Effect of NLRP3 Knockdown on LPS-induced Inflammatory Response in BV2 Microglia
10.3870/j.issn.1672-0741.23.09.009
- VernacularTitle:敲减NLRP3抑制LPS诱导BV2小胶质细胞的炎症反应
- Author:
Xinzi XU
1
,
2
;
Rui WANG
;
Yaming DU
Author Information
1. 华中科技大学同济医学院附属武汉中西医结合医院神经内科,武汉 430022
2. 湖北中医药大学中医临床学院,武汉 430065
- Keywords:
microglia;
NLRP3 inflammasome;
shRNA
- From:
Acta Medicinae Universitatis Scientiae et Technologiae Huazhong
2024;53(1):1-5
- CountryChina
- Language:Chinese
-
Abstract:
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.
