hFSSCs and human umbilical cord mesenchymal stem cells (hUCMSCs) were provided and isolated by following our previous study. 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. Briefly, cells were cultured and reached 70 ~ 80% confluence, placed in serum-free medium (SFM; Invitrogen, Shanghai, China), and incubated in 5% CO2 in a humidified condition. After cultured 24 h, the conditioned medium (CM) was collected and centrifuged to purify for 10 min at 4 °C, 4000 g. Next, 10 ml conditioned medium was re-centrifuged with Amicon Ultra Centrifugal Filters (Millipore Corp, Billerica, MA, USA) at 4 °C, 4000 g, 2 h. At last, 300 ~ 500 µl supernatant solution was collected as cell-free secretome each time. The protein content was measured using the BCA kit (Thermo Scientific, Rockford, IL, USA) according to the manufacturer’s instruction.
Ccl-induced Liver Fibrosis In Rats
Liver fibrosis was induced in Sprague Dawley (SD) rats (8-week old, female, 200 g). All protocols and procedures were approved by the Animal Experiment Ethics Committee of Changchun University of Traditional Chinese Medicine (Approval NO. XW201903167). Detailed procedures for the CCl4-induced liver fibrosis rat model were described in our previous published studies. Briefly, rats were administered with an intraperitoneal injection of 30% CCl4, 3 ml/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 (1 ml)/weekly): PBS group (receive sterile PBS); hUCMSC secretome group (receive 250 µg of hUCMSC secretome); hFSSC secretome group (receive hFSSC secretome of 250 µg). After 4 weeks, rats were sacrificed, liver tissue and serum were collected. Livers were divided into two parts. one part were preserved in 10% formalin (Histology studies) and other part flash frozen and kept in at -80 °C (biochemical studies).
Liver tissues were processed for paraffin embedding by slicing into 4 µm sections. Liver sections were stained with hematoxylin & eosin (H&E) and Masson and Sirius red according to standard protocols. Immunohistochemistry (IHC) was stained 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 100 °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), α-SMA (ab5694, 1:500 dilution, Abcam, Cambridge, UK), and HNF-4α(ab219610, 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 selected the liver section fields randomly (used 10 random fields) to analyze the intensity liver fibrosis. We used 10 sections in total (n = 10 rats) for quantification of IHC results. The percentage of collagen stained area was calculated via Image-Pro Plus software.
Immunofluorescence (if) Staining
When HSCs reached 60 ~ 70% confluence on 24-well plates, they were cultured with with either PBS, hUCMSC secretome, or hFFSC secretome (5 ng/ml) for 48 h. 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 1 h, 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.).
To determine the liver function, we processed serum samples to measure following markers, 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)
HSCs were co-cultured with either PBS, hUCMSC secretome, or hFFSC secretome (5 ng/ml) for 48 h before samples were collected for mRNA extraction. Total RNA was isolated from HSCs using Trizol reagent (Invitrogen, Shanghai, China) according to the manufacturer’s protocol. Then, 1 µg 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 levels of EMT related genes (E-cadherin, Snail1, Vimentin, FSP1 and α-SMA), TGF-β/Smad signaling pathway-related genes (TGF-β1, Smad2, Smad3, Smad7 and Collagen I), and the internal standard GAPDH mRNA were measured by qRT-PCR. 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.
HSCs were co-cultured with either PBS, hUCMSC secretome, or hFSSC secretome (5 µg/ml) for 48 h before samples were collected for protein extraction. Protein samples were mixed with SDS sample lysis 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 TGF-β1(ab92486), Smad2(ab40855), Smad3(ab40854), Smad7(ab216428), Collagen I (ab90395) andβ-actin(ab5694), (1:1000 dilution, Abcam, Cambridge, UK) 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 2 h, and visualized by chemiluminescent detection according to the manufacturer’s instructions (Immobilon western chemiluminescent HRP substrate, Millipore, Massachusetts, U.S.).
Statistical analysis was performed using GraphPad Prism Version 6. One-way ANOVA with post-hoc Dunnett’s multiple comparisons test was used to test for statistically significant differences among the groups. All quantitative data are expressed as mean ± SD. We performed at least three independent experiments, and p < 0.05 was considered to be statistically significant.