白三烯B4对巨噬细胞小凹蛋白-1表达的影响及其与冠状动脉斑块不稳定的关系

The role of leukotriene B4 on the expression of caveolin-1 in macrophage and its potential association with plaque unstable

目的研究小凹蛋白-1(Cav-1)及白三烯B4(LTB4)与冠状动脉斑块不稳定的关系,观察LTB4及其抑制剂对巨噬细胞Cav-1表达的影响并探讨其意义。方法免疫组化检测41例人尸检冠状动脉粥样斑块中巨噬细胞Cav-1蛋白的表达;以流式细胞技术检测167例入选患者外周血单核细胞Cav-1的表达水平,用ELISA检测血清中LB4的表达水平;分别用浓度为10-8mol/L、10-7mol/L的LTB4及10-7mol/LU75302处理THP-1源性的巨噬细胞,RT-PCR检测细胞内的TNF-αmRNA,Cav-1mRNA水平及Western Blot检测Cav-1蛋白表达的变化。结果与稳定斑块相比,不稳定斑块中巨噬细胞聚集处Cav-1表达下调或表达阴性。急性冠脉综合征(ACS)组中急性心梗(AMI)组及不稳定性心绞痛(UAP)组外周血单核细胞Cav-1表达水平皆显著低于稳定心绞痛(SAP)组、非冠心病组(99.2±40.8MnX比(176.1±90.3)MnX、(246.0±122.8)MnX(t=-4.786、5.112),(104.8±37.4)MnX比(176.1±90.3)MnX、(246.0±122.8)MnX(t=-4.508、-4.942,P<0.01);AMI组血清LTB4表达显著高于UAP组、SAP组及非冠心病组(209.8ng/L比40.7ng/L、59.5ng/L、63.8ng/L,Z=-3.771、-3.426、-2.849,P<0.01)。LTB4可下调Cav-1mRNA及蛋白表达水平(0.963、0.959比1.186,t=-5.079、-5.148),0.712比0.840(t=-4.050,P<0.01),上调肿瘤坏死因子-α(TNF-α)mRNA的表达(0.637比0.555)(t=5.148,P<0.01);U75302预处理组Cav-1mRNA及蛋白水平上调(1.089比0.959、0.963(t=3.018、3.087,0.799比0.712,t=2.849,P<0.05),TNF-αmRNA水平下降(0.529比0.637,t=-3.087,P<0.01)。结论冠状动脉不稳定斑块中巨噬细胞及ACS患者外周血单核细胞Cav-1表达降低,AMI患者血清中LTB4表达上调,提示高水平LTB4及低表达Cav-1与斑块不稳定有关。LTB4下调THP-1来源的巨噬细胞Cav-1mRNA及蛋白的表达,上调TNF-αmRNA水平,U75302预处理可拮抗上述LTB4的作用,提示使用拮抗LTB4的药物可能有稳定粥样斑块的作用。

Objective To study the effects of Caveolin-1（Cav-1）and Leukotriene B4（LTB4）on plaque destabilization,and to investigate the effects of LTB4 and LTB4 antagonist on the expression of Caveolin-1 in macrophage and its application.Methods The Cav-1 expression of macrophages in the coronary artery atherosclerotic plaques studied by immunohistochemisty was done in 41 cases of voluntary autopsy,Cav-1 expression on peripheral blood mononuclear cells measured by flow cytometry and serum LTB4 level by ELISA were done in 167 patients.Moreover,the macrophage differentiated from THP-1 was stimulated with LTB4 of different concentrations as 10 -8mol/L,10 -7mol/L and 10 -7mol/L of U75302,RT- PCR was performed to measure the mRNA level of TNF-α and Cav-1.The protein levels of Cav-1 was evaluated by Western Blot.Results Cav-1 expressions were lower or negative in macrophages from unstable plaques than stable ones.Compared with patients with Stable Angina Pectoris（SAP）or without coronary artery disease,the expression of Cav-1 in ACS patients was significantly lower（99.2 ± 40.8 MnX vs 176.1 ± 90.3 MnX and 246.0 ± 122.8 MnX（t = - 4.786 and 5.112）,104.8 ± 37.4 MnX vs 176.1 ± 90.3 MnX and 246.0 ± 122.8 MnX（t = - 4.508 and - 4.942,P 0.01）and the level of LTB4 in AMI patients was higher than the other patients or cases（209.8 ng/L vs 40.7 ng/L and 59.5 ng/L and 63.8 ng/L（Z = - 3.771 and - 3.426 and - 2.849）,P 0.01）.Furthermore,LTB4 down-regulated the expression of Cav-1 mRNA and protein translation in macrophages（0.963 and 0.959 vs 1.186（t = - 5.079 and - 5.148）,0.712 vs 0.840（t = - 4.050）,P 0.01）,but up-regulated the transcription of Tumor necrosis factor-alpha（TNF-α）mRNA（0.637 vs 0.555（t = 5.148）,P 0.01）.Moreover,preincubated with U75302 increased Cav-1 mRNA transcription or Protein translation（1.089 vs 0.959 and 0.963（t = 3.018 and 3.087）,0.799 vs 0.712（t = 2.849）,P 0.05）,but the transcription of TNF-α mRNA was decreased（0.529 vs 0.637,（t = - 3.087）,P 0.01）.Conclusions The expression of Cav-1 in macrophages from coronary artery atherosclerotic unstable plaques or on peripheral blood mononuclear cells from patients with ACS is down regulated,and the level of LTB4 in AMI is elevated.These results indicated that LTB4 abundance and Cav-1 poverty are associated with plaque unstability.LTB4 reduced Cav-1 mRNA transcription and protein translation but induced the production of TNF-α mRNA in macrophages,while LTB4 antagonist U75302 can inhibit the efforts of LTB4,suggesting LTB4 antagonist is a possible therapy to unstable plaque.