黄芩苷体外干扰IBV在CEK细胞内复制的转录组分析

Transcriptome analysis of baicalin interfering with IBV replication in CEK cells in vitro

为了解黄芩苷干扰禽传染性支气管炎病毒(IBV)感染鸡胚肾(CEK)细胞内复制前后细胞基因表达的差异性及表达情况,进一步揭示和分析黄芩苷体外干扰IBV在CEK细胞内复制的作用机制,IBV感染CEK细胞2h后,加入9.75mg/L的黄芩苷药液进行干扰,为黄芩苷(H-IBV)组,同时设立细胞对照(Cell)组和IBV感染(IBV)组各3个复孔,培养36h后收集细胞样品进行有参转录组测序。结果H-IBV组与IBV组相比共有102个显著差异表达基因,其中显著上调基因48个,显著下调基因54个;通过基因本体论(GO)和京都基因与基因组百科全书(KEGG)数据库中的功能注释和通路富集发现,差异表达基因主要注释在细胞过程、生物调节、代谢等生物过程和病毒性传染病、信号转导与相互作用等二级通路中,其中CEK细胞内视黄醇代谢通路、磷脂转移到膜、IL-27介导的信号通路、MDA5/RIG-I和Toll样受体信号通路均显著富集,并且Ⅰ型干扰素及干扰素α的产生、抗病毒感染过程也均得到了正向调节,富集到核酸催化、免疫系统与反应和生物间相互反应的差异基因数较多;通过STRING网络互作图发现大部分免疫相关基因能够组成一个有36个节点且以IRF7、TLR3、STAT1为中心的相互作用网络。故黄芩苷作用后与IBV组对比的差异表达基因主要注释和富集在生物过程中,免疫系统与反应也被加强,通过正向调节MDA5/RIG-I受体信号通路中的IRF7和抑制Toll样受体信号通路中的TLR3信号转导为主,正调IL-27介导的通路和调控JAKSTAT等信号通路为辅,激活IRF7、TLR3、STAT等相关基因并且与对应的下游蛋白相互作用,促进IFN-α和调控细胞因子的表达,再加上对病毒的(防御)反应和病毒过程的负调控,从而起到干扰IBV在CEK细胞内复制的作用。

In order to understand the difference and expression of genes in CEK cells before and after baicalin interferes with IBV,further reveal and analyze the mechanism of baicalin interfering IBV replication in CEK cells in vitro.After IBV infected CEK cells for 2h,9.75mg/L baicalin liquid was added to interfere with CEK cells,which was recorded as the baicalin(H-IBV)group,and three replicate wells were set in the control group and the IBV(IBV)infection group.After 36h culture,cell samples were collected and subject to transcriptome for sequencing.The results showed that there were 102differentially expressed genes in H-IBV group compared with IBV infection group,among which 48genes weresignificantly up-regulated and 54genes were significantly down-regulated.Through functional annotation in GO and KEGG databases,it was found that differentially expressed genes were mainly annotated in biological processes such as cellular processes,biological regulation,metabolism,and secondary pathways such as viral infectious diseases,signal transduction and interaction.Retinol metabolism pathway,phospholipid transfer to membrane,IL-27mediated signaling pathway,MDA5/RIG-I and Toll-like receptor signaling pathway were significantly enriched in CEK cells,and the production of typeⅠinterferon and interferon alpha and the process of antiviral infection were also positively regulated.There were more differential genes enriched in nucleic acid catalysis,immune system,and reaction,and interbiological reaction.Through the STRING network interaction map,it was found that most immune-related genes could form a 36-node interaction network centered on IRF7,TLR3and STAT1.Therefore,compared with IBV group,the differentially expressed genes after baicalin treatment were mainly annotated and enriched in the biological process,and the immune system and response were enhanced,mainly through the positive regulation of IRF7in the MDA5/RIG-I receptor signaling pathway and the inhibition of TLR3signal transduction in the Toll-like receptor signaling pathway.Positive regulation of IL-27mediated pathway and regulation of JAK-STAT signaling pathway were supplemented by activation of the expression of IRF7,TLR3,STAT1and other related genes,and interaction with corresponding downstream proteins to promote the expression of IFN-αand regulatory cytokines,coupled with negative regulation of viral(defense)response and viral processes.Thus,baicalin interferes with IBV replication in CEK cells.