title: Inhibitory effect of Phyllanthus urinaria L. extract on the replication of lamivudine-resistant hepatitis B virus in vitro.
abstract: BACKGROUND
  Long-term treatment of chronic hepatitis B (CHB) with nucleos(t)ide analogs results in the emergence of drug-resistant hepatitis B virus (HBV) harboring mutations in the polymerase (P) gene. The Phyllanthus extract has anti-HBV activity; however, its antiviral activity against lamivudine (LMV)-resistant mutants has not been examined.
  
  
  METHODS
  HBV harboring LMV-resistant mutations (rtM204I, rtM204V, and rtM204S) in the P gene at the YMDD ((203)tyrosine-methionine-aspartate-aspartate(206)) reverse transcriptase (RT) active site were generated and their sensitivity to Phyllanthus urinaria koreanis extract examined. Southern blotting and real-time PCR were used to determine the concentration of plant extract required to inhibit HBV DNA synthesis by 50 and 90% (EC50 and EC90, respectively). An enzyme-linked immunosorbent assay was used to measure the EC50 of HBV surface antigen (HBsAg) and HBV core antigen (HBcAg) secretion, and the 50% cytotoxic concentration of the extract was measured in a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Real-time RT-PCR was used to measure mRNA expression levels.
  
  
  RESULTS
  The expression of intracellular HBV DNAs in HBV WT- or mutant-transfected HepG2 cells decreased upon treatment with Phyllanthus extract. The secretion of HBsAg and HBcAg also fell in a dose-dependent manner. Phyllanthus extract induced interferon-beta (IFN-β), cyclooxygenase-2 (COX-2), and interleukin-6 (IL-6) mRNA expression in HBV WT-transfected HepG2 cells, possibly via activation of extracellular signal-regulated kinases and c-jun N-terminal kinases and the induction of retinoic acid inducible gene-I, toll-like receptor 3, myeloid differentiation primary response gene 88, and/or tumor necrosis factor receptor-associated factor 6 gene expression. HBV transfection in the absence of extract or exposure of cells to extract alone did not trigger these signaling cascades.
  
  
  CONCLUSIONS
  Phyllanthus extract inhibited HBV DNA synthesis and HBsAg and HBcAg secretion by replicating cells harboring HBV wild-type and LMV-resistant mutants, likely by inducing the expression of IFN-β, COX-2, and IL-6. These data indicate that Phyllanthus extract may be useful as an alternative therapeutic agent for the treatment of drug-resistant CHB patients.
authors.affiliation: Department of Microbiology, Ajou University School of Medicine, Woncheon-dong 5, Suwon, 443-721, Korea. jjsbio118@gmail.com.|Department of Biomedical Science, Graduate School of Ajou University, Woncheon-dong 5, Suwon, Korea. jjsbio118@gmail.com.
authors.forename: Jaesung
authors.initials: J
authors.lastname: Jung
authors.affiliation: Institute for Clinical Research, CHA Bundang Medical Center, CHA University, Seongnam, Gyeonggi-do, Korea. nkkim@cha.ac.kr.
authors.forename: Nam Keun
authors.initials: NK
authors.lastname: Kim
authors.affiliation: Department of Microbiology, Ajou University School of Medicine, Woncheon-dong 5, Suwon, 443-721, Korea. sinsun@ajou.ac.kr.|Department of Biomedical Science, Graduate School of Ajou University, Woncheon-dong 5, Suwon, Korea. sinsun@ajou.ac.kr.
authors.forename: Sun
authors.initials: S
authors.lastname: Park
authors.affiliation: Department of Microbiology, Ajou University School of Medicine, Woncheon-dong 5, Suwon, 443-721, Korea. hjshin@ajou.ac.kr.|Department of Biomedical Science, Graduate School of Ajou University, Woncheon-dong 5, Suwon, Korea. hjshin@ajou.ac.kr.
authors.forename: Ho-Joon
authors.initials: HJ
authors.lastname: Shin
authors.affiliation: Institute for Clinical Research, CHA Bundang Medical Center, CHA University, Seongnam, Gyeonggi-do, Korea. sghwang@cha.ac.kr.|Department of Internal Medicine, CHA Bundang Medical Center, CHA University, Seongnam, Gyeonggi-do, Korea. sghwang@cha.ac.kr.
authors.forename: Seong Gyu
authors.initials: SG
authors.lastname: Hwang
authors.affiliation: Department of Microbiology, Ajou University School of Medicine, Woncheon-dong 5, Suwon, 443-721, Korea. kimkm@ajou.ac.kr.|Department of Biomedical Science, Graduate School of Ajou University, Woncheon-dong 5, Suwon, Korea. kimkm@ajou.ac.kr.
authors.forename: Kyongmin
authors.initials: K
authors.lastname: Kim
mesh_terms: D000998:Antiviral Agents / Q000737:chemistry / Q000494:pharmacology; D024882:Drug Resistance, Viral / Q000187:drug effects*; D056945:Hep G2 Cells; D006515:Hepatitis B virus / Q000187:drug effects*; D006801:Humans; D019259:Lamivudine / Q000494:pharmacology*; D029803:Phyllanthus / Q000737:chemistry*; D010936:Plant Extracts / Q000737:chemistry / Q000494:pharmacology
chemical_list: D000998:Antiviral Agents; D010936:Plant Extracts; D019259:Lamivudine