摘要
为探讨细胞因子信号转导抑制因子3(suppressor of cytokine signaling 3,SOCS3)对高糖(high glucose,HG)诱导的大鼠肾小球系膜细胞RMC炎性因子表达的影响及机制,将RMC细胞分为6组:正常对照(normal control,NC)组、渗透压对照(osmotic control,OSM)组、HG 组、HG+TAK-242(TLR4 抑制剂)组、HG+sh-SOCS3 组和 HG+sh-SOCS3+TAK-242组.CCK-8法检测细胞的存活率;流式细胞术检测细胞的凋亡情况;ELISA检测细胞培养上清液中炎性因子TNF-α、IL-1β、IL-6和COX2表达水平;qPCR法检测RMC细胞TLR4、NF-κB p65和IKB-α mRNA的相对表达水平;Western blotting检测RMC细胞TLR4、NF-KBp65、p-p65、IKB-α和p-IκB-α蛋白的表达量.结果显示,与NC组相比,HG组细胞存活率显著降低(P＜0.01),细胞凋亡率,细胞上清液中TNF-α、IL-1β、IL-6和COX2表达水平,细胞中TLR4、p65、IKB-α mRNA相对表达水平和TLR4、p-p65、p-IKB-α蛋白表达水平均显著升高(均P＜0.01);与HG组相比,HG+TAK-242组和HG+sh-SOCS3组细胞存活率显著升高(均P＜0.05),细胞凋亡率,细胞上清液中TNF-α、IL-1β、IL-6和COX2表达水平,细胞中TLR4、p65、IκB-α mRNA 表达水平和 TLR4、p-p65、p-IKB-α 蛋白表达水平均显著降低(均 P＜0.05);HG+sh-SOCS3+TAK-242组较HG+TAK-242组效果更加显著(均P＜0.05).由此,SOCS3可通过促进TLR4/NF-KB信号通路的激活上调HG诱导的大鼠系膜细胞炎性因子表达,加重炎症反应.
Abstract
The aim of this study was to investigate the effect of suppressor of cytokine signaling 3(SOCS3)on the high glucose(HG)-induced inflammatory factors expression in rat glomerular mesangial cell(RMC)and the related mechanism.For this purpose,RMCs were divided into six groups:normal control(NC)group,osmotic control(OSM)group,HG group,HG+TAK-242(TLR4 inhibitor)group,HG+sh-SOCS3 group,and HG+sh-SOCS3+TAK-242 group.Cell viability was detected by CCK-8 assay and cell apoptosis was detected by FACS.The levels of inflammatory factors TNF-α,IL-1β,IL-6,and COX2 in cell culture supernatant were detected by ELISA.The mRNA expressions of TLR4,NF-κB p65,and IκB-α in RMC were detected by qPCR.The protein expressions of TLR4,NF-κB p65,p-p65,IκB-α,and p-IκB-α were detected by western blotting.The results showed that compared to those of NC group,the cell survival rate of the HG group was significantly decreased(P＜0.01),while the cell apoptosis rate,the levels of TNF-α,IL-1β,IL-6 and COX2 in cell culture supernatant,the mRNA expression of TLR4,p65 and IκBα and the protein expression of TLR4、p-p65 and p-IκB-α were significantly increased(all with P＜0.01).Compared to those of HG group,the cell survival rates of the HG+TAK-242 group and the HG+sh-SOCS3 group were significantly increased(both P＜0.05)while the apoptosis rate,the levels of TNF-α,IL-1β,IL-6 and COX2 in the cell culture supernatant,the mRNA expression of TLR4,p65,and IκB-α,and the protein expression of TLR4,p-p65,and p-IκB-α were significantly decreased(all with P＜0.05).The above changes were more significant in the HG+sh-SOCS3+TAK-242 group than the HG+TAK-242 group(all with P＜0.05).Taken together,SOCS3 up-regulates the expres-sion of inflammatory factors in RMCs cultured with HG by activating the TLR4/NF-κB signaling pathway,thus aggravates the inflammatory responses.
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