巨噬细胞中oxLDL对TLR4-Src信号通路激活的调控

OxLDL Promotes the Activation of TLR4-Src Signaling Pathway in Macrophages

目的探讨氧化型低密度脂蛋白(oxidized low-density lipoprotein,oxL DL)对于巨噬细胞中Toll样受体4(toll like receptor 4,TLR4)-Src信号通路激活的调控作用.方法 oxL DL(50 ug/mL)按不同时间(0、15、30和60 min)刺激巨噬细胞,同时采用特异性siR NA抑制巨噬细胞内TLR4表达后,Western blot检测Src-Y418位点磷酸化水平;并利用免疫共沉淀法及细胞免疫荧光法检测TLR4与Src及其家族相似蛋白Fyn和Lyn的体外结合情况.结果oxL DL诱导巨噬细胞内Src-Y418磷酸化水平显著性升高,且呈时间相关性.而抑制TLR4表达则可显著性削弱oxLDL对于Src的激活作用(P<0.01).此外,oxLDL诱导细胞内TLR4与磷酸化Src(p-Src)相互结合并共定位于细胞膜表面,而不与Src其他家族成员Fyn及Lyn结合.结论oxLDL通过诱导TLR4与Src相互结合,从而激活Src磷酸化水平,进而诱发动脉粥样硬化过程中脂质累积及炎症反应.

Objective To investigate the activation of toll-like receptor4(TLR4)-Src signaling pathway inoxidized low-density lipoprotein(oxLDL)-induced macrophages.Methods The macrophages were stimulated withoxLDL(50ug/ml)for different time points(0,15,30,60minutes),then the phosphorylation of SrcY418were detected by western blot.TLR4were knocked down via transfection with TLR4-specific siRNA,and theexpression of phosphorylated Src(p-Src)in mock and knock-down group was further tested after stimulated withoxLDL(50ug/ml)for60minutes;then macrophages were stimulated with oxLDL(50ug/ml)for different timegradient(0,15,30,60minutes),and the connection of TLR4and Src or other members of Src family such asFyn and Lyn in vitro were confirmed by immunoprecipitation,with the subcellular localization of TLR4and Src inoxLDL-stimulated macrophages by immunofluorescence assay.Results The phosphorylation of Src Y418wassignificantly increased in oxLDL-stimulated macrophages in a time-dependent manner.When the expression of TLR4was knocked down,the phosphorylation of Src in macrophages induced by oxLDL was decreased(P<0.01).With stimulation with oxLDL,TLR4connected with p-Src in a reciprocal manner and co-localized on the surface ofmacrophages,but not with Fyn or Lyn.Conclusions OxLDL can promote the activation of TLR4-Src signaling pathway by meditating the phosphorylation of Src through interaction with TLR4,and induce the lipid accumulationand inflammation in macrophages which take indispensable roles in the process of atherosclerosis.