The Anti-hepatitis B Virus Activity Of Zhenzhucaofufang In Vitro

BackgroundOn the basis of different clinical manifestations,viral hepatitis type B was defined as "the pain of upper part of the side of the human body"," choleplania" and" Ji-Ju" in traditional Chines Medicine.The pathogenesis of hepatitis B was heat disease virus invasion、liver, spleen and kidney deficiency、 disorder of Zang-fu and Qi-Xue. ZhenzhucaoFuFang is a self-mastery medicinal herb which has a relatively objective therapeutic effect on anti hepatitis B virus. This research project used its extract to treat the HepG2.2.15hepatic carcinoma cell line and the duck embryo liver cell and investigated in vitro its effective mechanism in fighting hepatitis B virus.Materials and Methods:To study the anti-hepatitis B virus of ZhenzhucaoFuFang in duck embryo liver cell, the duck embryo liver cell with congenital infection DHBV was given primary culture. During the experiment, the PCR tech was used to bolting the duck embryo with congenital infection DHBV. DHBV DNA load in culture supermatants was detected using FQ-PCR. The anti-hepatitis B virus effect (HBV-DNA, HBsAg and HBeAg, HBV-X gene, IP-10) of ZhenzhucaoFuFang in HepG2.2.15cell line was measured by PCR, ELLSA and Western blot tech.Results1. The anti-virual effection of ZhenzhucaoFuFang in duck embryo liver cell(l)The results showed that DHBV viral load of duck embryo liver cell with congenital infection DHBV was always in the order108copy/ml.(2)the14days old duck embryo, non-serum culture medium Medium William’s E was more appropriate for duck embryo liver cell than other condition of culture.(3)The TC50of duck embryo liver cells was59.95mg/ml with concentrations of ZhenzhucaoFuFang at100mg/ml,50mg/ml,25mg/ml,12.5mg/ml,6.25mg/ml,3.13mg/ml,1.56mg/ml. The rate of duck embryo liver cells was71.94%、41.33%、 %、19.39%、21.94%、15.82%、1.2%. Cells manifested vacuoles, atrophy and abscission, withdestruction rates of71.94%with an density of50mg/ml, the cell structure appeared roughly normal under the microscope.(4) The three Ievel of ZhenzhucaoFuFang supernating DHBV-DNA content (P<0.01). The Chinese medicine compound prescriptionZhenzhucaoFuFang has direct inhibitory effect on duck hepatitis B virus DNA(DHBV-DNA) in duck embryo cells.2. The anti--virual ef fection of ZhenzhucaoFuFang in HepG2.2.15cell line.(1) The TC50of HepG2.2.15cell was37.5mg/ml. Cells manifested vacuoles,atrophy and abscission,withdestruction rates of59.59%.With an density of25mg/ml, the cell structure appeared roughly normal under the microscope.(2) The IC50of HepG2.2.15cell inhibition the HBsAg was7.23mg/ml. and the IC50of HepG2.2.15cell inhibition the HBeAg was5.89mg/ml. The therapeutic index(TI) was4.49and6.06.(3) The result of fluorescence Polymerase Chain Reaction(PCR) indicated that ZhenzhucaoFuFang particle can inhibit the HBV-DNA that infect by hepatitis B virus compared with ACV.(4) The study of HBV-X gene and IP-10levels in culture supernatants effect by ZhenzhucaoFuFang showed the three different dose (high/middle and low) of ZhenzhucaoFuFang can inhibit the copy of HBV-X gene. At the same time reduce the communicate of IP-10.