Mycoplasma pneumoniae induces IL-1βproduction through activating NL-RP3 inflammasome by ROS in RAW264.7 cells / 中国病理生理杂志

ABSTRACT

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-THODSRAW264.7 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.