Eighty-five adult male C57BL/6 mice (weight 18-20 g) were obtained from the Shanghai Slack Laboratory Animal Center (license no. SCXK hu 2017-0005). All animals were housed in standard laboratory barrier facilities at 22-25 ˚C with a standard 12/12 light-dark cycle and 55-60% relative humidity. The mice had ad libitum access to food and autoclaved water. All animal procedures were approved by the Animal Ethics Committee of Mengchao Hepatobiliary Hospital of Fujian Medical University and Fuzhou General Hospital (Fuzhou, China).
Isolation and culture of ADSCs
Isolation of ADSCs was performed according to our previous reports [2-4]. Briefly, subcutaneous fat tissues were collected from the groin area of male C57BL/6 mice (n=10), and then cut into about 0.1mm3 size and digested with 0.1% type I collagenase (Sigma-Aldrich, USA) in HBSS (Hyclone, USA) at 37 °C for 1 h. Afterwards, the digestive solutions were neutralized by α-MEM (Hyclone, USA) containing 20%FBS (Gibco, USA), filtered through a 100-μm cell strainer, followed by eliminating the red blood cells with osmotic lysates (Biyuntian Biological Co., Ltd., Shanghai, China). Finally, the cells were collected and seeded into T-75 flasks at a density of 1×106/mL with complete medium (α-MEM containing 10% FBS). Once the cell confluence reached about 90%, they were detached using 0.25% trypsin–EDTA (Gibco, USA) and passaged at a ratio of 1:3. The ADSCs from passage 3 were applied for further usage.
Antioxidants including GSH and melatonin were obtained from Aladdin Chemical (Shanghai, China). Each antioxidant was firstly dissolved in DMSO (Sigma-Aldrich, USA) to the concentration of 1mM. For antioxidant pretreatment, the ADSCs were cultured with complete medium supplemented with 10μM antioxidant since the first passage. All preconditioned cells were rinsed with PBS for three times to remove any residual antioxidant before further analysis.
For ADSC cell labeling, the ADSCs were re-suspended with 1μM CellTracker™ Cm-dil (Qcbio Science&Technologies Co., Ltd., Shanghai, China) at a destiny of 1×106/mL with complete medium at 37 °C for 3 minutes, followed by incubating at 4 °C for 9 minutes, and rinsed with PBS for three times, and finally collected for cell transplantation.
Liver fibrosis model establishment and ADSC engraftment examination
Liver fibrosis model was established by intraperitoneal injection of 20% carbon tetrachloride (CCl4) with a dose of 5mL/kg diluted in olive oil, 2 times per week, for 12 weeks. Following the development of liver fibrosis, which was verified by pathological assessment, the mice were divided into three groups: the control group (n=15), mice with tail vein injection of ADSCs (1×106 cells/mouse); GSH group (n=15), mice with tail vein injection of GSH-pretreated ADSCs (1×106 cells/mouse); melatonin group (n=15), mice with tail vein injection of melatonin-pretreated ADSCs (1×106 cells/mouse). After ADSC transplantation for 1 hour, 4 hours, 1 day, 3 days and 7 days, mice were sacrificed with 2% pentobarbital sodium (100mg/kg; Sigma-Aldrich). Then, the major organs including heart, liver, spleen, lung and kidney were collected, and the distribution of ADSCs in these tissues was observed by using the IVIS system (PerkinElmer, USA). Liver tissues were collected to prepare paraffin section to further observe the hepatic engraftment efficiency of ADSCs using LSM780 confocal microscope (Zeiss, Germany).
ADSC therapy for liver fibrosis
Twenty-four liver fibrosis mice were divided into four groups: the model group (n=6), mice with tail vein injection of the same dose of normal saline (0.2mL); the ADSC group (n=6), mice with tail vein injection of ADSCs (1×106 cells/mouse); the G-ADSC group (n=6), mice with tail vein injection of GSH-pretreated ADSCs (1×106 cells/mouse); the M-ADSC group (n=6), mice with tail vein injection of melatonin-pretreated ADSCs (1×106 cells/mouse). Each group was treated with normal saline or ADSC transplantation once/week, for 2 weeks; after the last ADSC transplantation for 2 weeks, mice were sacrificed with 2% pentobarbital sodium (100mg/kg; Sigma-Aldrich), and the liver tissues and sera were collected for further investigation (Fig.1a).
Biochemical assays of liver function
To determine whether antioxidant preconditioning could accelerate liver function recovery after ADSC transplantation, the serum level of aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin (TBIL) and albumin (ALB), as well as the hydroxyproline content in liver tissues were analyzed by biochemical analysis using colorimetric assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to manufacture instructions, respectively.
Liver tissues were collected and fixed in 4% paraformaldehyde for 24 hours at room temperature, followed by the gradual dehydration of ethanol and embedding in paraffin. Then, the paraffin embedded tissues were sectioned into slices. Afterwards, the tissue sections were stained by hematoxylin and eosin (H&E) for histological analysis. To further observe fibrotic changes in liver tissues, Masson’s trichrome staining was performed using a commercial kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s protocol. The histological examination was independently performed by two pathological experts using an ortho-microscope (Zeiss, Germany), and the fibrotic area was analyzed using the ZEN 2012 Light Edition imaging analysis system (Zeiss, Germany).
Cell viability assay
In order to clarify the mechanism of anti-ROS oxidative injury of antioxidant preconditioning on ADSCs, the typical H2O2-induced oxidative injury model was established as previously described  with slight modification. Briefly, antioxidant pretreated ADSCs (passage 3) were cultured in a 96-well micro-plate (Corning, USA) at a density of 1 × 104 cells/well with 150 μL complete medium, and incubated at 37 °C under 5% CO2 atmosphere for 24 hours. After that, the cultured medium of each well was replaced by fresh complete medium supplied with 300 μM H2O2, and the cells incubated with complete medium were used as the negative control. After incubation for 24 hours, the cell viability was assessed by using a CCK-8 cell proliferation kit (Dojindo Molecular Technologies, Tokyo, Japan) following the manufacture’s protocol. Finally, the absorbance of each well at 450nm was measured in a micro-plate reader (Spectra Max M5).
Cell apoptosis assay
To determine the protective effect of antioxidant preconditioning on ADSC cell apoptosis, antioxidant pretreated ADSCs (passage 3) were cultured in a 6-well plate at a density of 1×105 cells/well for 24 hours, followed by the incubation with 300 μM H2O2 in fresh complete medium for another 24 hours, and finally analyzed using an Annexin V-FITC apoptosis assay kit (Dojindo Molecular Technologies, Tokyo, Japan) in Flow cytometry (Becton Dickinson, USA).
Cell migration assay
Transwell migration assay was use to explore the effects of antioxidant preconditioning on cell migration of ADSCs. Briefly, antioxidant-preconditioned ADSCs were treated with 300 μM H2O2 in fresh complete medium for 24 hours, then the cells were collected; afterwards, 5×105 treated cells were seeded into the upper compartment of the Transwell units (Millipore, USA) in α-MEM containing 2% FBS, and the lower compartment was filled with complete medium. 24 hours later, the filters were fixed with 4% PFA at room temperature for 20 minutes; then, the filters were washed with PBS for three times, and stained with crystal violet at room temperature for 3 hours; afterwards, the unmigrated cells were removed and the migrated cells on the lower surface of Transwell units were observed using an inverted microscope (Zeiss, Germany) at a magnification of 200x. To further quantify the migration rate, we randomly selected five fields to count the number of migrated ADSCs.
Western blot analysis
Cells were lysed with RIPA lysis buffer (0.5 M Tris-HCl, 10 mM EDTA, 1.5 M NaCl, 10% NP-40, 2.5% deoxycholic acid, pH=7.4) and the protein concentration was quantified using a BCA assay kit (TransGen Biotech, Beijing, China). Afterwards, equal amounts of protein lysate were separated by 10% SDS-PAGE electrophoresis and transferred to nitrocellulose membranes (PALL, USA) in transfer buffer (96 mM glycine, 12 mM Tris base, pH 8.3, and 20% methanol). Then, the membranes were blocked for 2 hours in the TBST buffer with 5% BSA and subsequently incubated with the CXCR4, Bax, Bcl-2 and Cyclin-D1 (all from Wuhan Boster Biological Technology Co., Ltd, Wuhan, China) at 4 °C overnight. Next, the membranes were washed with TBST buffer for three times and incubated with an anti-rabbit-conjugated secondary antibody (1:8000, Santa Cruz Biotechnology) for 1 hour at room temperature. Finally, the protein expression levels were detected by an enhanced chemiluminescence system, and the quantitative analysis was performed using Image J software.
Data were expressed as the mean ± standard deviation (SD). All the statistical analyses were performed with GraphPad Prism version 7.0 (GraphPad Software, CA, USA). T test was used to assess the statistical analysis between the two groups. P<0.05 was considered as statistically significant.