目的 探讨双特异性磷酸酶5(DUSP5)对卡介苗(BCG)介导的小鼠RAW264.7 巨噬细胞炎性反应的调控作用.方法 Western blot 法检测 BCG 感染 RAW264.7 巨噬细胞0.5、1、2、4、6、8、12、24 h,DUSP5 表达变化;采用小干扰 RNA(siRNA)技术下调RAW264.7巨噬细胞DUSP5水平,并设置siRNA阴性对照(si-NC)组、DUSP5敲低(si-DUSP5)组、si-NC联合BCG感染组、si-DUSP5联合BCG感染组.实时定量PCR检测细胞白细胞介素1 β(IL-1β)、IL-6、肿瘤坏死因子α(TNF-α)、IL-10的mRNA表达,ELISA检测细胞上清液IL-1β、IL-6、TNF-α、IL-10含量;Western blot法检测细胞核因子κB(NF-κB)与磷酸化的NF-κB(p-NF-κB)表达变化.结果 BCG感染上调RAW264.7巨噬细胞核DUSP5蛋白表达,并在BCG刺激4 h后,DUSP5表达量达到高峰;与si-NC联合BCG感染组相比,敲低DUSP5抑制细胞促炎因子IL-1β、IL-6与TNF-α表达与分泌,而抑炎因子IL-10表达不受DUSP5的影响;且敲低DUSP5抑制细胞NF-κB磷酸化.结论 敲低DUSP5通过阻断NF-κB信号抑制BCG介导的巨噬细胞炎性反应.
Knockdown of dual-specificity phosphatase 5(DUSP5)inhibits BCG-induced inflammatory response in RAW264.7 macrophages via blocking NF-κB signaling pathway
Objective To explore the regulatory role of dual-specificity phosphatase 5(DUSP5)in BCG-mediated inflammatory response in mouse RAW264.7 macrophages.Methods Western blot analysis was employed to detect the expression changes of DUSP5 in BCG-infected RAW264.7 macrophages at the period of 0.5,1,2,4,6,8,12 and 24 hours.Intracellular DUSP5 was reduced by small interfering RNA(siRNA)and transfected RAW264.7 macrophages were divided into siRNA-negative control(si-NC)group,DUSP5 knockdown(si-DUSP5)group,si-NC combined BCG infection group,and si-DUSP5 combined BCG infection group.Real-time quantitative PCR was conducted to measure the mRNA expression of interleukin 1β(IL-1β),IL-6,tumor necrosis factor α(TNF-α),and IL-10 in cells.ELISA was performed to measure the concentration of the cytokines in cell culture medium.Western blot analysis was performed to detect the expression changes of cellular nuclear factor κB(NF-κB)and phosphorylated NF-κB(p-NF-κB).Results BCG infection upregulated DUSP5 protein expression in RAW264.7 macrophages with the expression of DUSP5 reaching the peak after 4 hours'BCG stimulation.Comparing with si-NC combined BCG infection group,DUSP5 knockdown inhibited the expression and secretion of pro-inflammatory factors IL-1β,IL-6,and TNF-α,while the expression of the anti-inflammatory factor IL-10 was not affected by DUSP5.Moreover,knockdown of DUSP5 inhibited the phosphorylation of NF-κB in cells.Conclusion DUSP5 knockdown inhibites BCG-mediated macrophage inflammatory response via blocking NF-κB signaling activation.
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