Clinical samples.
All human normal liver tissues and cirrhotic liver tissues were provided by the Department of Liver Surgery and Liver Transplantation at Shandong Provincial Hospital. The tissues were obtained with informed consent, and the study was approved by the Ethical Committee of Shandong Provincial Hospital.
Cell culture.
LX-2 cells were purchased from Millipore Corporation. These cells were cultured in Dulbecco’s-modified Eagle medium (DMEM) supplemented with 10% foetal calf serum and 1% antibiotics at 37°C in a Thermo incubator containing 5% carbon dioxide.
Experimental animals.
All mice were purchased from Shandong University Laboratory Animal Centre and were housed and fed under specific pathogen-free (SPF) conditions. Among them, female BALB/c mice were prepared for neutralizing antibodies, and C57BL/6J mice were used for carbon tetrachloride and thioacetamide -induced liver fibrosis. We ensured that all animals in our study received humane care, and all study protocols complied with the institution’s guidelines. The animal experiments were approved by the Ethical Committee of Shandong Provincial Hospital and the approve No. was NSFC:No.2019-033.
Preparation of the carbon tetrachloride- and TAA-induced liver fibrosis model.
Eight C57BL/6J mice were injected with carbon tetrachloride (CCl4, 0.5 μL per gram body weight) in 25% olive oil twice per week at equal intervals for eight weeks. Another group of C57BL/6J mice (including eight mice) was injected with thioacetamide (TAA, 0.2 mg/g body weight) in double-distilled water three times per week at equal intervals for eight weeks. All chemical-inducible liver fibrosis mice were divided into two groups. The GDF15 mAb group mice (including eight mice) were treated with GDF15-neutralizing antibodies at a dose of 20 mg/kg twice a week by abdominal injection, and the control group mice (including eight mice) were injected with homologous IgG for six weeks.
Isolation of primary hepatic stellate cells from mice.
C57BL/6J mice were sacrificed using cervical dislocation method, and their livers were removed from the abdominal cavities. The fresh livers were perfused with a digestive solution consisting of 0.1% collagenase, 0.25% pronase E and 0.01% DNase and mechanically dissected. The digested livers were incubated in the same digestive solution at 37°C for approximately 25 to 30 minutes. Next, the suspension was removed and filtered through an iron mesh with 100-μm pores. Then, the filtered suspension was centrifuged through a Nycodenz gradient (Axis-Shield) at 8.2% concentration. The isolated primary HSCs were resuspended in DMEM supplemented with 20% foetal calf serum and 1% antibiotics (penicillin and streptomycin) and cultured at 37°C overnight in an environment containing 5% carbon dioxide. Twenty-four hours later, cell debris and non-adherent cells were removed.
Generation of GDF15-neutralizing antibodies.
GDF15-neutralizing antibody generation was performed using conventional hybridoma techniques. His-tag fusion constructs of GDF15 (which encoded Mouse GDF15 amino acids 10-303) were cloned into PET28 vectors and expressed according to previously established protocols. The purified His-tag fusion protein was used as an antigen to immunize female BALB/c mice for the generation of anti-GDF15 monoclonal antibodies. Afterwards, we identified one monoclonal antibody that effectively blocked TGF-β1-induced activation of the TGF-β/Smad2/3 signalling pathway and used this mAb as the primary GDF15-neutralizing antibody for all subsequent animal experiments.
Immunohistochemical and Sirius Red staining.
The paraffin-embedded liver tissues from mice and humans were sliced into 5-μm-thick sections followed by deparaffinization and stepwise rehydration in preparation for IHC and Sirius Red staining. For Sirius Red staining, the sections were stained with Sirius Red for approximately three minutes and dehydrated stepwise. For immunohistochemical staining, the sections were incubated with 0.3% hydrogen peroxide for thirty minutes and blocked with 10% bovine serum albumin. The sections were then incubated using an antibody targeting GDF15 (1:200, Abcam, ab189358) at 4ºC overnight and labelled with an HRP-conjugated secondary mouse antibody (Abcam) at room temperature for approximately one hour. Afterwards, the sections were incubated with a DAB substrate liquid (Thermo Fisher) and stained with haematoxylin for two minutes. All the sealed slides were imaged and recorded on a microscope manufactured by Carl Zeiss.
Masson staining.
The slides were deparaffinized and rehydrated stepwise before Masson staining. The slides were first stained with Weigert haematoxylin for 5 minutes and washed with water. The slides were then stained with acid ponceau for six minutes followed by quick immersion in 2% glacial acetic acid. Afterwards, the slides were immersed in 1% phosphomolybdic acid for approximately three minutes prior to immediate staining with aniline blue for five minutes. The slides were subsequently immersed in 0.2% glacial acetic acid briefly and dehydrated stepwise until they were sealed with neutral balsam.
Immunofluorescence staining.
For cell staining, primary mouse 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 phalloidin-FITC (Sigma) for seventy minutes at room temperature. For α-SMA staining, cells were incubated with α-SMA antibody (Sigma) for seventy-five minutes at room temperature and subsequently treated with Alexa Fluor 594-conjugated secondary antibody, avoiding light. The nuclei were stained using DAPI (Sigma), and the immunofluorescence images were recorded using a fluorescence microscope (Carl Zeiss).
Western blotting.
Cells were lysed in lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton-X 100, 1 mM MgCl2, 1 mM PMSF) and boiled for five minutes. The proteins were separated by SDS-PAGE and transferred onto a nitrocellulose membrane that was blocked using 1% bovine serum albumin in TBS. The NC membrane was incubated with antibodies targeting GDF15 (Abcam), Smad2 (Cell Signaling Technology, CST), phospho-Smad2 (CST), Smad3 (CST), phospho-Smad3 (CST), Smad4 (CST), and GAPDH (Abcam), followed by the addition of fluorescence-conjugated secondary antibodies. All the fluorescence signals were captured on an Odyssey imaging system (LI-COR).
Dual-luciferase reporter assay
We used the pGL4.19-TA-TGFBeta(V2) reporter vector (Promega) as a dual-luciferase reporter vector for TGF-beta signalling. pGL4.19-TA-TGFBeta was transfected into LX-2 cells and treated with GDF15, Ly364947, or TGF-β1. A Thermo Scientific Microplate Reader was used to detect the absorbance value of each group. Ly364947 is an inhibitor of the TGF-β signalling pathway.
Statistical analysis.
All the data are presented as the means ± standard error of the mean (SEM). Statistical analysis was performed using SPSS 16.0 software. One-way ANOVA method was used for multigroup comparison. The comparisons of absorbance value between different groups were carried out by using ANOVA method. Two-tailed Student’s t-test was used for comparisons between two groups. A P value less than 0.05 was considered statistically significant.