Fang H J, Cai J Y, Hou X X, Song J L, Peng L Y, Zhu C L
Department of Infectious Diseases, First Affiliated Hospital, Nanjing Medical University, Nanjing 210029, China.
Zhonghua Gan Zang Bing Za Zhi. 2024 Apr 20;32(4):318-324. doi: 10.3760/cma.j.cn501113-20230720-00012.
To explore the antiviral activity of the small-molecule compound AM679 in hepatitis B virus (HBV) replication and infection cell models. The positive regulatory effect of AM679 on EFTUD2 expression was validated by qPCR and Western blotting. HepAD38 and HepG2-NTCP cells were treated with AM679 (0.5, 1, and 2 nmol/L). Negative control, positive control, and AM679 combined with the entecavir group were set up. HBV DNA intra-and extracellularly, as well as the expression levels of intracellular HBV total RNAs and 3.5kb-RNA changes, were detected with qPCR. Hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) levels were measured in the cell supernatant by an enzyme-linked immunosorbent assay (ELISA). The t-test method was used for the statistical analysis of the mean difference between groups. EFTUD2 mRNA and protein expression levels were significantly increased in HepAD38 and HepG2-NTCP cells following AM679 treatment, with a statistically significant difference ( < 0.001). Intra-and extracellular indicators such as HBV DNA, HBV RNAs, HBV 3.5kb-RNA, HBsAg, and HBeAg were decreased to varying degrees in both cell models, and the decrease in these indicators was more pronounced with the increase in AM679 concentration and prolonged treatment duration, while the combined use of AM679 and entecavir had a more significant antiviral effect. The HBV DNA inhibition rates in the supernatant of HepAD38 cells with the use of 2 nmol/L AM679 were 21% and 48% on days three and nine, respectively. The AM679 combined with the ETV treatment group had the most significant inhibitory effect (62%), with a < 0.01. More active HBV replication was observed after silencing EFTUD2, while the antiviral activity of AM679 was significantly weakened. AM679 exerts anti-HBV activity in vitro by targeting the regulation of EFTUD2 expression.
探索小分子化合物AM679在乙型肝炎病毒（HBV）复制和感染细胞模型中的抗病毒活性。通过qPCR和蛋白质免疫印迹法验证AM679对EFTUD2表达的正向调节作用。用AM679（0.5、1和2 nmol/L）处理HepAD38和HepG2-NTCP细胞。设置阴性对照、阳性对照以及AM679与恩替卡韦联合用药组。采用qPCR检测细胞内、外HBV DNA以及细胞内HBV总RNA表达水平和3.5kb-RNA变化。用酶联免疫吸附测定（ELISA）法检测细胞上清液中乙型肝炎表面抗原（HBsAg）和乙型肝炎e抗原（HBeAg）水平。采用t检验方法对组间均值差异进行统计学分析。AM679处理后，HepAD38和HepG2-NTCP细胞中EFTUD2 mRNA和蛋白表达水平显著升高，差异有统计学意义（<0.001）。在两种细胞模型中，细胞内、外HBV DNA、HBV RNA、HBV 3.5kb-RNA、HBsAg和HBeAg等指标均有不同程度下降，且随着AM679浓度增加和处理时间延长，这些指标下降更明显，而AM679与恩替卡韦联合使用具有更显著的抗病毒效果。在HepAD38细胞上清液中，使用2 nmol/L AM679时，第3天和第9天的HBV DNA抑制率分别为21%和48%。AM679与ETV联合治疗组的抑制作用最显著（62%），P<0.01。沉默EFTUD2后观察到更活跃的HBV复制，而AM679的抗病毒活性显著减弱。AM679通过靶向调节EFTUD2表达在体外发挥抗HBV活性。
