P38MAPK对FKN诱导人单个核细胞NF-κB活化的影响和辛伐他汀的干预作用
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摘要
实验目的:通过对不规则趋化因子(fractalkine,FKN)诱导活化的人单个核细胞内核因子κB(nuclear factor kappa B,NF-κB)的测定,旨在研究以下内容:(1)FKN对单个核细胞NF-κB活化的影响;(2)FKN影响单个核细胞活化NF-κB过程与P38丝裂素活化蛋白激酶(p38 mitogen-activated protein kinase,P38MAPK)信号转导通路的相关性;(3)辛伐他汀可能的抗动脉粥样硬化机制及不同浓度的辛伐他汀与NF-κB级联信号转导途径的关系。
实验方法:采用密度梯度离心法分离人外周血单个核细胞(peripheral blood mononuclear cells,PBMC),实验分8组,空白组、FKN组、FKN+P38MAPK抑制剂(SB203580)组、FKN+辛伐他汀0.1μmol/L组、FKN+辛伐他汀1μmol/L组、FKN+辛伐他汀10μmol/L组、FKN+辛伐他汀20μmol/L组及FKN+辛伐他汀50μmol/L组,采用Western Blot法检测各组单个核细胞胞核内的NF-κB表达量。
实验结果:(1)FKN组核内NF-κB含量较空白组增加,统计学分析差异均有显著性;(2)FKN+P38MAPK抑制剂(SB203580)组核内NF-κB水平较FKN组降低,但较空白组增加,经统计学分析组间差异有显著性;(3)FKN+辛伐他汀各组核内NF-κB水平较FKN组降低,但较空白组增加,且以1μmol/L浓度下作用最强,经统计学分析组间差异有显著性。
实验结论:(1)FKN/CX3CR1可升高单个核细胞NF-κB的活化水平;(2)P38MAPK抑制剂(SB203580)可降低FKN/CX3CR1诱导的单个核细胞NF-κB的活性;(3)辛伐他汀可降低FKN/CX3CR1诱导的单个核细胞中NF-κB的活性,且1μmol/L辛伐他汀为可供选择的相对理想的预处理浓度,未表现出浓度依赖性。
The research about atherosclerosis has been going on for hundreds of years, so many hypotheses about its pathogenesis have been proposed. With in-depth study, inflammation receives much concern. Fractalkine (FKN), a new chemokine which is discovered in 1997, has both membranebinding and soluble forms, it can mediate capture, firm adhesion and migration of monocytes and lymphocytes to the vascular endothelial cells under physiologic flow conditions. Large number of studies have demonstrated that FKN and its receptor CX3CR1 are involved in the inflammatory response of atherosclerosis, but its mechanism remains to be clear. Mitogen-activated protein kinase (MAPK) is one of the key important components of the Signal Transduction Systems. P38 mitogen-activated protein kinase (P38MAPK), is a member of MAPK family, which is widely presented in the cytoplasm. its signaling pathway plays an important role in inflammation. Nuclear factorκB (NF-κB) has been found to be important transcription factors which participate in the gene regulation of inflammation, immunity, stress response, cell growth and oncogenesis, it plays an important role in inflammation and immune responses. Up to now, statins (3 - hydroxy - 3 - methyl coenzyme A (HMG - CoA) reductase inhibitors) are widely used in clinical activities, the clinical effect benefits not only from the classic effects of lipid modulation, but also from the anti-inflammatory effects, the antioxidation effects, the platelet aggregation inhibitory activity, and so on.Therefore, their non-lipid mechanisms may plays an important role in prevention and treatment of atherosclerosis.
We associated FKN/CX3CR1, P38MAPK, NF-κB as well as peripheral blood mononuclear cells (PBMC), studied relationship of them and anti-inflammatory mechanism of Simvastatin.
Objective:The aims of the study are as follows: (1) To research the effects of FKN on NF-κB activation in mononuclear cells. (2) To research the function of p38 mitogen-activated protein kinase(P38MAPK) in the signal transduction mechanism of FKN/CX3CR1 affecting atherosclerosis. (3) To research the intervention of 3-hydroxy-3-methyl coenzyme A reductase inhibitors Simvastatin in the signal transduction mechanism of Fractalkine/CX3CR1 affecting atherosclerosis.
Methods:(1)PBMC were isolated from fresh blood of healthy volunteers by Ficoll-Paque gradient centrifugation.(2)Divide the extractive Peripheral blood monocytes into eight groups:control group,FKN group,FKN+SB203580(P38MAPK inhibitor) group, FKN+1μmol/L Simvastatin group, FKN+0.1μmol/L Simvastatin group, FKN+10μmol/L Simvastatin group, FKN+20μmol/L Simvastatin group, FKN+50μmol/L Simvastatin group.(3)Measure the NF-κB actived by monocytes in each group by western blot method.
Result: The following are the integrated optical density valuesmeasured by western blot in each group: 1.00±0.00 in control group, 3.28±0.11 in FKN group, 1.26±0.14 in FKN+ SB203580 (P38MAPK inhibitor) group, 2.58±0.12 in FKN+0.1μM Simvastatin group, 1.69±0.09 in FKN+1μM Simvastatin group, 2.09±0.12 in FKN+10μM Simvastatin group, 2.40±0.11 FKN+20μM Simvastatin group, 2.94±0.08 in FKN+50μM Simvastatin group. Analyzed by statistics:(1)The expressions of NF-κB of FKN group increased remarkedly compared with the control group (P<0.01).(2)The expression of NF-κB of FKN+ SB203580 (P38MAPK inhibitor) group decreased obviously compared with the FKN group (P<0.01).(3)The difference of the integrated optical density values had the remarkable statistics meaning between control group and FKN+ SB203580 (P38MAPK inhibitor) group (P<0.01).(4)The expression of NF-κB of all of the Simvastatin group groups decreased remarkedly compared with FKN group (P<0.01). Comparing to the others, the role of the concentration of 1μmol/L Simvastatin is the strongest,they showed remarkable stastic difference (P<0.01),had shown no dose-dependent manner.(5)The expression of NF-κB of each Simvastatin group increased obviously compared with control group (P<0.01).
Conclusion: (1)FKN/CX3CR1 activates NF-κB in human PBMC(.2) P38MAPK plays an important role during the process that PBMC activate NF-κB induced by FKN/CX3CR1.(3)Simvastatin prevents FKN-induced NF-κB activation in human PBMC.Comparing to the others, the concentration of 1μmol/L Simvastatin is an ideal one chosen, has shown no dose-dependent manner.
实验方法:采用密度梯度离心法分离人外周血单个核细胞(peripheral blood mononuclear cells,PBMC),实验分8组,空白组、FKN组、FKN+P38MAPK抑制剂(SB203580)组、FKN+辛伐他汀0.1μmol/L组、FKN+辛伐他汀1μmol/L组、FKN+辛伐他汀10μmol/L组、FKN+辛伐他汀20μmol/L组及FKN+辛伐他汀50μmol/L组,采用Western Blot法检测各组单个核细胞胞核内的NF-κB表达量。
实验结果:(1)FKN组核内NF-κB含量较空白组增加,统计学分析差异均有显著性;(2)FKN+P38MAPK抑制剂(SB203580)组核内NF-κB水平较FKN组降低,但较空白组增加,经统计学分析组间差异有显著性;(3)FKN+辛伐他汀各组核内NF-κB水平较FKN组降低,但较空白组增加,且以1μmol/L浓度下作用最强,经统计学分析组间差异有显著性。
实验结论:(1)FKN/CX3CR1可升高单个核细胞NF-κB的活化水平;(2)P38MAPK抑制剂(SB203580)可降低FKN/CX3CR1诱导的单个核细胞NF-κB的活性;(3)辛伐他汀可降低FKN/CX3CR1诱导的单个核细胞中NF-κB的活性,且1μmol/L辛伐他汀为可供选择的相对理想的预处理浓度,未表现出浓度依赖性。
The research about atherosclerosis has been going on for hundreds of years, so many hypotheses about its pathogenesis have been proposed. With in-depth study, inflammation receives much concern. Fractalkine (FKN), a new chemokine which is discovered in 1997, has both membranebinding and soluble forms, it can mediate capture, firm adhesion and migration of monocytes and lymphocytes to the vascular endothelial cells under physiologic flow conditions. Large number of studies have demonstrated that FKN and its receptor CX3CR1 are involved in the inflammatory response of atherosclerosis, but its mechanism remains to be clear. Mitogen-activated protein kinase (MAPK) is one of the key important components of the Signal Transduction Systems. P38 mitogen-activated protein kinase (P38MAPK), is a member of MAPK family, which is widely presented in the cytoplasm. its signaling pathway plays an important role in inflammation. Nuclear factorκB (NF-κB) has been found to be important transcription factors which participate in the gene regulation of inflammation, immunity, stress response, cell growth and oncogenesis, it plays an important role in inflammation and immune responses. Up to now, statins (3 - hydroxy - 3 - methyl coenzyme A (HMG - CoA) reductase inhibitors) are widely used in clinical activities, the clinical effect benefits not only from the classic effects of lipid modulation, but also from the anti-inflammatory effects, the antioxidation effects, the platelet aggregation inhibitory activity, and so on.Therefore, their non-lipid mechanisms may plays an important role in prevention and treatment of atherosclerosis.
We associated FKN/CX3CR1, P38MAPK, NF-κB as well as peripheral blood mononuclear cells (PBMC), studied relationship of them and anti-inflammatory mechanism of Simvastatin.
Objective:The aims of the study are as follows: (1) To research the effects of FKN on NF-κB activation in mononuclear cells. (2) To research the function of p38 mitogen-activated protein kinase(P38MAPK) in the signal transduction mechanism of FKN/CX3CR1 affecting atherosclerosis. (3) To research the intervention of 3-hydroxy-3-methyl coenzyme A reductase inhibitors Simvastatin in the signal transduction mechanism of Fractalkine/CX3CR1 affecting atherosclerosis.
Methods:(1)PBMC were isolated from fresh blood of healthy volunteers by Ficoll-Paque gradient centrifugation.(2)Divide the extractive Peripheral blood monocytes into eight groups:control group,FKN group,FKN+SB203580(P38MAPK inhibitor) group, FKN+1μmol/L Simvastatin group, FKN+0.1μmol/L Simvastatin group, FKN+10μmol/L Simvastatin group, FKN+20μmol/L Simvastatin group, FKN+50μmol/L Simvastatin group.(3)Measure the NF-κB actived by monocytes in each group by western blot method.
Result: The following are the integrated optical density valuesmeasured by western blot in each group: 1.00±0.00 in control group, 3.28±0.11 in FKN group, 1.26±0.14 in FKN+ SB203580 (P38MAPK inhibitor) group, 2.58±0.12 in FKN+0.1μM Simvastatin group, 1.69±0.09 in FKN+1μM Simvastatin group, 2.09±0.12 in FKN+10μM Simvastatin group, 2.40±0.11 FKN+20μM Simvastatin group, 2.94±0.08 in FKN+50μM Simvastatin group. Analyzed by statistics:(1)The expressions of NF-κB of FKN group increased remarkedly compared with the control group (P<0.01).(2)The expression of NF-κB of FKN+ SB203580 (P38MAPK inhibitor) group decreased obviously compared with the FKN group (P<0.01).(3)The difference of the integrated optical density values had the remarkable statistics meaning between control group and FKN+ SB203580 (P38MAPK inhibitor) group (P<0.01).(4)The expression of NF-κB of all of the Simvastatin group groups decreased remarkedly compared with FKN group (P<0.01). Comparing to the others, the role of the concentration of 1μmol/L Simvastatin is the strongest,they showed remarkable stastic difference (P<0.01),had shown no dose-dependent manner.(5)The expression of NF-κB of each Simvastatin group increased obviously compared with control group (P<0.01).
Conclusion: (1)FKN/CX3CR1 activates NF-κB in human PBMC(.2) P38MAPK plays an important role during the process that PBMC activate NF-κB induced by FKN/CX3CR1.(3)Simvastatin prevents FKN-induced NF-κB activation in human PBMC.Comparing to the others, the concentration of 1μmol/L Simvastatin is an ideal one chosen, has shown no dose-dependent manner.
引文
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[2] Ross R. Atherosclerosis-An Inflammatory Disease[J]. New England Journal of Medicine,1999,340(2):115-126.
[3] Mcdermott D H, Halcox J, Schenke W H, et al. Association between poly- morphism in the chemokine receptor CX3CR1 and coronary vascular endothelial dysfunction and atherosclerosis[J]. Circulation Research,2001,89(5):401-407.
[4] Cominacini I, Garbin U, Pasini A F, et al. The expression of adhesion molecules on endothelial cells is inhibited by troglitazone through its anti- oxidant activity[J]. Cell Adhesion And Communication,1999,7(3):223-231.
[5] Ross R, Glomset J A. The pathogenesis of atherosclerosis (first of two parts)[J]. N Engl J Med,1976,295(8):369-377.
[6] Ross R. The pathogenesis of atherosclerosis--an update[J]. N Engl J Med, 1986,314(8):488-500.
[7] Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s [J]. Nature,1993,362(6423):801-809.
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