6-甲基香豆素对乙型肝炎病毒转录和复制的影响

Effect of 6-methylcoumarin on transcription and replication of hepatitis B virus

目的探讨6-甲基香豆素对乙型肝炎病毒(HBV)转录和复制的影响。方法MTT法检测6-甲基香豆素在HepG2.2.15和HepG2-NTCP细胞中的细胞毒性。使用含0.1%DMSO的生理盐水(阴性对照),6-甲基香豆素(5、10、20μmol/L)及恩替卡韦(25 nmol/L,阳性对照)处理HepG2.2.15和HepG2-NTCP细胞6 d。RT-qPCR检测细胞中HBV DNA和HBV RNA的水平,ELISA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。雄性HBV转基因小鼠每2天腹腔注射含0.1%DMSO的生理盐水(阴性对照),6-甲基香豆素(5 mg/kg)和恩替卡韦(0.02 mg/kg,阳性对照)1次,每6天采血及称重1次。药物处理24 d后,处死小鼠并解剖取肝脏。RT-qPCR检测小鼠血清和肝脏组织中HBV DNA水平及肝脏组织中HBV RNA水平,ELISA检测小鼠血清中丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)、HBsAg和HBeAg水平。结果与对照组相比,6-甲基香豆素可以呈浓度依赖性的降低HepG2.2.15和HepG2-NTCP细胞中HBV DNA和HBV RNA及细胞分泌的HBsAg和HBeAg水平(P<0.05)。6-甲基香豆素在不影响小鼠体重及AST、ALT的同时,可以显著降低HBV转基因小鼠血清中HBV DNA、HBsAg和HBeAg水平及小鼠肝脏组织中HBV DNA和HBV RNA水平(P<0.05)。结论在HepG2.2.15细胞、HepG2-NTCP细胞和HBV转基因小鼠模型中6-甲基香豆素均具有较强的抑制HBV转录和复制的作用。

Objective To study the effect of 6-methylcoumarin on the transcription and replication of hepatitis B virus(HBV).Methods The cytotoxicity of 6-methylcoumarin in HepG2.2.15 and HepG2-NTCP cells was detected by MTT assay.HepG2.2.15 and HepG2-NTCP cells were treated with normal saline containing 0.1%DMSO(negative control),6-methylcoumarin(5,10,20μmol/L)and entecavir(25nmol/L,positive control)for 6 d.The levels of HBV DNA and HBV RNA in the cells were detected by RT-PCR,and the levels of HBsAg and HBeAg in the supernatant were detected by ELISA.Male HBV transgenic mice were intraperitoneally injected with normal saline containing 0.1%DMSO(negative control),6-methylcoumarin(5 mg/kg)and entecavir(0.02 mg/kg,positive control)once every 2 d,blood collection and weighing once every 6 d.After 24 d of drug treatment,the mice were sacrificed and the liver was taken.The levels of HBV DNA in serum and liver and HBV RNA in liver tissue were detected by RT-PCR.The levels of alanine aminotransferase(ALT),aspartate aminotransferase(AST),HBsAg and HBeAg levels in serum were detected by ELISA.Results Compared with the control group,6-methylcoumarin could reduce the levels of HBV DNAand HBV RNA in HepG2.2.15 and HepG2-NTCP cells with concentration-dependent manner,as well as the levels of HBsAg and HBeAg secreted by cells(P<0.05).In addition,6-methylcoumarin could significantly reduce the levels of HBV DNA,HBsAg and HBeAg in serum and HBV DNA,HBV RNA in liver tissue of HBV transgenic mice without affecting the body weight,AST and ALT of mice(P<0.05).Conclusion 6-methylcoumarin has a strong inhibitory effect on HBV transcription and replication in HepG2.2.15 cells,HepG2-NTCP cells and HBV transgenic mice.