基于转录组测序技术分析穿心莲内酯抑制HepG2.CW细胞分泌乙型肝炎病毒表面抗原作用机制  

Mechanism of andrographolide for inhibiting secretion of HBsAg in HepG2.CW cells based on transcriptome sequencing technology

作　　者：黄曼琼 杨稳[1,2] 魏振桥 王晓龙 齐冬梅[3] 戚扬[1] 邢雅玲 王升启 HUANG Man-qiong;YANG Wen;WEI Zhen-qiao;WANG Xiao-long;QI Dong-mei;QI Yang;XING Ya-ling;WANG Sheng-qi(Institute of Traditional Chinese Medicine Innovation,Shandong University of Traditional Chinese Medicine,Jinan 250355,China;Institute of Microbial Epidemiology,Academy of Military Medical Sciences,Beijing 100850,China;Experimental Center,Shandong University of Traditional Chinese Medicine,Jinan 250355,China)

机构地区：[1]山东中医药大学中医药创新研究院,山东济南250355 [2]军事科学院军事医学研究院微生物流行病研究所,北京100850 [3]山东中医药大学实验中心,山东济南250355

出　　处：《中国药理学与毒理学杂志》2023年第6期426-434,共9页Chinese Journal of Pharmacology and Toxicology

基　　金：国家自然科学基金(81830101)

摘　　要：目的基于转录组测序技术分析穿心莲内酯(andrographolide)抑制乙型肝炎病毒表面抗原(HBsAg)分泌的作用机制。方法BALB/c小鼠ig给予穿心莲内酯0(正常对照组),6.25和18.75 mg·kg^(-1),24 h后取肝组织进行转录组测序,采用R包edgeR分析穿心莲内酯作用后的差异基因,用2个剂量组差异基因的交集作为穿心莲内酯作用后显著变化的基因。用含乙型肝炎病毒(HBV)基因组的HepG2.CW细胞作为验证模型,与不同浓度穿心莲内酯(1.5625,3.125,6.25和12.5μmol·L^(-1))分别作用12,24,48或72 h,采用CCK-8法检测细胞存活率;实时荧光定量PCR检测差异基因表达水平,对转录组测序结果进行验证;化学发光法检测HepG2.CW细胞培养上清中HBsAg和乙型肝炎病毒e抗原(HBeAg)含量。结果转录组测序结果显示,与正常对照组相比,穿心莲内酯6.25 mg·kg^(-1)组小鼠肝组织检出151个差异基因,其中72个基因上调,79个基因下调;穿心莲内酯18.75 mg·kg^(-1)组检出50个差异基因,其中25个基因上调,25个基因下调;2个剂量组同时显著变化的基因共17个,其中ADP核糖基化因子样蛋白4D(Arl4d)基因上调,肝细胞核因子6(HNF6)和Arl8a基因下调。与细胞对照组相比,穿心莲内酯1.5625,3.125和6.25μmol·L^(-1)组细胞存活率显著提高(P<0.01),12.5和25μmol·L^(-1)组细胞存活率无显著差异,50μmol·L^(-1)组细胞存活率显著降低(P<0.01);穿心莲内酯3.125,6.25和12.5μmol·L^(-1)组细胞内Arl4d mRNA表达水平均显著降低(P<0.01),穿心莲内酯各浓度组细胞培养上清中HBsAg含量均显著降低(P<0.01),细胞内Arl4d mRNA表达水平与细胞培养上清中HBsAg含量呈显著正相关(r=0.8525,P<0.01)。与细胞对照组相比,穿心莲内酯6.25μmol·L^(-1)作用HepG2.CW细胞12,24和48 h,细胞内Arl4d mRNA表达量均显著降低(P<0.05,P<0.01),作用72 h,细胞培养上清中HBsAg含量显著降低(P<0.01)。结论穿心莲内酯可能通过降低HepG2.CW细胞内Arl4d mRNA的表达,进�OBJECTIVE To investigate the mechanism by which andrographolide inhibits the secretion of hepatitis B virus surface antigen(HBsAg)based on gene transcriptome sequencing.METHODS Mice were ig given andrographolide(0,6.25 and 18.75 mg·kg^(-1))for 24 hours,and transcriptome sequencing technology was used to analyze and compare transcriptomes in the liver.The R package edgeR was used to analyze the differential genes in the three groups.The intersection of differential genes between andrographolide(6.25 and 18.75 mg·kg^(-1))groups was used as significantly differential genes in the mouse liver treated with andrographolide.As validation models,HepG2.CW cells containing the hepatitis B virus(HBV)genome were treated with andrographolide(1.5625,3.125,6.25 and 12.5μmol·L^(-1))for 2,24,48 or 72 h.CCK-8 assay was used to detect cell viability,RT-qPCR was used to detect differential gene expression levels and verify the transcriptome sequencing results,and the chemiluminescent method was employed to detect the contents of hepatitis B virus surface antigen(HBsAg)and hepatitis B virus e antigen(HBeAg)in cell culture medium.RESULTS The results of transcriptome sequencing showed that compared with the control group,151 differential genes were detected in the androgra⁃pholide 6.25 mg·kg^(-1) group,72 of which were up-regulated and 79 genes were down-regulated.50 genes were detected in the andrographolide 18.75 mg·kg^(-1) group,25 of which were up-regulated and 25 genes were down-regulated.The intersection of differential genes between the two groups obtained 17 genes as significantly differential genes,among which the ADP-ribosylation factor-like protein 4D(Arl4d)gene was up-regulated,while hepatocyte nuclear factor 6(HNF6)and Arl8a genes were down-regulated.Compared with the cell control group,the cell viability was significantly higher in the andrographolide(1.5625,3.125,6.25μmol·L^(-1))group(P<0.01),not significantly different in the(12.5,25μmol·L^(-1))group,but significantly lower in the 50μmol·L^(-1) group(P<0.0

关 键 词：穿心莲内酯 转录组测序 ADP核糖基化因子样蛋白4D 乙肝表面抗原 

分 类 号：R967[医药卫生—药理学] R285[医药卫生—药学] R512.6