过表达NLRC3抑制脂多糖诱导的小胶质 BV2细胞炎症反应实验研究  

NLRC3 overexpression inhibits the LPS-induced inflammatory reaction in microglial BV2 cells

作　　者：康涛[1] 郭晓敏[1] 王涛[1] 雷琦[1] 杨谦[1] 郭荷娜[1] KANG Tao;GUO Xiaomin;WANG Tao;LEI Qi;YANG Qian;GUO Hena(Second Department of Neurology,Shaanxi Provincial People’s Hospital,Xi’an 710068,China)

机构地区：[1]陕西省人民医院神经内二科,陕西西安710068

出　　处：《陕西医学杂志》2022年第10期1187-1190,1205,共5页Shaanxi Medical Journal

基　　金：陕西省重点研发计划项目(2020SF-129)。

摘　　要：目的:研究过表达NLRC3对脂多糖(LPS)诱导的小鼠小胶质细胞BV2炎症反应的影响,并探究其相关分子机制。方法:采用不同浓度LPS(0.1、1、2、5 g/ml)处理BV2细胞24 h,CCK-8法检测其细胞活力。以0.1 g/ml LPS诱导的BV2细胞作为帕金森病细胞炎症模型。实时定量PCR(qRT-PCR)和Western blot分别检测细胞中NLRC3的mRNA和蛋白质表达水平。用过表达NLRC3的腺病毒转染BV2细胞,然后用0.1 g/ml LPS处理24 h,用qRT-PCR和Western blot检测转染效率,利用比色法和酶联免疫吸附测定法(ELISA)分别检测一氧化氮(NO)含量和炎症因子浓度。Western blot检测磷脂酰肌醇-3-激酶(PI3K)/蛋白激酶B(AKT)和核因子κB(NF-κB)信号通路蛋白的相对表达量。结果:与未处理组相比,0.1 g/ml LPS对BV2的细胞活力无明显影响,说明0.1 g/ml LPS对BV2细胞没有细胞毒性作用。然而,LPS处理显著降低BV2细胞中NLRC3的表达水平(P<0.05)。过表达NLRC3后,NLRC3的mRNA与蛋白表达量显著上调(P<0.05),而LPS诱导的NO含量与炎症因子水平均显著降低(P<0.05),同时,PI3K/AKT和NF-κB信号通路蛋白的磷酸化水平也显著降低(P<0.05)。结论:NLRC3通过抑制NF-κB和PI3K/AKT通路的激活,从而抑制LPS诱导的BV2细胞的炎症反应。Objective:To study the effect of nucleotide-oligomerization domain(NOD)-like receptor subfamily C3(NLRC3) overexpression on the lipopolysaccharide(LPS)-induced inflammatory reaction in mouse microglial BV2 cells and explore its molecular mechanism.Methods:BV2 cells were treated with different concentrations of LPS(0.1,1,2,5 g/ml) for 24 hours,and the cell viability was detected by CCK-8 method.BV2 cells induced with 0.1 g/ml LPS were used as a cell model of Parkinson’s disease.Quantitative real-time PCR(qRT-PCR) and Western blot were used to detect the mRNA and protein expression levels of NLRC3.BV2 cells were transfected with adenovirus containing NLRC3 overexpression vector,and then treated with 0.1 g/ml LPS for 24 hours.Afterwards,the transfection efficiency was detected by qRT-PCR and Western blot.The content of nitric oxide(NO) and the concentrations of Inflammatory factors were measured by colorimetry and enzyme-linked immunosorbent assay(ELISA),respectively.The expression of nuclear factor κB(NF-κB) and phosphatidylinositol-3-kinase(PI3 K)/protein kinase B(AKT) pathway-related proteins were detected by Western blot.Results:Compared with the untreated group,0.1 g/ml LPS had no significant effect on the cell viability of BV2 cells,indicating that 0.1 g/ml LPS had no cytotoxic effect on BV2 cells.However,LPS treatment significantly decreased the expression level of NLRC3 in BV2 cells(P<0.05).After overexpressing NLRC3,the expression of mRNA and protein of NLRC3 were increased significantly,while NO content and inflammatory factors levels were decreased significantly,and the phosphorylation levels of PI3 K/AKT and NF-κB signaling pathway proteins were also significantly reduced(all P<0.05).Conclusion:NLRC3 inhibits LPS-induced inflammation in BV2 cells by inhibiting activation of NF-κB and PI3 K/AKT pathways.

关 键 词：NLRC3 小胶质细胞 炎症 磷脂酰肌醇-3-激酶/蛋白激酶B信号通路 核因子ΚB 

分 类 号：R739.41[医药卫生—肿瘤]