Cell culture
hFSSCs and human umbilical cord mesenchymal stem cell (hUCMSCs) were provided and extracted by our previous study[13]. HSCs were purchased from the Chinese Academy of Medical Sciences, China. In brief, hFSSCs, hUCMSCs and HSCs were cultured in DMEM (Gibco, Grand island, U.S.) supplemented with 500 U/ml penicillin and 500 μg/ml streptomycin (Invitrogen, Shanghai, China), and 10% FBS (Gibco, Grand island, U.S.) at 37°C, with saturated humidity and 5% CO2. hFSSCs and hUCMSCs at the P5 were used for this study, and hFSSCs and hUCMSCs) secretome was collected as reported in our previous study[13].
CCl4-induced liver fibrosis in rats
Liver fibrosis was induced in Sprague Dawley (SD) rats (8-week old, female, 200g). All protocols and procedures were approved by the Animal Experiment Ethic Committee of Changchun University of Traditional Chinese Medicine (Approval NO. XW201903167). Detailed procedures for CCl4-induced have been described in our published studies[6]. Briefly, rats were administered with an intraperitoneal injection of 30% CCl4, 3ml/kg body weight twice weekly in olive oil. After eight weeks, CCl4 treated rats were randomly assigned into three groups (n=10 rats, tail vein injection/weekly): PBS group (1ml); hUCMSC secretome group (250μg, 1ml); hFSSC secretome group (250μg, 1ml). After 4 weeks, liver tissue and serum were collected. Livers were divided into two parts of preservation in 10% formalin and freezing at -80 °C.
Histopathological analysis
Liver tissues were processed for paraffin embedding by slicing into 4μm sections. Liver sections were stained with hematoxylin & eosin (H&E) and Masson according to standard protocols. We selected the liver section fields randomly to analyze the liver fibrosis. The percentage of collagen stained area was calculated via Image-Pro Plus. Immunohistochemistry (IHC) was measured with the Kit (Maixin KIT-9710, Fuzhou, China) in accordance with the manufacturer’s instructions. In brief, the liver sections were deparaffinized, rehydrated, and incubated in a 99 °C water bath for 15 minutes. Then, the slide was incubated with 3% H2O2 for 15 minutes, and blocked with 10% normal goat serum for 1 h at 37 °C. Following with the incubation of primary antibody against PCNA (ab15497, 1:500 dilution, Abcam, Cambridge, UK) and α-SMA (ab5694, 1:500 dilution, Abcam, Cambridge, UK) overnight at 4 °C. Next, slides were incubated with biotinylated goat-anti-rabbit IgG antibody. Add diaminobenzidine solution for 15 minutes at 37 °C, then incubated with avidin peroxidase reagent, and hematoxylin for counterstaining. Lastly, slides were photographed using an optical microscope (Olympus, Tokyo Metropolitan, Japan). We used 10 random fields per section and 10 sections in total (n=10 rats) for quantification of IHC results. The IHC results were calculated via Image-Pro Plus.
Biochemical analysis
The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein (TP), total bilirubin (TBIL), alkaline phosphatase (ALP), gamma glutamyl transpeptidase (γ-GT) were assessed using the Automated Biochemical Analyzer (AU-680, Beckman, California, U.S.) according to the procedure. Liver homogenate (10%, w/v) was prepared by homogenizing the right lobe of liver on ice in 150 mM Tris-HCl buffered saline (pH 7.2) using a polytron homogenizer (PT3100D; Kinematical, Lucerne, Switzerland). The levels of Malondialdehyde (MDA) and Hydroxyproline (Hyp) in liver tissue were measured using kits (NanJing JianCheng Bio., Nanjing, China) according to the manufacturer’s instructions.
Quantitative real-time PCR (qRT-PCR)
Total RNA was isolated from liver tissue using Trizol reagent (Invitrgen, Shanghai, China) according to the manufacturer’s protocol. Then, 1μg total RNA was reverse-transcribed to give cDNA, which was used as the template, and combined with standard SYBR premix Ex Taq (Invitrogen, Shanghai, China) on the Real-Time PCR Detection System (Roche, Basel, Switzerland), and experiments were conducted in triplicate. The primers are listed in Table S1, and GAPDH served as the internal control. All reactions were performed in triplicate and the data were analyzed using the 2-ΔΔCt method.
Immunofluorescence (IF) staining
When HSCs reached 60~70% confluence on 24-well plates, they were cultured with hBM-MSCs-Ex (5ng/ml) for 48h. Next, HSCs were incubated with 4% paraformaldehyde at room temperature for 10 minutes, and then incubated with 1% bovine serum albumin (BSA, Biosharp, Wuhan, China) for 30 minutes. Cells were incubated with a primary antibody against α-SMA (ab5694, 1:100 dilution, Abcam, Cambridge, UK) for 1h, followed by incubation with a secondary antibody (goat anti-rabbit IgG, ab15007, 1:500 dilution, Abcam, Cambridge, UK) for 30 minutes at room temperature. Rhodamine phalloidin (Thermal Scientific, Waltham, U.S.) was stained for cytoskeleton. The nuclei were labeled with DAPI (Thermal Scientific, Waltham, U.S.). Fluorescent images were captured using an EVOS Cell Imaging System (Thermo Scientific, Waltham, U.S.).
Western blotting
HSCs were co-cultured with either SFM, hBM-MSCs, hBM-MSCs-Ex (5ng/ml) for 48h before samples were collected for protein extraction. Liver tissue was collected from each treatment group (liver fibrosis, hBM-MSCs, and hBM-MSCs-Ex group) for protein extraction. Protein samples were mixed with SDS sample buffer and heated to 95 °C for 10 minutes, followed by separation on SDS-polyacrylamide gels. Resolved proteins were electro-blotted onto nitrocellulose membrane and probed with antibodies against PPARγ (ab 23673), Wnt3a (ab 248472), Wnt10b (ab70816), β-catenin (ab32572), WISP1 (ab50041), Cyclin D1 (ab16663), α-SMA (ab5694), Collagen I (ab138492) and GAPDH (ab 8245) overnight at 4℃ (1:1000 dilution, Abcam, Cambridge,UK). Nitrocellulose membranes were then incubated with a secondary antibody, HRP-conjugated goat anti-rabbit IgG (ab15007), at room temperature for 2h, and visualized by chemiluminescent detection according to the manufacturer’s instructions (Immobilon western chemiluminescent HRP substrate, Millipore, Massachusetts, U.S.).
Statistical analysis
Statistical analysis was performed using GraphPad Prism Version 6. One-way ANOVA with Dunnett’s multiple comparisons test was used to test for statistically significant differences. All quantitative data are expressed as mean ± SD. p < 0.05 was considered to be statistically significant.