白细胞介素-1受体相关激酶1在脂多糖诱导的足细胞损伤中的作用及其机制

Role and its mechanism of interleukin-1 receptor associated kinase 1 in lipopolysaccharide-induced podocyte injury

目的探讨白细胞介素(IL)-1受体相关激酶1(IRAK1)在脂多糖(LPS)诱导的足细胞损伤中的作用及其机制。方法将体外培养的永生化人足细胞分为5组:正常对照组(control组)、LPS组、LPS+空载转染组(LPS+si-NC组)、LPS+IRAK1干扰质粒转染组(LPS+si-IRAK1组)、LPS+肿瘤坏死因子受体相关因子6(TRAF6)干扰质粒转染组(LPS+si-TRAF6组)。采用蛋白质免疫印迹法(Western Blot)及实时荧光定量聚合酶链反应(RT-PCR)分别检测5组足细胞IRAK1和TRAF6蛋白及其mRNA的相对表达水平;采用免疫荧光检测5组足细胞紧密连接蛋白(ZO)-1、结蛋白(Desmin)的定位及相对表达水平;采用Western Blot检测5组足细胞IL-1β、IL-6、肿瘤坏死因子(TNF)-α、人核因子(NF)-κB抑制蛋白(IKB)α、NF-κB(p65)、磷酸化NF-κB(p-p65)蛋白的相对表达水平;采用免疫共沉淀(Co-IP)实验检测IRAK1和TRAF6结合情况。结果LPS组足细胞IRAK1、TRAF6蛋白及mRNA相对表达水平均高于control组,LPS+si-IRAK1组足细胞上述指标均低于LPS组(P<0.05)。LPS组、LPS+si-NC组足细胞Desmin、IL-1β、IL-6、TNF-α、p65、p-p65蛋白相对表达水平均高于control组,ZO-1、IKB-α蛋白相对表达水平均低于control组;LPS+si-IRAK1组、LPS+si-TRAF6组足细胞Desmin、IL-1β、IL-6、TNF-α、p65、p-p65蛋白相对表达水平均低于LPS组、LPS+si-NC组,ZO-1、IKB-α蛋白相对表达水平均高于LPS组、LPS+si-NC组(P<0.05)。Co-IP实验结果显示IRAK1与TRAF6存在结合。结论LPS诱导足细胞发生损伤并激活炎症反应,调控IRAK1通过靶向TRAF6/NF-κB通路减轻LPS导致的足细胞损伤。

Objective To explore the role and its mechanism of interleukin(IL)-1 receptor associated kinase(IRAK)1 in lipopolysaccharide(LPS)-induced podocyte injury.Methods Immortalized human podocytes cultured in vitro were divided into 5 groups:normal control group(control group),LPS group,LPS+null transfection group(LPS+si-NC group),LPS+IRAK1 interference plasmid transfection group(LPS+si-IRAK1 group),LPS+tumor necrosis factor receptor-associated factor 6(TRAF6)interference plasmid transfection group(LPS+si-TRAF6 group).Western blotting and real-time quantitative polymerase chain reaction(RT-PCR)were used to detect relative expression levels of IRAK1 and TRAF6 protein and mRNA in podocytes of 5 groups.Localization and relative expression levels of zonula occludens(ZO)-1 and Desmin in podocytes of 5 groups were detected by immunofluorescence.Relative expression levels of IL-1β,IL-6,tumour necrosis factor(TNF)-α,human nuclear factor(NF)-κB inhibitor(IKB)α,NF-κB(p65)and phosphorylated NF-κB(p-p65)in podocytes of 5 groups were detected by Western blotting.CO-Immunoprecipitation(Co-IP)experiment was used to detect the combination of IRAK1 and TRAF6.Results Relative expression levels of IRAK1 and TRAF6 protein and mRNA in podocytes of LPS group were higher than those in control group,and above indexes in podocytes of LPS+si-IRAK1 group were higher than those in LPS group(P<0.05).Relative expression levels of Desmin,IL-1β,IL-6,TNF-α,p65,p-p65 protein in podocytes of LPS group,LPS+si-NC group were higher than those in control group,relative expression levels of ZO-1 and IKB-αprotein were lower than those in control group;relative expression levels of Desmin,IL-1β,IL-6,TNF-α,p65,p-p65 protein in podocytes of LPS+si-IRAK1 group,LPS+si-TRAF6 group were lower than those in LPS group and LPS+si-NC group,relative expression levels of ZO-1 and IKB-αprotein were higher than those in LPS group and LPS+si-NC group(P<0.05).Co-IP experiment showed that IRAK1 and TRAF6 were combined.Conclusion LPS induces injury to podocyte and activates inflammatory response,and regulation of IRAK1 attenuates lipopolysaccharide-induced podocyte injury by targeting TRAF6/NF-κB pathway.