2.1. Isolation and identification of HuMSCs
This study was approved by the Institutional Review Board and Human Ethics Committee of Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China. All umbilical cords used in this study were obtained from healthy full-term cesarean section fetuse at Renji Hospital affiliated to Shanghai Jiao Tong University School of Medicine. Written consent for the use of samples for research purposes was obtained from all patients. HuMSCs were treated as previously described [14]. HuMSCs differentiated into chondrogenic (Cyagen Biosciences, Guangzhou, China), osteogenic (Cyagen Biosciences, Guangzhou, China), and adipogenic lineages (Cyagen Biosciences, Guangzhou, China), as indicated by positive type II collagen (Abcam, Cambridge, MA, USA, Cat. #ab34712), Alizarin red, and Oil Red O staining, respectively, after induced with a conditioned medium for 2-3 weeks. The expressioin patterns of CD73, CD90, CD105, CD34, CD45, major histocompatibility complex, class II, DR (HLA-DR) were detected by flow cytometry to evaluate the phenotypes of HuMSCs.
2.2. Establishment and identification of HuMSC-HNF4α
HuMSCs with stable overexpression of HNF4α, called HuMSC-HNF4α, were constructed as previously described [14]. HuMSCs transfected with a lentiviral vector containing only green fluorescent protein (HuMSC-CON) were treated as controls. The lentiviral transduction efficiency was detected by Western bot and Real-time quantitative reverse transcription–PCR.
2.3. Flow cytometry
Flow cytometry (BD Sciences) was performed to characterize the phenotypes of HuMSCs and macrophages. Antibodies against the human antigens CD11b (Cat. #562721), CD68 (Cat. #565594), CD80 (Cat. #561134), CD206 (Cat. #550889), CD73 (Cat. #562430), CD90 (Cat. #563070), CD105 (Cat. #563803), CD34 (Cat. #562383), CD45 (Cat. #563792), and HLA-DR (Cat. #562804) were purchased from BD Sciences, San Diego, CA, USA. Further, antibodies against the mouse antigens CD86 (BD Sciences, San Diego, CA, USA, Cat. #553690) and CD206 (BD Sciences, San Diego, CA, USA, Cat. #565250) were used to observe the effect of in vivo on macrophages phenotype. The data were analyzed using the BD CellQuest Pro software (V1.0.2).
2.4. Animal
Wild-type mice (C57BL/6J) were purchased from Shanghai Slark Experimental Animal Co., Ltd., and managed by the Animal Experimental Center of Renji Hospital, Shanghai Jiaotong University (Specific Pathogen Free grade). The experimental procedure was approved by the Animal Ethics Committee of Renji Hospital, Shanghai Jiao Tong University School of Medicine. Further, 2 × 106 HuMSC-HNF4α and HuMSC-CON were added to phosphate-buffered saline (PBS) solution. Mice aged 6 weeks were intraperitoneally injected with HuMSC-HNF4α cells, HuMSC-CON, or PBS (10 mL/kg). D-GalN (700 mg/kg, Sigma-Aldrich, St. Louis, MO, Cat. #G0500) and LPS (10 μg/kg, Sigma-Aldrich, St. Louis, MO, Cat. #L2630) were administered via the abdominal cavity after 24 h of pretreatment. The survival status of mice was observed and recorded 4 h after LPS/D-GalN injection for 48 h. It was recorded every half hour from 4 h to 12 h and every 6 hours between 12 h and 48 h. The mice were anesthetized with 1% sodium 3 h after intraperitoneal injection of LPS/D-GalN. Blood and livers were collected subsequently. The blood samples were centrifuged at 5000 rpm for 15 min to collect plasma. The plasma and liver tissues were frozen immediately in liquid nitrogen and stored at −80°C until analysis.
2.5. Real-time quantitative reverse transcription-PCR
Total RNA was isolated from cultured HuMSCs using TRIzol reagent (Invitrogen Inc, CA, USA) and reverse-transcribed into cDNA using a reverse transcription kit (Takara Bio USA, CA, USA). The PCR reaction was carried out in 20 μL of a final volume containing 0.1 μM of each forward and reverse primer, cDNA and 10 μL of SYBR Green PCR Master Mix (Takara Bio USA, CA, USA). DNA was amplified and analyzed using a StepOnePlus real-time PCR machine (Thermo‐Fisher Scientific, Inc., Rochford, IL, USA).
2.6. Western blot analysis
To determine the expression of HNF4α, total protein was lysed with radio immunoprecipitation assay (RIPA) peptide lysis buffer (Beyotime Biotechnology, Jiangsu, China, Cat. #P0013B) containing 1% protease inhibitors (Thermo Fisher Scientific, Waltham, USA , Cat. # A32953 ). Western blot analysis were performed according to anti-β-actin (Santa Cruz, CA, USA, Cat. #sc-8432) and anti-HNF4α (Santa Cruz, CA, USA, Cat. #sc-374229).
2.7. Detection of serological indicators
The enzyme activities of aspartate aminotransferase (AST) and alanine transaminase (ALT) in plasma were ditected with assay kits (Nanjing Jiancheng Bioengineering Institute, China). Enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN) were used to evaluate TNF-α and IL-1β levels in plasma. HuMSC-CON and HuMSC-HNF4α were seeded in six-well plates at a density of 5 × 105/well. When the cells reached 70% confluence, the remaining supernatant was washed with PBS. Fetal bovine serum (FBS)-free F12 (Gibco, MA, USA ) was used to continue culture routinely. After 48 h, the supernatant was collected into 15 mL centrifuge tubes and tested with ELISA kits to assess the levels of various immune factor indicators such as IL-10 and macrophage colony stimulating factor (M-CSF).
2.8. Histopathological examination
The tissue samples were cut into appropriate size and fixed with 4% paraformaldehyde for hematoxylin-eosin staining (H&E) and immunohistochemical staining. Antibodies used for immunohistochemical analysis were used as follows: F4/80 (Abcam, Cambridge, UK, Cat. #ab6640), MPO (Abcam, Cambridge, UK, Cat. #ab208670), TNF-α (Abcam, Cambridge, UK, Cat. #ab9579). Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay (TUNEL) was performed for apoptosis.
2.9. THP-1 cell culture and differentiation into macrophages
The 1640 complete medium (Gibco, MA, USA) with 1% double antibody and 10% FBS (Gibco, MA, USA) were used. THP-1 cells with ideal growth activity were seeded in six-well plates at a density of 1×106/well, and 100ng/mL phorbol 12-myristate 13-acetate (PMA, Sigma, MA,USA, Cat. #P1585) was added to the medium to induce differentiation for 24h. Then, the cells were cultured for 4 days, and the morphology of cells was observed under a light microscope. The induced macrophages were collected, and the surface antigens CD11b and CD68 were detected by flow cytometry.
THP-1 cells were induced to differentiate into macrophages and cultured in six-well plates. A 0.4-μm Transwell chamber (Corning, NY) was placed on a six-well plate. Further, 5×105 HuMSC-HNF4α were seeded into the chamber (control group with HuMSC-CON and negative control group without cells) and co-cultured with macrophages in 1640 medium for 24 h to construct co-culture systems. The MTT assay was used to detect the viability of macrophages. The Transwell chamber was discarded after collecting cells of the three groups. The mixed cells were cultured and stimulated in 1640 medium containing LPS (1 µg/mL) for 6 h. The negative control group cultured for another 6 h. The cell culture medium of macrophages in the three groups was collected. Immune factors secreted by macrophages, such as TNF-α and IL-1β, were detected using ELISA kits. The expression patterns of macrophage CD80 and CD206 were detected by flow cytometry.
2.10. Statistical analysis
The data were expressed as mean ± standard deviation (mean ± SD). SPSS 19.0 statistical software was used for data analysis. The comparison of indicators depended on a single-factor analysis of variance. The t-test was used for comparison between groups. A P value < 0.05 indicated a statistically difference .