Figure 9. Inhibition of liver fibrosis by HSC-specific deletion of FPN.

(a) Immunoblottings for p-Smad3. LX-2 cells were treated with 100 nM Hep-20 or Hep-25 for 3 h, and continuously treated with 5 ng ml⁻¹ TGFβ1 for 20 min. (b) Immunoblottings for p-Akt and FPN in LX-2 cells treated with Hep-20 or Hep-25. (c) qRT–PCR assays for α-SMA and TGFβ1. Mice were treated with vehicle or CCl₄ (0.6 ml kg⁻¹ body weight, i.p., 24 h) 3 h after an intraperitoneal injection of 50 μg Hep-20 or Hep-25 (N=4 or 6 each). Details for treatment schedule are provided in the Methods section. (d) HSC-specific FPN knockout mouse model. FPN-floxed mice were treated with vehicle or CCl₄ (0.6 ml kg⁻¹ body weight, i.p., twice a week, for 4 weeks) after a tail vein injection of PBS, LV-Control or LV-αSMA-Cre (N=4 or 6 each). Details for treatment schedule are provided in the Methods section. Immunoblottings for FPN confirmed HSC-specific silencing of FPN. (e) H&E, Masson's trichrome stainings and IHC for α-SMA or collagen type 1 on the liver tissues of mice treated as in d (scale bar, 100 μm). (f) qRT–PCR assays for α-SMA and TGFβ1 in the liver of mice as in d. For c and f, data represent the mean±s.e.m (N=4 or 6). Statistical significance of the differences between each treatment group and vehicle (**P<0.01) was determined by analysis of variance (Bonferroni's or LSD method). (g) A proposed scheme illustrating the effect of hepcidin on FPN-mediated Smad3 activation in HSCs. Hepcidin inhibits TGFβ1-mediated Smad3 phosphorylation by degrading FPN in HSCs, which relies on Akt signalling. The inhibition of HSC response to TGFβ1 may contribute to anti-fibrotic effect of hepcidin.