多黏菌素B通过下调NF-κB抑制HBV转录复制的机制研究  

Polymyxin B inhibits HBV transcription and replication by down-regu⁃lating NF-κB

作　　者：孟凡荣[1] 陈琛 潘红丽 李雪冰[2] MENG Fanrong;CHEN Chen;PAN Hongli;LI Xuebing(Department of Gynecology and Obstetrics,Tianjin Key Laboratory of Female Reproductive Health and Eugenics,Tianjin Medical University General Hospital,Tianjin 300052,China;Tianjin Key Laboratory of Lung Cancer Metastasis and Tumor Microenvironment,Tianjin Lung Cancer Institute,Department of Lung Cancer Surgery,Tianjin Medical University General Hospital,Tianjin 300052,China.)

机构地区：[1]天津医科大学总医院妇产科,天津市女性生殖健康与优生重点实验室,天津300052 [2]天津市肺癌转移与肿瘤微环境重点实验室,天津市肺癌研究所,天津医科大学总医院肺部肿瘤外科,天津300052

出　　处：《中国病理生理杂志》2023年第5期827-837,共11页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金面上项目(No.82273019);中国癌症基金会重点项目(No.CFC2020kyxm003);天津市医学重点学科(专科)建设项目(No.TJYXZDXK-061B;No.TJYXZDXK-031A);天津市肺癌研究所面上项目(No.TJLCMS2021-03);天津医科大学总医院孵育基金(No.ZYYFY2019022)。

摘　　要：目的:研究多黏菌素B通过下调核因子κB(nuclear factor-κB,NF-κB)对乙型肝炎病毒(hepatitis B virus,HBV)转录和复制的影响。方法:通过MTT法检测多黏菌素B在HepG2-NTCP、HepAD38、HepG2.2.15、HepG2、Huh-7和PLC/PRF/5细胞中的细胞毒性;使用10、50和100 nmol/L的多黏菌素B处理HBV感染的HepG2-NTCP细胞和HBV稳定复制的HepG2.2.15细胞,通过RT-qPCR方法检测细胞中HBV RNA和HBV DNA水平,ELISA方法检测细胞上清中乙型肝炎表面抗原(hepatitis B surface antigen,HBsAg)和乙型肝炎e抗原(hepatitis B e antigen,HBeAg)水平,明确多黏菌素B对HBV的调控作用。利用转录组测序筛选多黏菌素B调控HBV转录复制的效应分子NF-κB;同时检测敲减及过表达NF-κB对HBV的调控作用;利用双萤光素酶报告基因实验检测NF-κB对Cp、Xp、Sp1和Sp2启动子活性的影响;利用回补实验探究多黏菌素B调控HBV转录复制是否依赖于NF-κB。结果:MTT实验结果显示,浓度低于100μmol/L时,多黏菌素B对HepG2-NTCP、HepAD38、HepG2.2.15、HepG2、Huh-7和PLC/PRF/5细胞无明显毒性。在10、50和100 nmol/L的浓度下,多黏菌素B呈浓度依赖性抑制HBV感染的HepG2-NTCP细胞和HBV稳定复制的HepG2.2.15细胞中HBV RNA、HBV DNA、HBsAg和HBeAg水平。通过转录组测序发现,多黏菌素B可以显著抑制HBV感染的HepG2-NTCP细胞中NF-κB的表达水平;进一步,过表达NF-κB显著上调HBV感染的HepG2-NTCP细胞中HBV RNA、HBV DNA、HBsAg和HBeAg水平,沉默时则相反;同时,双萤光素酶报告基因实验显示NF-κB可增强HBV Sp2启动子活性。回补实验发现多黏菌素B调控HBV转录复制依赖于NF-κB。结论:多黏菌素B通过下调NF-κB进而抑制HBV Sp2启动子活性,最终抑制HBV转录复制。AIM:To investigate the effect of polymyxin B on hepatitis B virus(HBV)transcription and repli⁃cation.METHODS:The cytotoxicity of polymyxin B in HepG2-NTCP,HepAD38,HepG2.2.15,HepG2,Huh-7 and PLC/PRF/5 cells was examined by MTT assay.The HBV-infected HepG2-NTCP cells and HepG2.2.15 cells with stable HBV replication were treated with 10,50 and 100 nmol/L polymyxin B,and the mRNA and DNA levels of HBV in the cells were measured by RT-qPCR.The levels of hepatitis B surface antigen(HBsAg)and hepatitis B e antigen(HBeAg)in the cell supernatants were quantified by ELISA.The regulatory effect of polymyxin B on HBV transcription and replica⁃tion was clarified.Subsequently,we used transcriptome sequencing to screen nuclear factor-κB(NF-κB)as the effector molecule of polymyxin B regulating HBV transcription and replication,and we determined the regulatory effect of knock⁃down and overexpression of NF-κB on HBV transcription and replication.We used dual-luciferase reporter assay to deter⁃mine the effect of NF-κB on the activity of Cp,Xp,Sp1 and Sp2 promoters.Finally,a reverse complementation assay was used to investigate whether the regulation of HBV transcription and replication by polymyxin B is dependent on NF-κB.RESULTS:The MTT assay results showed that polymyxin B did not exhibit significant toxicity in HepG2-NTCP,HepAD38,HepG2.2.15,HepG2,Huh-7 and PLC/PRF/5 cells when used at concentrations less than 100μmol/L.Polymyxin B de⁃creased the HBV RNA,HBV DNA,HBsAg and HBeAg levels in HBV-infected HepG2-NTCP cells and HepG2.2.15 cells with stable HBV replication in a concentration-dependent manner.Transcriptome sequencing revealed that polymyxin B significantly decreased the expression level of NF-κB in HBV-infected HepG2-NTCP cells.Furthermore,overexpres⁃sion of NF-κB significantly increased the levels of HBV RNA,HBV DNA,HBsAg and HBeAg in HBV-infected HepG2-NTCP cells,and the opposite effect was found when NF-κB was knocked down.Moreover,the results of dual-luciferase re⁃porter assay showed that NF-κB

关 键 词：多黏菌素B 乙型肝炎病毒 核因子ΚB 转录 启动子 

分 类 号：R512.62[医药卫生—内科学] R363.2[医药卫生—临床医学]