The Effect Of Atorvastatin On TNF-α And IL-1β In Peripheral Blood Monocytes Stimulated By LPS

BackgroundAtherosclerosis 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 atherosclerosisIn 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 vitroMaterials 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 conditionThe 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...