TRIM31过表达改善脂多糖诱导下神经细胞的炎症损伤  

TRIM31 overexpression improved neurocellular inflammatory injury induced by lipopolysaccharide

作　　者：王涛 梁日初 周佳[2] 罗炜 WANG Tao;LIANG Ri-Chu;ZHOU Jia;LUO Wei(Neurosurgery Department,the Second Affiliated Hospital of South China University,Hengyang 421001,China)

机构地区：[1]南华大学附属第二医院神经外科,衡阳421001 [2]南华大学附属第一医院急诊科,衡阳421001 [3]南华大学附属第一医院心血管内科,衡阳421001

出　　处：《中国免疫学杂志》2020年第16期1935-1940,共6页Chinese Journal of Immunology

基　　金：湖南省卫生健康委2020年度科研立项课题(No.20201912);衡阳市科技局指导性课题(No.132)。

摘　　要：目的:探讨E3泛素连接酶31(TRIM31)对LPS诱导PC12细胞炎症性损伤的保护作用和机制。方法:用不同浓度LPS处理PC12细胞24 h,MTT法检测细胞增殖活性,Western blot检测LPS最佳浓度处理后PC12细胞TRIM蛋白表达水平。将TRIM31过表达质粒(pcDNA3.1-TRIM31)及其阴性对照质粒(pcDNA3.1-NC)转染至PC12细胞,qRT-PCR和Western blot检测细胞TRIM31 mRNA和蛋白表达水平。PC12细胞分为对照组(Control)、LPS组、LPS+NC组和LPS+TRIM31组,分组干预后,ELISA检测细胞上清IL-6、TNF-α、IL-1β和IL-18含量,流式细胞术检测细胞凋亡率,免疫荧光检测NLRP3蛋白表达,qRT-PCR检测NLRP3、caspase-1 mRNA表达,Western blot检测NLRP3、caspase-1、Cleaved-caspase-3、Bcl-2和Bax蛋白表达。结果:LPS剂量增加,PC12细胞增殖活性逐渐降低(P<0.05),LPS处理可降低PC12细胞TRIM31蛋白表达水平(P<0.01),TRIM31过表达,PC12细胞TRIM31 mRNA和蛋白表达水平显著提高(P<0.05)。与Control组相比,LPS组细胞上清中IL-6、TNF-α、IL-1β和IL-18含量、细胞凋亡率及细胞Cleaved-caspase-3和Bax蛋白水平显著提高(P<0.05),Bcl-2和TRIM31蛋白水平显著降低(P<0.05),NLRP3蛋白荧光强度及NLRP3、caspase-1 mRNA和蛋白表达水平显著提高(P<0.05);与LPS组相比,LPS+TRIM31组细胞上清中IL-6、TNF-α、IL-1β和IL-18含量、细胞凋亡率及细胞Cleaved-caspase-3和Bax蛋白水平显著降低(P<0.05),Bcl-2蛋白水平显著提高(P<0.05),NLRP3荧光强度及NLRP3、caspase-1 mRNA和蛋白表达水平显著降低(P<0.05)。结论:过表达TRIM31能通过抑制NLRP3炎症小体活化,改善LPS诱导的PC12细胞炎症损伤。Objective:To investigate protective effect and mechanism of E3 ubiquitin ligase 31(TRIM31)on LPS-induced inflammatory injury of PC12 cells.Methods:PC12 cells were treated with different concentrations of LPS for 24 h.Cell proliferation activity was detected by MTT,and Western blot to detect expression of TRIM31 protein in PC12 cells treated with optimal LPS.TRIM31 overexpressed plasmid(pcDNA3.1-TRIM31)and its negative control plasmid(pcDNA3.1-NC)were transfected into PC12 cells,and mRNA and protein expression levels of TRIM31 were detected by qRT-PCR and Western blot.PC12 cells were divided into Control group,LPS group,LPS+NC group and LPS+TRIM31 group.After intervention,contents of IL-6,TNF-α,IL-1βand IL-18 in supernatant were detected by ELISA.Apoptosis rate was detected by flow cytometry.NLRP3 protein expression was detected by immunofluorescence.mRNA expression of NLRP3 and caspase-1 were detected by qRT-PCR.Protein expressions of NLRP3,caspase-1,Cleaved caspase-3,Bcl-2 and Bax were detected by Western blot.Results:With increasing of LPS dose,proliferation activity of PC12 cells decreased gradually(P<0.05).LPS treatment reduced expression of TRIM31 protein in PC12 cells(P<0.01).After TRIM31 was overexpressed,mRNA and protein expression levels of TRIM31 were significantly increased in PC12 cells(P<0.05).Compared with Control group,contents of IL-6,TNF-α,IL-1β,IL-18,apoptosis rate,Cleaved caspase-3 and Bax protein levels in cell supernatant of LPS group were significantly increased(P<0.05),and levels of Bcl-2 and TRIM31 protein were significantly decreased(P<0.05).Fluorescence intensity of NLRP3 protein and expression levels of NLRP3,caspase-1 mRNA and protein in cell supernatant of LPS group were significantly increased(P<0.05).Compared with LPS group,contents of IL-6,TNF-α,IL-1β,IL-18,apoptosis rate,Cleaved-caspase-3 and Bax protein levels in cell supernatant of LPS+TRIM31 group were significantly decreased(P<0.05),and levels of Bcl-2 protein were significantly increased(P<0.05).Fluorescence intensity o

关 键 词：TRIM31 NLRP3炎症小体 脂多糖 神经损伤 

分 类 号：Q291[生物学—细胞生物学]