RESUMO
Objective:To investigate the effects of Helicobacter pylori on NLRP3 inflammasomes activation in THP-1 ( human monocytic cell line) -derived macrophages and evaluate the role of ROS.Methods:H.pylori strain SS1 was co-cultured with the THP-1-derived macrophages at a multiplicity of infection (MOI) of 1∶100 based on trial results with different MOIs (ratios of THP-1 cells to bacteria ranging from 1∶25 to 1∶200).The co-culture supernatants and THP-1 cells were collected at various time points (3 h,6 h,12 h,24 h) and cytokine production was quantitated using ELISA analysis.The generation of intracellular ROS was detected by FCM,and the mRNA transcript levels of NLRP3 and caspase-1 were measured by Real-time PCR.Western blot was employed to analyze the expression of active caspase-1 subunit ( p10).Then we observed the inhibitory effects of NAC and siRNA specific for NLRP3 on the ex-pression of NLRP3 inflammasome-related components and the secretion of cytokines induced by H.pylori.Results:We found that H.pylori SS1 induced IL-1βand IL-18 production in human acute monocytic leukemia cell line THP-1 in a time-and dose-dependent manner.We further showed that H.pylori could induce the mRNA expressions of NLRP3 and caspase-1 in THP-1 cells.Moreover, release of IL-1βand IL-18 from H.pylori-infected THP-1 cells was suppressed by the ROS scavenger NAC,which was an agent known to inhibit NLRP3 inflammasome formation.NAC administration also resulted in a significant decrease in the level of H.pylori-induced caspase-1 protein expression in THP-1 cells.Additionally,secretion of IL-1βand IL-18 in response to H.pylori infection was remarkably reduced by NLRP3-siRNA.Conclusion:The induction of IL-1βand IL-18 secretion by H.pylori strain SS1 in THP-1 cells could be mediated through activation of NLRP3 inflammasome via ROS signaling pathway, which may be involved in the host innate immune defence and the pathogenesis of the bacteria.
