title: Fuzheng Huayu recipe alleviates hepatic fibrosis via inhibiting TNF-α induced hepatocyte apoptosis.
abstract: BACKGROUND
  What was the relationship of Fuzheng Huayu recipe (FZHY) inhibiting hepatocyte apoptosis and HSC activation at different stage of liver fibrosis? In order to answer this question, the study was carried out to dynamically observe FZHY's effect on hepatocyte apoptosis and HSC activation and further explored underling mechanism of FZHY against hepatocyte apoptosis.
  
  
  METHODS
  Mice were randomly divided into four groups: normal, model, FZHY, and N-acetylcystein (NAC) groups. Acute hepatic injury and liver fibrosis in mice were induced by CCl4. Three days before the first CCl4 injection, treatment with FZHY powder or NAC respectively was started. In vitro, primary hepatocytes were pretreated with FZHY medicated serum or Z-VAD-FMK and then incubated with ActD and TNF-α. Primary HSCs were treated with DNA from apoptotic hepatocytes incubated by Act D/TNF-α or FZHY medicated. Liver sections were analyzed for HE staining and immunohistochemical evaluation of apoptosis. Serum ALT and AST, Alb content and TNF-α expression in liver tissue were detected. Hyp content was assayed and collagen deposition was visualized. Expressions of α-SMA and type I collagen were analyzed by immunofluorescence and immunoblotting. Flow cytometry, immunofluorescence, and DNA ladder for hepatocyte apoptosis and immunoblotting for TNF-R1, Bcl-2 and Bax were also analyzed.
  
  
  RESULTS
  Mice showed characteristic features of massive hepatocytes apoptosis in early stage of liver injury and developed severe hepatic fibrosis in later phase. FZHY treatment significantly alleviated acute liver injury and hepatocyte apoptosis, and inhibited liver fibrosis by decreasing α-SMA expression and hepatic Hyp content. In vitro, primary hepatocytes were induced by TNF-α and Act D. The anti-apoptotic effect of FZHY was generated by reducing TNFR1 expression and balancing the expressions of Bcl-2 and Bax. Meanwhile, the nuclear DNA from apoptotic hepatocytes stimulated HSC activation in a dose dependent manner, and the DNA from apoptotic hepatocytes treated with FZHY or Z-VAD-FMK reduced HSC activation and type I collagen expression.
  
  
  CONCLUSIONS
  These findings suggested that FZHY suppressed hepatocyte apoptosis through regulating mediators in death receptor and mitochondrial pathways, and the effect of FZHY on hepatocyte apoptosis might play an important role in inhibiting liver fibrosis.
authors.forename: Yan-yan
authors.initials: YY
authors.lastname: Tao
authors.forename: Xiu-chuan
authors.initials: XC
authors.lastname: Yan
authors.forename: Tao
authors.initials: T
authors.lastname: Zhou
authors.forename: Li
authors.initials: L
authors.lastname: Shen
authors.forename: Zu-long
authors.initials: ZL
authors.lastname: Liu
authors.affiliation: Institute of Liver Diseases, Shuguang Hospital affiliated to Shanghai University of Traditional Chinese Medicine, 528 Zhangheng Road, Pudong New Area, Shanghai 201203, China. chenghailiu@hotmail.com.
authors.forename: Cheng-hai
authors.initials: CH
authors.lastname: Liu
mesh_terms: D000199:Actins / Q000378:metabolism; D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002251:Carbon Tetrachloride; D056486:Chemical and Drug Induced Liver Injury / Q000188:drug therapy* / Q000378:metabolism; D004365:Drugs, Chinese Herbal / Q000494:pharmacology / Q000627:therapeutic use*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D055166:Hepatic Stellate Cells / Q000187:drug effects; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism; D006909:Hydroxyproline / Q000378:metabolism; D008099:Liver / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D008103:Liver Cirrhosis / Q000139:chemically induced / Q000188:drug therapy* / Q000378:metabolism; D008297:Male; D008810:Mice, Inbred C57BL; D008517:Phytotherapy; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*; D051028:bcl-2-Associated X Protein / Q000378:metabolism
chemical_list: C558396:Acta2 protein, mouse; D000199:Actins; D000590:Amino Acid Chloromethyl Ketones; C494827:Bax protein, mouse; D004365:Drugs, Chinese Herbal; D004791:Enzyme Inhibitors; D019253:Proto-Oncogene Proteins c-bcl-2; D047888:Receptors, Tumor Necrosis Factor, Type I; C506236:Tnfrsf1a protein, mouse; D014409:Tumor Necrosis Factor-alpha; D051028:bcl-2-Associated X Protein; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; C532829:fuzheng huayu; C052617:Bcl2 protein, mouse; D002251:Carbon Tetrachloride; D006909:Hydroxyproline