All animal procedures in this study were conducted in accordance with the committee guidelines of Shenzhen People’s Hospital for animal experiments, which met the NIH guidelines for the care and use of laboratory animals (NIH Pub. No. 85–23, revised 1996).
hBMSCs were purchased from American Type Culture Collection (ATCC, Manassas, USA). mBMSCs were obtained from Cyagen Biosciences Company (Guangzhou, China). These cells were cultured with a Mesenchymal Stem Cell Growth Kit for Bone Marrow-derived MSCs (ATCC PCS-500-041) (supplemented with rh FGF basic: 125 pg/mL, rh IGF-1: 15 ng/mL, 7% foetal bovine serum (FBS) and L-Alanyl-L-Glutamine: 2.4 mM.) Hypoxic preconditioning was induced in an oxygen control incubator (Smartor 118, Zhejiang, China) filled with 5% CO2 and 90~94% N2. The oxygen concentrations and hypoxic incubation times were indicated as shown. Cells cultured under normoxic conditions (21% O2) served as a control.
The human keratinocyte (HaCaT) cell line was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) (originally from American Type Culture Collection (Manassas, USA)) and were cultured in Minimum Essential Medium (MEM; Procell Life Science & Technology, PM150410) containing 10% FBS (Gibco, A3160802) and 1% penicillin and streptomycin (Gibco). HaCaT cells were treated with high glucose (HG) (25 mM glucose) and 1% O2 hypoxia.
Preparation of CM
To collect CM from hypoxia-preconditioned BMSCs, 3×105 BMSCs were seeded into 10-cm dishes and grown to 80% confluence. The culture medium was then replaced with DMEM containing 0.1% FBS, and the cells were cultured under hypoxic or normoxic conditions. Then, the media was collected.
Western blot analysis
Cells were harvested and washed twice with ice-cold PBS and lysed with protein extraction agent (Beyotime, Beijing, China). Proteins (25–50 μg) were loaded and run on a 6–10% SDS/PAGE gel and then transferred onto PVDF membranes (Millipore, Billerica, MA). After PVDF membranes were blocked at room temperature for 1 h, the membranes were incubated with primary antibodies overnight at 4 °C. The primary antibodies used in the study included：HIF-1α (1:5000, Proteintech, 20960-1-AP), TGF-β1 (1:1000, Abcam, ab215715), TGF-β2 (1:1000, Abcam, ab36495), TGF-β3 (1:1000, Abcam, ab15537), ATG5 (1:1000, HUABIO, ET1611-38), ATG7 (1:1000, HUABIO, ET1610-53), LC3B (1:1000, HUABIO, ET1701-65), BECN1 (1:1000, HUABIO, R1509-1), SQSTM1 (1:500, HUABIO, R1309-8), SMAD2 (1:1000, CST, #5339), phospho-SMAD2 (1:1000, CST, #18338), SMAD4 (1:1000, CST, #8685), mTOR (1:1000, CST, #2983), p-mTOR(ser2448) (1:1000, CST, #5536), ERK1/2 (1:1000, CST, #4695), p-ERK1/2 (1:2000, CST, #4370), β-Actin (#3700s, 1:1000, CST), GAPDH (1:1000, CST, #5174S), and Lamin B1 (1:1000, CST, #13435S). After that, the membranes were incubated with secondary antibodies for 1 h at room temperature. The protein bands were visualized using Millipore’s enhanced chemiluminescence (ECL) system and detected using MultiImage Light Cabinet Filter Positions (Alpha Innotech, San Leandro, CA, USA). The intensity of each band was analyzed by ImageJ software (NIH, Bethesda, MD, USA).
Immunofluorescence (IF) staining
After being treated, the cells on glass coverslips were fixed in 4% paraformaldehyde for 30 min, and mouse skin was fixed in 4% paraformaldehyde for 2 days and then resected for frozen sectioning. The samples were washed three times with PBS, blocked with 10% goat serum for 1 h, nested, incubated with primary antibodies at 4 °C overnight and washed three times with phosphate-buffered saline (PBS). Next, the samples were stained with fluorescent secondary antibodies for 1 h at 37 °C. The following primary antibodies were used: HIF-1α (1:200, Proteintech, 20960-1-AP), ATG5 (1:200, HUABIO, ET1611-38), ATG7 (1:200, HUABIO, ET1610-53), K14 (1:200, Proteintech, 60320-1-Ig), and SMAD4 (1:200, CST, #8685). Nuclei were then counterstained with DAPI (Abcam) before imaging. IF images were captured using fluorescence microscopy and confocal microscopy (Leica Microsystems, Germany).
Cell viability assay
Cell viability was determined after 12, 24, 36, 48 and 72 hours of incubation with different media at 37 °C by a Cell Titer 96® Aqueous One Solution Cell Proliferation Assay (MTS) from Promega (Madison, WI, USA), a colorimetric method for determinating the number of viable cells in proliferation assays. All experiments were repeated in triplicate.
CM from normoxic and hypoxic BMSCs were collected and subjected to ELISA analysis to determine the TGF-β1, TGF-β2, and TGF-β3 contents. ELISA kits from R&D Systems (Minneapolis, MN, USA) were used according to the manufacturer’s protocols. Concentrations were normalized to the total protein content.
Ethynyl-2’-deoxyuridine (EdU) assay
Cell proliferation was also measured using EdU assay kit EdU assay kit (RiboBio, C10310-1) according to the manufacturer’s instructions. Briefly, cells were seeded into 24-well plates at a density of 5.0 × 104 cells/well and cultured for 24 h before the administration of Edu (50 mM). Then, Apollo and DNA stains were added. Finally, proliferation images were acquired and analysed by fluorescence microscopy (Leica Microsystems, Germany).
We utilized a scratch assay to measure HaCaT cell migration. HaCaT cells were plated on 6-well plates. Cell monolayers were scratched with 200 µl plastic pipets. At 24 hours after wounding, cells that migrated into the cell-free area were monitored with an inverted light microscope (Olympus, Japan). Cell migration was assessed by the residual wound rate: (residual scratch width / original scratch width) × 100%.
On the day prior to transfection, cells were plated to the required cell density (at least 70% confluence). Small interfering RNAs (siRNAs) specific for ATG5, ATG7, TGF-β1, SMAD2, HIF-1α and the corresponding scrambled siRNA (siNC) were diluted in Opti-MEM (Life Technologies) and incubated for 5 min at room temperature. The diluted siRNAs were added to diluted Lipofectamine 2000 (Invitrogen, USA) and further incubated for 20 min. The complex was added according to the manufacturer's protocol.
Quantitative real-time reverse transcription PCR (qRT-PCR)
AG RNAex Pro Reagent (AG21102, Accurate Biotechnology, Hunan) was used to extract total RNA from cells. Complementary DNA (cDNA) was synthesized using the PrimeScript RT reagent kit (RR037A, Takara, Japan) and real-time PCR was performed with TB Green Premix Ex Taq (RR420A, Takara, Japan) on a StepOnePlus quantitative PCR system (Applied Biosystems, USA). Expression levels were normalized to the internal control (GAPDH) and the relative expression levels were evaluated using the comparative 2-ΔΔCT method. The specific primers for ATG5, ATG7 and GAPDH were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). The primer sequences are listed in Supplementary Fig. S1.
Full-thickness cutaneous wound model
A total of thirty-six 8-week-old male type-2 diabetic mice (db/db) were acquired from Shanghai Slac Labortory Animal Co., Ltd. (Shanghai, China). The full-thickness cutaneous wound model was established as previously described. Briefly, the mice were anaesthetized by 5% pelltobarbitalum natricum (25 mg/kg) and Sumianxin (0.1 ml/kg), and an 8-mm diameter wound was created by a punch biopsy instrument with moderate force on the back of the mouse. Next, the middle of the outlined region of skin was sharply excised along the outline with a pair of scissors. The excised tissues were full-thickness in depth, leaving subcutaneous dorsal muscle exposed after the excision. Before being injected, mBMSCs were transfected with TGF-β1 siRNA or negative control siRNA, 2 × 106 cells were suspended in 4 ml of PBS, and then the cells were intradermally injected around each wound. PBS (4 ml) was used as a control.
Haematoxylin and eosin (H&E)
For histological analysis, the excised skin samples were fixed in 4% paraformaldehyde for 24 h, embedded in paraffin, and then stained with H&E (Sigma, Poole, UK). The stained sections were scanned for digital imaging by light microscopy (Olympus BX51, Olympus, Japan).
All experiments were performed at least in triplicate to be eligible for the indicated statistical analysis. Statistical comparisons were then performed with GraphPad Prism 9.0 (GraphPad Software). All data are presented as the mean ± SD. The Shapiro-Wilk test was used to check whether the data were normally distributed. Unpaired t test was used to examine the difference if the data satisfied the normality requirement, otherwise, Wilcoxon test was used. Statistical significance among three or more groups was assessed by one-way analysis of variance (ANOVA), and differences between two or more groups at different time points were estimated using two-way ANOVA. The Bonferroni method was used for multiple-group comparisons after ANOVA. A two-sided P value of less than 0.05 was considered statistical significance (*P < 0.05, **P < 0.01).