Isolation and identification of AMSCs
C57BL/6J mice were purchased from Nanjing BioMedical Research Institute of Nanjing University. The experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee in our institution. Inguinal adipose tissues were obtained from male mice (8 weeks old as young mice, 20 months old as aged mice). Adipose tissues were digested with 0.075% collagenase type I (Sigma-Aldrich, St Louis, MO, USA) for 30 min, then centrifuged and washed with PBS for 2 times. Finally, the isolated cells were cultured in murine MesenCultTM Expansion Kit (Stemcell) containing 2 mM L-glutamine (Thermo Fisher Scientific) and 1% antibiotic-antimycotic. Cells were maintained and expanded by 2–10 passages before usage.
ALF mouse model and AMSCs treatment
C57BL/6J Mice (aged 5–6 weeks) were intraperitoneally injected with Lipopolysaccharide (LPS, 10 µg/kg, Sigma-Aldrich) and D-galactosamine (GalN, 400 mg/kg, Sigma-Aldrich) to establish a mouse model of ALF. AMSCs from young mice (yAMSC) or aged mice (oAMSC), P2, P6 or P10 AMSCs from a culture-induced senescent cell model, and miR-17 or miR-20a-modified oAMSCs (oAMSC-M17, oAMSC-M20a) or control miRNA-modified oAMSCs (oAMSC-M67) were administered via the tail vein (2×105, n = 6) immediately after LPS/GalN injection. The control group was administered with vehicle alone (n = 6). At 6 h after LPS/GalN injection, the mice were sacrificed, and serum and liver samples were collected. Serum was evaluated for biochemical parameters. The liver samples were evaluated for histochemistry and Western blot analysis.
Isolation and detection of miRNA
Total RNA enriched with miRNAs was isolated from yAMSCs, oAMSCs and different passaged AMSCs by using a miRVana miRNA isolation kit (Thermo Fisher) according to the manufacturer’s instructions. Complementary DNA was synthesized from the isolated miRNAs by using TaqMan™ mmu-miRNA-speciଁc primers including miR-17-19 cluster (Thermo Fisher) and TaqMan™ MicroRNA Reverse Transcription Kit (Thermo Fisher). Real-time PCR was then performed following the manufacturer’s instructions (Thermo Fisher) to examine the expression of miR-17-19 cluster, especially miR-17 and miR-20a. Data were normalized over the average cycle threshold (CT) value of U6, and the 2−ΔΔCT method was used to determine the relative miRNA expression.
RNA isolation and qPCR
Total RNA was isolated from AMSCs by using TRIzol. First-strand cDNA was subsequently synthesized followed by qPCR analysis by using ABI Prism 7900 (Applied Biosystems) to examine the expression levels of TNF-ɑ, IL-6, MCP1, PAI-1, Mmp3 and Mmp13, β-actin were used as internal controls. The 2−ΔΔCT method was used to determine the relative mRNA expression levels of these genes.
Liver histological and serum biochemical analysis
Liver tissues were processed for paraffin embedding and were sectioned into 4 µm sections. The sections were then routinely stained with hematoxylin and eosin (H&E staining). The serum levels of alanine aminotransferase aspartate (ALT) and aspartate aminotransferase (AST) were analyzed by standard analyzer DRICHEM 4000ie (FUJIFILM). Total bilirubin (TBiL) was measured using standard clinical chemistry techniques (Integra II; Roche, UK).
Cytokine detection by ELISA
The murine serum levels of the cytokines including TNF-α, IL-6, and MCP-1 (MultiSciences, Shanghai, China) were assayed by commercial ELISA kits following the manufacturer’s instructions.
SA-β-gal staining was used to determine cell senescence of AMSCs. Briefly, yAMSCs, oAMSCs and different passaged AMSCs were seeded at a density of 5 × 104/well in a 6-well plate with complete medium and incubated for 24 h at 37°C in 5% CO2. Afterwards, the cells were fixed with 4% paraformaldehyde (PFA, Sigma-Aldrich, USA) at room temperature for 20 min and subsequently stained by SA-β-gal staining kit (Beyotime, Shanghai, China) following the manufacturer’s instructions. Positive senescent cells stained in blue were observed using an inverted microscope (AxioCam ERc 5s, Carl Zeiss, Germany).
Western blot analysis
AMSCs were lysed in ice-cold RIPA buffer (Beyotime Biotechnology, Jiangsu, China) containing 1% protease inhibitors (Pierce) for 30 min at 4ºC. Lysates (25 µg of protein) were analyzed by 10% sodium-dodecyl sulfate-polyacrylamide gel electrophoresis (Sigma-Aldrich) and transferred to nitrocellulose membranes (Merck Millipore, Billerica, MA, USA) according to standard procedures. Primary antibodies were used as follows: p21 (1:1000, Abcam), p16 (1:2000, Abcam), TXNIP (1:2000, Abcam), TRX (1:1000, Cell Signaling Technology), c-Myc (1:1000, Abcam), and GAPDH (1:3000, HuaBio). Protein bands were developed using the enhanced chemiluminescence (ECL) system and were visualized using the ChemiScope Western Blot Imaging System (Clinx Science Instruments Co., Ltd). The gray value assay was performed by using Image J software (Rawak Software, Inc. Germany).
Determination of reactive oxygen species (ROS) generation
AMSCs were stained with 5 µM H2DCF-DA (Life Technologies) for 10 min and then washed twice with ice-cold PBS and ROS generation was determined by FACS analysis. ROS generation was quantified as the increase in mean fluorescence intensity (MFI).
Transfection of small interfering RNA (siRNA) and miRNA
SiRNA against c-Myc, TXNIP and scrambled siRNA were purchased from Guangzhou RiboBio Co., Ltd. and miR‑17 and miR-20a mimics, miRNA mimic negative control (M-Ctrl), miR‑17 and miR-20a inhibitors, and miRNA inhibitor negative control (I-Ctrl) were also purchased from RiboBio. RNAiMAX (Thermo Fisher Scientific) was used for siRNA and miRNA transfection. At 75% confluence, AMSCs were transfected with siRNA (100 nm) and/or miRNA (50 nM) at 37˚C. At 48 h after transfection, the cells were harvested for qPCR and Western blot analysis.
Thioredoxin (TRX) activity assay
TRX activity was measured by the Thioredoxin Fluorometric Activity Assay Kit (Cayman, Item No.500228) according to the manufacturer’s instruction. Briefly, 0.5-1 mg/ml of cell lysates were prepared in cold buffer (100 mM Tris-HCl pH 7.5, 1 mM EDTA), supplemented with protease inhibitor. Then 20µl cell lysates were mixed with 10 µl TRX reductase, 10 µl NADPH and 40 µl assay buffer. After the reaction was performed at 37°C for 30 min, 20µl reconstituted Eosin-Labeled Insulin was added. Measure the fluorescence at excitation and emission wavelength of 520 nm and 560 nm, once every minute for 60 min by Synergy Neo2 (BioTek).
Construction of miRNA-modified AMSCs
The miR-17 (LV-miR-17) and miR-20a (LV-miR-20a) expression vectors and the control vector cel-miR-67 (LV-cel-miR-67) were constructed by cloning their precursor sequences, together with a short upstream and a short downstream flanking region, into lentiviral expression vectors. Before infection, 1 × 106 AMSCs were seeded in 10 ml complete medium overnight and then transfected with 100 nM LV-miR-17, LV-miR-20a or LV-cel-miR-67 at a multiplicity of infection of 10 in the presence of polybrene (8 µg/ml; Sigma-Aldrich, USA) for 24 h. Stable transfectants were used in the subsequent experiments.
Statistical evaluation was performed using independent samples t-tests between two groups and using one-way analysis of variance with Tukey's post hoc test between three or more groups. All data are presented as the mean ± SD. P < 0.05 was considered significant.