TLR4/MAPKs信号通路在ox-LDL诱导的血管平滑肌细胞分泌MCP-1中的作用

Role of Toll-like receptor 4/MAPKs pathway on monocyte chemoattractant protein-1 secretion induced by oxidized low density lipoprotein in vascular smooth muscle cells

目的:探讨Toll样受体4/丝裂原活化蛋白激酶(TLR4/MAPKs)信号通路在氧化性低密度脂蛋白(ox-LDL)诱导的血管平滑肌细胞分泌单核细胞趋化因子-1(MCP-1)中的作用。方法:在ox-LDL刺激下采用逆转录聚合酶链技术(RT-PCR)和酶联免疫吸附试验(ELISA)检测血管平滑肌细胞MCP-1的表达,用Western blotting检测细胞外信号调节激酶(ERK1/2)、p38促分裂原活化蛋白激酶(p38MAPK)磷酸化水平的变化。同时,分别应用TLR4中和抗体(TLR4单克隆抗体、TLR4阻断剂)、PD98059(ERK1/2特异性抑制剂)、SB23015(p38MAPK特异性抑制剂)、SP600125(JNK特异性抑制剂),观察其对ox-LDL诱导的MCP-1的表达和ERK1/2、p38MAPK磷酸化水平的影响。结果:ox-LDL刺激血管平滑肌细胞上调MCP-1mRNA和其蛋白的表达(P<0.05);用TLR4中和抗体、PD98059、SB23015预孵育后MCP-1mRNA和其蛋白的表达较单独ox-LDL刺激情况下降低(P<0.05),而用SP600125预孵育后降低不明显(P>0.05);TLR4调节了ERK1/2和p38MAPKs的磷酸化水平。结论:ox-LDL是TLR4的内源性配体;ox-LDL通过或部分通过TLR4/ERK1/2和TLR4/p38MAPK信号通路介导血管平滑肌细胞MCP-1的表达。

AIM：To investigate the role of Toll-like receptor 4/MAPKs pathway on the secretion of monocyte chemoattractant protein-1 （MCP-1） induced by oxidized low density lipoprotein （ox-LDL） in the vascular smooth muscle cells （VSMCs）. METHODS：mRNA and protein expressions of MCP-1 in VSMCs stimulated with oxidized low density lipoprotein were determined by RTPCR and ELISA,respectively. The phosphorylated forms of ERK1/2 and p38MAPK were determined by Western blotting. TLR4 neutralizing antibodies （ a specific TLR4 inhibitor）,PD98059 （ERK1/2 specific inhibitor）,SB23015 （p38MAPK specific inhibitor） and SP600125 （JNK specific inhibitor） were used to investigate the underlying mechanisms. RESULTS：The mRNA and protein expressions of MCP -1 in VSMCs were upregulated by ox-LDL （P 0. 05）,while those were inhibited by TLR4 neutralizing antibodies,PD98059 or SB23015 （P 0. 05）,but not by SP600125 （P 0. 05）. TLR4 had regulatory effect on the phosphorylation of ERK1/2 and p38MAPK. CONCLUSION：ox -LDL is an endogenous ligand of TLR4. The secretion of MCP-1 induced by ox-LDL in VSMCs is at least in part via TLR4 /ERK1 /2 and TLR4 /p38MAPKs pathways.