PAF对BV2细胞炎症因子分泌的影响及转录组学分析

Effects of Platelet Activating Factor on Secretion of Inflammatory Factors from BV2 Cells and Transcriptomics Analysis

探究血小板活化因子(Platelet activating factor,PAF)对BV2细胞炎症因子分泌的影响及初步推测其调控机制。外源PAF(2×10^(-5)M)作用于BV2细胞24 h,36 h,48 h后,(1)CCK8实验检测空白对照组,Mock组和PAF处理组细胞活性;(2)ELISA实验测炎症因子TNFα、IL-6、Cxcl2、IL-1a的分泌;(3)Western Blot检测炎症介质COX-2蛋白水平;(4)对PAF处理24h的BV2细胞以及对应的Mock组细胞进行RNA测序得到转录组测序结果;(5)GO和KEGG富集分析后,运用String数据库配合Cytoscape软件找出炎症相关的Hub基因并挑选出以下基因(Ccl4、Ccl3、Ccl7、IL-1a、Cxcl2、IL1f9、IL34、COX-2、Serpinb1a)进行qPCR验证。实验结果表明,PAF作用时间为24 h,36 h,48 h后,相比对照组:(1)BV2细胞活性降低;(2)上清液中的炎症因子TNFα,IL-6,IL-1a,Cxcl2分泌增加;(3)炎症介质COX-2蛋白表达水平上调;(4)从组学中筛选出炎症反应相关的Hub基因有:COX-2、Ccl3、Ccl4、Cxcl2、Ccl7、IL-1a、Mapk14、Cd80、Casp1、Src、Nos2、Serpinb1a、Serpinb9、Il1r1;(5)qPCR验证表达上调的基因包括Ccl3、Ccl4、Ccl7、IL-1a、Cxcl2、IL1f9、IL34、COX-2,表达下调的基因Serpinb1a。说明PAF可以诱导BV2细胞释放炎症因子,PAF可能是促进“细胞因子风暴”的重要介质,根据转录组学分析结果我们锁定了炎症反应密切相关的14种Hub基因,并对产生炎症因子的分子机制提出了初步推测。

To explore regulation of secretion of inflammatory cytokines in BV2 cells by platelet activating factor(PAF) and to carry out transcriptomics analysis to understand the regulatory mechanisms.BV2 cells were treated with exogenous PAF(2×10^(-5)M) for 24 h,36 h,or 48 h.First,the Cell Counting Kit-8 assay was undertaken to detect cell viability in the blank control,mock control,and PAF-treated cells.Second,enzyme-linked immunosorbent assays were used to measure secretion of the inflammatory cytokines tumor necrosis factor(TNF)α,interleukin(IL)-6,chemokine(C-X-C motif)ligand 2(Cxcl2),and IL-1a.Third,protein expression of the inflammatory mediator cyclooxygenase(COX)-2 was measured by Western Blotting.Fourth,after PAF treatment of BV2 cells for 24 h,RNA sequencing was done on mock control and PAF-treated cells.Fifth,enrichment analyses using the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases were done based on transcriptomics analysis.Then,the Search Tool for the Retrieval of Interacting Genes/Proteins(STRING) database was used in combination with Cytoscape to discover the hub genes related to inflammation.Then,the genes of interest(chemokine(C-C motif)ligands 4(Ccl4),Ccl3,Ccl7,IL-1a,Cxcl2,IL1f9,IL34,COX-2,Serpinb1a)were selected for quantitative polymerase chain reaction(qPCR)validation.BV2 cells were treated with PAF for 24 h,36 h,or 48 h,and compared with the control groups.BV2 cells showed decreased activity.Increased secretion of the inflammatory cytokines TNFα,IL-6,IL-1a,and Cxcl2 were found in the cell supernatant.Upregulated protein expression of the inflammatory mediator COX-2 was observed.The inflammatory response-related hub genes according to transcriptomics analysis were COX-2,Ccl3,Ccl4,Cxcl2,Ccl7,IL-1a,Mapk14,Cd80,Casp1,Src,Nos2,Serpinb1a,Serpinb9,and IL1R1.qPCR validated upregulated expression of the genes of interest(Ccl3,Ccl4,Ccl7,IL-1a,Cxcl2,IL1f9,IL34,COX-2)and downregulated expression of serpinb1a.PAF can induce the release of inflammatory factors from BV2 cells.PAF may be an important mediator in promoting the “cytokine storm”.Transcriptomic analysis revealed 14 hub genes closely related to the inflammatory response.