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.


 

Keywords: bile duct ligation GCSF Biliary atresia liver fibrosis


INTRODUCTION

Biliary atresia (BA) is a rare neonatal congenital disease with a prevalence of 1/10,000 to 1/25,000 births and is characterized by obstruction to bile flow from injuries to the bile ducts. It is the most common indication for pediatric liver transplantation. Contributing factors of BA are both congenital and environmental, yet specific causes of the disease remain unknown 1,2. Untreated BA is fatal, with a median survival of 8 months of age. In BA, fibro-obliterative bile duct injuries cause intra-hepatic and extra-hepatic bile obstruction, persistent liver inflammation, fibrosis, and advanced liver failure 3,4,5. The hepatoportoenterostomy (also known as Kasai) procedure is offered to improve bile flow, but the success rate decreases with time and the majority of patients require liver transplantation for long — term survival. The transplantation option is limited in developing countries.

Granulocyte colony-stimulating factor (GCSF) has been used to reduce the risk of infection for innate or acquired neutropenia by enhancing neutrophil production with minimal adverse effects 6,7,8. GCSF was exploited as a mobilizing agent of hematopoietic stem cells (HSC s) to increase peripheral blood stem cell harvest efficiency during allogeneic or autologous stem cell transplantation 9,10 for the treatment of many diseases, such as liver disease 11,12. Moreover, compared to the labor-intensive procedure of harvesting and reinfusing stem cells, GSCF injection alone has been shown to have multiple beneficial effects on liver disease 13,14. According to systematic reviews, GCSF treatment of patients with advanced liver failure 15,16 and acute-on-chronic liver failure 17 significantly improved survival or significantly reduced short-term mortality compared to placebo. The in vivo mechanisms include the following: GCSF mobilization of HSC homing and differentiating into hepatocytes in CCl4 mice to improve liver functions 18; increase of neutrophil circulation in the peripheral blood and neutrophil maturation to decrease the risk of infection in patients with end-stage liver disease 13,19; reduction of CD8 T cells and increase of regulatory T cells (T-regs) 19; and Kupffer cell activation to reduce inflammation in the injured liver 20. Furthermore, GCSF promotes endogenous repair mechanisms, increasing the number of proliferating hepatocytes in CCl4 mice 21 and enhanc ing proliferation of liver progenitor cells (oval cells) 22. These lines of evidence suggest that GCSF could be used to support the failing liver in BA.

In this study, we performed bile duct ligation (BDL) of Balb/c mice as a model of cholestatic liver disease to test the hypothesis that GCSF improves liver function and decreases fibrosis of intrahepatic biliary diseases, such as BA.

MATERIALS AND METHODS

Mouse strain

The study was approved by our Institutional Ethics Committee (Laboratory of Stem Cell Research and Application, University of Science, Vietnam National University (VNU)-Ho Chi Minh City (HCMC)). Healthy, 8-week old male Balb/c mice from Pasteur Institute in HCMC (Vietnam) were kept in a stable environment of 12 hours light-dark cycle in the micro-ventilation cage system (THREE-SHINE Inc., Korea) with ad libitum access to food and water, and were acclimated for 1 week prior to the operation.

Bile duct ligation (BDL) and experiment design

In this study, the BDL procedure was performed as described by Carmen Tag et al. 23. The common bile duct was ligated between sutures and divided with care to avoid injury to the portal vein and the pancreas. Intramuscular (i.m.) administration of 10 mg/kg Ilium xylazil-20 (Troy Laboratories, Australia) was used as a sedative, followed by 7 mg/kg of Zoletil (Virbac, France). Lincomycin (Vemedim, Vietnam) at 20 mg/kg x 2 doses/day was given for 3 days i.m. to prevent post-surgery infections. BDL mice with visible jaundice at 7 days post-operation (d.p.o) were divided into three treatment groups (n=5):

(1) Sham-surgery treated with 200 μl/day of NaCl (0.9% solution);

(2) BDL with Placebo as 200 μl/day of NaCl (0.9% solution); and

(3) BDL + GCSF (10 µg/kg/day; Neupogen® Syringe (Filgrastim) (Roche, Switzerland)).

All treatments were administered subcutaneously (s.c.) starting on d.p.o 8 for 5 consecutive days, and mice were humanely sacrificed the day after the last dose of GCSF (on day 13 post surgery).

Blood sample preparation

Blood samples were taken from the facial vein. Leukocyte and neutrophil counts were calculated by trained and blinded observers using the blood film technique. In brief, a drop of the blood sample was smeared upon a glass slide, air-dried for 3 minutes, fixed