Ren Chao, Liu Wenmiao, Luo Jia
Institute of Medical Sciences, Department of Medical Record Statistics, General Hospital of Ningxia Medical University, Yinchuan 750001, China.
The Center of Laboratory Medicine, General Hospital of Ningxia Medical University, Yinchuan 750001, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2024 May;40(5):404-410.
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.
目的 探讨双特异性磷酸酶5（DUSP5）在卡介苗介导的小鼠RAW264.7巨噬细胞炎症反应中的调控作用。方法 采用蛋白质免疫印迹法检测卡介苗感染RAW264.7巨噬细胞0.5、1、2、4、6、8、12和24小时时DUSP5的表达变化。利用小干扰RNA（siRNA）降低细胞内DUSP5表达，将转染后的RAW264.7巨噬细胞分为siRNA阴性对照组（si-NC组）、DUSP5基因敲低组（si-DUSP5组）、si-NC联合卡介苗感染组和si-DUSP5联合卡介苗感染组。采用实时定量聚合酶链反应检测细胞中白细胞介素1β（IL-1β）、IL-6、肿瘤坏死因子α（TNF-α）和IL-10的mRNA表达。采用酶联免疫吸附测定法检测细胞培养基中细胞因子的浓度。采用蛋白质免疫印迹法检测细胞核因子κB（NF-κB）和磷酸化核因子κB（p-NF-κB）的表达变化。结果 卡介苗感染上调RAW264.7巨噬细胞中DUSP5蛋白表达，卡介苗刺激4小时后DUSP5表达达峰值。与si-NC联合卡介苗感染组相比，DUSP5基因敲低抑制促炎因子IL-1β、IL-6和TNF-α的表达与分泌，而抗炎因子IL-10的表达不受DUSP5影响。此外，DUSP5基因敲低抑制细胞中NF-κB的磷酸化。结论 DUSP5基因敲低通过阻断NF-κB信号激活抑制卡介苗介导的巨噬细胞炎症反应。
