title: [Establishment of a complex alcoholic liver fibrosis mouse model and investigation of OPN and TGF-beta1 hepatic expression].
abstract: OBJECTIVE
  To create a convenient method to establish an alcoholic liver fibrosis model in mice and use it to explore the putative pathogenic mechanisms involving the immunomodulatory proteins osteopontin (OPN) and transforming growth factor-betal (TGF-beta1).
  
  
  METHODS
  Forty C57BLI6J mice were fed the Lieber-DeCarli 4% ethanol-containing liquid diet for four weeks, followed by an additional four weeks of the 4% ethanol diet combined with intraperitoneal injection of carbon tetrachloride (CC14 5% solution in olive oil; 2ml/ kg body weight, 2 times/week) to induce alcoholic liver fibrosis. Control groups (n = 6 each) included: normal diet; normal diet plus CCl4 injections; ethanol diet alone; ethanol diet plus solvent (olive oil) injections. Model establishment was monitored by sacrificing six mice at model inception (week 0), and weeks 4, 5, 6, 7, and 8 of modeling to collect liver tissues and blood for histological and biochemical analyses. Extent of hepatic steatosis, inflammation, and fibrosis was assessed by hematoxylin-eosin and Masson staining. Liver function markers, serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, were tested by automated enzymatic assays. Alpha-smooth muscle actin (alpha-SMA) expression was detected by immunohistochemistry. The mRNA and protein expression of OPN and TGF-beta1 was detected by real-time quantitative reverse transcription-PCR and western blotting, respectively. Significance of differences between multiple groups was assessed by one-way ANOVA analysis followed by least significant difference t-test or Kruskal-Wallis H test followed by the Mann-Whitney U test.
  
  
  RESULTS
  Compared to the control groups, the group of mice administrated ethanol and CCl4 developed mild to moderate hepatic steatosis at week 4 of modeling, progressive necroinflammation and perisinusoidal and portal fibrosis from weeks 5-8, and irregular necrosis and bridging fibrosis at week 8. In addition, the model group showed progressive up-regulation of a-SMA expression in the activated hepatic stellate cells (HSCs) and fibrotic areas from weeks 5-8. Both hepatic OPN and TGF-beta1 showed significantly increasing trends in mRNA and protein expressions from weeks 5-8 (OPN mRNA: 1.83 +/- 0.25, 2.94 +/- 0.19, 3.45 +/- 0.31, and 5.99 +/- 0.17 (F= 476.27, P < 0.001); OPN protein: 0.52 +/- 0.06, 1.02 +/- 0.10, 1.52 +/- 0.11 and 1.50 +/- 0.08 (F= 298.03, P< 0.001); TGF-beta1 mRNA: 13.19 +/- 0.40, 3.31 +/- 0.28, 1.58 +/- 0.18 and 2.08 +/- 0.26 (F= 85.55, P < 0.001); TGF-P31 protein: 1.26 +/- 0.16, 0.96 +/- 0.12, 1.09 +/- 0.25 and 1.10 +/- 0.20 (F = 43.64, P < 0.001).
  
  
  CONCLUSIONS
  Feeding C57BL/6J mice the Lieber-DeCarli ethanol-containing liquid diet combined with CCl4 intraperitoneal injection is a convenient method to establish a model of alcoholic liver fibrosis within a relatively short amount of time (eight weeks). Progression of alcoholic liver fibrosis is accompanied by increased hepatic expression of OPN and TGF-beta1, which may contribute to the pathogenic mechanism of this disease and may be targets of future molecular therapies.
authors: Department of Traditional and Western Medical Hepatology, Third Hospital of Hebei Medical University, Shijiazhuang 050051, China.
authors: Ling-bo
authors: LB
authors: Kong
authors: Wei-guang
authors: WG
authors: Ren
authors: Hong-mei
authors: HM
authors: Mi
authors: Su-xian
authors: SX
authors: Zhao
authors: Yu-guo
authors: YG
authors: Zhang
authors: Yue-min
authors: YM
authors: Nan
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D004195:Disease Models, Animal; D008099:Liver / Q000378:metabolism*; D008104:Liver Cirrhosis, Alcoholic / Q000378:metabolism*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D053495:Osteopontin / Q000378:metabolism*; D053773:Transforming Growth Factor beta1 / Q000378:metabolism*
chemical_list: D000199:Actins; C506896:Spp1 protein, mouse; C508961:Tgfb1 protein, mouse; D053773:Transforming Growth Factor beta1; C544013:alpha-smooth muscle actin, mouse; D053495:Osteopontin