Effect Of Herb Medicine Gan-fu-kang On The Expression Of Mitogen Activated Protein Kinases/active Protein-1 In Liver Fibrotic Rats

Background and objective:Liver fibrosis is a major consequence that causes death in a variety of chronic liver diseases. Liver fibrosis is characterized by the activation of hepatic stellate cells (HSCs) which in turn produce an excess of extra-cellular matrix (ECM) and fibrosis associated factors, such as tissue inhibitor metal proteinase (TIMP-1).Active protein-1 (AP-1) is an inducible transcription factor implicated in diverse cellular processes, including cell proliferation, differentiation, apoptosis, and neoplastic transformation. Once activated, AP-1 binds to a palindromic sequence (5'-TGAGTCA-3') to activate downstream target genes, including type I collagen, transforming growth factor-beta 1 (TGF-β1), and TIMP-1, which in turn could promote the deposition of ECM.Platelet derived growth factor (PDGF) is the most potent mitogenic and proliferative cytokine described for HSCs. PDGF-BB is the most potent PDGF isoform. MAPK/AP-1 signal pathway has been identified as an intermediary in PDGF-BB-induced TGF-β1, MCP-1, and PAI-1 genes expression in human mesangial cells. Previous study indicated that PDGF-BB induced AP-1 DNA-binding activity in vascular smooth muscle cells of SD rats.Using a rat model of liver fibrosis, we determined:1) effect of GFK on liver function of CCl4-induced liver fibrosis in rats; 2) effect of GFK treatment on the expression of AP-1 in rat liver tissues; 3) effect of GFK on PDGF-BB/PDGFRβand MAPKs signal pathway and the correlation analysis among these cytokines. Methods:Liver fibrosis was established by 12 weeks of carbon tetrachloride (CC14) treatment (0.5 mg/kg, twice per week) followed by 8 weeks of "recovery" in rats. Rats randomly received GFK (31.25,312.5 and 3125 mg/kg/day, p.o.) or vehicle from weeks 9 to 20, and were sacrificed at the end of week 20 for histological, biochemical, and molecular biological examinations. In a separate set of experiments, rats received 12 weeks of CCl4 treatment, concomitant with GFK (312.5 mg/kg/day, p.o.) during the same period in some subjects, but were then sacrificed immediately. An additional group of rats receiving no CCl4 treatment served as normal controls.Results:(1) CCl4 treatment resulted in severe liver damage and fibrosis. GFK attenuated liver damage and fibrosis.Liver fibrosis was established by 12 weeks of carbon tetrachloride (CCl4) treatment followed by 8 weeks of "recovery" in rats. CCl4 treatment significantly increased serum ALT, AST, and A/G (the ratio between ALB and globulin). GFK treatment significantly attenuated the increase of serum ALT, AST, and A/G induced by CC14.(2) Expressions of c-fos and c-jun and AP-1 DNA-binding ability in CCl4-treated rats:Immunohistochemistry and RT-PCR showed, significantly increased positive staining of c-Fos and c-Jun in the cell nuclei in rats receiving CCl4 treatment, and attenuation by GFK prevention treatment。EMSA showed that AP-1 DNA-binding activity was significantly higher at 20 weeks after initiation of the CC14 treatment than that of the normal controls.(3) GFK suppressed the expression of TIMP-1, type I collagen, platelet-derived growth factor-BB (PDGF-BB)/PDGF receptor-βchains (PDGFRβ) and mitogen activated protein kinases (MAPKs)/AP-1 signal pathways. Correlation analysis revealed that both PDGF-BB and PDGFRp were significantly correlated with the following measures:phosphorylated MAPKs, AP-1 DNA-binding activity, procollagenα1(Ⅰ), and TIMP-1.Conclusions:To sum up, (1) GFK could attenuate hepatic dysfunction in a rat model of liver fibrogenesis; GFK also has prevention effects against direct hepatic injury; (2) PDGF-BB/PDGFRβand MAPK/AP-1 signal pathways contribute to increased TIMP-1 and type I collagen genes expression in the CCl4-injured liver fibrotic rats; and (3) AP-1 may be a novel therapeutic target for treating liver fibrosis.