Effect of NLRP3 Knockdown on LPS-induced Inflammatory Response in BV2 Microglia
Objective To evaluate the impact and clinical significance of NOD-like receptor pyrin domain-containing protein 3(NLRP3)in the activation of lipopolysaccharide(LPS)-induced BV2 microglia cells.Methods shRNA plasmids were devised for BV2 microglia transfection,and the most effective transfection segment was identified via RT-PCR and Western blot assays.NLRP3 expression in the cell line was detected by Western blotting,while light microscopy was used to observe morpho-logical alterations in BV2 cells transfected with NLRP3-shRNA.Furthermore,enzyme-linked immunosorbent assay(ELISA)was used to quantify levels of inflammatory cytokines IL-18,IL-1β,TNF-α and NO in cell supernatants.Immunofluorescence staining was used to observe the expression and localization of microglial activation markers iNOS,Arg-1,Iba1,and NLRP3.Results NLRP3 was highly expressed in LPS-induced BV2 cells.The Western blot result showed that the mRNA expression level was the lowest and transfection was the least effective in NLRP3-mus-727 group.Microscopic examination revealed a round or short spindle-shaped morphology in BV2 cells transfected with shNLRP3,which was akin to resting state cells in the blank control group.ELISA showed that pro-inflammatory mediators IL-18,IL-1β,TNF-α and NO levels were decreased in BV2 cells trans-fected with shNLRP3(all P＜0.05).Immunofluorescence staining indicated a relative decrease in iNOS and Iba1 expression,with an upregulation of Arg-1 in BV2 cells transfected with shNLRP3.Conclusion NLRP3 is highly expressed in LPS-induced BV2 cells.Inhibition of NLRP3 expression can suppress the inflammatory response of BV2 cells induced by LPS,promoting their polarization towards the M2 phenotype.
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