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

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。

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 random 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 concentration 1μg/ml).Group L+E was incubated for 24 h with Lac-EVs(final concentration 2.5μg/ml)after being treated with LPS for 24 h.The expression of CD86 and CD206 was detected using immunofluorescence staining.Cell precipitates were taken from L and L+E groups,and proteomics were used to screen for differentially 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.Results 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 significantly 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 involved in biological processes such as endothelial cell proliferation,SDNA damage detection,and lipoprotein transport.The results of KEGG analysis indicated that there were differences in PPAR signaling pathway,endocytosis,metabolic pathway,MAPK signaling pathway,etc.The expression trends of the differentially 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 proteins apolipoprotein A1 and G protein-coupled receptor kinase 2.