摘要
目的探究小分子化合物AM679在乙型肝炎病毒(HBV)复制及感染细胞模型中的抗病毒活性。方法通过qPCR及蛋白质免疫印迹法验证AM679对延伸因子结合蛋白2(EFTUD2)表达的正向调节作用;用AM679(0.5、1、2nmol/L)处理HepAD38、HepG2-NTCP细胞,设置阴性对照组、阳性对照组及AM679与恩替卡韦联合用药组。通过qPCR检测细胞内外HBVDNA水平变化,以及细胞内HBVRNAs与3.5kb-RNA的表达水平;通过酶联免疫吸附试验检测细胞上清液中乙型肝炎表面抗原(HBsAg)、乙型肝炎e抗原(HBeAg)水平。组间均数差异采用t检验方法进行统计学分析。结果AM679处理后的HepAD38、HepG2-NTCP细胞中EFTUD2mRNA及蛋白表达水平均明显升高,差异有统计学意义(P<0.001);在两种细胞模型中,HBVDNA、HBVRNAs、HBV3.5kb-RNA、HBsAg、HBeAg等细胞内外指标均有不同程度的下降,且随AM679浓度的增加、处理时间的延长,上述指标下降更为明显;联合使用AM679与恩替卡韦则具有更显著的抗病毒效果。HepAD38细胞上清液中,2nmol/L AM679作用第3、9天对HBVDNA抑制抑制率分别为21%对比48%,而AM679联合ETV治疗组,抑制作用最显著(62%),P值均<0.01。沉默EFTUD2后可观察到HBV复制更活跃,AM679的抗病毒活性被显著削弱。结论AM679通过靶向调节EFTUD2的表达发挥体外抗HBV活性。
Objective To explore the antiviral activity of the small-molecule compound AM679 in hepatitis B virus(HBV)replication and infection cell models.Methods The positive regulatory effect ofAM679 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.Results EFTUD2 mRNA and protein expression levels were significantly increased in HepAD38 and HepG2-NTCP cells following AM679 treatment,with a statistically significant difference(P<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 P<0.01.More active HBV replication was observed after silencing EFTUD2,while the antiviral activity of AM679 was significantly weakened.Conclusion AM679 exerts anti-HBV activity in vitro by targeting the regulation of EFTUD2 expression.
作者
方慧晶
蔡金原
侯晓雪
宋佳逦
彭林雅
朱传龙
Fang Huijing;Cai Jinyuan;Hou Xiaoxue;Song Jiali;Peng Linya;Zhu Chuanlong(Department of Infectious Diseases,First Affiliated Hospital,Nanjing Medical University,Nanjing 210029,China)
出处
《中华肝脏病杂志》
CAS
CSCD
北大核心
2024年第4期318-324,共7页
Chinese Journal of Hepatology
基金
国家自然科学基金(81770591)
江苏省科教能力提升工程(JSDW202207)。
