Wistar rats weighing 120–150 g were obtained from the Experimental Animal Research Center of Hubei Province (Wuhan, China). Animal care and all experimental procedures were approved by ethical review by the Laboratory Animal Management and Use Committee of the Center for Disease Control of Hubei Province.
Preparation and flow cytometry phenotyping of hUC-MSCs
hUC-MSCs were provided by Shenzhen Beike Cell Engineering Research Institute. hUC-MSCs from Wharton’s jelly were isolated by a non-enzymatic method and culture-expanded as described in previous report. The infused hUC-MSCs were harvested at passage 4 and stained with trypan blue to evaluate the vitality using an automated cell counter. The vitality of final infused hUC-MSCs was ≥90%. The phenotypes of hUC-MSCs were analyzed by cytometry (Figure S1). hUC-MSCs were stained with antibodies specific for MSCs markers CD105 (BD, USA, 562408), CD90 (BD, USA, 555595), and CD73 (BD, USA, 550257) and hematopoietic cell markers CD45 (BD, USA, 555482) and CD34 (BD, USA, 550619), respectively.
Establishment of ACLI and ACLF models and hUC-MSC transplantation
Rats were intraperitoneally administered porcine serum (PS) (Solarbio, China, S9060) at a dose of 0.5 mL twice per week for 11 weeks to generate an immune liver fibrosis model. After 11 weeks, rats with immune liver fibrosis were intravenously injected with lipopolysaccharide (LPS) (Sigma-Aldrich, USA, L2880) at a dose of 50 μg/kg to generate an ACLI model. After 1 h, the rats were divided into three groups. In the hUC-MSC groups, the rats underwent intravenous tail vein transplantation of hUC-MSCs at a concentration of 2×106 cells/mL per rat (n = 12) or hUC-MSCs at a concentration of 4×106 cells/mL per rat (n = 12). In the control group, the rats received 1 mL of 0.9% sodium chloride (n = 12). Three rats in each group were sacrificed at 1, 2, 4, and 6 weeks after hUC-MSCs or 0.9% sodium chloride injection, and blood samples and liver tissues were collected for biochemical and histological investigation (Fig. 1a).
Rats were intraperitoneally administered 0.5 mL PS (Solarbio, China, S9060) twice per week for 11 weeks to generate an immune liver fibrosis model. After 11 weeks, rats with immune liver fibrosis were intravenously injected with LPS at a dose of 50 μg/kg. Thirty minutes later, D-galactosamine (D-GalN) (Sigma-Aldrich, USA, G0500) was intraperitoneally injected at a dose of 600 mg/kg to induce an ACLF model . After 1 h, the rats were divided into three groups. In the hUC-MSC groups, the rats underwent intravenous tail vein transplantation of hUC-MSCs at a concentration of 2×106 cells/mL per rat (n = 20) or 4×106 cells/mL per rat (n = 20). In the control group, control rats received 1 mL of 0.9% sodium chloride (n = 20). Three to four rats in each group were sacrificed at 4, 12, and 24 h after hUC-MSC injection, and blood samples and liver tissues were collected for biochemical and histological investigation (Fig. 1b).
Determination of blood biochemical indices
The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBiL), direct bilirubin (DBiL), alkaline phosphatase (ALP), and albumin (ALB), and plasma level of ammonia were detected with an automatic analyzer (Rayto, China).
Determination of coagulation function
The plasma prothrombin time (PT) and international normalized ratio (INR) were detected with an automatic analyzer (ACL TOP700, Spain).
Liver tissues were fixed in 4% paraformaldehyde solution. The liver tissue samples were processed using the paraffin block technique in wax, deparaffinization in xylene, and dehydration in alcohol. The samples were then sectioned (5 μm) and stained with hematoxylin-eosin (HE) to evaluate the pathological changes and Masson staining to demonstrate the collagen deposition that had occurred in the liver. The sections were then examined and photographed by light microscopy (Olympus, Japan, 200×).
Immunohistochemistry staining was conducted using a previously described method . The following primary antibodies were used: rabbit anti-alpha smooth muscle actin (α-SMA) mAb (1:200, Abcam, UK, ab32575), rabbit anti-desmin pAb (1:200, Abcam, UK, ab15200), rabbit anti-matrix metalloproteinase 9 (MMP9) mAb (1:1000, Abcam, UK, ab76003), rabbit anti-CD90 mAb (1:50, Abcam, UK, ab133350), rabbit anti-tissue inhibitor of metalloproteinase 1 (TIMP1) pAb (1:100, Proteintech, China, 16644-1-AP), rabbit anti-cytokeratin 18 (CK18) pAb (1:200, Proteintech, China, 10830-1-AP), rabbit anti-alpha fetoprotein (AFP) pAb (1:100, Affinity, USA, AF5134), rabbit anti-HGF pAb (1:100, Affinity, USA, DF6326), rabbit anti-proliferating cell nuclear antigen (PCNA) pAb (1:100, Affinity, USA, AF0239), rabbit anti-Phospho-Stat1 mAb (1:100, Cell Signaling Technology, USA, 8826S), and rabbit anti-Phospho-Stat3 mAb (1:100, Cell Signaling Technology, USA, 9145S). Goat anti-rabbit polyclonal antibody (1:200, Affinity, USA, S0001) was used as the secondary antibody. Three areas are randomly selected for each slice under high magnification to take pictures. ImageJ software was used to calculate the area of positive cells and the total area of the image. The area of positive staining (percent) = (area of positive cells/total area of the image) × 100%.
Detection of fibrotic markers and cytokines
The serum levels of hyaluronic acid (HA) and N-procollagen type III peptide (PIIINP) were detected by ELISA kits (CUSABIO, China). The serum levels of TNF-α, IFN-γ, IL-6, interleukin-1β (IL-1β), transforming growth factor-β1 (TGF-β1), interleukin-4 (IL-4), IL-10, and HGF were detected by ELISA kits (RayBiotech, China).
Quantitative real-time PCR analysis
The total RNA was isolated from liver tissues with TRIzol regent (Invitrogen, USA, 15596026) according to the manufacturer’s instructions. Approximately 1 μg of total RNA from each sample was used to synthesize cDNA using the HiScript II Q RT SuperMix for qPCR (Vazyme, China, R223-01) according to the manufacturer’s specification. Then, quantitative real-time PCR was performed by using SYBR Green Master Mix (Vazyme, China, Q111-02), with 15 s at 95°C and 60 s at 60°C for 40 cycles. GAPDH was used as the reference gene for calculations. The 2-△△Ct method was used to analyze the real-time PCR data. The primers were provided in Table S1.
Protein was extracted from liver tissues with RIPA lysis buffer (beyotime, China, P0013B) containing protease inhibitor (Roche, Germany, 4693116001) and phosphatase inhibitor (beyotime, China, S1873). Equal protein extracts were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred onto polyvinylidene fluoride membranes. The membranes were incubated sequentially with appropriate primary antibodies and secondary antibodies. Immune complexes were visualized by ECL detection reagent (Applygen, China, P1050), and band intensities were determined using the BandScan software. Primary antibodies used in this study were as follows: rabbit anti-GAPDH mAb (1:1000, Cell Signaling Technology, USA, 5174S), mouse anti-Stat3 mAb (1:1000, Cell Signaling Technology, USA, 9139S), rabbit anti-Phospho-Stat3 mAb (1:2000, Cell Signaling Technology, USA, 9145S), rabbit anti-Stat1 mAb (1:1000, Cell Signaling Technology, USA, 14994S), rabbit anti-Phospho-Stat1 mAb (1:1000, Cell Signaling Technology, USA, 8826S), rabbit anti-CyclinD1 mAb (1:1000, Cell Signaling Technology, USA, 55506S), rabbit anti-c-Myc mAb (1:1000, Cell Signaling Technology, USA, 18583S), and mouse anti-Bcl2 mAb (1:200, Santa cruz, USA, sc-7382). Secondary antibodies used in this study were horseradish peroxidase-conjugated anti-mouse IgG (1:10000, Boster, China, BA1051) and anti-rabbit IgG (1:10000, Boster, China, BA1054).
All data are expressed as the mean ± standard deviation (SD). Comparisons between two groups were assessed by the Mann-Whitney U test. Kruskal-Wallis test with Dunn’s multiple comparisons post-test analysis was used to compare among three groups. P <0.05 was considered statistically significant.