ABSTRACT
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.
ABSTRACT
To observe the effect of livin gene-specific siRNA interference on proliferation and apoptosis of nasopharyngeal carcinoma (NPC) cell line CNE-2Z.Methods: siRNA expression vectors pGPU6/GFP/Neo-livin were transfected into NPC cell line CNE-2Z by using Lipofectamine 2000.The expressions of livin mRNA and protein were detected by semi-quantitative reverse transcriptase-polymerase chain reaction ( RT-PCR) and Western blot.The changes of caspase-3 activity were assessed by kinase semi-quantitative activity test.The proliferation activity and apoptosis of CNE-2Z cells were examined by MTT and flow cytometry respectively.Results:The expression levels of livin mRNA and protein in pGPU 6/GPF/Neo-livin transfected CNE-2Z cells were significantly lower than those in untreated and pGPU 6/GFP/Neo-shNC ( control non-target siRNA ) transfected cells with the expression inhibitory rate of 64.38% and 61.43% respectively.The caspase-3 activity and the apoptotic rate of experimental group cells were increased obviously.The growth of CNE-2Z cells was inhibited by siRNA recombinant expression vector transfection.Conclusion:siRNA targeting livin gene inhibits proliferation and induce apoptosis of CNE-2Z cells.