AIM:To investigate whether Mycoplasma pneumoniae ( Mp)-induced interleukin-1β( IL-1β) pro-duction in RAW264.7 cells is through the activation of NLRP3 inflammasome via reactive oxygen species (ROS).ME-THODS:RAW264.7 cells were randomly divided into 3 groups.In normal group , RAW264.7 cells were treated without Mp.In model group, RAW264.7 cells were treated with 1∶10 multiplicity of infection ( MOI) of Mp.In NAC group, RAW264.7 cells were pretreated with N-acetylcysteine ( NAC) at a concentration of 5 mmol/L for 30 min before infection with Mp.The RAW264.7cells were infected with Mp (1∶10 MOI) for 4, 8, 16 and 24 h in model group and NAC group , respectively.The intracellular ROS level was analyzed by flow cytometry .The mRNA expressions of NLRP3, ASC and caspase-1 were detected by real-time PCR.The protein levels of NLRP3, ASC and caspase-1 p20 were determined by Western blot.The levels of pro-inflammatory cytokine IL-1βin the supernatant were measured by ELISA .RESULTS:Compared with normal group , the production of ROS were significantly increased at 4, 8, 16 and 24 h after infection, the mRNA expression of NLRP3, ASC and caspase-1 were increased at 8, 16 and 24 h after infection, the protein levels of NL-RP3, ASC and caspase-1 p20 were increased at 16 and 24 h after infection, and the releases of IL-1βwere increased at 24 h after infection in model group (P<0.01).Compared with the model group, the level of ROS in NAC group decreased, so as the expression of NLRP3, ASC and caspase-1 at mRNA and protein levels and the releases of IL-1βin the superna-tant at the corresponding time points .CONCLUSION:Mp may stimulate the ROS production to activate NLRP 3 inflam-masome in RAW264.7 cells.%目的：观察肺炎支原体（ Mycoplasma pneunoniae，Mp）促进NLRP3炎性体的活化及下游促炎性细胞因子白细胞介素－1β（ IL－1β）的表达是否与活性氧簇（ ROS）有关。方法：预先用5 mmol／L ROS清除剂N－乙酰半胱氨酸（ NAC）预处理RAW264．7细胞30 min，用10 MOI Mp分别感染RAW264．7细胞4、8、16和24 h。流式细胞术检测细胞内ROS水平；real－time PCR法检测细胞NLRP3、ASC和caspase－1 mRNA的表达；Western blot 法检测NL－RP3、ASC和caspase－1 p20的蛋白水平；ELISA法检测细胞上清液IL－1β的分泌水平。结果：与正常组比较，感染后4、8、16和24 h模型组ROS生成显著增加（ P＜0．01）；感染后8、16和24 h模型组NLRP3、ASC和caspase－1 mRNA的表达显著增加（P＜0．01），感染后16和24 h NLRP3、ASC和caspase－1 p20的蛋白水平上调（P＜0．01），感染后24 h 细胞上清液IL－1β含量显著增加（P＜0．01）；与模型组比较，NAC组在上述相应时点ROS生成降低，NLRP3、ASC和caspase－1 mRNA的表达下调，蛋白水平下降，细胞上清液IL－1β的含量减少。结论： Mp可能通过刺激RAW264．7细胞生成ROS而激活NLRP3炎性体。
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