乳杆菌源性外囊泡对LPS诱导小胶质细胞活化的影响及蛋白质组学分析
Effect of Lactobacillus-derived outer vesicles on lipopolysaccharide-induced activation of microglia and proteomic analysis
杨彦芳 1徐反宁 1倪新莉1
作者信息
- 1. 宁夏医科大学总医院麻醉与围术期医学科,银川 750004
- 折叠
摘要
目的 评价乳杆菌源性细胞外囊泡(Lac-EVs)对脂多糖(LPS)诱导小胶质细胞活化的影响及蛋白质组学分析.方法 取生长状态较好的小鼠BV2小胶质细胞,采用随机数字表法分为3组(n=12):对照组(C组)、LPS组(L组)和LPS+Lac-EVs组(L+E组).C组正常培养;L组LPS(终浓度1 μg/ml)孵育24 h;L+E组于LPS处理后24 h加入Lac-EVs(终浓度2.5 μg/ml)孵育24 h.随后采用免疫荧光染色法检测CD86和CD206的表达.取L组和L+E组细胞沉淀,采用蛋白质组学方法筛选两组差异表达蛋白.对鉴定得到的差异表达蛋白进行生物信息学分析并采用RT-qPCR和Western blot法对载脂蛋白1(Apoa1)和G蛋白偶联受体激酶相互作用蛋白2(Git2)两个差异表达蛋白进行验证.结果 与C组相比,L组CD86表达上调,CD206表达下调(P＜0.05);与L组相比,L+E组CD86表达下调,CD206表达上调(P＜0.05).采用蛋白质组学法筛选出125个差异表达蛋白(FC=2.0,P＜0.05),其中66个蛋白表达上调,59个蛋白表达下调,其中Apoa1和Git2表达上调并且排名较前.GO分析结果表明,这些差异表达蛋白主要参与内皮细胞增殖、SDNA损伤检查以及脂蛋白运输等生物过程.KEGG分析结果表明,PPAR信号通路、内吞作用、代谢途径、MAPK信号通路等存在差异.Western blot法和RT-qPCR法测定上述差异蛋白的表达趋势与蛋白质组学结果一致.结论 Lac-EVs可抑制LPS诱导小胶质细胞向M1型极化,其机制可能与上调的差异表达蛋白Apoa1和Git2有关.
Abstract
Objective To evaluate the effect of Lactobacillus-derived extracellular vesicles(Lac-EVs)on lipopolysaccharide(LPS)-induced activation of microglia and proteomic analysis.Methods BV2 microglia obtained from mice with good growth status were divided into 3 groups(n=12 each)using a ran-dom number table method:control group(group C),LPS group(group L)and LPS+Lac-EVs group(group L+E).Group C was commonly cultured.Group L was incubated for 24 h with LPS(final concentra-tion 1 µg/ml).Group L+E was incubated for 24 h with Lac-EVs(final concentration 2.5 μg/ml)after being treated with LPS for 24 h.The expression of CD86 and CD206 was detected using immunofluorescence stai-ning.Cell precipitates were taken from L and L+E groups,and proteomics were used to screen for differenti-ally expressed proteins between the two groups.The differentially expressed proteins were analyzed by the bioinformatics analysis,and two differentially expressed proteins,apolipoprotein A1 and G protein-coupled receptor kinase 2,were verified by quantitative real-time polymerase chain reaction and Western blot.Re-sults Compared with group C,the expression of CD86 was significantly up-regulated,and the expression of CD206 was down-regulated in group L(P＜0.05).Compared with group L,the expression of CD86 was sig-nificantly down-regulated,and the expression of CD206 was up-regulated in L+E group(P＜0.05).One hundred and twenty-five differentially expressed proteins were identified using proteomics(FC=2.0,P＜0.05),of which the expression of 66 proteins was up-regulated and the expression of 59 proteins was down-regulated.The results of GO analysis indicated that these differentially expressed proteins were mainly in-volved in biological processes such as endothelial cell proliferation,SDNA damage detection,and lipopro-tein transport.The results of KEGG analysis indicated that there were differences in PPAR signaling path-way,endocytosis,metabolic pathway,MAPK signaling pathway,etc.The expression trends of the differen-tially expressed proteins determined by Western blot and quantitative real-time polymerase chain reaction were consistent with the results of proteomics.Conclusions Lac-EVs can inhibit LPS-induced microglial polarization toward M1 phenotype,and the mechanism may be related to the up-regulated differential pro-teins apolipoprotein A1 and G protein-coupled receptor kinase 2.
关键词
乳杆菌属/细胞外囊泡/脂多糖类/小神经胶质细胞/蛋白质组学Key words
Lactobacillus/Extracellular vesicles/Lipopolysaccharides/Microglia/Pro-teomics引用本文复制引用
基金项目
国家自然科学基金地区科学基金(82260238)
出版年
2024
NETL
NSTL
万方数据