Cell culture
Human embryonic skin fibroblasts CCC-ESF-1 (ESF) (generation 10, G10), human embryonic lung fibroblast CCC-HPF-1 (HPF) (G10), human immortal keratinocyte line (HaCaT) (G8) and human Umbilical Vein Endothelial Cells (HUVECs) (G3) were obtained from the National Infrastructure of Cell Line Resource Center, Beijing, China. Primary fibroblasts derived from newborn umbilical cords (Human Umbilical Cord Primary Fibroblasts (HUCPFs)) were donated by Beijing Obstetrics and Gynecology Hospital. Cells were routinely cultured at 5% CO2 and 37°C in DMEM medium (for ESF, HPF, HaCaT and HUVEC) (Gibco, NY, USA) supplemented with 10% fetal calf serum (Gibco, NY, USA) and 1% penicillin and streptomycin (Gibco, NY, USA).
Animal experiments
All animal studies were carried out according to protocols approved by the Committee on Ethics of Biomedicine Research, the Sixth Medical Center, PLAGH, China, and all procedures were conducted in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals (China). Male C57BL/6J mice (7 weeks old, 20–24 g) were purchased from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). Animals were maintained under standard conditions at 22°C to 25°C with a 12 h light-dark cycle and were fed a normal diet. Oral enrofloxacin (0.17 mg/mL) intake was administered in daily drinking water, and the antibiotic was prepared with chlorinate-free and acid-free water. For the NAC treatment group, 3.3 mg NAC (the ROS scavenger acetylcysteine; #S1623, Selleckchem, China) in 260 μL distilled water was injected in each mouse with 22 (± 2) g of body weight.
Full-thickness cutaneous wound healing model
The full-thickness cutaneous wound healing model was established according to the Murine Model of Wound Healing 91 with some modifications (Fig. 1A). Briefly, mice were anesthetized with tribromoethyl alcohol (20 mg/mL, 10 µL/g injection), and the hair was shaved and cleaned with 70% ethanol. We split the back skin with a ring of medical silica gel membrane (to prevent the wound closure due to loose skin of mice), and then seamed it well with medical thread and removed the center skin to create 10 mm full-thickness excisional dorsal skin wounds using a sterile scissor. Wound areas were calculated using ImageJ software, and the wound closure percentage was obtained as follows:
Wound closure percentage (%) =
$$\frac{\text{w}\text{o}\text{u}\text{n}\text{d} \text{a}\text{r}\text{e}\text{a} \text{o}\text{n} \text{d}\text{a}\text{y} 0-\text{w}\text{o}\text{u}\text{n}\text{d} \text{a}\text{r}\text{e}\text{a} \text{o}\text{n} \text{d}\text{a}\text{y} \text{n}}{\text{w}\text{o}\text{u}\text{n}\text{d} \text{a}\text{r}\text{e}\text{a} \text{o}\text{n} \text{d}\text{a}\text{y} 0} \times 100\%$$
H2 Chamber treatment
A transparent closed box (20 cm × 18 cm × 15 cm) was connected to a hydrogen generator (KLE-H7, Kelieng Biomedical Co. Ltd., Shenzhen, China) (Figure 1A), which produces 66% H2 and 33% O2 (V/V), 5% H2 (V/V) mixed with air, or 33% O2 (V/V) mixed with air. Hydrogen treatment was given immediately after establishment of the mouse cutaneous wound healing establishment, and then daily administration was given until the end of the experiment. Animals were placed in the box with the mixed air for 1 h each day. During this inhalation period, mice were awake and freely moving. Thermal trace GC ultra-gas chromatography (Thermo Fisher, MA, USA) was used to monitor the concentration of hydrogen gas in the closed box.
H2 rich medium preparation
H2 rich medium was produced by injecting gas using the same hydrogen generator used for H2 Chamber treatment (KLE-H7, Kelieng Biomedical Co. Ltd., Shenzhen, China) into DMEM medium (Gibco) for 30 minutes Hydrogen dissolved in reaction solution was detected with a needle-type H2 sensor (Unisense, Aarhus N, Denmark). To measure the H2 concentration in the reaction system, the sensor was inserted below the liquid surface.
Blood perfusion and blood oxygen detection
Blood perfusion at the wound site during the healing processes was detected by using a moorFLPI-2 (Moor Instruments Limited, UK) according to manufacturer instructions. Images were further selected from videos and analyzed by moorFLPIReview V50 software (Moor Instruments Limited, UK). Blood oxygen levels were tested by the moorVMS-OXY Tissue Oxygen Monitor (Moor Instruments Limited, UK) according to the instructions. Five sites (top, bottom, left, right and middle) of the wound area were chosen, and an average was calculated to create the oxygen kinetic curve.
In vitro tube formation assay
Tube formation assay was performed as described 92. Firstly, Matrigel was thawed at 4°C overnight to avoid bubble formation. Briefly, 15-well μ-Slides (ibidi, Germany) were coated with 10 μl of Matrigel, which was allowed to solidify at 37°C. HUVECs before generation 10 were harvested and the cell number and viability were determined by Trypan blue staining before seeding. A cell suspension was prepared in DMEM and 15,000 cells/well were seeded in 50 μl medium on top of the matrigel. HUVECs were seeded in conditioned medium for 6 h, and the enclosed networks of complete tubes were counted and photographed under an inverted microscope. The tubular loops of the cells were measured and calculated for each well.
Immunohistochemistry (IHC) and immunofluorescence (IF)
2.8.1 Tissue immunostaining:
Skin tissue at the wound site was harvested and fixed with formalin and embedded in paraffin blocks, and then 4 μm thick paraffin sections were mounted on glass slides for histological staining. IHC and IF of the paraffin-embedded tissue sections were performed as previously indicated 93. Briefly, sections were dewaxed and rehydrated by subsequent immersion in xylene, ethanol (100%, 95%, 70%, and 50%) and deionized H2O. Antigen was then retrieved in citrate buffer, and non-specific staining between the primary antibodies (Table S1A) and the tissue was blocked by incubation in 1% goat serum in PBS for 60 minutes at RT. The sections were incubated with the primary antibodies listed in the Table S8A at 37°C for 60 minutes for IHC or at 4°C overnight for IF. Further labeling with specific secondary antibodies for IHC or IF (Table S1B) was performed according to the manufacturer’s instructions. For IF, nuclei were stained with NucBlue Live cell stain (R37605, Invitrogen). DAB, hematoxylin and neutral balsam mounting reagent used in IHC processes were obtained from ZSGB-BIO (Beijing, China).
2.8.2 Cells immunofluorescence staining:
Cells suspensions in a 96 well plates at 10,000 cells/well were allowed to adhere for 24 h and then switched to full medium. Wells containing the seeded cells were washed with 1X PBS immediately at the end of the incubation times and fixed in 4% paraformaldehyde for 30 minutes. Subsequent to the fixation, the cells were permeabilized with 0.3% PBS-Tween for 15 minutes, washed, and then blocked with normal sheep serum blocking buffer (ZSGB-BIO, China) for at least 1 h at room temperature. Primary antibodies (Table S1A) were added at the dilution recommended by the manufacturers and were incubated overnight at 4°C. Appropriate fluorescent dye-labeled secondary antibodies (Table S1B) were used, and cell nuclei were stained with NucBlue Live cell stain (R37605, Invitrogen).
Hematoxylin and eosin and Masson trichrome staining
The protocol described by Fischer et al was used for the Hematoxylin and eosin (H&E) staining 94, while Masson trichrome staining was performed in strict accordance with the manufacturer's protocol (Masson Stain Kit (60532ES58), Yeasen, China).
Flow cytometric staining
For analysis of Th1, Th2, Treg and Th17 cells, single-cell suspensions were prepared from the spleens of mice 24 hours after wounding. The cells were labeled with CD8-APC, CD4-FITC and CD3-PECy5. For T-bet and GATA-3, protein amounts were normalized and cell surface staining was performed using APC-conjugated anti-CD4 followed by fixation with 1× Fixation/Permeabilization buffer and intracellular staining with PE-conjugated anti–T-bet and APC–conjugated anti–GATA-3 in 1× permeabilization buffer. Cells were washed in 1× permeabilization buffer and analyzed by flow cytometry (B53009, CytoFLEX Flow Cytometer, Biotek).
For the analysis of Treg cells, cell surface staining was performed using FITC-conjugated anti-CD4, PE-conjugated anti-CD25, PB450-conjugated anti-CD127 and appropriate isotype controls. Cells were incubated with antibodies for 20 min at room temperature in the dark, followed by washing in phosphate buffered solution (PBS) and analysis by flow cytometry. For the analysis of Th17 cells, cells were stained with FITC anti-CD4 for surface expression of CD4, and intracellular cytokine IL-17 was detected by staining with APC-conjugated Anti-IL-17. Finally, cells were analyzed by flow cytometry. All antibodies and reagents were purchased from Biolegend Inc., USA (Table S1).
Enzyme-linked immunosorbent assay
Murine serum was carefully collected from whole blood and stored in −80°C. Wound edge tissue was harvested after the mice were sacrificed, washed twice in distilled PBS, and cut into small pieces in lysis buffer containing protease inhibitor and 0.1 mM PMSF (Sangon Biotech, Shanghai, China), followed by homogenization and centrifugation (12,000 rpm, 20 min). The supernatant was then collected for further assessment of EGF (MM-0043M), bFGF (MM-0050M1), PDGF (MM-0070M1) and TGF-β1 (MM-0921M) levels using mouse ELISA kits (Jiangsu Meimian industrial, China). For tissue detection, amounts of target growth factors were normalized to the total amount of whole protein.
Non-targeted metabolomics analysis
The Non-targeted metabolomics analysis was performed by IGENECODE Company, Beijing, China. Thermo Scientific™ Dionex™ UltiMate™ 3000 Rapid Separation LC (RSLC) system. UHPLC separation was achieved with reverse phase C18 or hydrophilic interaction liquid chromatography columns. For C18 separation, the mobile phase A was acetonitrile/water (60/40) and mobile phase B was isopropanol/ acetonitrile (90/10); both A and B contained 0.1% formic acid and 10 mmol/L ammonium acetate. The gradient conditions for reverse phase C18 separation are shown in Table S2. The HSS T3 column (2.1 x 100 mm, 1.8 µm, waters) operated at 45°C. The flow rate was 300 µL/min, and the injection volume was 1 µL.
For HILIC separation, the mobile phase A was acetonitrile, and the mobile phase B was water; both A and B contained 0.1% formic acid and 10 mmol/L ammonium acetate. A BEH Amide column (2.1×100 mm, 1.7 µm, waters) was operated at 40°C. The flow rate was 300 µL/min, and the injection volume was 1 µL (See Table S3).
A Thermo Scientific™ Q Exactive™ hybrid quadrupole Orbitrap mass spectrometer equipped with a HESI-II probe was employed. The pos HESI-II spray voltage was 3.7 kV, the heated capillary temperature was 320°C, the sheath gas pressure was 30 psi, the auxiliary gas setting was 10 psi, and the heated vaporizer temperature was 300°C. Both the sheath gas and auxiliary gas were nitrogen. The collision gas was also nitrogen at a pressure of 1.5 mTorr. The parameters of the full mass scans were as follows: a resolution of 70,000, an auto gain control target under 1 × 106, a maximum isolation time of 50 ms, and an m/z range 50–1500. The LC-MS system was controlled using Xcalibur 2.2 SP1.48 software (Thermo Fisher Scientific), and data were collected and processed with the same software.
All data obtained from the four assays in the two systems in both pos and neg ion modes were processed using Progenesis QI data analysis software (Nonlinear Dynamics, Newcastle, UK) for imputing raw data, peak alignment, picking, and normalization to produce peak intensities for retention time (tR) and m/z data pairs. The ranges of automatic peak picking for the C18 were between 1 and 16 min and between 1 and 12 min, respectively; Adduct ions of each “feature” (m/z, tR) were deconvoluted, and these features were identified in the human metabolome database (HMDB) and Lipidmaps.
Fibroblast movement and migration and keratinocyte cell epithelialization ability test by a live cell imaging system
HPF cells were seeded at 5,000 cells/well in a 96 well plate with a clear bottom, stained with Actin-GFP (C10506, CellLingt, Invitrogen, US) and cultured for another 24 hours to observe movement. ESF cells were seeded at 20,000 cells/well, scratched by the high throughput scratcher of Cytation 5 (Biotek), and then observed for another 24 hours to determine the conditioned medium in fibroblast migration function. Cell movement and migration were scanned using a Cytation 5 Cell Imaging Multi-mode reader (Biotek), and migration was quantified automatically according to the manufacturer’s instructions. HaCaT cells were seeded at 30,000 cells/well and then cultured for another 24 hours to observed and calculate the area of colony formation (epithelialization). The images in each small field were captured every hour in 3×3 montage frames at 10× magnification. For cell movement video footage, both bright field and fluorescent channel were used.
Statistical analysis
For comparison between two groups, two-tailed paired and unpaired Student’s t tests were performed to calculate P values and determine statistically significant differences (significance was set at P < 0.05, as detailed in the figure legends). For comparison among more than two groups, ordinary one- or two-way analysis of variance (ANOVA) tests were followed by the appropriate multiple comparison tests (as detailed in the figure legends). All experiments were repeated twice with the same results. All statistical analyses were performed with GraphPad Prism 8 software.