Int J Clin Exp Pathol 2018;11(3):1112-1122 www.ijcep.com /ISSN:1936-2625/IJCEP0069421 Original Article Blocking follistatin-like 1 attenuates liver fibrosis in mice by regulating transforming growth factor-beta signaling Xiao-Hua Zhang1*, Yong Chen1*, Bin Li1, Ji-Yong Liu1, Chong-Mei Yang1, Ming-Ze Ma2 Departments of 1Gastroenterology, 2Infectious Diseases, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, Shandong Province, China. *Equal contributors. Received November 19, 2017; Accepted December 2, 2017; Epub March 1, 2018; Published March 15, 2018 Abstract: Aim: To elucidate the effect of inhibiting follistatin-like 1 on liver fibrosis and activation of hepatic stel- late cells in mice. Methods: We generated a follistatin-like 1 neutralizing antibody that can inhibit TGF-β 1-induced expression of collagen1α1 in primary mouse liver fibroblasts. All of the mice in our study were induced with carbon tetrachloride and thioacetamide. In addition, primary hepatic stellate cells from mice were isolated from fresh livers using density gradient separation. The degree and extent of fibrosis in mouse livers from the different groups were evaluated by Sirius Red and Masson staining. The effect of the follistatin-like 1 neutralizing antibody on proliferation and migration of hepatic stellate cells was detected using CCK-8 and Transwell assays, respectively. Results: Expres- sion of follistatin-like 1 in human cirrhotic liver tissue was higher than that in normal liver tissue. Blocking follistatin- like 1 resulted in a delay of primary hepatic stellate cell activation and down-regulation of the migratory capacity of hepatic stellate cells. Blocking follistatin-like 1 also down-regulated TGF-beta signaling in primary hepatic stellate cells from mice. Finally, inhibition of follistatin-like 1 attenuated liver fibrosis and liver function damagein vivo. Con- clusions: Inhibiting follistatin-like 1 attenuates liver fibrosis and causes a delay in hepatic stellate cell activation. The effect of follistatin-like 1 on liver fibrosis is mainly attributed to its role in regulating TGF-beta signaling. Keywords: Follistatin-like 1, liver fibrosis, transforming growth factor-beta, hepatic stellate cells Introduction cause many unacceptable adverse effects in animal trials [4]. Based on this, it is important Liver fibrosis is a common chronic liver disease to find new targets that can influence TGF-beta that is characterized by the accumulation of signaling as well as liver fibrosis progression. collagen and activation of Hepatic Stellate Cells (HSCs) in the liver [1]. Liver fibrosis is a common Follistatin-like 1 (FSTL1) was initially identified precursor of liver cirrhosis and even hepatocel- as a TGF-beta-induced gene that encodes the lular carcinoma [1]. Liver cirrhosis is irreversible secreted protein Fstl1 [5]. Dong et al. reported that FSTL1 can regulate fibroblast activation by and can only be effectively cured with liver affecting TGF-beta signaling and bone morpho- implantation [2]. Therefore, preventing progres- genetic protein signaling [6]. Lung fibrosis in sion from liver fibrosis to liver cirrhosis can be mice was attenuated in vivo by a FSTL1 neutral- highly beneficial to patients with chronic liver izing antibody [7]. The results above suggested disease. that FSTL1 may play a role in the progression of liver fibrosis due to similar mechanisms in the TGF-beta signaling plays a central role in the pathogenesis of pulmonary fibrosis and liver progression of liver fibrosis and activation of fibrosis. HSCs [3]. Thus, preventing TGF-beta signaling may theoretically result in the blockage of liver We conducted this study and found that block- fibrosis progression [3]. Nevertheless, directly ing FSTL1 led to attenuation of liver fibrosis in blocking TGF-beta signaling has been shown to carbon tetrachloride-treated mice. In addition, Blocking follistatin-like 1 attenuates liver fibrosis we confirmed the effect of FSTL1 on TGF-beta abdominal cavities. The fresh livers were per- signaling in mouse liver tissues. fused with a digestive solution comprising 0.1% collagenase, 0.25% pronase E and 0.01% Materials and methods DNase and mechanically dissected. The digest- ed livers were incubated in the same digestive Clinical samples solution at 37°C for approximately 25 to 30 minutes. The suspension was then removed All human normal liver tissues and cirrhotic and filtered through an iron mesh with 100 μm liver tissues were provided by the Department pores. The filtered suspension was centrifuged of Liver Surgery at Shandong Provincial through a Nycodenz gradient (Axis-Shield) at Hospital. All the human tissues were obtained 8.2% concentration. The isolated primary HSCs with informed content, and approved by the were resuspended in DMEM supplemented Ethical Committee of Shandong Provincial with 20% fetal calf serum and 1% antibiotics Hospital. and cultured at 37°C overnight in an environ- ment containing 5% carbon dioxide. After 24 Cell culture hours, cell debris and non-adherent cells were Rat HSC-T6 cells were purchased from Millipore removed. Corporation. These cells were cultured in Du- Generation of FSTL1-neutralizing antibodies lbecco’s-modified Eagle medium (DMEM) sup- plemented with 10% fetal calf serum and 1% FSTL1 neutralizing antibody generation was antibiotics at 37°C in a Thermo incubator con- performed using conventional hybridoma tech- taining 5% carbon dioxide. niques. His-tag fusion constructs of FSTL1 (which encoded amino acids 37-306 of the full- Experimental animals length mouse FSTL1 protein, NP_009016) were cloned into PET28 vectors and expressed Female BALB/c mice were purchased from according to previously established protocols. Shandong University Laboratory Animal Center The purified His-tag fusion protein was used as and used to produce neutralizing antibodies. In an antigen to immunize BALB/c mice for gener- addition, C57BL/6J mice were purchased from ation of anti-FSTL1 monoclonal antibodies. Shandong University Laboratory Animal Center Afterwards, one monoclonal antibody was iden- and used for carbon tetrachloride-induced liver tified that effectively blocked TGF-beta1-in- fibrosis. All of the mice in our study were housed duced expression of Collagen1α1 in hepatic and fed in specific-pathogen free (SPF) condi- fibroblasts and this mAb was used as the pri- tions and received humane care. We ensured mary FSTL1 neutralizing antibody for all subse- that all animals in our study received humane quent animal experiments. care, and all study protocols complied with the institution’s guidelines. Immunohistochemical and Sirius Red staining Carbon tetrachloride- and TAA-induced liver Paraffin-embedded liver tissue from mice and fibrosis model humans were sliced into 5-μm thick sections followed by deparaffinization and step-wise C57BL/6J mice were injected with carbon tetra- rehydration in preparation for IHC and Sirius chloride (CCl4) (0.5 μL per gram body weight) in Red staining. For Sirius Red staining, the sec- 25% olive oil twice per week at equal intervals tions were stained by Sirius Red for approxi- for eight weeks. Another group of C57BL/6J mately three minutes and dehydrated step- mice were injected with thioacetamide (TAA) wise. For immunohistochemical staining, the (0.2 mg/g body weight) in double distilled water sections were incubated with 0.3% hydrogen three times per week at equal intervals for eight peroxide for thirty minutes and blocked with weeks. 10% bovine serum albumin. The sections were then incubated using antibody targeting FSTL1 Primary hepatic stellate cell isolation from (1:200, Abcam) at 4°C overnight and labeled by mice HRP-conjugated secondary mouse antibody (Abcam) at room temperature for approximately C57BL/6J mice were sacrificed after anesthe- one hour. Afterwards, the sections were incu- sia, and their livers were removed from the bated with a DAB substrate liquid (Thermo) and 1113 Int J Clin Exp Pathol 2018;11(3):1112-1122 Blocking follistatin-like 1 attenuates liver fibrosis minutes followed by a quick immersion into 2% glacial ace- tic acid. Afterwards, the slides were immersed in 1% phos- phomolybdic acid for approxi- mately three minutes prior to immediate staining with An- iline Blue for five minutes. The slides were subsequently im- mersed into 0.2% glacial ace- tic acid briefly and dehydrated step-wise until they were sealed with neutral balsam. Immunofluorescence staining For cell staining, primary mo- use HSCs were seeded onto rounded coverslips in 24-well plates and incubated at 37°C for approximately seven days in an environment containing 5% CO2. For F-actin staining, cells were incubated with ph- alloidin-FITC (Sigma) for sev- enty minutes at room temper- ature. For α-SMA staining, cells were incubated with α-SMA antibody (Sigma) for seventy-five minutes at room temperature and subsequent- ly treated with Alexa Flu- or 594-conjugated secondary antibody. The nuclei were stained using DAPI (Sigma), and the immunofluorescence images were recorded using a fluorescence microscope (Carl Figure 1. (A) IHC images of FSTL1 expression in human cirrhotic liver tissues Zeiss). (a) and normal liver tissues (b). FSTL1 expression was mainly localized to cirrhotic lesions of liver tissues; (B) Western blot assay showed that FSTL1 Western blotting protein expression was higher in human cirrhotic liver tissue than in normal liver tissue; (C) ICC assay showed that localization of FSTL1 expression (a) in liver tissue was highly similar to that of α-SMA expression (b). Cells were lysed in lysis solu- tion (50 mM Tris-HCl, 150 mM NaCl, 1% Triton-X 100, 1 mM stained with hematoxylin for two minutes. All MgCl2, 1 mM PMSF) and boiled for five minutes. the sealed slides were imaged and recorded on The proteins were separated by SDS-PAGE and a microscope manufactured by Carl Zeiss. transferred onto a nitrocellulose membrane that was blocked using in 1% bovine serum Masson staining albumin in TBS. The NC membrane was incu- bated with antibodies targeting FSTL1 (Abcam), The slides were deparaffinized and rehydrated Smad2 (Cell Signaling Technology, CST), phos- step-wise before Masson staining.