Inhibition Of Hepatitis B Virus Replication By Oxymatrine And Its Mechanism In Vitro

This research was composed of two parts:Ⅰ. Inhibitory effect of oxymatrine and adefovir dipivoxil on the replication of hepatitis B virus in vitro;Ⅱ. Effect of oxymatrine on the activity of HBV polymeraseⅠ. Inhibitory effect of oxymatrine and adefovir dipivoxil on the replication of hepatitis B virus in vitroObjective To compare the inhibitory effect of oxymatrine and adefovir dipivoxil on the replication of hepatitis B virus in vitro. Methods The cell line HepG2.2.15 was maintained in DMEM culture medium. Forty-eight hours after culturing, new medium containing oxymatrine (1000μg /ml, 500μg /ml and 250μg /ml) or adefovir dipivoxil (20μg /ml , 10μg /ml and 5μg /ml) were added, and new medium without antiviral drug was used as normal control. At different time (48h, 96h) after agent added, supernatants from 5 wells of each group were harvested .HBV DNA was determined by real-time fluorescent quantitative PCR, and HBsAg and HBeAg were determined by enzyme-linked immunosorbent Assay. Results At different time after drugs were added, HBV DNA, HBsAg, HBeAg declined significantly both in oxymatrine groups and adefovir dipivoxil groups comparing with normal control group. After incubation for 48 h and 96 h, the maximum inhibitory effect was observed when 1000μg /ml of oxymatrine and 20μg /ml of adefovir dipivoxil were added. HBV DNA, HBsAg and HBeAg in supernatant were reduced by 24.28%, 29.85% and 18.48% respectively when cell lines were incubated with 1000μg /ml of oxymatrine for 48 h, and with 20μg /ml of adefovir dipivoxil they were reduced by 23.80%, 34.50% and 19.74% respectively. After incubation for 96 h, 1000μg /ml of oxymatrine reduced HBV DNA, HBsAg and HBeAg in supernatant by 29.06%, 43.32% and 25.13% respectively, and 20 μg /ml of adefovir dipivoxil reduced them by 32.98%, 47.65% and 25.40%, respectively. Conclusions Rplication of hepatitis B virus could be inhibited by oxymatrine, and inhibitory effect by 1000μg /ml of oxymatrine was similar to that by 20μg /ml of adefovir dipivoxil.ⅡEffect of oxymatrine on the activity of HBV polymeraseObjective To determine whether the activity of HBV DNA polymerase could be inhibited by oxymatrine in vitro. Methods Hepatitis B virus particles were purified from supernatant of HepG2.2.15 cell lines by ultracentrifugation, and then were mixed with reaction buffer which contained NP-40,β-mercaptoethanol, nucleoside triphosphates (dCTP were labelled with 32P) , MgCl2 , and different inbitors including oxymatrine (1000μg/ml, 800μg/ml, 600μg/ml, 400μg/ml and 200μg/ml) or adefovir dipivoxil (100μg /ml, 80μg /ml and 60μg /ml, 40μg /ml and 20μg /ml). After incubations at 37℃overnight, proteinase K was added to the reaction system for digestion and 35μl of samples were spotted onto DE81 paper. Acitivity of endogenous polymerase in HBV paricles treated with different doses of oxymatrine or adefovir dipivoxil were assessed by determining the radioactivity of 32P-labelled dCTP incorporated in the plus-strain of viral DNA. Results Comparing to the normal control, the acitivity of endogenous polymerase in HBV paricles treated with different doses of oxymatrine varied from 103% to 107%, and when treated with different doses of adefovir dipivoxil varied from 91% to 101%. No significant difference was observed between different groups. Conclusions No direct inhibitory effect of oxymatrine and adefovir dipivoxil on the activity of HBV polymerase was observed in vitro.