基于RNA-Seq对淫羊藿次苷Ⅱ体内抗HBV时胰岛素信号通路基因的研究  

Transcriptome sequencing analysis of insulin signaling pathway in icarisideⅡanti-HBV treated mice

作　　者：刘正芸 廖晏蔺 易世豪 罗果 秦颖 龚其海 王欢 Liu Zhengyun;Liao Yanlin;Yi Shihao;Luo Guo;Qin Ying;Gong Qihai;Wang Huan(Key Laboratory of infectious disease&Biosafety,Provincial Department of Education,Zunyi Medical University,Zunyi Guizhou 563099,China;Key Laboratory of Basic Pharmacology of Ministry of Education and Joint International Research Laboratory of Ethnomedicine of Ministry of Education,Zunyi Medical University,Zunyi Guizhou 563099,China)

机构地区：[1]遵义医科大学贵州省普通高等学校传染病与生物安全特色重点实验室,贵州遵义563099 [2]遵义医科大学基础药理教育部重点实验室暨特色民族药教育部国际合作联合实验室,贵州遵义563099

出　　处：《遵义医科大学学报》2023年第6期562-567,共6页Journal of Zunyi Medical University

基　　金：遵义市科技计划项目[NO:遵市科合HZ字(2020)76号];贵州省教育厅资助项目[NO:黔教技(2022)027号];贵州省高层次创新人才“百”层次项目[NO:黔科合平台人才20165684号]。

摘　　要：目的利用转录组测序(RNA-Seq)技术筛选淫羊藿次苷Ⅱ(ICSⅡ)作用于乙型肝炎病毒(HBV)复制型C57BL/6小鼠后肝脏的差异基因,探索ICSⅡ在抗HBV中对胰岛素通路相关基因的影响。方法用高压水动力法建立HBV复制型C57BL/6小鼠模型,建模后随机进行分组:模型(Model)组、生理盐水(NS)组、阳性药物恩替卡韦(ENT)对照组(0.5 mg/kg)和ICSⅡ组(20 mg/kg),每组5只,灌胃给药(10 mL/kg),1d 1次,连续给药20 d。采用荧光定量PCR技术检测血清样本中HBV DNA的拷贝量,确认ICSⅡ对HBV DNA有抑制作用后,用高通量Illumina HiSeq 2500测序平台对小鼠肝脏样本进行转录组测序。对测序结果进行GO富集和KEGG通路注释,通过KEGG通路注释分析筛选出多条与ICSⅡ抗HBV相关的通路,并使用荧光定量PCR对胰岛素通路相关基因Hras、Gck、Phka2、Prkacb、Akt2、Grb2进行验证分析。结果ICSⅡ能明显抑制HBV DNA的复制,差异基因分析显示,Model组和ICSⅡ组相比,982个基因上调,756个基因下调(P<0.05)。注释到GO数据库中的差异基因共有3675个,有493个差异基因被注释到226条通路中。荧光定量PCR结果显示,在HBV感染的小鼠肝脏中ICSⅡ下调胰岛素通路相关基因Hras、Gck、Phka2、Prkacb、Akt2、Grb2的转录水平(P<0.05),和测序结果一致。结论ICSⅡ可能通过下调胰岛素信号通路相关基因Hras、Gck、Phka2、Prkacb、Akt2、Grb2发挥抗HBV作用。Objective To identify differentially expressed genes(DEGs)in the liver of HBV-replicative C57BL/6 mice following treatment with ICSⅡby employing transcriptome sequencing(RNA-Seq)technology to investigate the impact of icarisideⅡ(ICSⅡ)on hepatitis B virus(HBV)through the insulin signaling pathway.Methods HBV replicative C57BL/6 mice models were established using high-pressure hydrodynamic method.Following modeling,mice were randomly divided into four groups:model group(model),normal saline group(NS),positive drug entecavir group(ENT,0.5 mg/kg)and ICSⅡgroup(20 mg/kg).Each group consisted of 5 mice,which were orally administered a dose of 10 mL/kg once a day for 20 days.The copy numbers of HBV DNA in serum were detected by fluorescence quantitative PCR.After confirming that ICSⅡhad an inhibitory effect on HBV DNA,the liver samples of mice were sequenced by Hiseq PE150.GO enrichment and KEGG pathway annotation were performed on the sequencing results.Multiple pathways of ICSⅡantagonizing HBV were screened through KEGG pathway annotation analysis,and insulin pathway-related genes(Hras,Gck,Phka2,Prkacb,Akt2,Grb2)were verified and analyzed by fluorescence quantitative PCR.Results ICSⅡwas found to have a significantly inhibitory effect on copies of HBV DNA,and DEG analysis showed that 982 genes were up-regulated and 756 genes down-regulated in model group compared with the ICSⅡgroup.3,675 DEG were annotated in the GO database,493 DEG were annotated in 226 pathways.Fluorescence quantitative PCR results showed that ICSⅡdown-regulated the transcription levels of insulin pathway related genes Hras,Gck,Phka2,Prkacb,Akt2,Grb2 in the liver of HBV infected mice,which were consistent with the sequencing results.Conclusion ICSⅡmight exert its antagonistic effects on HBV through down-regulating insulin signaling pathway-related genes(Hras,Gck,Phka2,Prkacb,Akt2,Grb2).

关 键 词：淫羊藿次苷Ⅱ 乙型肝炎病毒 转录组测序 胰岛素信号通路 

分 类 号：R373.9[医药卫生—病原生物学]