Abstract：
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.
摘要：
目的探讨双特异性磷酸酶5(DUSP5)在BCG介导的小鼠RAW264.7巨噬细胞炎症反应中的调控作用.方法采用Westernblot方法检测BCG感染的RAW264.7巨噬细胞在0.5、1、2、4、6、8、12和24小时时DUSP5的表达变化。小干扰RNA(siRNA)降低细胞内DUSP5,转染RAW264.7巨噬细胞分为siRNA阴性对照(si-NC)组,DUSP5敲除(si-DUSP5)组,si-NC联合卡介苗感染组,和si-DUSP5联合卡介苗感染组。实时定量PCR检测白细胞介素1β(IL-1β)mRNA表达,IL-6,肿瘤坏死因子α(TNF-α),和细胞中的IL-10。进行ELISA以测量细胞培养基中细胞因子的浓度。Westernblot检测细胞核因子κB(NF-κB)和磷酸化NF-κB(p-NF-κB)的表达变化。结果BCG感染上调RAW264.7巨噬细胞中DUSP5蛋白的表达，BCG刺激4小时后DUSP5的表达达到峰值。与si-NC联合卡介苗感染组相比，DUSP5敲低抑制促炎因子IL-1β的表达和分泌,IL-6和TNF-α,而抗炎因子IL-10的表达不受DUSP5的影响。此外，DUSP5敲除抑制细胞中NF-κB的磷酸化。结论DUSP5敲低可通过阻断NF-κB信号通路的激活抑制卡介苗介导的巨噬细胞炎症反应。
