Recombinant human granulocyte colony-stimulating factor alleviates liver fibrosis in bile duct-ligated mice

Abstract

Introduction: Biliary atresia (BA) is the leading cause of liver fibrosis and failure in neonates with surgical jaundice, leading to poor outcome. Clinical and animal studies showing that granulocyte colony-stimulating factor (GCSF) treatment could improve liver fibrosis and cirrhosis suggest that GCSF may be offered as a low-cost intervention to improve the course of BA. This study aims to test the hypothesis that 10 µg/kg/day x 5 days of GCSF could improve liver function, reduce molecular pro-fibrotic markers and decrease liver fibrosis in a mouse model of bile duct ligation (BDL). Methods: Balb/c mice underwent Sham surgery, or BDL for seven days followed by subcutaneous GCSF administration at 10 µg/kg/day for five consecutive days. Twelve days post-operation, blood samples were taken from the facial vein for leukocyte/neutrophil count and for measurement of serum enzymatic activities. The median lobe of the liver was acquired for total RNA and protein extraction. Moreover, the median liver lobe was used for hematoxylin-eosin staining, sirius red staining, and for visualization by immunohistochemistry (IHC). Results: Twelve days post-operation, GCSF-treated bile duct-ligated (BDL) mice had a higher survival rate than that of placebo-treated mice (hazard ratio=1.88, p=0.084). The GCSF-treated mice had diminished liver serum transaminase activities (AST: 228.92 ± 222.67 vs. 313.46 ± 164.80 IU/L; ALP: 573.24 ± 177.89 IU/L vs. 471.75 ± 117.92 IU/L). GCSF treatment also reduced fibrosis with down-regulation of expression of pro-fibrotic markers including TGF-β1 (-2.61-fold mRNA), α-SMA (-2.46-fold mRNA; -1.88-fold protein, p<0.001) and collagen (-3.28-fold mRNA; -1.79-fold collagen deposit, p=0.0055). Moreover, GCSF treatment led to an improvement of histological grade and a reduction of extension of ductular structures caused by cholestasis (-1.77-fold CK7-positive bile ducts, p<0.0001; -2.33-fold CK7 positivity, p<0.0001). Conclusion: Administration of GCSF (10 μg/kg/day) for five consecutive days improved the pathological condition of BDL mice. In this study, the positive effect of GCSF could be eventually surpassed due to end-stage liver disease caused from BDL in the mouse model. Further experiments are required to elucidate the effects and mechanisms of GCSF on bile obstruction.