【作者】 张翼冠；

【导师】 李晓辉；

【作者基本信息】 第三军医大学 ， 药理学， 2008， 博士

【摘要】 背景与目的:心血管疾病每年导致的死亡人数位列各类疾病之首,尤其是动脉粥样硬化(Atherosclerosis, AS)及其并发症不仅占有很大比例,也是大多数心血管疾病的共同病理基础和关键发病环节。既往研究认为血清总胆固醇增高等是致AS的主要因素,但新近临床流行病学调查发现大约50%的AS患者的血脂水平是正常的。全球在努力控制血脂水平的情况下,AS及其他心血管疾病的发病率仍逐年增加,提示有其他重要致病因素存在?最近大规模的临床研究发现,慢性支气管炎、前列腺炎、牙龈炎等慢性炎症性疾病患者AS发病率显著高于正常人群,其缺血性脑中风的发生率也显著高于非感染/炎症患者(48% vs 24%)。另一方面,心血管疾病患者单核细胞趋化蛋白(monocyte chemoattractant protein-1, MCP-1)及C反应蛋白(C-reactive protein, CRP)等炎症因子水平显著升高,并与心血管疾病(即使在血脂水平“正常”的人)发病率也呈显著正相关,这些强烈提示炎症免疫因素在AS形成中可能具有重要作用。中医学根据AS受累部位、病理特征等,认为该病属于“瘀血”范畴,在辨证上多属气虚血瘀,治则上多选用活血化瘀等药物。三七总皂苷(total saponins of Panax notoginseng,PNS)是三七的主要有效成分,研究证明其可降血脂,并对多种心血管疾病尤其是AS有良好的防治作用。运用高通量基因表达谱芯片对药物治疗前后基因表达差异分析,是研究中药多环节、多靶点治疗的有效手段之一。为进一步明确炎症免疫因素诱发AS的信号转导机制以及PNS作用的具体分子靶点,我们用高脂饮食喂饲大鼠的同时叠加酵母多糖(zymosan, Zym)成功建立炎症免疫因素诱发SD大鼠AS模型,利用AS功能分类基因芯片收集PNS作用相关信号转导信息,希望相对明确PNS在防治炎症免疫因素诱发AS过程中所涉及的关键调节基因、蛋白及其功能,从而在一定程度上揭示AS发病机制以及PNS调节机制,也为从中药中筛选控制AS或炎症免疫性疾病新药提供可靠理论依据。方法:1.实验分为对照组、模型组和治疗组3组,分别腹腔注射给予无菌医用液体石蜡、酵母多糖(Zym,20mg/kg,1次/3天)、Zym (20mg/kg,1次/3天)+PNS (100mg/kg,1次/天)。所有大鼠均喂食含3%胆固醇的高脂饲料9周。最后一次给药后3天禁食12小时,腹腔注入l%戊巴比妥钠(30mg/kg)麻醉,从左心室取血,测定血脂水平和血液粘度;解剖后取下整段主动脉,置于液氮保存备用。2.血清总胆固醇和甘油三酯水平测定采用酶法,全血粘度(高切和低切)以及血浆比粘度测定采用旋转法,在全自动血液流变分析仪上进行。电镜分析PNS对病变组织改变作用。3.第二部分实验分对照组、模型组和治疗组3组(对照组喂食正常饲料,其余同第一部分),采用动脉粥样硬化及信号转导通路两种功能分类基因芯片检测免疫炎症因素刺激9周后3组AS病变相关基因表达差异,应用实时定量PCR及Western blot对芯片结果进行验证。4.根据上述结果线索,整合素是PNS调节的关键环节,因此选用LPS、LPS+PNS10μg组、LPS+PNS30μg组、LPS+PNS100μg分别作用于小鼠腹腔巨噬细胞,采用免疫组化方法、Western blot技术检测P397 FAK表达水平。结果:1. Zym组TC、TG水平显著增加,分别是对照组的2.62、5.56倍(P<0.01),经PNS治疗后,TC、TG水平比起对照组明显降低。另外,Zym组刺激后血粘度也显著增加,PNS对其有上述类似效应。电镜结果显示高脂对照组内皮损伤较轻,仅有少量内皮细胞脱落,内皮下未见泡沫化的单核细胞,Zym组动脉壁内皮细胞明显破坏,在动脉壁还可见穿过弹力板游走至内膜下的平滑肌细胞及血源性吞噬脂质的单核细胞,单核细胞、平滑肌细胞游走至内皮下是AS病变的特征性超微结构的改变。2.动脉粥样硬化及信号转导通路两种功能功能分类基因芯片结果显示:在Zym组,炎症相关因子MMP2、MMP9、MMP7、IL-18、IL-1b表达显著上调,而PNS显著降低这些基因的表达;另外,一些与血脂调节及血管活性相关的基因如ANF、iNOS、在经PNS治疗后显著上调,而Edn、凝固因子Ⅲ(coagulation factor III ,TF)则下调。在本实验中,PNS对整合素家族的调节作用尤为明显,与对照组相比,Zym组90%以上整合素家族成员表达上调;而与Zym相比,PNS组90%以上整合素家族表达下调。之后挑选ANF、iNOS、IκBa、MMP7、COX-2基因做实时定量PCR分析,结果与芯片结果一致。Western blot结果进一步证实Zym刺激引起大鼠NFκB蛋白表达升高,IκBα蛋白表达降低;与Zym组相比,PNS则明显抑制NFκB蛋白表达,促进IκBα蛋白表达。3. PNS显著降低LPS诱导的P397 FAK蛋白表达,免疫组化显示FAK活化主要在胞浆,对照组少量表达P397 FAK ,LPS组P397 FAK表达明显高于对照组。PNS各治疗组P397 FAK表达量明显减少。结论:1.我们在成功建立大鼠炎性AS模型基础上,观察到炎症刺激可加重脂代谢紊乱,明显升高血液粘度,损伤动脉血管内皮细胞,加速AS病变。PNS可通过改善脂代谢紊乱及降低血液粘度等多条途径预防AS。2.炎症可明显增加动脉壁组织MMP2、7、9,VCAM-1, ICAM-1, Edn2、3表达。而PNS可显著抑制这些基因表达,其机制除了抑制NFκB表达及核转移外,还可以通过增加IκB的表达实现,而后者表现出一定的类糖皮质激素效应。3.炎症刺激对整合素家族表达上调作用尤为明显,由于整合素胞内效应分子为FAK,而FAK是多种信号调节通路上游调控分子(包括NFκB等),因此推测炎症免疫因素诱发AS重要途径是通过增加整合素表达激活FAK实现,相反PNS可显著抑制整合素的表达,以至抑制FAK第397位磷酸化,此途径可能与增加IκB的表达途径一起共同干预AS病变过程中的炎症、粘附反应与增殖反应。4. PNS血管活性调节作用在防治AS发病过程中也发挥重要作用, PNS可显著增加iNOS、ANF等基因表达,并可抑制Edn 1、2、3及TF mRNA表达。以上这些发现为筛选、创制新型抗AS药物提供了新的理论依据。更多还原

【Abstract】 Background and Aims:Atherosclerosis (AS) is one of the serious diseases that threaten the health of human being. Traditionally, high serum total cholesterol (TCL) is the major factor for AS. However, recent clinical epidemiological study have revealed that the TCL level is among the normal range for about 50 percent of AS patient, indicating that other etiological factors maybe contribute to AS. In addition, some inflammation diseases such as chronic bronchitis, prostatitis and gingivitis are positive related to high AS morbility. And the content of monocyte chemoattractant protein-1 (MCP-1) and C-reactive protein (CRP) increased in the patients with cardiovascular diseases. Taken together, inflammation and immunological factors play very important roles in the progress of AS.In Chinese Medicine, AS is the stagnated blood disease and promoting blood flow is the effective therapy. Panax notoginseng saponins (PNS) is the principal ingredient extracted from the traditional Chinese herb medicinal P. notoginseng and has extensive effects on the cardiovascular system, such as inhibiting platelet aggregation, increasing the blood flow of the coronary arteries, improving left ventricular diastolic function in hypertension patients, protecting the damage resulted from myocardial ischemia, showing an obvious anti-inflammatory effect due to reduction of the level of the intracellular free calcium concentration in neutrophils, reducing the myocardial oxygen consumption, and anti-arrhythmia, etc. Our previous experiments indicated that inflammation stimulation could induce the formation of foam cells, which can be inhibited through the anti-inflammation action of PNS.To investigate the mechanism of therapeutic action of PNS on AS, AS model of rats was employed in our study. Differential gene expression analysis by gene chip is the major method to study mechanism of Chinese medicine. The key molecules for the progress of AS were determined in this study, which provide a solid evidence for further applied study.Methods:1. Thirty rats were divided in to three groups randomly, i.e. Control (injected with sterilitas liquid paraffin, ip), Zym group (injected with Zymosan,20mg/kg,once every 3 days) and PNS group (treated with Zym ip +PNS 100mg/kg, once daily). All the animals were fed with high fat diet containing 3% cholesterol for 9 weeks.2. Serum TCL level and TGL were measured by enzymatic method and tissue slides were observed under electron microscopy.3. Three experiment groups: a, control, sterilitas liquid paraffin, ip; b, model, Zym,20mg/kg,one time every 3 days; c, treatment group, one time every 3 days +PNS (100mg/kg, once a day), The animals in Zym group and treatment group were fed with high fat diet containing 3% cholesterol for 9 weeks. The profiles of gene expression were determined by functional gene chip and were confirmed by real-time PCR and western blot.4. Macrophages were harvested from abdominal cavity of rats by stimulating with 1640 medium and cultured in vitro with LPS. Base on LPS, macrophages were treated with PNS (10, 30 or 100μg/ml, respectively). P397 FAK phosphorylation level in macrophages was detected by western blotting technique.Results:1. Endothelial cells in Zym group were damaged seriously, desquamated from artery walls and covered by adjacent endothelial cells. Swellened cytoplasm, phagotrophic lipid droplets and twisted nuclei presented in the endotheliocytes. Subendothelial space widened and subintimal smooth muscle cells permeated through elastic plank. After treated with PNS, the damage of endotheliocytes and smooth muscle cells relieved significantly.2. PNS obviously decreased the expression of MMP2, MMP9, MMP7, IL-18, IL-1b, Edn and TF, which are increased in the Zym group by comparison of control group. However, the expression of ANF and iNOS increased after treatment with PNS. The expressions of integrin family members increased markedly in Zym group and were reduced by PNS.3. The western blots showed that the expression of NFκB increased and IκBαdecreased significantly in Zym group, whereas PNS inhibited these changes obviously.4. Activated FAK was mainly expressed in the cytoplasm of macrophages. PNS could inhibit the phosphorylation of P397 FAK induced by LPS in macrophages significantly.Conclusion:1. PNS can markedly decrease the serum lipid level, improve the function of endothelial cells and inhibit the expression of endothelin 1, 2, 3 and coagulation factor III.2. PNS down-regulate inflammation-related genes, especially adhesion molecules, such as integrin. And PNS inhibit the expression of NFκB and its activity. In addition, PNS up-regulate IκB expression and directly inhibit FAK activation.3. PNS significantly increases the expression of iNOS and ANF. iNOS and ANF can obviously relax blood vessels, which is consistent with the traditional function of PNS.4. The effects of PNS on atherosclerosis are related with its anti-inflammatory and lipidemic-modulating actions.更多还原