基于二代测序技术探究葛根素治疗酒精性肝炎的潜在分子靶点

Exploration of the molecular targets of puerarin in the treatment of alcoholic hepatitis based on second-generation sequencing technology

目的基于二代测序技术筛选葛根素作用于酒精性肝炎(AH)的潜在药物靶点和信号通路,探讨葛根素的作用机制。方法将8周龄健康雄性C57BL/6小鼠9只随机均分为对照组、AH组(构建AH小鼠)、葛根素组(构建AH小鼠并葛根素处理)。检测各组小鼠肝功能指标。HE染色及油红O染色评估肝脏组织活动性及脂肪变性。基于二代测序技术对AH组及葛根素组进行转录组测序,采用Trimmomatic软件获取质控数据和edgeR软件识别组间差异表达基因。利用KOBAS软件进行GO分析和KEGG分析。结果与AH组比较,葛根素组转氨酶明显降低,肝组织炎症评分及脂肪变性评分均明显下降。二代测序结果显示,AH组、葛根素组分别平均获得55601831个和52934410个校正后短测序片段,共筛出790个GO成功注释(生物过程574个,分子功能141个,细胞成分64个)。两组间差异表达基因上调178个,下调258个,主要富集于有机酸代谢、细胞骨架的结构组成、微管。KEGG通路富集分析显示45条通路。结论葛根素治疗酒精性肝炎的作用靶点功能富集在有机酸代谢、细胞骨架的结构组成、微管;可能通过氨基酸代谢通路起到治疗作用。

Aim To screen the potential therapeutic molecular targets and signaling pathways,and to preliminarily explore the protective mechanism of puerarin in the treatment of alcoholic hepatitis(AH),based on second-generation sequencing technology.Methods Nine healthy male C57BL/6 mice(8-week old)were randomly divided into the control group,the AH group,and the puerarin group(AH mice treated with puerarin).Liver function of mice in each group were measured.Liver tissue activity and steatosis were measured by using HE staining and oil red O staining.Second-generation sequencing was performed on AH model group and puerarin treatment group.Quality control data were obtained by Trimmomatic software and differentially expressed genes were identified by edgeR software.KOBAS software was used to analyze the GO function and KEGG signaling pathway enrichment.Results The puerarin group showed a significant decrease in transaminase,as well as a significant decrease in liver tissue inflammation and steatosis scores,compared with the AH group.The second-generation sequencing results show that,an average of 55601831 and 52934410 corrected short sequencing fragments were obtained in AH group and puerarin group,respectively.A total of 790 GO functional enrichment genes were screened successfully(574 biological processes,141 molecular functions and 64 cellular components).There were 178 up-regulated genes and 258 down-regulated genes differentially expressed between the two groups,which were enriched in organic acid metabolism,cytoskeletal structure and microtubule.KEGG pathway enrichment analysis showed 45 pathways.Conclusion The molecular targets of puerarin in the treatment of AH are functionally enriched in organic acid metabolism,cytoskeletal structure and microtubule.Puerarin may play a role in protecting AH by the modulation of amino acid metabolism pathway.