Resistin mediates the hepatic stellate cell phenotype

Figure 5 Resistin indirectly enhances hepatic stellate cells collagen I expression through a transforming growth factor β1 dependent mechanism via the action of Kupffer cells. Rat Kupffer cells (KCs) at day 2 were cultured with resistin (500 ng/mL) for 24 h. Total RNA was extracted and quantitative polymerase chain reaction was performed. Transforming growth factor β1 (TGFβ1) protein expression in KC conditioned medium (KM) was quantified by immunoblotting. Sirius red was used for collagen I protein quantification in the medium. For the KC-hepatic stellate cell (HSC) co-culture experiment, KCs at day 2 were incubated with resistin for 24 h and then washed three times with phosphate-buffered saline, fresh medium was subsequently added to the culture for another 24 h. KM was then transferred to HSCs at day 4 for 24 h. HSC collagen I, TGFβ1, tissue inhibitor of metalloproteinase 1 (TIMP1), connective tissue growth factor (CTGF) and α smooth muscle actin (αSMA) expression were determined. For inhibition experiments, TGFβ1 monoclonal antibody (10 μg/mL), SP600125 (50 μmol/L) and SB203580 (20 μmol/L) were added to KCs for 24 h. A: Resistin promoted KC TGFβ1 and CTGF gene expression; B: Resistin augmented TGFβ1 expression in KM medium; C: HSC collagen I and CTGF mRNA were augmented by resistin conditioned KM reversed by TGFβ1 neutralization (10 μg/mL); D: HSC collagen protein was increased by resistin conditioned KM but reversed by TGFβ1 neutralization (10 μg/mL); E: Resistin increased KC JNK and p38 phosphor-protein; F: pJNK inhibitor (50 μmol/L, SP600125) and p-p38 inhibitor (20 μmol/L, SB203580) partially, but significantly reversed resistin-induced TGFβ1 and CTGF expression by KCs. Data are expressed as mean ± SD. At least three independent experiments were conducted in triplicate for data analysis. ^aP < 0.05 and ^bP < 0.01 vs controls (untreated); ^cP < 0.05 vs resistin treatment alone.