| Summary: | 碩士 === 國立陽明大學 === 生物化學研究所 === 92 === Infection by hepatitis B virus (HBV) frequently results in acute and chronic hepatitis and is associated with a high risk of developing primary hepatocellular carcinomas in human. Immunization and nucleoside analogs as drugs against HBV have been shown to be effective. Owing to the efficacy and resistance of drugs, effective drugs to eradicate HBV in chronic carriers are still not available. There is an urgent need to develop novel drug screening system to search for new anti-HBV therapeutics.
Recently, cell culture system has been used to study the mechanisms of Chinese medicinal herbs, which are widely used as a resource for the development of new drugs. Human hepatoma cell lines, HepG2/A2 and Hep3B/T2, contain integrated HBV gnomes and continually secrete HBsAg. These cell lines provide an assay system for screening of anti-HBV agents from Chinese herb medicines. This study has examined the anti-HBV activities of several pure compounds extracted from traditional Chinese herbs. Clausena excavate has been used for snake biting. Schizandrae fructus has exhibited protective effect in the liver injury. TS95 and KP-19-1-401 were extracted from Clausena excavate and Schizandrae fructus respectively.
Both TS95 and KP-19-1-401 suppressed the HBsAg production in a dose-dependent manner in Hep3B/T2 cells. In HepG2/A2 cells, these two pure compounds suppressed the HBsAg and HBeAg production both in time- and dose-dependent manners but had no obvious effect on cell growth. The cell line HepES2 is a clonal derivative of human hepatoblastoma, HepG2 in which the 1.3 copies of the entire HBV genome is stably transfected into the host genome, and in which HBV is replicated, resulting in the production of infectious virus. Northern blotting analysis was performed to study the HBV RNA transcripts. The HBV-specific transcripts both on 3.5/3.6 and 2.1/2.4 kb were reduced by 2 ug/ml TS95 and 1 ug/ml KP-19-1-401. We further investigated the regulatory mechanism by transiently transfecting various plasmids that contain a HBV promoter region and a luciferase reporter gene into Hep3B/T2 cells. The promoter activity was analyzed by luciferase assay. TS95 and KP-19-1-401 suppressed both CP-luciferase and SPII-luciferase activity in a dose-dependent manner. KP-19-1-401, but not TS95, suppressed SPI-luciferase activity in a dose-dependent manner. TS95 and KP-19-1-401 showed no obvious effect on X-luciferase activity.
Furthermore, KP-19-1-401 suppressed cyclin A-luciferase activity in a dose-dependent manner in Hep3B/T2 cells. The protein level of cyclin A but not cyclin B1 and cyclin D was reduced by 1 ug/ml KP-19-1-401-treated Hep3B/T2 cells. 1.0 ug/ml KP-19-1-401 induced the occurrence of cell cycle arrest at the G1 phase in Hep3B/T2 cells.
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