活血解毒中药干预动脉粥样硬化易损斑块的作用及机理研究

【作者】 周明学；

【导师】 陈可冀； 徐浩；

【作者基本信息】 中国中医科学院 ， 中西医结合临床， 2008， 博士

【摘要】 目前,动脉粥样硬化（AS）易损斑块在导致急性心血管事件方面的重要性已受广泛重视。改善易损斑块内部成分,使其趋于稳定,成为防治急性心血管事件的重要内容。随着“AS炎症反应”学说的公认,为中医产生新的冠心病病机学说、更新治疗观念提供了良好契机。我们在既往动物实验研究的基础上,提出“活血解毒——抑制炎症反应——稳定斑块”的假说,并从基础研究和临床试验两个方面展开研究。1.基础研究:活血解毒中药对ApoE基因敲除(ApoE^-/-)小鼠斑块稳定性的影响及机制探讨。目的:观察活血（三七总皂苷）、解毒（黄连提取物）、活血解毒（虎杖提取物、大黄醇提物）中药有效部位对ApoE^-/-小鼠主动脉粥样斑块稳定性的影响,并从抑制炎症反应、调节脂质代谢和改善胶原沉积及代谢三个角度探讨其作用机制。方法:70只ApoE^-/-小鼠经“西方膳食”饲料喂养13周后,随机取4只小鼠观察基础AS病变水平。其余小鼠随机分为6组:虎杖提取物组、大黄醇提物组、黄连提取物组、三七总皂苷组、辛伐他汀组、模型组各11只,并以同品系C57BL/6J小鼠10只作为正常对照。继续高脂喂养并按体重比折算的临床推荐剂量给予相应药物治疗13周后,处死取主动脉根部的4个切面,分别行HE染色、Movat染色、苦味酸天狼星红染色以及免疫组织化学染色法观察斑块内部成分,同时取血清用酶联免疫吸附法（ELISA）检测炎性相关指标,实时荧光定量PCR法检测小鼠主动脉内相关基因表达,观察并计算:1)主动脉斑块形态变化:包括斑块面积,细胞外脂质、泡沫细胞、胶原成分各自占斑块面积的百分比以及斑块内脂质成分与胶原成分比值,埋藏纤维帽数目以及纤维帽占斑块表层比值,并结合免疫组化染色半定量观察斑块内平滑肌细胞的阳性面积,最后采用易损指数综合评价药物对小鼠主动脉斑块稳定性的影响;2)血脂改变及对脂质代谢相关基因周脂素和清道夫受体CD36的基因表达影响;3)炎性指标:血清超敏C反应蛋白（hs-CRP）和可溶性白细胞分化抗原配体（sCD40L）水平、斑块内粒细胞-巨噬细胞集落细胞刺激因子（GM-CSF）和肿瘤坏死因子α（TNF-α）的表达、过氧化物酶体增殖物激活受体γ（PPAR-γ）和核转录因子-κB（NF-κB）的基因表达;4)胶原的合成代谢:Ⅰ、Ⅲ型胶原的组成、基质金属蛋白酶9（MMP-9）及组织金属蛋白酶抑制剂1（TIMP-1）的表达。结果:（1）无论是在改善斑块易损指数方面,还是在修复斑块纤维帽的能力方面,活血解毒中药虎杖提取物及大黄醇提物组,均优于单纯活血中药三七总皂苷和单纯解毒中药黄连提取物组。综合评价药物稳定斑块的作用,上述药物表现出的趋势是活血解毒中药＞清热解毒中药＞活血化瘀中药;（2）虎杖提取物和大黄醇提物还可明显降低ApoE^-/-小鼠血清炎症标志物hsCRP及sCD40L水平,优于活血对照药三七总皂苷和解毒对照药黄连提取物,这与稳定斑块的作用相平行。各中药组对斑块内炎性因子GM-CSF表达均有一定抑制作用,大黄醇提物还可明显抑制TNF-α表达,而黄连提取物组则明显促进斑块内PPAR-γmRNA和蛋白表达;（3）各中药组可不同程度调节ApoE^-/-小鼠血清脂蛋白水平,减少斑块内细胞外脂质成分,其中大黄醇提物、黄连提取物还可明显抑制斑块内perilipin mRNA表达,但各中药组清道夫受体CD36的基因表达无显著影响;（4）各中药组均可明显增加斑块内胶原纤维成分,降低Ⅲ/Ⅰ型胶原比值,其中虎杖提取物、三七总皂苷、黄连提取物组MMP-9/TIMP-1比值亦明显降低。结论:活血、解毒及活血解毒中药有效部位三七总皂苷、黄连提取物、虎杖提取物、大黄醇提物在临床推荐剂量上均可通过改善斑块内部成分发挥稳定易损斑块的作用,但其作用环节有所差异。其中,兼有活血和解毒作用的中药虎杖提取物、大黄醇提物效果最为显著。其机制可能与抑制炎症反应、调节脂质代谢和改善胶原沉积及代谢有关,尤以抑制炎症反应为最主要机制,验证了“活血解毒——抑制炎症反应——稳定斑块”的假说。2.临床研究:活血解毒中药对冠心病稳定期患者血清炎症标记物的影响。目的:以新清宁片（熟大黄）为活血解毒中药的代表药物、以丹七片为活血对照药,观察在常规他汀类药物治疗基础上加用新清宁或丹七片对冠心病稳定期患者血清炎症标志物的影响。方法:将30例冠心病稳定期患者随机分为三组:他汀对照组,予他汀类降脂药常规治疗;活血中药治疗组,在他汀类药物常规治疗基础上加用丹七片,每次三片（0.3g╱片）,每天3次;活血解毒中药治疗组,在他汀类药物常规治疗基础上加用新清宁片,每次三片（0.3g╱片）,每天3次。每组10例。治疗1个月后,观察患者治疗前后血清hs-CRP、TNF-α、MMP-9和CD40L浓度变化,血脂水平,心绞痛症状和血瘀证积分变化。结果:无论是降低冠心病患者血清hs-CRP浓度、调节血脂还是降低患者心绞痛症状和血瘀证积分方面,活血解毒中药治疗组（他汀药常规治疗加用新清宁片）均表现出良好作用,优于他汀药对照组及活血中药治疗组（他汀药常规治疗加丹七片）。但各组对血清TNF-α、MMP-9和CD40L水平影响无显著差异。结论:对于稳定期冠心病患者,他汀药常规治疗加用活血解毒中药在抗炎、调脂等方面优于加用活血中药,与实验研究结果一致,为“活血解毒——抑制炎症反应——稳定斑块”的假说提供了进一步支持。虽然在改善心绞痛症状和血瘀证积分方面,加用活血解毒药亦比加用活血中药作用明显,但本研究仅为小样本预试验,在急性心血管事件预防方面有无更大获益尚需大样本、长期随访的临床试验加以证实。更多还原

【Abstract】 Nowadays,vulnerable atherosclerotic plaque（VAP） has been highlighted for its role in leading to acute cardiovascular events.Thus,it is regarded as an important strategy to prevent the incidence of acute cardiovascular events by changing the composition of VAP to make it more stable.The theory of inflammation reaction in atherosclerosis（AS） provides innovation of pathogenesis theory and refreshment of therapeutic concept on coronary heart disease（CHD） in view of traditional Chinese medicine（TCM） with a good opportunity.Based on the results of our previous experiment study,we proposed a hypothesis that the method of activating blood circulation and detoxicating（ABCD） could stabilize atherosclerotic plaque by inhibiting inflammatory reaction,and try to confirm it in the following experimental research and clinical study.1.Experimental research:Study on the effect and underlying mechanism of herbs with function of activating blood circulation（ABC）,detoxicating（D） as well as ABCD on plaque stability of ApoE gene-knockout(ApoE^-/-) mice.Objective:To observe the effects of herbal extractives with function of ABC （saponins of Panax pseudo-ginseng,SPP）,D（Coptis chinensis extractive,CCE） and ABCD（giant knotweed rhizome,GKR;rhubarb extractives,RE） on the stability of VAP in the aortic roots of fat-fed ApoE^-/- mice and investigate the mechanisms in views of inhibiting inflammatory reaction,modulating lipid metabolism and improving collagen deposition and metabolism.Methods:Seventy ApoE^-/- mice were fed a high-fat diet for 13 weeks until the mature atherosclerotic plaques formed.Thereafter,they were randomized to six groups（11 in each group） and fed a high-fat diet for subsequent 13 weeks:GKR group,RE group, CCE group,SPP group,simvastatin group（positive control）,and control group.Moreover, another 10 mice（C57BL/6J） was served as a normal control group.The drug-treated groups were medicated with respective drug in a clinically relevant dose for another 13 weeks.Then all the mice were sacrificed at the end of experiment.Their blood was collected for determination of blood lipids and inflammatory indices by Enzyme-Linked Immuno Srbent Assay.The morphology and composition of atherosclerotic plaques in four sections of aortic roots treated with Movat stain,HE stain and Sirius red picro-polarization method and immunohistochemistry stain respectively.The related gene expressions in the aorta of mice were determined by real-time fluorescent quantitative PCR technology.The indices includes:1) The morphology and composition of atherosclerotic plaques in aortic roots:plaque area,the percentage of extracelluar lipids,foam cells and collagen in plaque, the ratio of lipid to collagen content in plaque,the number of buried fibrous caps and the ratio of the length of fibrous cap to plaque intima.The effect on plaque stability was evaluated by using the vulnerability index（area of foam cells and extracellular lipids/area of collagens and smooth muscle cells）;2) The blood lipids level and the gene expressions of perilipin and scavenger receptor CD36 associated with lipid metabolism;3 ) Inflammatory indices:serum high-sensitivity C reaction protein（hs-CRP） and soluble leukocyte differentiation Ag-40 ligand（sCD40L）,the expressions of granulocyte-macrophage colony-stimulating factor（GM-CSF） and tumor necrosis factor-alpha （TNF-a）,the gene expressions of peroxisome proliferator-activated receptor-γ（PPAR-γ） and nuclear factor-kappa B（NF-KB）.4) Collagen metabolism:the ratio of collagenⅢtoⅠin plaque,the protein expressions of matrix metalloprotease-9（MMP-9） and tissue inhibitor-1 of metalloproteinases（TIMP-1）.Results:（1） The herbal extractives with the effect of ABCD（GKR and RE） were significantly superior to ABC group（SPP） and D group（CCE） not only in improving the vulnerability index in plaque,but also in the capability of restoring fibrous cap.The comprehensive effects of the above herbs was manifested as a trend:ABCD＞D＞ABC. （2） Compared with ABC and D group,the concentrations of hsCRP and sCD40L in serum were significantly decreased in ABCD group treated with either GKR or RE,which is parallel to their effects on stabilizing atherosclerotic plaque.All of the above herbs could inhibit the expressions of GM-CSF in plaque.RE also significantly inhibited the protein expression of TNF-a,while CCE could significantly promote the mRNA and protein expression of PPAR-γ.（3） The above herbs could modulate serum lipid level to different degree and diminish extracellular lipids content in plaque.RE and CCE also remarkably inhibit the gene expression of perilipin.However,none of the above herbs showed effect on the gene expression of scavenger receptor CD36;（4） All of the above herbs could significantly increase collagen contents in plaque and reduce the ratio of collagenⅢ/Ⅰ, GKR,SPP and CCE could also reduce the ratio of MMP-9 to TIMP-1.Conclusions:The herbal extractives with function of ABC（SPP）,D（CCE） and ABCD（GKR and RE） could stabilize VAP by changing plaque composition in clinically relevant doses with different mechanisms of action.The herbal extractives with function of ABCD（GKR and RE） showed superior effect on stabilizing VAP and the mechanisms of action might be related to inhibiting inflammatory reaction,modulating lipids metabolism and improving collagen deposition and metabolism.The inhibition of inflammatory reaction as the major mechanism also verified our hypothesis.2.Clinical study:The effects of the herbs with function of ABCD on the serum inflammatory markers in stable CHD patients.Objective:To observe the additional effects of Xin Qing-ning Tablets,（a representative herb with the effect of ABCD,consisting of RE）,or Dan Qi Tablets（as a control with the effect of ABC,consisting of Salvia miltiorrhiza and Panax pseudo-ginseng） on the serum inflammatory markers in stable CHD patients receiving standardized statins treatment.Methods:Thirty stable CHD patients were randomized to three groups（10 in each group）:the control group treated with standardized statins;the ABC group,treated with Dan Qi Tablets in addition to standardized statins treatment;the ABCD group,treated with Xin Qing-ning Tablets based on standardized statins treatment.After one month treatment, the concentrations of hs-CRP、TNF-a、MMP-9 and CD40L in serum of CHD patients before and after treatment were determined.Moreover,the blood lipids level,the scores of angina pectoris and blood stasis syndrome were also examined.Results:The ABCD group showed better effects in reducing the concentration of hs-CRP in serum,modulating blood lipid level,and improving the scores of angina pectoris and blood stasis syndrome,as compared with the control group and the ABC group.As for the concentrations of TNF-a、MMP-9 and CD40L in serum,there were no significant difference among groups.Conclusions:For stable CHD patients,the treatment with the herb with function of ABCD was superior to the treatment with function of ABC based on stabdardized statins treatment in both inhibiting inflammatory reaction and modulating blood lipids,which is consistent with our experimental results.The results also showed the better effect of ABCD group on improving the scores of angina pectoris and blood stasis syndrome as compared to ABC group.However,this is only a small-sample pilot study,whether the herbs with function of ABCD has additional benefit on preventing acute cardiovascular events still needs further demonstration with a large-scale and long-term follow-up clinical trial.更多还原