祛湿活血方作用非酒精性脂肪肝肝细胞转录组分析及验证  

Transcriptome Analysis and Verification of the Effect of Qushi Huoxue Recipe on Non-alcoholic Fatty Liver Cells

作　　者：庞华珍 李辉[2] 刘旭东[1] 赵晓芳[1] 唐艳芳[1] PANG Hua-zhen;LI Hui;LIU Xu-dong;ZHAO Xiao-fang;TANG Yan-fang(Department of Liver Diseases,Ruikang Hospital Affiliated to Guangxi University of Chinese Medicine,Nanning,530011;College of Animal Science and Technology,Guangxi University,Nanning,530004)

机构地区：[1]广西中医药大学附属瑞康医院肝病科,南宁530011 [2]广西大学动物科学技术学院,南宁530004

出　　处：《中国中西医结合杂志》2024年第2期208-215,共8页Chinese Journal of Integrated Traditional and Western Medicine

基　　金：国家自然科学基金资助项目(No.82160837);2019年广西一流学科建设课题项目(No.2019XK139);广西中医药大学岐黄工程高层次人才培育项目(No.2021007);2019年中医药传承与创新人才培养平台建设项目(No.国中医药人教函[2019]41号)。

摘　　要：目的获取和发掘祛湿活血方作用于非酒精性脂肪肝(NAFLD)肝细胞后的转录组数据库和肝细胞炎症环境相关细胞焦亡差异表达基因。方法研究用棕榈酸诱导的人肝细胞系L-02建立NAFLD的细胞模型,采用去除核糖体RNA的转录组测序法(Ribo-Zero RNA-Seq)对中药祛湿活血方处理的L-02细胞模型进行转录组测序,并进行系统的生物信息学分析,筛选出与炎症环境相关细胞焦亡的差异表达基因。然后通过实时荧光定量PCR(qRT-PCR)技术和蛋白免疫印迹(Western Blot)验证核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)及其诱导的肝细胞焦亡相关分子的表达。结果本次测序分析共获得3000多个显著性差异表达的基因,其中差异基因的数量在空白处理组/祛湿活血方组最多,1435个上调,658个下调。差异表达基因主要富集在炎症信号、细胞命运和脂肪代谢等相关通路,NLRP3、消皮素D(GSDMD)和半胱氨酸天冬氨酸蛋白酶1(Caspase-1)等焦亡相关基因被筛选出。qRT-PCR、Western Blot等验证结果表明,祛湿活血方处理肝细胞可以降低以上分子在基因和蛋白水平的表达。结论祛湿活血方可调控炎症相关焦亡信号分子NLRP3、GSDMD和Caspase-1基因及蛋白的表达,从而降低炎症反应,抑制肝细胞焦亡,这可能是祛湿活血方治疗NAFLD的一个有效靶点。Objective To obtain and dig out the transcriptome database of Qushi Huoxue Formula acting on nonalcoholic fatty liver disease(NAFLD)hepatocytes and the differentially expressed genes of pyrolysis related to the inflammatory environment of hepatocytes.Methods The research used palmitic acidinduced human liver cell line L-02 to establish the cell model of NAFLD.Transcriptome sequencing with Ribosome RNA removed(Ribo-Zero RNA-Seq)was used to transcriptome sequence the L-02 cell model treated by Qushi Huoxue Recipe.Systematic bioinformatics analysis was performed and differentially expressed genes related to inflammatory environment pyroptosis were screened out.Then the expression of nucleotides bind oligomerized domain-like receptor proteins 3(NLRP3)and its induced liver pyroptosis related molecules were verified by real-time PCR quantitative(qRT-PCR)and Western Blot.Results More than 3000 significantly differentially expressed genes were obtained in this sequencing analysis.Of them,the number of differentially expressed genes was the highest in the blank treatment group/Qushi Huoxue Formula group,including 1435 up-regulated and 658 down-regulated genes.Differentially expressed genes were mainly enriched in related pathways such as inflammation signals,cell fate,and fat metabolism.Pyroptosis related genes such as NLRP3,gasdermin D(GSDMD),and cysteinyl aspartate specific proteinase-1(Caspase-1)were screened out.Results of qRT-PCR and Western Blot proved that the treatment of hepatocyte by Qushi Huoxue Formula lowered the expressions of the above molecules at gene and protein levels.Conclusions Qushi Huoxue Formula regulated the gene and protein expressions of inflammation-related pyroptosis signal molecules NLRP3,GSDMD,and Caspase-1,attenuated inflammatory reactions and inhibited hepatocyte pyrolysis.This might be an effective target of Qushi Huoxue Formula in the treatment of NAFLD.

关 键 词：祛湿活血方 转录组测序 肝细胞 非酒精性脂肪肝 差异表达基因 中药复方 

分 类 号：R285[医药卫生—中药学]