汉黄芩素对LPS和ATP联合诱导的巨噬细胞炎症反应的抑制作用

Wogonin-mediated inhibition of LPS and ATP-induced inflammation in mouse macrophages

目的:探究汉黄芩素对巨噬细胞炎症反应的抑制作用。方法:设正常对照组,LPS和ATP联合刺激组(LPS刺激4 h后加ATP刺激30 min),汉黄芩素干预组(刺激的同时加入汉黄芩素)。利用免疫荧光法观察各组细胞NF-κB的核定位,荧光定量PCR法检测各组细胞IL-1β、IL-18、IL-6、TNF-α、NLRP3及Caspase-1的mRNA水平,流式细胞术检测各组细胞活性氧(ROS)水平,酶联免疫吸附法检测胞外IL-6和TNF-α水平。结果:与LPS和ATP刺激组相比,汉黄芩素干预组细胞胞内NF-κB核定位明显减少,胞内IL-1β、IL-18、IL-6、TNF-α、NLRP3和Caspase-1的mRNA,胞内ROS及胞外IL-6和TNF-α水平都显著降低(P<0. 01)。结论:汉黄芩素可能通过减少巨噬细胞ROS的产生,降低NF-κB的核定位,进而弱化NF-κB调控的炎症相关分子的基因转录来干预巨噬细胞的炎症反应。

Objective:To investigate the wogonin-mediated inhibition of LPS and ATP-induced inflammation in mouse macrophages. Methods: The macrophages (RAW264.7) were divided into three groups,including the control group (macrophages unchallenged),both LPS and ATP stimulation induced group (macrophages were induced with ATP for 30 minutes after 4 hours-induction with LPS) and wogonin intervention group (macrophages challenged with LPS and ATP in the wogonin-contained medium).Immunofluorescence assay (IFA) were adopted to observe nuclear location of NF-κB in different macrophages,reverse transcription quantitative polymerase chain reaction (RT-QPCR) was used to identify the mRNAs (IL-1β,IL-18,IL-6,TNF-α,NLRP3 and Caspase-1),fluorescence-activated cell sorting (FACS) was used to analyze the intracellular ROS,and enzyme linked immunosorbent assay (ELISA) was used to check the extracellular secreted IL-6 and TNF-α. Results: Compared with the cells from both LPS and ATP stimulation induced group,the reduced nuclear location of NF-κB was observed in cells from wogonin intervention group.Moreover,the intracellular ROS and mRNAs (IL-1β,IL-18,IL-6,TNF-α,NLRP3 and Caspase-1) were significantly decreased,including the extracellular IL-6 and TNF-α. Conclusion: Wogonin may prevent ROS generation and decrease nuclear translocation of NF-κB,attenuating the gene transcription mediated by NF-κB factor to inhibit the inflammation in challenged macrophages.