【作者】 何雪峰；

【导师】 李晓辉；

【作者基本信息】 第三军医大学 ， 药理学， 2007， 博士

【摘要】 动脉粥样硬化（AS）导致的心脑血管疾病是当今人类死亡的首要原因。虽然AS的概念已提出近百年,但其发病机制尚未完全明了。AS的发生是一个多种因素在多层次上综合作用的过程。血脂水平的升高伴随着促炎因子的表达和免疫反应的激活,单核细胞活化成为巨噬细胞,血管壁脂质过氧化导致大量ox-LDL生成,巨噬细胞通过吞噬ox-LDL及其一系列信号分子的表达导致脂质代谢的障碍,并最终形成泡沫细胞。泡沫细胞的形成是AS早期变化的特征性标志,巨噬泡沫细胞的形成及其作用是动脉粥样硬化斑块损伤形成、发展和崩解的关键。因此,在调脂抗炎的同时,影响巨噬泡沫细胞的形成作为AS治疗的新策略日益受到重视。三七皂苷（PNS）是从三七根部提取的有效成分,主要含人参皂苷Rg1、Rb1、三七皂苷R1等。我室既往的研究证实,PNS能通过炎症免疫调节、增加动脉粥样硬化斑块稳定性、抗自由基等机制发挥防治AS的作用。但是药物作用的物质基础及机制的不明确成为制约PNS充分、合理应用的重要因素。因此,立足于PNS的有效性,以现代药理研究方法和新技术为平台,我们以巨噬泡沫细胞形成这一脂质代谢和炎症反应相互交汇的AS早期事件为切入点,研究了PNS单体及其单体配伍对AS过程中泡沫细胞形成的影响,并探讨其可能的作用机制。方法1.32只雄性日本大白兔,随机分成4组:对照组、AS模型组、PNS低剂量组、PNS高剂量组,分别用下列饲料喂养:（1）基础饲料,（2）高脂饲料,（3）高脂饲料+低剂量PNS（60mg/kg）,（4）高脂饲料+高剂量PNS（120mg/kg）。实验周期12w。通过测定血清中TC、TG、LDL、MDA水平、SOD活性、主动脉苏丹Ⅳ染色及主动脉组织切片HE染色确定家兔AS模型的建立、泡沫细胞的形成及PNS的影响。2.收集昆明种小鼠腹腔巨噬细胞,培养与纯化后,分为5组:（1）对照组,（2）模型组,（3）PNS低剂量组(20μg·ml^-1),（4）PNS中剂量组(40μg·ml^-1),（5）PNS高剂量组(80μg·ml^-1)。各组细胞置5%CO₂、37℃孵箱培养72h。油红O染色进行形态学观察,用酶法测定细胞内TC及FC,Lowry法测定细胞内蛋白质。3.正交设计实验:根据预实验结果,选取皂苷单体Rg₁、Rb₁、R₁为考察因素,每个因素各选择10^-4、10^-5、10^-6M三个剂量为考察水平,以胆固醇酯为指标,进行正交实验,分别确定皂苷单体Rg1、Rb1、R1及其配伍对泡沫细胞形成的影响。4.收集昆明种小鼠腹腔巨噬细胞,培养与纯化后,分为7组:对照组、模型组、PNS(80μg·ml^-1)组、单体配伍组(含10^-5M R1、10^-5M Rg1、10^-6M Rb1)、R1(10^-5M)组、Rg1(10^-5M)组和Rb1(10^-6M)组。各组细胞置5%CO₂、37℃孵箱培养72h。RT-PCR方法测定巨噬细胞内ACAT-1 mRNA、Adipophilin mRNA、ABCA1 mRNA、CD36 mRNA和LXRαmRNA表达;用Western blot方法测定巨噬细胞内CD36蛋白表达。结果1.AS模型兔主动脉内膜下大量泡沫细胞积聚,动脉粥样硬化斑块面积显著增加;高剂量PNS可明显减少泡沫细胞的形成,降低动脉粥样硬化斑块面积（p＜0.01）。2.AS模型组家兔血清中TC、TG、LDL-C水平均显著高于对照组（p＜0.01）;与AS模型组相比,高剂量PNS能非常显著降低TC、TG、LDL-C水平（p＜0.01）,低剂量PNS能显著降低家兔血清中TC水平（p＜0.05）,TG、LDL-C水平无统计学差异（p＞0.05）。3.AS模型组动物血清中MDA水平明显升高（p＜0.01）,SOD活性明显下降（p＜0.01）;PNS高剂量组血清中MDA水平明显低于AS模型组（p＜0.01）,而SOD活性显著升高（p＜0.01）;PNS低剂量组血清中MDA水平低于AS模型组（p＜0.05）,而SOD活性无明显升高。4.体外培养的巨噬细胞,未经处理的细胞形态多呈梭形,油红O染色无着色;与ox-LDL共培养后,油红O染色阳性细胞遍布,且细胞体积明显增大,形态多呈圆形或不规则形;PNS中、高剂量组细胞油红O染色阳性细胞比模型组明显减少,形态渐趋正常,而PNS低剂量组细胞与模型组相比无明显变化。5.模型组细胞内TC、FC、CE水平呈显著高于对照组（p＜0.01）,且CE/TC有显著性差异（p＜0.01）;与模型组相比,PNS高、中剂量组细胞内TC、FC、CE水平及CE/TC均有显著性降低（p＜0.01）,PNS低剂量组除TC、CE显著降低外（p＜0.01）,其它无明显变化;PNS各剂量组间细胞内TC、FC、CE水平及CE/TC均有显著性差异（p＜0.01）。6.正交实验表明,皂苷单体R1、Rg1、Rb1效应均显著,并且R1×Rb1和Rg1×Rb1的交互作用显著（p＜0.01）,其最佳单体配伍组合是:Rg1:R1:Rb1=10^-5:10^-5:10^-6M。7.PNS单体配伍能显著降低与ox-LDL共培养巨噬细胞中CE/TC（%）值,与高剂量PNS相比无显著差异。8.ox-LDL诱导巨噬细胞CD36 mRNA表达显著增加（p＜0.01）;PNS组和单体配伍组均能非常显著地降低CD36 mRNA表达（p＜0.01）,Rg1、R1组可显著降低CD36 mRNA表达（p＜0.05）,Rb1组无显著性差异（p＞0.05）;与PNS组相比,单体配伍组、Rg1组、R1组无显著性差异。9.ox-LDL诱导巨噬细胞Adipophilin mRNA表达显著增加（p＜0.01）;与模型组相比,PNS组、单体配伍组、Rg1、Rb1、R1组Adipophilin mRNA的表达与模型组无显著差异。10.模型组LXRαmRNA表达显著增加（p＜0.05）;PNS组和单体配伍组、Rg1、R1组均能非常显著地增加LXRαmRNA表达（p＜0.01）,Rb1组无显著性差异（p＞0.05）;与PNS组相比,单体配伍组无显著性差异（p＞0.05）,Rg1组有显著性差异（p＜0.05）,R1、Rb1组有非常显著差异（p＜0.01）。11.模型组ABCA1 mRNA表达较对照组显著增加（p＜0.05）;与模型组相比,PNS组、单体配伍组均能非常显著地增强ABCA1 mRNA表达（p＜0.01）,R1组可显著增加ABCA1 mRNA表达（p＜0.05）,且三组之间无显著差异。12.模型组ACAT-1 mRNA表达显著增加（p＜0.01）;PNS组、单体配伍组和Rb1组均能非常显著地降低ACAT-1 mRNA表达（p＜0.01）,且三组之间无显著性差异。13.模型组CD36蛋白表达明显高于对照组（p＜0.01）;PNS组与单体配伍组CD36蛋白表达量较模型组有显著性降低（p＜0.01）,Rg1、R1组CD36蛋白表达量较模型组明显减少（p＜0.05）;与PNS组相比,单体配伍组、Rg1组和R1组无显著性差异（p＞0.05）。结论1.PNS通过减轻氧化应激、调节血脂水平和抑制巨噬细胞吞噬功能,防治家兔实验性AS和泡沫细胞的形成。2.PNS、PNS单体配伍均能抑制ox-LDL诱导的巨噬细胞源性泡沫细胞形成。3.PNS与PNS单体配伍下调ox-LDL诱导的CD36 mRNA、ACAT-1 mRNA表达及CD36蛋白表达,上调ABCA1 mRNA、LXRαmRNA的表达;Rg1可下调CD36mRNA及CD36蛋白表达、上调LXRαmRNA表达;Rb1下调ACAT-1 mRNA表达;R1下调CD36 mRNA及CD36蛋白表达、上调ABCA1 mRNA、LXRαmRNA的表达,从而减少巨噬细胞内胆固醇蓄积,促进其外流,从不同环节上影响泡沫细胞的生成。4.本实验中,三七皂苷单体Rg1、Rb1、R1最佳配伍是:R1:Rg1:Rb1=10^-5:10^-5:10^-6,单体配伍对泡沫细胞的作用与PNS相当。因此,推测Rg1、Rb1、R1是PNS影响泡沫细胞形成的主要物质基础。更多还原

【Abstract】 It is well known that cardiovascular diseases resulted from atherosclerosis（AS） is the principal cause of death in the world.Although the concept of AS had been proposed for nearly 100 years,the onset mechanism of AS remains unclear.It has been clear that the development of AS is an integration process with multiple factors at multilevel.Following expressions of pro-inflammatory factors and activation of immune response,lipid level was increased.At the same time,lipid peroxidation aggravated production of ox-LDL,and macrophage swallow ox-LDL.All these resulted in lipid metabolism disorder and foam cell formation ultimately.The occurrence of macrophage-derived foam cell is the key marker of AS at the early stage.Along with the deepening to the mechanism of AS,we pay more attention to inhibiting foam cell formation as a new policy to cure AS.Panax Notoginseng saponin（PNS） is regarded as the main active constituents, including Rg1,Rb1,R1,etc.Our research indicated that PNS could prevent and cure AS through immunological regulation,anti-inflammation,anti-free radical,etc.Because less study on material foundation and mechanism had been operated,the applications of PNS were restricted.Based on the effectiveness of PNS on AS prevention and curing,we used the modern pharmacological methods and techniques to explore the effects of PNS and monomer combination on the foam cell formation during AS.Methods:1.32 New Japanese male rabbits,divided into 4 groups（including control group, AS model group,high-dose PNS group and low-dose PNS group） randomly,were fed with forages as follows respectively:（1） base forage,（2） high-fat forage（containing cholesterol 0.5%）,（3） high-fat forage with low-dose PNS（60mg/kg）,（4） high-fat forage with high-dose PNS（120mg/kg）.The experiment lasted 12 weeks.Rabbit AS models were confirmed by measuring levels of TC,TG,LDL,MDA and SOD activity in rabbit serum,thoracic aortas staining in SudanⅣsolution and tissue section oil red O staining.2.Macrophages,collected from mouse peritoneal,were divided into five groups after cultured and purified.（1） control group,（2） model group,（3） low-dose PNS group(20μg·ml^-1),（4） medium-dose PNS group(40μg·ml^-1),（5） high-dose PNS group(80μg·ml^-1).Cells were maintained at 37℃in 5%CO₂-95%air in an incubator for 72 hours.The cellular lipid accumulation was examined by oil red staining.The cellular contents of total cholesterol（TC） and free cholesterol（FC） were detected by enzymatic colorimetry.The cellular protein was measured by Lowry method.3.According to cholesteryl ester as the index and Rg1,Rb₁ and R₁ as investigation factors(every factor containing 3 levels:10^-4、10^-5、10^-6M),the effects of Rg1,Rb₁ and R1 on the foam cell formation of and their interaction were obtained by orthogonal design experiment.4.Expressions of CD36 mRNA,Adipophilin mRNA,LXRαmRNA,ABCA1 mRNA and ACAT-1 mRNA in macrophage were analyzed by RT-PCR.Expression of CD36 protein in macrophage was determined by western blotting.Results:1.Amount of foam cells accumulated under aortic tunica intima of rabbits in AS model rabbits,and the atherosclerotic plaque square obviously increased.High-dose PNS could reduce AS plague area and decrease foam cell formation under rabbit aortic tunica intimas（p＜0.01）).2.The serum concentrations of TC,TG and LDL-C in AS model group were significantly higher than that in normal control group（p＜0.01）.Compared to AS group, high-dose PNS could reduce the serum concentrations of TC,TG and LDL-C markedly （p＜0.01）.The level of TC in low-dose PNS group is lower than that in AS group （p＜0.05）.3.In AS model group,the serum concentration of MDA was increased,but the activity of SOD was decreased.High-dose PNS could reduce the serum concentration of MDA and enhance the activity of SOD in rabbits（p＜0.01）,and low-dose PNS could decreased the level of MDA（p＜0.05）.4.Macrophage in vitro culture was almost fusiform and there was no oil red-staining positive cell.Because of uptaking amount of lipid,oil red-staining positive cells were filled with macrophages and the shapes were round or irregular after co-cultured with ox-LDL.In high-dose and medium PNS groups,obvious reduction of oil red-staining positive cells was observed.Compared to model group,there was no significant difference in low-dose PNS group. 5.The contents of TC,FC,CE（cholesteryl ester） and CE/TC ratio in model group were greatly higher than that in control group（p＜0.01）.Compared with model group,the contents of TC,FC,CE and CE/TC ratio were significantly reduced in PNS high and medium dose groups（p＜0.01）.Meanwhile,there were obvious differences among PNS groups.6.Orthogonal experiment indicated that Rg1,Rb1and R1 were key responsive factors,and there were interaction between Rg1×Rb1 and R1×Rb1（p＜0.01）.The optimized monomer combination was Rg1:R1:Rb1=10^-5:10^-5:10^-6M。7.Compared with model group,PNS and monomer combination could reduce the ratio of CE/CT significantly.There were no obvious differences between PNS group and monomer combination group.8.CD36 mRNA expression in macrophages was increased remarkably after treated with ox-LDL（p＜0.01）.Compared with model group,PNS（p＜0.01）,single substrates compatibility（p＜0.01）,Rg1（p＜0.05） and R1（p＜0.05） could decrease CD36 mRNA expression in macrophages induced by ox-LDL.There were no obvious differences among PNS group,monomer combination group,Rg1 group and R1 group.9.Treated with ox-LDL,Adipophilin mRNA expression increased remarkably in macrophages（p＜0.01）.Compared with model group,PNS,monomer combination,Rg1, Rb1 and R1 didn’t affect Adipophilin mRNA expression induced by ox-LDL.10.Treated with ox-LDL,LXRαmRNA expression was increased in macrophages （p＜0.05）.Compared with model group,PNS,monomer combination,Rg1 and R1 could increased LXRαmRNA expression induced by ox-LDL（p＜0.01）.LXRαmRNA expressions in PNS group and monomer combination group had no significant differences. To some extent,LXRαmRNA expressions in Rg1 group（p＜0.05） and R1 group（p＜0.05） were lower than that in PNS group.11.Treated with ox-LDL,ABCA1 mRNA expression was increased in macrophage （p＜0.05）.Compared with model group,PNS（p＜0.01）,monomer combination group （p＜0.01） and R1（p＜0.05） could enhanced ABCA1 mRNA expression induced by ox-LDL in its degree.Compared with PNS group,there were no difference in monomer combination group and R1group.12.Treated with ox-LDL,macrophages ACAT-1 mRNA expression increased remarkably（p＜0.01）.Compared with model group,PNS,monomer combination group and Rb1 could decreased ACAT-1 mRNA expression induced by ox-LDL（p＜0.01）,and there was no significant difference among them.13.CD36 protein expression in model group macrophages was much higher than that in control group（p＜0.01）.PNS（p＜0.01）,monomer combination group（p＜0.01）, Rg1（p＜0.05） and R1（p＜0.05） remarkably reduced CD36 protein expression induced by ox-LDL,and there was no significant difference among them.Conclusions1.PNS could cure AS and prevent formation of foam cell through the pathway of relieving oxidative stress,modulating blood lipid and inhibiting phagocytose function of macrophage.2.PNS and monomer combination could inhibit macrophage-derived foam cell formation induced by ox-LDL.3.PNS and monomer combination could down-regulate the expressions of CD36 mRNA,ACAT-1 mRNA and CD36 protein,and up-regulate the expressions of ABCA1 mRNA and LXRαmRNA.Rg1 could down-regulate CD36 mRNA and CD36 protein expressions and up-regulate LXRαmRNA expression.Rb1 could down-regulate ACAT-1 mRNA expression.R1 could down-regulate CD36 mRNA and protein expressions and up-regulate ABCA1 mRNA and LXRαmRNA expressions.So, cholesterol ingestion into macrophage was decreased and the excretion was increased. Above all,PNS,monomer combination and monomer had effects on the foam cell formation at multi -targets.4.The optimized monomer combination ratio is Rg1:R1:Rb1=10^-5:10^-5:10^-6 in this experiment.The effect of monomer combination on foam cell formation is the same as that of PNS.So,we can preliminary predict that Rg1,Rb1 and R1 may be the indispensable material foundation for PNS preventing and curing AS.更多还原