基于核糖核酸测序探讨马尔尼菲篮状菌感染致小鼠脑皮层损伤的机制

Mechanism of cerebral cortex injury in mice induced by Talaromyces marneffei infection:an exploration based on RNA sequencing

目的应用核糖核酸测序(RNA-seq)技术探讨马尔尼菲篮状菌(TM)致小鼠脑皮层损伤的可能机制。方法(1)将10只C57/BL6小鼠随机分为TM感染组和健康对照组(5只/组)后,分别经尾静脉注射TM分生孢子悬液、生理盐水。将BV2细胞和N2a细胞均随机分为TM感染组和空白对照组,TM感染组加入TM分生孢子悬液,空白对照组加入等量培养液。(2)建模成功后,取小鼠脑皮层组织进行RNA-seq后分析差异表达基因(DEGs);采用R 4.1.2软件对DEGs进行京都基因与基因组百科全书(KEGG)通路富集分析和基因本体论(GO)功能富集分析等。取两组小鼠脑皮层组织及两组BV2细胞、N2a细胞,采用实时荧光定量PCR对富集通路相关DEGs、促炎因子进行体内体外验证。结果(1)共获得871个DEGs,其中上调基因373个、下调基因498个。KEGG通路富集分析结果显示,显著性最大的通路为氧化磷酸化通路(Q=9.60×10^(-3)),基因数目比例最大的通路为神经退行性病变-多种疾病通路;GO功能富集分析结果显示,DEGs主要涉及细胞因子产生的正向调节、突触组织等生物学过程和组织。(2)体内体外验证结果表明,与对应对照组相比,TM感染组小鼠脑皮层组织和N2a细胞中Atp4a、Abcg3的mRNA表达水平均上调,Med8 mRNA表达水平下调,N2a细胞中Ifi47、AA388235的mRNA表达水平上调(均P<0.05),TM感染组小鼠脑皮层组织及BV2细胞中白细胞介素6、白细胞介素1β和肿瘤坏死因子α的mRNA表达水平均上调(均P<0.05),均与RNA-seq结果一致。结论TM感染可能通过直接改变神经元细胞糖代谢,或间接激活小胶质细胞炎症反应,从而导致脑皮层损伤。

Objective To explore the possible mechanism of cerebral cortex injury in mice induced by Talaromyces marneffei(TM)infection using RNA sequencing(RNA-seq)technique.Methods(1)A total of 10 C57/BL6 mice were randomly assigned to TM infection group or healthy control group,with 5 mice in each group,after that they received injection of TM conidial suspension or normal saline through the caudal vein,respectively.BV2 cells and N2a cells were randomly assigned to TM infection group or blank control group,and TM conidial suspension was added to the TM infection group,as well as culture solution with equivalent volume was added to the blank control group.(2)After successful modeling,the differentially expressed genes(DEGs)were analyzed after performing RNA-seq by obtaining cerebral cortex tissues in mice.The Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis and Gene Oncology(GO)functional enrichment analysis,etc.on DEGs were performed by the R.4.1.2 software.The cerebral cortex tissues of mice in both groups,and BV2 and N2a cells in both groups were obtained,and enrichment pathway-related DEGs and proinflammatory cytokines were verified in vivo and in vitro by the real-time fluorescent quantitative PCR.Results(1)A total of 871 DEGs were obtained,therein,there were 373 up-regulated genes and 498 down-regulated genes.The results of KEGG pathway enrichment analysis revealed that the most significant pathway was oxidative phosphorylation pathway(Q=9.60×10^(-3)),and the pathway with the largest proportion of gene number was neurodegenerative-multiple diseases pathway.The results of GO analysis interpreted that DEGs were mainly involved in the biological processes and tissues in terms of positive regulation produced by cytokines,and synaptic tissues,etc.(2)The results of verification in vivo and in vitro implied that the TM infection group exhibited up-regulated mRNA expressions of Atp4a,Abcg3 in mice cerebral cortex tissues and in N2a cells,and a down-regulated mRNA expression of Med8 in mice cerebral cortex tissues and in N2a cells,as well as up-regulated mRNA expressions of Ifi47 and AA388235 in N2a cells as compared with the corresponding control groups(all P<0.05).The TM infection group yielded up-regulated mRNA expressions of interleukin 6,interleukin 1β,and tumor necrosis factorαin mice cerebral cortex tissues and in BV2 cells as compared with the corresponding control groups(all P<0.05).All results were consistent with RNA-seq results.Conclusion TM infection may directly change the glucose metabolism of neuron cells,or indirectly activate the microglial inflammatory response,so as to lead to cerebral cortex injury.