白酶抑制因子-2（tissue inhibitors of metalloproteinase-2，TIMP-2）的免疫组化检测表达水平和MMP-2/TIMP-2表达水平比的影响。④探讨软坚散结配方对CAS的斑块ICAM-1、MMP-2、TIMP-2的蛋白表达水平的影响。⑤探讨软坚散结配方对CAS的斑块ICAM-1、MMP-2、TIMP-2的mRNA的表达水平的影响。研究方法：①理论探讨：从“脉微积”理论探讨CAS的发病机制及治疗，从中医方剂理论和现代医学药理阐述软坚散结配方（制鳖甲、莪术、法半夏、陈皮及葛根按2:1:1:1:1比例配伍研粉均匀混合）防治CAS的立题依据。②新西兰雄性白兔48只随机数字法分为空白对照组（以下简称空白组）、模型组、辛伐他汀组（以下简称他汀组）及软坚散结配方低、中、高剂量组（以下简称低、中、高剂量组）各8只，空白组喂养普通饲料组，余喂养高脂饲料组4周后，除空白组外，其余行颈动脉内膜空气干燥术，术后空白组、模型组喂养普通饲料，低、中、高剂量组分别予0.5、1、2g/kg软坚散结配方放入温水后灌胃及喂养普通饲料。他汀组用辛伐他汀片,按0.94mg/kg剂量灌服及喂养普通饲料。8周后，检测兔血清TG、TC、LDL-C、HDL-C水平，后处死兔子，取右侧颈动脉截取远端，分3部分，一部分为免疫组化样品，一部分为PCR样品，一部分为Western样品。③HE染色检测血管病理形态。通过成像系统检测各组颈动脉内膜、中膜的厚度及其比值。④免疫组化测定各组兔颈动脉斑块处ICAM-1、MMP-2、TIMP-2表达水平。⑤Western实验检测兔颈动脉斑块处ICAM-1、MMP-2、TIMP-2的蛋白表达水平。⑥荧光定量PCR检测各组兔颈动脉斑块处ICAM-1、MMP-2、TIMP-2的mRNA的表达水平。结果：①颈动脉HE染色结果：低剂量组兔颈动脉处均有粥样硬化斑块,内膜下有明显的泡沫细胞堆积，与模型组相比程度减轻，内膜增厚减轻（P＜0.05）,具有统计学意义，中层平滑肌细胞和细胞间质减少，代之明显的泡沫细胞，纤维组织增生，粥样斑块形成，内膜、中膜均可见较多炎性细胞侵润，内膜/中膜厚度比与模型组比较（P＜0.05），具有统计学意义；中剂量组兔颈动脉处均粥样硬化斑块形成，程度轻于低剂量组,内膜下有较多的泡沫细胞堆积，与模型组相比程度明显减轻，内膜增厚减轻（P＜0.01）,具有统计学意义，中层平滑肌细胞和细胞间质减少，代之少量的泡沫细胞，纤维组织增生，粥样斑块形成，内膜、中膜均可见炎性细胞侵润，内膜/中膜厚度比与模型组比较（P＜0.05），具有统计学意义；他汀组、高剂量组兔颈动脉处内膜均有粥样硬化斑块形成，程度轻于中剂量组,内膜下极少量的泡沫细胞堆积，与模型组相比程度明显减轻，内膜增厚减轻（P＜0.01）,具有统计学意义，中层平滑肌细胞和细胞间质减少，代之极少量的泡沫细胞，纤维组织增生，粥样斑块形成，内膜、中膜均可见散在的炎性细胞侵润，内膜/中膜厚度比与模型组比较（P＜0.01），具有统计学意义。高、中、低剂量组及他汀组与模型组CAS家兔血清内TC、TG、LDL-C水平相比较降低，而HDL-C水平升高（低剂量组P＜0.05，高、中剂量组及他汀组P＜0.01）。中、高剂量组与他汀组比较，HDL-C水平升高（P＜0.05）。②颈动脉处粥样斑块免疫组织化学染色结果：与模型组比较，高、中、低剂量组及他汀组细胞内ICAM-1、MMP-2免疫沉淀物积分吸光度(IA)值以及MMP-2/TIMP-2比值均明显低于模型组，而TIMP-2免疫沉淀物积分吸光度(IA)值显著高于模型组，差异显著，具有统计学意义（高、中剂量组及他汀组P＜0.01，低剂量组P＜0.05）。③软坚散结配方高、中、低剂量组及他汀组与模型组比较颈动脉处动脉粥样斑块ICAM-1、MMP-2蛋白表达度均有明显降低，TIMP-2蛋白表达均明显升高（高、中剂量组及他汀组P＜0.01，低剂量组P＜0.05），软坚散结配方高、中、低剂量组随着药物剂量加大，ICAM-1、MMP-2蛋白表达降低，TIMP-2蛋白表达均明显升高，经统计学处理，具有统计学意义（P＜0.05）。④高、中、低剂量组及他汀组与模型组比较，颈动脉处粥样硬化斑块的ICAM-1、MMP-2的mRNA表达水平均降低（高、中剂量组及他汀组P＜0.01，低剂量组P＜0.05），而TIMP-2蛋白表达显著高于模型组，差异显著，具有统计学意义（高、中剂量组及他汀组P＜0.01，低剂量组P＜0.05）。高、中、低剂量组随着药物剂量加大，组间比较颈动脉斑块ICAM-1、MMP-2的mRNA表达水平也降低而TIMP-2的mRNA水平升高有显著差异，经统计学处理，具有统计学意义（P＜0.05）。结论：①软坚散结配方高、中、低剂量组及他汀组与模型组相比较，能明显降低颈动脉内膜厚度及内膜/中膜的比值，具有统计学意义，说明软坚散结配方能够显著改善CAS的动脉粥样硬化程度，具有消退颈动脉斑块的作用。②软坚散结配方高、中、低剂量组均能降低CAS家兔血清中TG、TC及LDL-C水平，而在升高HDL-C血清水平方面，与他汀组相比，具有潜在的优势。推论软坚散结配方通过调节血浆血脂水平，特别是升高HDL-C血清水平方面起到祛除痰浊从而达到抑制和延缓CAS斑块进展作用。③软坚散结配方各组能降低CAS的颈动脉处粥样硬化斑块ICAM-1、MMP-2免疫沉淀物表达，而强化TIMP-2免疫沉淀物表达。说明软坚散结配方可以通过滋阴熄风，软坚散结，化痰消积治法，从而达到痰浊瘀血消失，“脉微积”消失，现代作用机理可以通过降低ICAM-1、MMP-2而强化TIMP-2免疫沉淀物表达水平，干预MMP-2/TIMP-2免疫沉淀物表达平衡，从而达到干预CAS粥样硬化斑块形成、促进斑块稳定性和防治斑块进一步发展的可能。④软坚散结配方各组均能明显降低颈动脉粥样斑块中ICAM-1、MMP-2蛋白的表达水平，强化颈动脉AS斑块TIMP-2的蛋白表达，从而推论软坚散结配方可以通过降低CAS中颈动脉处粥样硬化斑块中的ICAM-1、MMP-2的蛋白表达，强化TIMP-2的蛋白表达而起到抑制颈动脉硬化斑块的形成及促进粥样斑块的稳定性。在一定的范围内成正相关性。这与中医脾胃升降气机，使浊气降清气升恢复平衡，使“脉微积”消散机理相似。⑤从实验结果分析可以看出软坚散结配方通过降低颈动脉硬化斑块中ICAM-1、MMP-2的mRNA的表达水平，同时强化斑块中TIMP-2的mRNA的表达水平来干预CAS的硬化斑块的形成、延缓斑块的炎性进展及促进斑块的稳定性，其效果与药物的剂量在一定的范围内成正相关性，与中医调整心火和肾水的制衡，心火和肝木的相生有相似之处。更多还原

【Abstract】 Objective：Reaserch of mechanism on Ruanjiansanjie formula in treatmentof carotid atherosclerotic(CAS) plaques.①To observe theinfluence of Ruanjiansanjie formula on the pathological morphologyand intcima-media thickness （IMT） in carotid arteryatherosclerotic （AS） plaques of rabbits.②To observe theinfluence of Ruanjiansanjie formula Plasma Total cholesterol （TG）Triglyceride （TC）、High-density lipoprotein （HCL-C） and Lowdensity lipoprotein （LCL-C） levels of each rabbits CAS model.③To observe the influence of Ruanjiansanjie formula on theimmunohistochemical detection expression lever of intercellular-adhesion molecule-1(ICAM-1)、matrix metalloproteinase-2(MMP-2)、tissue inhibitors of metalloproteinase-2(TIMP-2) and the ratio ofMMP-2/TIMP-2in AS plaques of rabbits.④To observe the influenceof Ruanjiansanjie formula on the protein expression lever ofICAM-1、MMP-2and TIMP-2in AS plaques of rabbits.⑤To observe theinfluence of Ruanjiansanjie formula on the expression mRNA leverof ICAM-1、MMP-2and TIMP-2in AS plaques of rabbits. Methods：①Theoretical discussion：To investigate the athogenesisand treatment of CAS from “micro mass of vascular” of TraditionalChinese medicine theory. and modern medical research，to expatiatetheory of Runjiansanjie formula in establishing the project basisfor the prevention and control of CAS from the prescriptions ofTraditional Chinese medicine theory and modern pharmacologicalresearch of medicine，which is the mixing powder from turtle shell、curcuma zedoary、the tuber of pinellia、dried tangerine and theroot of kudzu vine according to the proportion of2:1:1:1:1.②48male New Zealand white rabbits were randomly divided into the blankcontrol group（the blank group）+the model group+the simvastatingroup（the statin group）+the low dosage of Ruanjiansanjie formulagroup（the low dosage group）+the medium dosage of Ruanjiansanjieformula group（the medium dosage group）+the high dosage ofRuanjiansanjie formula group（the high dosage group）,8rabbitsin each group.Rabbits in the blank group were put on a normal diet，and others were fed with a high-fat diet.After4weeks Carotidartery intima air drying operation except the blank group anddifferent interventions，the blank group and the model group wereput on a normal diet，Rabbits in the low dosage group、the mediumdosage group and the high dosage group were put on0.5,1,2g/kgRuanjiansanjie formula with the warm water and a normal dietrespectively， the statin group were put on simvastatintablets according to0.94mg/kg dose and a normal diet. Eight weekslater,to detect the serum lipid levels of TG, TC, HDL-C and LDL-C,executed after the rabbit, take the right carotid artery tointercept the distal, three parts, part for immunohistochemicalsamples, part of the samples for PCR, part of Western immunohistochemical fixed sample, sample extraction of RNA PCRsamples, measured concentration. Western protein concentrationafter the extraction of total protein test sample.③Organizationafter dehydration, transparent, paraffin embedding, sectioning, HEdyeing photo records. Imaging system tests each carotid intima, thefilm thickness.④Immunohistochemical detection of each group testICAM-1、MMP-2and TIMP-2expression level in Carotid plaques.⑤Western experimental testing, ICAM-1、 MMP-2and TIMP-2proteinexpression level in Carotid plaques.⑥Fluorescence quantitativePCR detection between groups, ICAM-1、MMP-2and TIMP-2of the mRNAexpression level value in Carotid plaques.Results:①The results of carotid artery HE staining:the low dosage groupof rabbits reduce atherosclerotic plaque formation, lining undera significant accumulation of foam cells, compared with the modelgroup can alleviate, intimal thickening reduce (P <0.05),sistati-cally significant, the middle of smooth muscle cells and stromalcells decreased, generation of obvious foam cells and fibroustissue hyperplasia, atheromatous plaque formation, lining, in thefilm are seen more inflammatory cells invasion, lining/in the filmthickness ratio compared with model group (P <0.05), statisticallysignificant; the medium dosage group of rabbits reduce AS plaqueformation, the degree of light in low dose group, lining under morefoam cell accumulation, compared with the model group was signi-ficantly reduced, intimal thickening reduce (P <0.01),statisti-cally significant, the middle of smooth muscle cells and stromalcells, instead of a small amount of foam cells and fibrous tissuehyperplasia, atheromatous plaque forma-ation, lining, membrane are visible in the inflammatory cells invasion, film thickness thanin endometrium/compared with model group (P <0.05), statisticallysignificant; the statin group and the high dosage group of rabbitcarotid intima are both in AS plaque formation and the degree oflight in the medium dose group, lining under extremely small amountof foam cell accumulation, compared with the model group was signi-ficantly reduced, intimal thickening reduce (P <0.01),statisti-cally significant, The high、 medium、low dosage group and statingroup are lower level of CAS in rabbit serum TC, TG, LDL-C comparedto the model, elevated levels of HDL-C (the low dosage group, P<0.05; the high dose group、the medium and the statin group,P<0.01). the high and medium dosage group are higher compared withstatin group about improving HDL-C levels (P <0.05)②Carotidartery AS plaque immunohistochemical staining results: Comparedwith model group, high, medium and low dose group of intracellularICAM-1, MMP-2positive cells expression level and MMP-2/TIMP-2ratio are significantly lower than the model group, and TIMP-2positive cells expression is significantly higher than model group,significant difference, statistically significant (P <0.01, highdose group, low dose group (P <0.05).③Ruanjiansanjie formula ofhigh, medium and low dose group AS compared with model group carotidartery plaque ICAM-1, MMP-2protein expression were significantlydecreased, TIMP-2protein expression were significantly higher (P<0.01, high dose group, low dose group (P <0.05), Ruanjiansanjieformula of high, medium and low dose group with increasing dose ofdrug, ICAM-1, MMP-2protein expression decreased, TIMP-2proteinexpression were significantly elevated, after statistics process-ing, with statistical significance (P <0.05).l④High, medium and low dose group and statin group compared with model group, thecarotid artery AS plaques, MMP-2、ICAM-1mrna expression levelswere lower (P <0.01, high dose group, low dose group (P <0.05),and TIMP-2protein expression was significantly higher than thatof model group, significant difference, statistically significant(P <0.01, high dose group, low dose group (P <0.05). High, mediumand low dose group with increasing dose of drug, carotid arteryplaque is compared between groups, MMP-2、ICAM-1mRNA expressionlevel decreased and TIMP-2mRNA levels have significant differenceafter statistics processing, with statistical significance (P <0.05).Conclusion：①The high, medium、 low dosage group of Ruanjiansanjie formulaand the statin group can obviously decrease the thickness of thecarotid artery intima and the ratio of membrane compared with themodel group, membrane/statistically significant, so thatRuanjiansanjie formula formula can significantly reduce the degreeof hardening of early atherosclerosis of CAS and has the effectof removing carotid plaques.②Ruanjiansanjie formula of the high、medium、 low dosage group all with statins can reduce the CAS rabbitserum TG, TC and HDL levels, and elevated LDL in serum levels. Inrespect of higher HDL-C serum levels, compared with the statingroup, has a potential advantage. Corollary Ruanjiansanjie formulaby regulating serum lipid levels, especially higher HDL-C serumlevels rise to dispel phlegm turbidity to inhibit and delay the CASplaque progression.③Ruanjiansanjie formula of the high、 medium、low dosage group can reduce the carotid artery of CAS, the AS plaqueImmune deposits expressing of ICAM-1、 MMP-2, and strengthen the deposits expressing of TIMP-2. Ruanjiansanjie formula can nourishYin extinguish wind, soft firm fights, phlegm elimination treatment,so as to achieve phlegm turbid blood stasis disappear, disappearcalculus of arteries and veins, modern mechanism can reduceimmunohistochemical deposits expressing of ICAM-1、 MMP-2andimprove immunohistochemical deposits expressing TIMP-2,intervention of MMP-2/TIMP-2immunohistochemical depositsexpression of balance, so as to achieve intervention CASatherosclerotic plaque formation and to intervene the CASatherosclerotic plaque formation, promote plaque stability, andcontrol the possibility of further development.④Ruanjiansanjieformula between groups can obviously decrease carotid atheromatousplaque in the expression of ICAM-1protein level, strengthen thecarotid artery AS plaque TIMP-2protein expression, deduce thatRuanjiansanjie formula can reduce carotid atherosclerotic plaquesin CAS ICAM-1, the protein expression of MMP-2, improved TIMP-2protein expression and inhibiting the formation of carotidatherosclerotic plaques. With the TCM spleen qi activity, make thematerial drop, clear Qi lift to restore balance mechanism ofdissipate " micro mass of vascular " is similar.⑤Ruanjiansanjieformula of high, medium and low dose group can obviously reduceICAM-1and carotid atherosclerosis plaque in mRNA expression ofMMP-2, at the same time can be significantly improved carotidplaques in the mRNA expression of TIMP-2levels, can be seen fromthe analysis of experimental results is Ruanjiansanjie formulaformula by reducing carotid atherosclerosis plaque in the mRNAexpression of ICAM-1and MMP-2levels, at the same time, improvedplaques TIMP-2mRNA expression level of intervention of CAS hardened plaque formation and delay the progress of inflammatorypatches. It is similar to adjust XinHuo and ShenShui of TCM torestrict or XinHuo and GanMu of TCM to beget.更多还原