Immortalized human HSC cell line (LX-2) was purchased from Chinese Academy of Sciences Cell Bank (Shanghai, China) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, China), supplemented with 10% fetal bovine serum (FBS) (Gibco, USA) and 1% Penicillin/Streptomycin at 37 °C humidified chamber with 5% CO2. All cell experiments were performed in strict accordance with cell culture protocols.
For in vitro experiments, Ruxolitinib (INCB018424, Selleck chemicals, USA) was dissolved in DMSO and further diluted to the required concentration. For in vivo experiments, Ruxolitinib suspension was prepared in 0.5% carboxymethyl cellulose sodium normal saline solution.
Small interfering RNA (siRNA) Transfection
The specific siRNA for JAK1 and JAK2 were designed and synthesized by RiboBio (Guangzhou, China). The siRNA sequences were siJAK1: CCACATAGCTGATCTGAAA; siJAK2: ATGACTTTGTCATGTCTTA. siRNAs were transfected into cells using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s protocol. Cells were collected after 48–72 h for further experiments.
Human liver tissue microarray was purchased from Biomax, including 5 liver samples with normal liver tissue, 22 liver samples with liver cirrhosis, and 53 liver samples with liver cancer which were collected in US.
Histology and immunohistochemistry
Formalin-fixed, paraffin-embedded liver tissue samples were cut into 4 µm-thick sections and stained with hematoxylin eosin (H&E), Sirius red, and immunohistochemistry (IHC) according to standard procedures. Fibrosis was scored according to the Ishak scoring system . The amount of Sirius red staining was quantified with ImageJ. For IHC, liver sections were stained with the following antibodies: JAK1 (sc-1677; Santa Cruz Biotechnology), JAK2 (#3230; Cell signaling Technology), Alpha-Smooth muscle actin (α-SMA,1A4, ab7817; Abcam), Both the intensity and extent of immunostaining were taken into consideration when analyzing the data. The intensity of staining was determined by the following rules: 0 for negative; 1 for weak staining; 2 for moderate staining; 3 for strong staining. The staining extent was determined by the following rules: 0 for no staining; 1 for less than 10%; 2 for 10% to 50%; 3 for 51% to 75%; 4 for more than 75%. We randomly selected 5 areas from each area to count the intensity and extent of staining and to calculate the mean staining extent. The score was obtained by plus these two values (intensity score + extent score).
Liver function assay
Serum levels of several biochemical markers were measured to assess liver function and liver injury. The measured biochemical markers included alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL) and albumin (Alb) were measured using standard laboratory assays .
Western blotting was performed as previously described . The primary antibodies were purchased from Santa Cruz Biotechnology [JAK1 (sc-1677)], Cell signaling Technology [JAK2 (#3230), phosho-JAK1 (Tyr1022/1023, #3331), phosho-JAK2 (Tyr1007/1008, #3771), STAT3(#9139), phosho-STAT3 (Tyr705,#9145), phosho-STAT3 (Ser705, #9134), STAT5 (#94205), phosho-STAT5 (Tyr694, #9359)], Abcam [α-SMA (1A4, ab7817)], Bimake [PDGFRβ (A5541), PAI-1 (A5396)], Bioworld [β-actin (#64132)]. β-actin was used as a loading control for all blots.
Quantitative Real-time RT-PCR
Total RNA was extracted from cells and tissues using Trizol (Takara, Japan), and was reverse transcribed into cDNA according to the manufacturer’s instructions, and the GAPDH gene was used as gene control. The relative gene expression ratio was calculated by the ΔΔCt method. The specific primers were listed as follows: JAK1: 5’-CCACTACCGGATGAGGTTCTA-3’ (forward) and 5’-GGGTCTCGAATAGGAGCCAG-3’ (reverse); JAK2: 5’-GCCGGGTTTCAGAAGCAGG-3’ (forward) and 5’- GTAAGGCAGGCCATTCCCAT -3’ (reverse); ACTA2: 5’-GACAATGGCTCTGGGCTCTGTAA -3’ (forward) and 5’-CTGTGCTTCGTCACCCACGTA-3’ (reverse); PAI-1: 5’- AGTGGACTTTTCAGAGGTGGA-3’ (forward) and 5’- GCCGTTGAAGTAGAGGGCATT -3’ (reverse); PDGFRβ: 5’-GCCCTTATGTCGGAGCTGAAGA-3’ (forward) and 5’-GTTGCGGTGCAGGTAGTCCA-3’ (reverse); COL1A1: 5’-CAGCCGCTTCACCTACAGC-3’ (forward) and 5’-TCAATCACTGTCTTGCCCCA-3’ (reverse); GAPDH: 5’-TGTTGCCATCAATGACCCCTT -3’ (forward) and 5’-CTCCACGACGTACTCAGCG-3’ (reverse). Experiments were performed according to the manufacturer’s instructions (Takara, Japan). Independent experiments were done in triplicate.
Cell proliferation assay
LX-2 cells were seeded at a density of 3000 cells/well in 96-well microplates. The cells were treated with siRNA transfection or varying concentrations of Ruxolitinib as designed (0.1–100 μM). Cell viability was measured using the Cell Counting Assay Kit-8 (CCK-8; Dojindo, Japan) according to the manufacturer’s instructions. For each experimental condition, five parallel wells were assigned to each group. Experiments were performed in triplicate.
Cell migration assay
LX-2 cells were seeded in a 6-well plate treated with siRNA transfection or drug for 24 h. Migration assays were conducted using 24-well Boyden chambers containing inserts (8 μm pores; BD Biosciences,USA). The lower chamber was filled with medium containing 10% serum, whereas the top chamber contained 1 × 105 cells without serum. The plates were incubated at 37 °C in 5% CO2 for 10 h. After migration, the cells that had migrated to the underside of the membrane were fixed with paraformaldehyde and stained with 0.1% crystal violet. Migrated cells on each insert were counted in five randomly selected fields and quantified using the ImageJ software.
Cell apoptosis assay
LX-2 cells were seeded in 6-well plates and treated with different concentrations of Ruxolitinib for 24 h. For cells were then harvested and stained with Annexin V-FITC Apoptosis Detection Kit (BD Pharmingen, CA) according to the manufacture’s protocol. FACS caliber flow cytometry (BD Biosciences, USA) was used to assess apoptotic rate. The sum of early and late apoptotic cells was measured.
All mice were housed in the Animal Facility at Southern Medical University under standard pathogen-free conditions, and were maintained at 16–27℃, 30–70% humidity and a 12-h light/dark cycle. All animal experiments were conducted in accordance with the National Institutes of Health guide for the care and use of Laboratory animals s and approved by the Animal Care and Use Committee of Southern Medical University. Each treatment group comprised 6–10 mice (N = 6–10).
Liver fibrosis progression and reversal model induced by CCl4
For the Liver fibrosis progression model, 4–6 weeks old male C57BL/6 mice (Vital River, Beijing, China) were treated three times a week with or without 0.1 ml of a 40% CCl4 in olive oil by oral gavage for 8 weeks and mice were treated with or without Ruxolitinib (30 mg/kg, oral gavage, each day) from 5 to 8 weeks. For Liver fibrosis reversal model, 4–6 weeks old male C57BL/6 mice (Vital River, Beijing, China) were treated three times a week with or without 0.1 ml of a 40% CCl4 in olive oil by oral gavage for 6 weeks, and then mice allowed to recover from 6 to 8 weeks, with or without treatment with Ruxoltinib (30 mg/kg, oral gavage, each day).
Panlobular liver fibrosis model induced by TAA
6–8 weeks old male C57BL/6 mice were accepted an optimistic dose-escalating TAA as described . And then mice were allowed to recover from 6 to 10 weeks, with or without treatment with Ruxolitinib (30 mg/kg, oral gavage, each day).
All statistical analyses were performed with GraphPad Prism V.5.00. Data are expressed as mean ± standard deviation (SD). Differences between two groups were compared using a two-tailed unpaired Student’s t-test. One-way ANOVA was used for sample comparison among multiple groups. Statistical significance was defined as P < 0.05.