阿托伐他汀对脂多糖刺激下外周血单核细胞分泌TNF-α、IL-1β的影响
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摘要
研究背景
动脉粥样硬化是通过脂质的累积、平滑肌细胞的增殖和钙化而引起的一种动脉损伤为特征的疾病,血浆总胆固醇水平升高是动脉粥样硬化(AS)的主要危险因素,低密度脂蛋白(LDL)尤其是LDL亚型中的小而致密的低密度脂蛋白是AS的重要致病因素。LDL经氧化修饰而生成的氧化LDL(OX-LDL)是损伤内皮细胞及其下的平滑肌细胞的主要原因。大量研究结果已充分表明,血浆TC或LDL-C水平升高在动脉粥样硬化的发生和发展过程中起着很重要的作用,与人群中冠心病的患病率和死亡率呈显著的正相关;血浆HDL-C水平低下已被公认为是冠心病的危险因素;血浆甘油三酯浓度升高也逐渐被认为是冠心病的独立危险因素。人体内的总胆固醇(TC)1/3来源于食物,2/3由肝脏合成,肝脏合成胆固醇需要经历3个阶段25个步骤,3-羟-3甲基戊二酰辅酶A(HMG-CoA)还原酶是肝脏生物合成胆固醇的限速酶,其活性受体内胆固醇代谢的调节,当体内胆固醇排空时,其活性增强而促使胆固醇合成,而当胆固醇合成或摄入增加时,其活性降低,负反馈使胆固醇生物合成减少,HMG-CoA还原酶抑制剂(他汀)属HMG-CoA还原酶抑制剂,其作用为竞争性抑制HMG-CoA还原酶,使其活性显著降低,肝内胆固醇合成量骤减,结果使血胆固醇和低密度脂蛋白胆固醇水平降低,中度降低血清甘油三酯水平和增高血高密度脂蛋白水平,且增加肝细胞表面低密度脂蛋白受体浓度,降低LDL-C能力,在临床上对高脂血症特别是对中、高度高胆固醇血症疗效显著,是目前预防及治疗动脉粥样硬化性心脑血管疾病的首选药物。他汀类药物的应用显著降低了动脉粥样硬化引起的心脑血管疾病的发病率及病死率。
近年来随着研究的深入,临床研究和药物实验研究发现了他汀类药物还具有降脂以外的治疗作用,血管内皮功能失调是动脉粥样硬化早期形成的始动因素,他汀类药物可在短期内使血流介导的内皮依赖性舒张功能明显迅速改善,其机制是动员骨髓内皮原组细胞入血并粘附于受损部位,另外,还通过改善一氧化氮合成酶(eNOS)活性使NO水平增加,血管扩张。他汀类药物可以减轻“犯罪”血管病变部位的炎症反应,稳定粥样硬化斑块,此处所说的炎症包括物理性、化学性和感染性。炎症细胞可产生粘附分子、白介素、细胞因子等,对细胞增殖、脂质沉积和形成不稳定粥样硬化斑块起了重要作用。C-反应蛋白(CRP)是炎症标志物,其水平增高可作为预测冠心病患者发生冠脉事件的标志之一,他汀可以降低C-反应蛋白(CRP),并且此作用独立于LDL-C水平及多种危险因素,此外研究还发现他汀可以下调可溶性黏附分子ICAM-1、VCAM-1、E选择素、P选择素的水平,通过抑制组织因子、纤溶酶原激活物抑制剂-1表达、凝血酶的生成及血小板的活化发挥抗凝作用,等等,通过这些机制他汀类药物发挥了改善内皮功能、稳定斑块,抗炎、抗氧化作用、免疫调节等多方面的功能,他汀类药物治疗作用的“多效性”日益受到人们的重视,甚至于他汀类药物的应用也突破主要治疗冠心病、高脂血症的局限,开始应用于心力衰竭、肾功能不全、类风湿关节炎等疾病的治疗,在临床上取得了不错的效果。
目前对他汀类药物作用机制的研究大多通过人体内或动物体内实验来展开,临床病人口服他汀类药物或者是动物经消化道给药,在不同的时间点抽出人体或动物血标本,通过各种不同技术检测研究设定的实验指标,从而探索他汀类药物的可能作用。本研究试图从体外细胞培养的角度,来研究他汀药物的作用,相对于体内试验来说,体外细胞培养的研究结果更为直观,可能具有更好的说服力。此外,研究还发现感染引起的炎症也参与了冠心病的发生及发病。脂多糖(LPS)是革兰阴性菌细胞外膜中活性成分。可刺激机体防御系统过度释放炎性因子、包括肿瘤坏死因子(TNF)、白介素(IL)及一氧化氮(NO)等。故本实验,以脂多糖刺激外周血单核细胞,再以阿托伐他汀加以干预,比较干预前后TNF-α、IL-1β的分泌量,探讨阿托伐他汀在炎症反应和免疫调节中的作用,同时从感染的角度探讨他汀抗动脉粥样硬化作用。
目的
观察阿托伐他汀对脂多糖(LPS)刺激下外周血单核细胞(PBMC)分泌TNF-α、IL-1β的影响,实验结果有助于加深他汀类药物抗炎、免疫调节等非降脂作用的认识,同时从感染的角度探讨他汀抗动脉粥样硬化作用。
材料和方法
1.材料:
1.1研究对象:外周血单核细胞
2.研究方法:
2.1主要溶液的配制
(1)阿托伐他汀溶液配制:
阿托伐他汀原料药60.47mg,溶于1ml无水乙醇,加入0.1mol/LNaOH1.5ml,55℃水浴锅中静置2h,以0.1mmol/L HCl调pH至7.2,加PBS至总体积5ml,过滤后分装,-70℃冻存,贮存浓度为10mmol/L
(2)脂多糖溶液的配制:
在超净台内进行,脂多糖100mg,加入1mlDMSO辅助其溶解,吸取0.5ml溶入无菌蒸馏水中,总体积50ml,待其完全溶解后过滤后分装,-70℃冻存。
2.2 PBMC分离、培养:
空腹、无菌条件下抽取健康人外周静脉血,肝素抗凝,以无菌的PBS液等比释稀,缓慢加入人淋巴细胞分离液上,两者体积比约2:1,按Ficoll-gradient密度梯度离心法分离人PBMC,2000r/min,离心20min,小心吸取白膜状的单核细胞层,以无菌的PBS释稀,离心10min,弃上清,重复洗涤2次,细胞接种于培养皿中,置于37℃、5%CO2培养箱2h,而后用37℃预热的培养基清洗2次,将未贴壁细胞洗脱,贴壁的为单核细胞。将贴壁细胞用酶消化法消化下来,重悬于含有2mmol/L谷氨酰胺,100U/ml青、链霉素双抗,10%胎牛血清的RPMI1640培养基中,调整细胞数为1×10~6备用。
2.3 PBMC活性测定
各组在下一步实验前进行细胞活性测定。方法:制备单细胞悬液,稀释为1×10~6个/ml,取1滴细胞悬液移入试管中,加一滴0.4%台盼蓝液混匀。在3分钟内用血球计数板分别计数,死细胞被染成淡蓝色,活细胞拒染。根据下式求细胞活力:
活细胞率(%)=活细胞总数/(活细胞总数+死细胞总数)
2.4实验分组:
以移液器将将培养的细胞接种于24孔培养板上,每孔2ml,共分4组,每组6个复孔,具体如下:
①空白组,不加任何干预,培养板细胞继续培养;
②LPS组:加入LPS使得其终浓度为10ng╱ml,并加入PBS作为对照组;
③LPS+1 umol/l阿托伐他汀组:LPS终浓度为10ng/ml和阿托伐他汀终浓度1μmol/L;
④LPS+10μmol/l阿托伐他汀组:LPS终浓度为10ng/ml和阿托伐他汀终浓度10μmol/L。
2.5 TNF-α、IL-1β含量检测:
分别收集各组单核细胞培养液上清,Elisa法测量各分组上清液中TNF-α、IL-1β的含量,按免疫酶标试剂盒说明书操作步骤进行检测,大致如下:将不同浓度的TNF-α、IL-1β标准品、及待测品以每孔100μL加入酶标孔,室温下孵育120 min,然后洗板4次,加生物素标记物,室温下孵育60min,再洗板4次。加底物显色剂避光室温孵育30min,加终止液。在450nm波长下读取每孔吸光度值。以浓度值为横坐标,所测各吸光度均值为纵坐标,绘制标准曲线。利用各样本孔测得的吸光度均值,从标准曲线上查得相应的浓度值。再乘以各自释稀的倍数,就可得实际值。每个样本及标准品各浓度均做2个复孔。2.6数据的处理及统计学分析方法:
实验结果所测指标的数据都纳入分析,无缺失数据,因测量指标的样本较少n=6,总体分布未知,实验结果均采用中位数(median)表示,统计分析采用了非参数检验的方法,两组间比较用两个独立样本的willcoxon秩和检验,多个独立样本比较的用多个独立样本Kruskal-wallis H检验,采用SPSS13.0统计软件进行数据处理,计算各组平均秩次及检验统计量Z或卡方值(x~2),检验水准α=0.05。
结果
1mmol/l阿托伐他汀分别均减少LPS刺激下PBMC分泌TNF-α53.3%、IL-1β35.4%,10mmol/l阿托伐他汀减少TNF-α82.0%、IL-1β65.6%,经多个独立样的的Kruskal-wallis H检验,分泌TNF-α的LPS+PBS组、LPS+1mmol/l托伐他汀组、LPS+10mmol/l阿托伐他汀组的平均秩次分别为:15.17、9.83、3.50,x~2=14.363,P=0.001,分泌IL-1β的LPS+PBS组、LPS+1mmol/l托伐他汀组、LPS+10mmol/l阿托伐他汀组的平均秩次分别为:15.00、9.50、4.50,x~2=12.737,P=0.002,可以认为阿托伐他汀显著下调脂多糖刺激下外周血单核细胞TNF-α、IL-1β的分泌量,且在一定范围内阿托伐他汀能浓度越大,作用越明显:
结论
1.体外细胞培养实验结果显示阿托伐他汀具有显著的抗炎、免疫调节作用,
2.他汀可能通过抑制感染炎症参与了抗动脉粥样硬化进程。
Background
Atherosclerosis is an arterial injury disease characterized by the accumulation of lipid,smooth muscle cell proliferation and calcification.Plasma total cholesterol levels are the main risk factors of atherosclerosis(AS).Low density lipoprotein(LDL) in particular subtypes of small dense low-density lipoprotein is an important pathogenic factor in AS.Oxidized LDL generated by the oxidation of LDL (OXLDL) are the main reasons of injury of endothelial cells and smooth muscle cells. Substantial results of the studies amply that the plasma TC or LDL-C level plays a very important role in the occurrence and development of atherosclerosis,and they both are significantly positive related with the crowd of coronary heart disease morbidity and mortality:plasma low level of HDL-C has also been recognized as risk factors for coronary heart disease;increased plasma triglyceride concentration gradually is considered an independent risk factor for coronary heart disease.1/3 of Human body's total cholesterol(TC) come from food and 2/3 is synthesized in liver. The synthesis of cholesterol in liver include three stages and 25 steps.3-hydroxy-3methyl-Coenzyme A(HMG-CoA) reductase is a rate-limiting enzyme in the cholesterol biosynthesis in liver,its activity is affected by the regulation of cholesterol metabolism in vivo.When the body's cholesterol get empty,HMG-CoA is activated and the synthesis of cholesterol increase;when cholesterol is ingested or the synthesis of cholesterol increase,its activity decreased negative feedback to reduce the biosynthesis of cholesterol.HMG-CoA reductase inhibitors(statins) are inhibitor for HMG-CoA reductase,they role as a competitive to HMG-CoA reductase result to a significant reduction in its activity,so the synthesis of cholesterol in liver sharply decreases,with the results that the blood cholesterol and the level of low-density lipoprotein cholesterol are reduced,serum triglyceride level moderately is decreased and blood high density lipoprotein level is elevated,and result to more cell surface low density lipoprotein receptor concentration in liver and lower ability of LDL-C.In clinical practice statins take significant effect on the therapy of hyperlipemia especially on a high degree of hypercholesterolemia.Statins have become the first important drug for the prevention and treatment atherosclerotic cardiovascular and cerebrovascular diseases.And they have greatly reduced cardiovascular and cerebrovascular morbidity and mortality caused by atherosclerosis
In recent years,with in-depth research,clinical researches and drug experiments have show that statins also have effect out of lipid-lowering.For example,the dysfunction of vascular endothelial is a early factor to the formation of atherosclerosis, statins can rapidly improved flow-mediated endothelium-dependent relaxation function in the short term.Its mechanism is to mobilized the original bone marrow endothelial cells into the blood to adhesion to the damaged parts and to improve the nitric oxide synthase(eNOS) activity so the level of NO increasese and blood vassel is vasodilator.Statins can also reduce the inflammatory response of the "crime" of vascular lesion and stabilize atherosclerotic plaque.Inflammation used here include the physical,chemical and infectious.Inflammatory cells produce adhesion molecules、interleukin、cytokines,etc,those play an important role on cell proliferation, lipid deposition and the formation of atherosclerotic plaque instability.C-reactive protein(CRP) are markers of inflammation,and the increase of its level in patients with coronary heart disease can be used as one of the hallmarks to predict the occurrence of coronary artery disease.Statins can reduce C-reactive protein(CRP), this effect is independent of lipid-lowering and other multiple risk factors.In addition to,studies also found that statins may lower the soluble adhesion molecule ICAM-1, VCAM-1,E-selectin,P-selectin levels.By inhibiting the expression of the tissue factor、plasminogen activator inhibitor - 1、thrombin generation and platelet activation play a important role in anticoagulation,and so on.The "multi-effect" nature of statin drugs have get much attention.Even breaking its major application in the treatment of coronary heart disease and hyperlipidemia statins began to be used in the treatment of heart failure,renal insufficiency,diseases such as rheumatoid arthritis in clinical practice,it turn out to be a good method.
The current experiments of statins are mostly carried out in vivo of human or animals.Clinical patients or animals are take medicine of statins by the digestive tract and their blood samples are collected at different points in time,then detecting the indicators in the blood samples set before the experiment by various technical the possible role of statins will be explored.This study attempt to study the role of statins in vitro.Compared with the experiment in vivo,the results of cell culture in vitro are more indenpent and have a better convincing.In addition,the researches also discovered that the inflammation caused by the infectiones also participated in the occurrence and the morbidity of coronary disease.Lipopolysaccharide(LPS) is a Gram-negative bacteria outer membrane of cells,it is the major active ingredient of Gram-negative bacteria.Stimulated by LPS defense system of the body can release excessive inflammatory factors,including tumor necrosis factor(TNF),interleukin (IL) and nitric oxide(NO) and so on.So in this experiment PBMC was stimulate by LPS and was intervened by atorvastatin.Compared TNF-αand IL-1βbefore the intervention by atorvastatin with them after the intervention,we could found the possible role of atorvastatin in inflammation and immunity,meanwhile discussed it' s anti-atherosclerosis function from the infection angle.
Objective:
To observe the level of TNF-αand IL-1βin human peripheral blood monocytes (PBMC) which were stimulated by LPS and the effects of atorvastatin on it.The results of this experiment contribute to understand statins possible effects on inflammatory and the immune regulation,meanwhile discussed its anti-atherosclerosis function from the infection angle.
Subject:Cultured human peripheral blood mononuclear cells in vitro
Materials and Methods:
1.Materials:
1.1 Subjects The cultured human PBMCs.
2.Methods:
2.1 Preparation of the main solution
(1) Preparation of atorvastatin:
Crude drug ofatorvastatin(60.47 mg) was dissolved in 1ml pure alcohol, 0.1 mol/L NaOH of 1.5 ml was added.Then the mixture was heated at 55℃for 2 hours,and PH value was regulated with 0.1 mol/L HCl to be 7.2,then PBS was added to 5 ml and 10mmol/L reserved atorvastatin solution was prepared and stored at-70℃.
(2)Preparation of lipopolysaccharide:
Prepared the solution of LPS in super-clean bench.Lipopolysaccharide (100mg) was dissolved into 1ml DMSO solution,then took out 0.5ml and added it into sterile distilled water,the total volume were 50ml,degermed by filtration,and stored at -70℃.
(3) Isolation of monocytes from human peripheral blood:
Monocytes were isolated from human peripheral blood by Ficoll-Hypaque density gradient centrifugation.Collected peripheral blood under sterile conditions, added in heparin and tha same volume pbs,then slowly added into lymphocyte separation medium,the two volume ratio were 2:1.Separated human PBMCs by Density gradient centrifugation,centrifuged at 2000r/min for 20min,carefully draw out white membranous layer of mononuclear cells,diluted cells by sterile PBS, centrifuged for 10min,discarded the supernatant,did it twice.Then the cells were inoculated in culture bottles,placed in 37℃and 5%CO2 incubator for 2h,then washed twice with RMPI 1640 at 37℃,descarded non-adherent cells,got adherent mononuclear cells by Enzymatic digestion.Re-suspended into RPMI1640 medium with 2mmol/L L-glutamine、100U/mlpenicillin and streptomycin、10%FBS,adjusted the cell number to 1×10~6/ml.Trypan blue staining was applied to access the survival rate.
1.2.Groups and cultural condition
The cells were divided into four groups:blank group,10ng/mlLPS + PBS group, 10ng/mlLPS+1umol/l atorvastatin group,10ng/ml LPS+10umol/l atorvastatin group, there were six wells each group.
2.1 Detection of TNF-αand IL-1β:
In this experiment TNF-αand IL-1βin supernatant was detected by Human TNF-αand IL-1βELISA KIT.Did it step by step according to the instructions of the ELISA KIT.The major steps of detection were as follows:different concentrations of standard preparation of TNF-αand IL- 1βand test samples were added in holes of enzyme marker board by 100 ul each hole,incubated in normal temperature for 120 min,and then washed the board 4 times,added in four drops of biotin markers, incubated in normal temperature for another 120 min,washed the board 4 times,added in substrates(for colour display),avoided light to incubate for 30min in normal temperature,finally added in terminated liquid.Then read each hole absorbency value under 45 nm wavelength in elisa reader.Based upon the concentrations and absorbency value of Standard preparationdrawn drawn standard curve.Use the standard curve and absorbency value of test samples to calculate concentrations of test samples.Each test sample and standard preparation detected twice.
2.2 Data analysis and statistical methods:
Because samples were only 6 and the overall distribution of the data were unknown,the date analysis used non-parametric test statistical methods,experimental data are presented as median.Willcoxon and Kruskal-wallis H of rank-sum test were used to analyze the date,SPSS13.0 statistical software was used to calculate chi-square value or Z value and P value.P value＜0.05 was considered significant.
Resusts:
1mmol/l atorvastatin reduced secretion of TNF-α53.3%and IL-1β35.4% released by PBMC stimulated by LPS in this experiment,while 10mmol/l atorvastatin reduced TNF-α82.0%and IL-1β65.6%.Date were analyzed by Kruskal-wallis H test.χ~2 of TNF-αgroup was 14.363 and P value was 0.001,whileχ~2 of IL-1βgroup was 12.737 and P value was 0.002,so it can say that atorvastatin significantly reduced secretion of TNF-αand IL-1βreleased by PBMC stimulated by LPS,at a certain scope the greater atorvastatin is,the more obvious its effect is.
Conclusion:
1.In vitro cell culture experiments showed that atorvastatin have significant effects in anti-inflammatory and immunomodulatory.
2.Statins may involve in anti-atherosclerosis through suppressing infection inflammation
动脉粥样硬化是通过脂质的累积、平滑肌细胞的增殖和钙化而引起的一种动脉损伤为特征的疾病,血浆总胆固醇水平升高是动脉粥样硬化(AS)的主要危险因素,低密度脂蛋白(LDL)尤其是LDL亚型中的小而致密的低密度脂蛋白是AS的重要致病因素。LDL经氧化修饰而生成的氧化LDL(OX-LDL)是损伤内皮细胞及其下的平滑肌细胞的主要原因。大量研究结果已充分表明,血浆TC或LDL-C水平升高在动脉粥样硬化的发生和发展过程中起着很重要的作用,与人群中冠心病的患病率和死亡率呈显著的正相关;血浆HDL-C水平低下已被公认为是冠心病的危险因素;血浆甘油三酯浓度升高也逐渐被认为是冠心病的独立危险因素。人体内的总胆固醇(TC)1/3来源于食物,2/3由肝脏合成,肝脏合成胆固醇需要经历3个阶段25个步骤,3-羟-3甲基戊二酰辅酶A(HMG-CoA)还原酶是肝脏生物合成胆固醇的限速酶,其活性受体内胆固醇代谢的调节,当体内胆固醇排空时,其活性增强而促使胆固醇合成,而当胆固醇合成或摄入增加时,其活性降低,负反馈使胆固醇生物合成减少,HMG-CoA还原酶抑制剂(他汀)属HMG-CoA还原酶抑制剂,其作用为竞争性抑制HMG-CoA还原酶,使其活性显著降低,肝内胆固醇合成量骤减,结果使血胆固醇和低密度脂蛋白胆固醇水平降低,中度降低血清甘油三酯水平和增高血高密度脂蛋白水平,且增加肝细胞表面低密度脂蛋白受体浓度,降低LDL-C能力,在临床上对高脂血症特别是对中、高度高胆固醇血症疗效显著,是目前预防及治疗动脉粥样硬化性心脑血管疾病的首选药物。他汀类药物的应用显著降低了动脉粥样硬化引起的心脑血管疾病的发病率及病死率。
近年来随着研究的深入,临床研究和药物实验研究发现了他汀类药物还具有降脂以外的治疗作用,血管内皮功能失调是动脉粥样硬化早期形成的始动因素,他汀类药物可在短期内使血流介导的内皮依赖性舒张功能明显迅速改善,其机制是动员骨髓内皮原组细胞入血并粘附于受损部位,另外,还通过改善一氧化氮合成酶(eNOS)活性使NO水平增加,血管扩张。他汀类药物可以减轻“犯罪”血管病变部位的炎症反应,稳定粥样硬化斑块,此处所说的炎症包括物理性、化学性和感染性。炎症细胞可产生粘附分子、白介素、细胞因子等,对细胞增殖、脂质沉积和形成不稳定粥样硬化斑块起了重要作用。C-反应蛋白(CRP)是炎症标志物,其水平增高可作为预测冠心病患者发生冠脉事件的标志之一,他汀可以降低C-反应蛋白(CRP),并且此作用独立于LDL-C水平及多种危险因素,此外研究还发现他汀可以下调可溶性黏附分子ICAM-1、VCAM-1、E选择素、P选择素的水平,通过抑制组织因子、纤溶酶原激活物抑制剂-1表达、凝血酶的生成及血小板的活化发挥抗凝作用,等等,通过这些机制他汀类药物发挥了改善内皮功能、稳定斑块,抗炎、抗氧化作用、免疫调节等多方面的功能,他汀类药物治疗作用的“多效性”日益受到人们的重视,甚至于他汀类药物的应用也突破主要治疗冠心病、高脂血症的局限,开始应用于心力衰竭、肾功能不全、类风湿关节炎等疾病的治疗,在临床上取得了不错的效果。
目前对他汀类药物作用机制的研究大多通过人体内或动物体内实验来展开,临床病人口服他汀类药物或者是动物经消化道给药,在不同的时间点抽出人体或动物血标本,通过各种不同技术检测研究设定的实验指标,从而探索他汀类药物的可能作用。本研究试图从体外细胞培养的角度,来研究他汀药物的作用,相对于体内试验来说,体外细胞培养的研究结果更为直观,可能具有更好的说服力。此外,研究还发现感染引起的炎症也参与了冠心病的发生及发病。脂多糖(LPS)是革兰阴性菌细胞外膜中活性成分。可刺激机体防御系统过度释放炎性因子、包括肿瘤坏死因子(TNF)、白介素(IL)及一氧化氮(NO)等。故本实验,以脂多糖刺激外周血单核细胞,再以阿托伐他汀加以干预,比较干预前后TNF-α、IL-1β的分泌量,探讨阿托伐他汀在炎症反应和免疫调节中的作用,同时从感染的角度探讨他汀抗动脉粥样硬化作用。
目的
观察阿托伐他汀对脂多糖(LPS)刺激下外周血单核细胞(PBMC)分泌TNF-α、IL-1β的影响,实验结果有助于加深他汀类药物抗炎、免疫调节等非降脂作用的认识,同时从感染的角度探讨他汀抗动脉粥样硬化作用。
材料和方法
1.材料:
1.1研究对象:外周血单核细胞
2.研究方法:
2.1主要溶液的配制
(1)阿托伐他汀溶液配制:
阿托伐他汀原料药60.47mg,溶于1ml无水乙醇,加入0.1mol/LNaOH1.5ml,55℃水浴锅中静置2h,以0.1mmol/L HCl调pH至7.2,加PBS至总体积5ml,过滤后分装,-70℃冻存,贮存浓度为10mmol/L
(2)脂多糖溶液的配制:
在超净台内进行,脂多糖100mg,加入1mlDMSO辅助其溶解,吸取0.5ml溶入无菌蒸馏水中,总体积50ml,待其完全溶解后过滤后分装,-70℃冻存。
2.2 PBMC分离、培养:
空腹、无菌条件下抽取健康人外周静脉血,肝素抗凝,以无菌的PBS液等比释稀,缓慢加入人淋巴细胞分离液上,两者体积比约2:1,按Ficoll-gradient密度梯度离心法分离人PBMC,2000r/min,离心20min,小心吸取白膜状的单核细胞层,以无菌的PBS释稀,离心10min,弃上清,重复洗涤2次,细胞接种于培养皿中,置于37℃、5%CO2培养箱2h,而后用37℃预热的培养基清洗2次,将未贴壁细胞洗脱,贴壁的为单核细胞。将贴壁细胞用酶消化法消化下来,重悬于含有2mmol/L谷氨酰胺,100U/ml青、链霉素双抗,10%胎牛血清的RPMI1640培养基中,调整细胞数为1×10~6备用。
2.3 PBMC活性测定
各组在下一步实验前进行细胞活性测定。方法:制备单细胞悬液,稀释为1×10~6个/ml,取1滴细胞悬液移入试管中,加一滴0.4%台盼蓝液混匀。在3分钟内用血球计数板分别计数,死细胞被染成淡蓝色,活细胞拒染。根据下式求细胞活力:
活细胞率(%)=活细胞总数/(活细胞总数+死细胞总数)
2.4实验分组:
以移液器将将培养的细胞接种于24孔培养板上,每孔2ml,共分4组,每组6个复孔,具体如下:
①空白组,不加任何干预,培养板细胞继续培养;
②LPS组:加入LPS使得其终浓度为10ng╱ml,并加入PBS作为对照组;
③LPS+1 umol/l阿托伐他汀组:LPS终浓度为10ng/ml和阿托伐他汀终浓度1μmol/L;
④LPS+10μmol/l阿托伐他汀组:LPS终浓度为10ng/ml和阿托伐他汀终浓度10μmol/L。
2.5 TNF-α、IL-1β含量检测:
分别收集各组单核细胞培养液上清,Elisa法测量各分组上清液中TNF-α、IL-1β的含量,按免疫酶标试剂盒说明书操作步骤进行检测,大致如下:将不同浓度的TNF-α、IL-1β标准品、及待测品以每孔100μL加入酶标孔,室温下孵育120 min,然后洗板4次,加生物素标记物,室温下孵育60min,再洗板4次。加底物显色剂避光室温孵育30min,加终止液。在450nm波长下读取每孔吸光度值。以浓度值为横坐标,所测各吸光度均值为纵坐标,绘制标准曲线。利用各样本孔测得的吸光度均值,从标准曲线上查得相应的浓度值。再乘以各自释稀的倍数,就可得实际值。每个样本及标准品各浓度均做2个复孔。2.6数据的处理及统计学分析方法:
实验结果所测指标的数据都纳入分析,无缺失数据,因测量指标的样本较少n=6,总体分布未知,实验结果均采用中位数(median)表示,统计分析采用了非参数检验的方法,两组间比较用两个独立样本的willcoxon秩和检验,多个独立样本比较的用多个独立样本Kruskal-wallis H检验,采用SPSS13.0统计软件进行数据处理,计算各组平均秩次及检验统计量Z或卡方值(x~2),检验水准α=0.05。
结果
1mmol/l阿托伐他汀分别均减少LPS刺激下PBMC分泌TNF-α53.3%、IL-1β35.4%,10mmol/l阿托伐他汀减少TNF-α82.0%、IL-1β65.6%,经多个独立样的的Kruskal-wallis H检验,分泌TNF-α的LPS+PBS组、LPS+1mmol/l托伐他汀组、LPS+10mmol/l阿托伐他汀组的平均秩次分别为:15.17、9.83、3.50,x~2=14.363,P=0.001,分泌IL-1β的LPS+PBS组、LPS+1mmol/l托伐他汀组、LPS+10mmol/l阿托伐他汀组的平均秩次分别为:15.00、9.50、4.50,x~2=12.737,P=0.002,可以认为阿托伐他汀显著下调脂多糖刺激下外周血单核细胞TNF-α、IL-1β的分泌量,且在一定范围内阿托伐他汀能浓度越大,作用越明显:
结论
1.体外细胞培养实验结果显示阿托伐他汀具有显著的抗炎、免疫调节作用,
2.他汀可能通过抑制感染炎症参与了抗动脉粥样硬化进程。
Background
Atherosclerosis is an arterial injury disease characterized by the accumulation of lipid,smooth muscle cell proliferation and calcification.Plasma total cholesterol levels are the main risk factors of atherosclerosis(AS).Low density lipoprotein(LDL) in particular subtypes of small dense low-density lipoprotein is an important pathogenic factor in AS.Oxidized LDL generated by the oxidation of LDL (OXLDL) are the main reasons of injury of endothelial cells and smooth muscle cells. Substantial results of the studies amply that the plasma TC or LDL-C level plays a very important role in the occurrence and development of atherosclerosis,and they both are significantly positive related with the crowd of coronary heart disease morbidity and mortality:plasma low level of HDL-C has also been recognized as risk factors for coronary heart disease;increased plasma triglyceride concentration gradually is considered an independent risk factor for coronary heart disease.1/3 of Human body's total cholesterol(TC) come from food and 2/3 is synthesized in liver. The synthesis of cholesterol in liver include three stages and 25 steps.3-hydroxy-3methyl-Coenzyme A(HMG-CoA) reductase is a rate-limiting enzyme in the cholesterol biosynthesis in liver,its activity is affected by the regulation of cholesterol metabolism in vivo.When the body's cholesterol get empty,HMG-CoA is activated and the synthesis of cholesterol increase;when cholesterol is ingested or the synthesis of cholesterol increase,its activity decreased negative feedback to reduce the biosynthesis of cholesterol.HMG-CoA reductase inhibitors(statins) are inhibitor for HMG-CoA reductase,they role as a competitive to HMG-CoA reductase result to a significant reduction in its activity,so the synthesis of cholesterol in liver sharply decreases,with the results that the blood cholesterol and the level of low-density lipoprotein cholesterol are reduced,serum triglyceride level moderately is decreased and blood high density lipoprotein level is elevated,and result to more cell surface low density lipoprotein receptor concentration in liver and lower ability of LDL-C.In clinical practice statins take significant effect on the therapy of hyperlipemia especially on a high degree of hypercholesterolemia.Statins have become the first important drug for the prevention and treatment atherosclerotic cardiovascular and cerebrovascular diseases.And they have greatly reduced cardiovascular and cerebrovascular morbidity and mortality caused by atherosclerosis
In recent years,with in-depth research,clinical researches and drug experiments have show that statins also have effect out of lipid-lowering.For example,the dysfunction of vascular endothelial is a early factor to the formation of atherosclerosis, statins can rapidly improved flow-mediated endothelium-dependent relaxation function in the short term.Its mechanism is to mobilized the original bone marrow endothelial cells into the blood to adhesion to the damaged parts and to improve the nitric oxide synthase(eNOS) activity so the level of NO increasese and blood vassel is vasodilator.Statins can also reduce the inflammatory response of the "crime" of vascular lesion and stabilize atherosclerotic plaque.Inflammation used here include the physical,chemical and infectious.Inflammatory cells produce adhesion molecules、interleukin、cytokines,etc,those play an important role on cell proliferation, lipid deposition and the formation of atherosclerotic plaque instability.C-reactive protein(CRP) are markers of inflammation,and the increase of its level in patients with coronary heart disease can be used as one of the hallmarks to predict the occurrence of coronary artery disease.Statins can reduce C-reactive protein(CRP), this effect is independent of lipid-lowering and other multiple risk factors.In addition to,studies also found that statins may lower the soluble adhesion molecule ICAM-1, VCAM-1,E-selectin,P-selectin levels.By inhibiting the expression of the tissue factor、plasminogen activator inhibitor - 1、thrombin generation and platelet activation play a important role in anticoagulation,and so on.The "multi-effect" nature of statin drugs have get much attention.Even breaking its major application in the treatment of coronary heart disease and hyperlipidemia statins began to be used in the treatment of heart failure,renal insufficiency,diseases such as rheumatoid arthritis in clinical practice,it turn out to be a good method.
The current experiments of statins are mostly carried out in vivo of human or animals.Clinical patients or animals are take medicine of statins by the digestive tract and their blood samples are collected at different points in time,then detecting the indicators in the blood samples set before the experiment by various technical the possible role of statins will be explored.This study attempt to study the role of statins in vitro.Compared with the experiment in vivo,the results of cell culture in vitro are more indenpent and have a better convincing.In addition,the researches also discovered that the inflammation caused by the infectiones also participated in the occurrence and the morbidity of coronary disease.Lipopolysaccharide(LPS) is a Gram-negative bacteria outer membrane of cells,it is the major active ingredient of Gram-negative bacteria.Stimulated by LPS defense system of the body can release excessive inflammatory factors,including tumor necrosis factor(TNF),interleukin (IL) and nitric oxide(NO) and so on.So in this experiment PBMC was stimulate by LPS and was intervened by atorvastatin.Compared TNF-αand IL-1βbefore the intervention by atorvastatin with them after the intervention,we could found the possible role of atorvastatin in inflammation and immunity,meanwhile discussed it' s anti-atherosclerosis function from the infection angle.
Objective:
To observe the level of TNF-αand IL-1βin human peripheral blood monocytes (PBMC) which were stimulated by LPS and the effects of atorvastatin on it.The results of this experiment contribute to understand statins possible effects on inflammatory and the immune regulation,meanwhile discussed its anti-atherosclerosis function from the infection angle.
Subject:Cultured human peripheral blood mononuclear cells in vitro
Materials and Methods:
1.Materials:
1.1 Subjects The cultured human PBMCs.
2.Methods:
2.1 Preparation of the main solution
(1) Preparation of atorvastatin:
Crude drug ofatorvastatin(60.47 mg) was dissolved in 1ml pure alcohol, 0.1 mol/L NaOH of 1.5 ml was added.Then the mixture was heated at 55℃for 2 hours,and PH value was regulated with 0.1 mol/L HCl to be 7.2,then PBS was added to 5 ml and 10mmol/L reserved atorvastatin solution was prepared and stored at-70℃.
(2)Preparation of lipopolysaccharide:
Prepared the solution of LPS in super-clean bench.Lipopolysaccharide (100mg) was dissolved into 1ml DMSO solution,then took out 0.5ml and added it into sterile distilled water,the total volume were 50ml,degermed by filtration,and stored at -70℃.
(3) Isolation of monocytes from human peripheral blood:
Monocytes were isolated from human peripheral blood by Ficoll-Hypaque density gradient centrifugation.Collected peripheral blood under sterile conditions, added in heparin and tha same volume pbs,then slowly added into lymphocyte separation medium,the two volume ratio were 2:1.Separated human PBMCs by Density gradient centrifugation,centrifuged at 2000r/min for 20min,carefully draw out white membranous layer of mononuclear cells,diluted cells by sterile PBS, centrifuged for 10min,discarded the supernatant,did it twice.Then the cells were inoculated in culture bottles,placed in 37℃and 5%CO2 incubator for 2h,then washed twice with RMPI 1640 at 37℃,descarded non-adherent cells,got adherent mononuclear cells by Enzymatic digestion.Re-suspended into RPMI1640 medium with 2mmol/L L-glutamine、100U/mlpenicillin and streptomycin、10%FBS,adjusted the cell number to 1×10~6/ml.Trypan blue staining was applied to access the survival rate.
1.2.Groups and cultural condition
The cells were divided into four groups:blank group,10ng/mlLPS + PBS group, 10ng/mlLPS+1umol/l atorvastatin group,10ng/ml LPS+10umol/l atorvastatin group, there were six wells each group.
2.1 Detection of TNF-αand IL-1β:
In this experiment TNF-αand IL-1βin supernatant was detected by Human TNF-αand IL-1βELISA KIT.Did it step by step according to the instructions of the ELISA KIT.The major steps of detection were as follows:different concentrations of standard preparation of TNF-αand IL- 1βand test samples were added in holes of enzyme marker board by 100 ul each hole,incubated in normal temperature for 120 min,and then washed the board 4 times,added in four drops of biotin markers, incubated in normal temperature for another 120 min,washed the board 4 times,added in substrates(for colour display),avoided light to incubate for 30min in normal temperature,finally added in terminated liquid.Then read each hole absorbency value under 45 nm wavelength in elisa reader.Based upon the concentrations and absorbency value of Standard preparationdrawn drawn standard curve.Use the standard curve and absorbency value of test samples to calculate concentrations of test samples.Each test sample and standard preparation detected twice.
2.2 Data analysis and statistical methods:
Because samples were only 6 and the overall distribution of the data were unknown,the date analysis used non-parametric test statistical methods,experimental data are presented as median.Willcoxon and Kruskal-wallis H of rank-sum test were used to analyze the date,SPSS13.0 statistical software was used to calculate chi-square value or Z value and P value.P value＜0.05 was considered significant.
Resusts:
1mmol/l atorvastatin reduced secretion of TNF-α53.3%and IL-1β35.4% released by PBMC stimulated by LPS in this experiment,while 10mmol/l atorvastatin reduced TNF-α82.0%and IL-1β65.6%.Date were analyzed by Kruskal-wallis H test.χ~2 of TNF-αgroup was 14.363 and P value was 0.001,whileχ~2 of IL-1βgroup was 12.737 and P value was 0.002,so it can say that atorvastatin significantly reduced secretion of TNF-αand IL-1βreleased by PBMC stimulated by LPS,at a certain scope the greater atorvastatin is,the more obvious its effect is.
Conclusion:
1.In vitro cell culture experiments showed that atorvastatin have significant effects in anti-inflammatory and immunomodulatory.
2.Statins may involve in anti-atherosclerosis through suppressing infection inflammation
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[21]Pliquet t RU,Cornish KG,Peuler JD,et al 1Simvastatin normalizes autonomic neural control in experimental heart failure[J].Ci rcul at ion,2003,107(19):2493.
[22]Planavila A,Laguna JC,Vazquez- Carrera M,et al.Atorvastrtin improves peroxisome proliferator - activated receptor signaling in cardiac hypertrophy by preventing nuclear factor -κB activation.Biochimica Biophysica Acta,2005,1687:76- 83
[23]Ogata Y,Takahshi M,Takeuchi K,et al.Simvastatin induces apoptosis in rat neonatal cardiac myocytes:a possible mechanism of statin - attenuated cardiac hypertrophy cardiomyopathy[J].J Cardiovasc Pharmacol,2002,40:907 - 915.
[24]Mozaffarian D,Nye R,LevyWC.Statin therapy is associated withlowermortality among patients with severe heart failure[J].Am J Cardiol,2004,93(9):1124-1129.
[25]Kumagai K,Nakashima H,Saku K.The HMG-CoA reductase inhibitor atorvastatin prevents atrial fibrillation by inhibiting inflammation in a canine sterile pericarditismodel[J].Cadiovasc Res,2004,62(1):105-111.
[26]Mark L,Katona A.Effect of fluvastatin on QT dispersion:a new pleitropic effect?[J].Am J Cardiol,2000,85(7):919-920.
[27]Vrtovec B,Okrajsek R,Golicnik A,et al.Atorvastatin therapy increase heart rate variability,decreases QT variability,and shortens QTc interval duration in patientswith advanced chronic heart failure[J].J Card Fail,2005,11(9):691.
[28]Hackam DG,MamdaniM,Li P,et al.Statins and sep sis in Patients with card iovascular disease a population - based cohort analysis[J].Lancet,2006,367 (9508): 413-418.
[29]Thorn sen RW, Hundborg HH, Johnsen SP, et al.Statin use and mortality within 180 days after bacteremia A population - based cohort study[J].Cirt CareMed,2006,34(4): 1080-1086.
[30]Merx MW ,Liehn EA, Graf J , et al 1 Statin t reatment after onset of sep sis in a murine model improves sur-vival [J].Circulation ,2005 ,112 (2): 1171
[31]Schmidt H ,Hennen R , Keller A , etal .Association of statint herapy and increased survival in patients with multiple organ dysf unction syndrome [J].Intensive Care Med ,2006 ,32 (8) :1 2481
[32]Palinski W.Immunomodulation : a new role of statins [J].Nat Med , 2000 , 6:1311-1312.
[33]WeitZ2Schmidt G,WelZenbach K, Brinkmann V , et al .Statins selectively inhibit Leukocyte function antigen21 by binding to a novel regulatory intergrin site[J].Ibid, 2001,7:6872692.
[34]Simons M ,Schwarzler F ,Lut johann D , etal .Treatment with simvastatin in normcholesterolemic patients with Alzheimer's disease : A 26 - week randomized,placebo - cont rolled , double - blind t rial [J].Ann Neurol ,2002 ,52 (3) :3461
[35]Petanceska SS ,DeRosa S ,01mV, et al .Statin t herapy for Alzheimer's disease : Will it work[J]J Mo.Neu-rosci ,2002 ,19 (1) :1551
[36]SparksDL ,Sabbagh M ,Connor D , et al .Statin t herapy in Alzheimer's disease [J].Act a Neurol Scand Suppl ,2006 ,185 (1) :781
[37]Pahan K, Sheikh FC , Namboodiri AM , etal .Lovastatin and phenylacetate inhibit the induction of nitric oxide synt hase and cytokines in rat primary ast rocytes , microglia and macrophages [J].JClin Invest ,1997 ,100 :2671-2679.
[38]Vollmer T , Key L , Durkalski V , etal.Oral simvastatin t reat-ment in relapsing remitting multiple Sclerosis[J].Lancet,2004,363(9426):1607-1616.
[39]Klaveskog L.Hamsten A,Satins in rheumatoid art hritis-two birds wit h one stone[J].Ibid,2004,363(9426):2011-2012.
[40]Abud.Mendoza C,De La Fuente H,Cuevas2Orta E,etal.Therapy wit h statins in Patient s with refractory rheumatic disease:a prelimimary study[J].Lupus,2003,12:607-611.
[41]Park HJ,Kong D,Iruela-ArispeL,etal.3-hydroxy-3-Methyl-glutayl coenzyme A reductase inhibitors interfere with angio-genesis by inhibiting the geranyL-geranylation of Rho A[J].Circ Res,2002,19:143-150.
[42]Leung BP,Sattar N,Crilly A,etal.A novel anti-inflammatory role for Simvastatin in inflammatory art hritis[J].J Immune,2003,170:1524-1530.
[43]McCarey DW,McInnes IB,Madhok K,et al.Trial of Atorvastatin Rheumatoid Art hritis(TARA):double-blind,randomized placebo-controlled trial[J].Lancet,2004,363(9426):2015-2021.
[44]Mckay A,Leung BP,Mc Innes IB,etal.A novel anti -inflammatory role of simvastatin in a murine model of allergic asthma[J].Immunol,2004,172(5):2903.
[45]李毅,戚好文,刘颖格,等氟伐他汀对大鼠气道平滑肌增生的影响[J].第四军医大学学报,2003,24(6):513.
[46]宋立强,戚好文,李研,等.氟伐他汀对哮喘气道重塑的抑制作用及机制研究[J].中国病理生理杂志,2002,18(12):1 478.
[47]Danesh FR,Sadeghi MM,Amro N,etal.3 - Hydroxy - 3 -Methylglutary.CoA reductase inhibitors prevent high glucose - induced proliferation of mesangial cells via modulation of Rho GTPase/p21 signaling pathway:Implications for diabetic nephropathy.Proc Natl Acad Sci U S A,2002,99(12):8301-8305.
[48]Nogaki.Direct inhibitory effects of simvastatin on matrix accumulation in cultured murine mesangial cells.Kidney Int Suppl, 1999 , 71 : 198～201.
[49]Nakamura T, Chifuyu U.Effect of cerivastatin on p roteinuria and urinary podocytes in patients with chronic glomeru - lonephritis [J].Nephrol Dial Transp lant, 2002, 17:798.
[50]Tsung - Ming Lee, Sheng - Fang Su, etal.Effect of p ravastatin on proterinuria in patients with well - controlled hypertension [J].Hypertension, 2002, 67.
[51]Van Aelst L, D'Souza-Schorey C.Rho GTPases and signaling networks [J].Genes Dev, 1997,ll(18):2295-2322.
[52]Mackay DJ, Hall A.Rho GTPases [J].J Biol Chem, 1998,273(33):20685-20688.
[53]Blank N, Schiller M, Krienke S, et al.Atorvastatin inhibits T cell activation through 3-Hydroxy-3-methylglutaryl coenzyme A reductase without decreasing cholesterol synthesis [J].The Journal of Immunology, 2007,179(6):3613-3621.
[54]Wibaut-Berlaimont V, Randi AM, Mandryko V, etal.Atorvastatin affects leukocyte gene expression in dyslipidemia patients: in vivo regulation of hemostasis, inflammation and apoptosis [J].J Thromb Haemost, 2005,3(4):677-685.
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[20]韦立新.不稳定斑块破裂的形态学及发生机制的病理学研究进展[J].国外医学:生理、病理科学与临床分册,2003,23(5):441-443.
[21]Pliquet t RU,Cornish KG,Peuler JD,et al 1Simvastatin normalizes autonomic neural control in experimental heart failure[J].Ci rcul at ion,2003,107(19):2493.
[22]Planavila A,Laguna JC,Vazquez- Carrera M,et al.Atorvastrtin improves peroxisome proliferator - activated receptor signaling in cardiac hypertrophy by preventing nuclear factor -κB activation.Biochimica Biophysica Acta,2005,1687:76- 83
[23]Ogata Y,Takahshi M,Takeuchi K,et al.Simvastatin induces apoptosis in rat neonatal cardiac myocytes:a possible mechanism of statin - attenuated cardiac hypertrophy cardiomyopathy[J].J Cardiovasc Pharmacol,2002,40:907 - 915.
[24]Mozaffarian D,Nye R,LevyWC.Statin therapy is associated withlowermortality among patients with severe heart failure[J].Am J Cardiol,2004,93(9):1124-1129.
[25]Kumagai K,Nakashima H,Saku K.The HMG-CoA reductase inhibitor atorvastatin prevents atrial fibrillation by inhibiting inflammation in a canine sterile pericarditismodel[J].Cadiovasc Res,2004,62(1):105-111.
[26]Mark L,Katona A.Effect of fluvastatin on QT dispersion:a new pleitropic effect?[J].Am J Cardiol,2000,85(7):919-920.
[27]Vrtovec B,Okrajsek R,Golicnik A,et al.Atorvastatin therapy increase heart rate variability,decreases QT variability,and shortens QTc interval duration in patientswith advanced chronic heart failure[J].J Card Fail,2005,11(9):691.
[28]Hackam DG,MamdaniM,Li P,et al.Statins and sep sis in Patients with card iovascular disease a population - based cohort analysis[J].Lancet,2006,367 (9508): 413-418.
[29]Thorn sen RW, Hundborg HH, Johnsen SP, et al.Statin use and mortality within 180 days after bacteremia A population - based cohort study[J].Cirt CareMed,2006,34(4): 1080-1086.
[30]Merx MW ,Liehn EA, Graf J , et al 1 Statin t reatment after onset of sep sis in a murine model improves sur-vival [J].Circulation ,2005 ,112 (2): 1171
[31]Schmidt H ,Hennen R , Keller A , etal .Association of statint herapy and increased survival in patients with multiple organ dysf unction syndrome [J].Intensive Care Med ,2006 ,32 (8) :1 2481
[32]Palinski W.Immunomodulation : a new role of statins [J].Nat Med , 2000 , 6:1311-1312.
[33]WeitZ2Schmidt G,WelZenbach K, Brinkmann V , et al .Statins selectively inhibit Leukocyte function antigen21 by binding to a novel regulatory intergrin site[J].Ibid, 2001,7:6872692.
[34]Simons M ,Schwarzler F ,Lut johann D , etal .Treatment with simvastatin in normcholesterolemic patients with Alzheimer's disease : A 26 - week randomized,placebo - cont rolled , double - blind t rial [J].Ann Neurol ,2002 ,52 (3) :3461
[35]Petanceska SS ,DeRosa S ,01mV, et al .Statin t herapy for Alzheimer's disease : Will it work[J]J Mo.Neu-rosci ,2002 ,19 (1) :1551
[36]SparksDL ,Sabbagh M ,Connor D , et al .Statin t herapy in Alzheimer's disease [J].Act a Neurol Scand Suppl ,2006 ,185 (1) :781
[37]Pahan K, Sheikh FC , Namboodiri AM , etal .Lovastatin and phenylacetate inhibit the induction of nitric oxide synt hase and cytokines in rat primary ast rocytes , microglia and macrophages [J].JClin Invest ,1997 ,100 :2671-2679.
[38]Vollmer T , Key L , Durkalski V , etal.Oral simvastatin t reat-ment in relapsing remitting multiple Sclerosis[J].Lancet,2004,363(9426):1607-1616.
[39]Klaveskog L.Hamsten A,Satins in rheumatoid art hritis-two birds wit h one stone[J].Ibid,2004,363(9426):2011-2012.
[40]Abud.Mendoza C,De La Fuente H,Cuevas2Orta E,etal.Therapy wit h statins in Patient s with refractory rheumatic disease:a prelimimary study[J].Lupus,2003,12:607-611.
[41]Park HJ,Kong D,Iruela-ArispeL,etal.3-hydroxy-3-Methyl-glutayl coenzyme A reductase inhibitors interfere with angio-genesis by inhibiting the geranyL-geranylation of Rho A[J].Circ Res,2002,19:143-150.
[42]Leung BP,Sattar N,Crilly A,etal.A novel anti-inflammatory role for Simvastatin in inflammatory art hritis[J].J Immune,2003,170:1524-1530.
[43]McCarey DW,McInnes IB,Madhok K,et al.Trial of Atorvastatin Rheumatoid Art hritis(TARA):double-blind,randomized placebo-controlled trial[J].Lancet,2004,363(9426):2015-2021.
[44]Mckay A,Leung BP,Mc Innes IB,etal.A novel anti -inflammatory role of simvastatin in a murine model of allergic asthma[J].Immunol,2004,172(5):2903.
[45]李毅,戚好文,刘颖格,等氟伐他汀对大鼠气道平滑肌增生的影响[J].第四军医大学学报,2003,24(6):513.
[46]宋立强,戚好文,李研,等.氟伐他汀对哮喘气道重塑的抑制作用及机制研究[J].中国病理生理杂志,2002,18(12):1 478.
[47]Danesh FR,Sadeghi MM,Amro N,etal.3 - Hydroxy - 3 -Methylglutary.CoA reductase inhibitors prevent high glucose - induced proliferation of mesangial cells via modulation of Rho GTPase/p21 signaling pathway:Implications for diabetic nephropathy.Proc Natl Acad Sci U S A,2002,99(12):8301-8305.
[48]Nogaki.Direct inhibitory effects of simvastatin on matrix accumulation in cultured murine mesangial cells.Kidney Int Suppl, 1999 , 71 : 198～201.
[49]Nakamura T, Chifuyu U.Effect of cerivastatin on p roteinuria and urinary podocytes in patients with chronic glomeru - lonephritis [J].Nephrol Dial Transp lant, 2002, 17:798.
[50]Tsung - Ming Lee, Sheng - Fang Su, etal.Effect of p ravastatin on proterinuria in patients with well - controlled hypertension [J].Hypertension, 2002, 67.
[51]Van Aelst L, D'Souza-Schorey C.Rho GTPases and signaling networks [J].Genes Dev, 1997,ll(18):2295-2322.
[52]Mackay DJ, Hall A.Rho GTPases [J].J Biol Chem, 1998,273(33):20685-20688.
[53]Blank N, Schiller M, Krienke S, et al.Atorvastatin inhibits T cell activation through 3-Hydroxy-3-methylglutaryl coenzyme A reductase without decreasing cholesterol synthesis [J].The Journal of Immunology, 2007,179(6):3613-3621.
[54]Wibaut-Berlaimont V, Randi AM, Mandryko V, etal.Atorvastatin affects leukocyte gene expression in dyslipidemia patients: in vivo regulation of hemostasis, inflammation and apoptosis [J].J Thromb Haemost, 2005,3(4):677-685.
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