Additional file 1 of Molecular hydrogen promotes wound healing by inducing early epidermal stem cell proliferation and extracellular matrix deposition

Additional file 1: Supplemental Figure S1. H2 slightly increases growth factors concentration at wound sites at day 1-3 after wounding. A, B. Profiles of tissue growth factors PDGF and EGF among the three groups at time points of day 1, day 2 and day 3 post-wounding. C, D. Concentrations of tissue growth factors bFGF and TGFβ-1 among all three groups at day 3. Data in A and B processed Two-way ANOVA test, and data in C and D processed unpaired t test. All data were plotted as Mean±SEM. * P-value < 0.05; ** P-value <0.01; *** P-value < 0.001; no stars for P-value > 0.05. Supplemental Figure S2. Whole-mount scanning showing H2 promoted early tube formation at the first 2 days after wounding. A and B. Panoramic scanning of wound edge and the representative immunofluorescence images of CD31 (green) and k14 (red) expression in the leading edge (L, 0-1 mm from wound edge), mid-end (M, 1-2 mm from wound edge) and distal (D, 2-3 mm from wound edge) of the wound at day 1 and day 2 post wounding respectively. White dotted line indicates the boundary between the epithelium and dermis. White arrowhead indicates tube formation. Scale bar = 100 μM. Supplemental Figure S3. Whole-mount scanning showing H2 promoted early tube formation at the first 2 days after wounding. A and B. Representative microscopic images and the quantification of in vitro blood vessel formation of Human Umbilical Vein Endothelial Cells (HUVEC) at 12 h after different treatments. Red hatched line outlines the newly formed tubes. Data in B were processed unpaired multiple T test. All of the data are plotted as Mean±SEM. * P-value < 0.05; ** P-value <0.01; *** P-value < 0.001; no stars for P-value > 0.05. Scale bar = 100 μM. Supplemental Figure S4. Comprehensive gene set function enrichment analysis of differential gene expression induced by 66% H2 treatment at the first 3 days post wounding. A. Heat map showing the differentially expressed genes (DEGs) among the three time points (D1H vs D1C, D2H vs D2C, D3H vs D3C). B. Venn diagrams showing the overlapping number of DEGs among the comparative data of three time points. C. Counting of total, up- and downregulated DEGs among the comparative data of three time points. D. Heat map showing four clusters identified from all the DEGs. E. GO-BP analysis in each individual cluster, the GO terms for genes related with extracellular matrix organization and muscle cell differentiation were significantly enriched in D1H and D3H, separately. F. Top 10 enriched up and down GO-BP in D1H by GSEA. G. Top 10 enriched up and down GO-BP in D2H by GSEA. H. Top 10 enriched up and down GO-BP in D1H by GSEA. Supplemental Figure S5. Transcriptomics and metabolomics correlation analysis reveal that differential genes and metabolites of the D3H group are enriched in tight junction and focal adhesion pathways. A. Correlation plot of DEGs and differential metabolites between D3H and D3C. B. Count of D3H vs D3C DEGs enriched in KEGG pathways. Supplemental Figure S6. ECM components col-I, fibronectin, laminin, and integrin deposition 2 days after wounding are increased in the H2 group especially in the proximal wound edge. IHC staining showing the difference between D2H and D2C in ECM deposit of day 2 post wounding. A-D indicated Col-1, fibronectin, laminin and integrin expression in D2H and D2H, separately. Scale bar = 100 μm. Supplemental Figure S7. H2 conditioned medium promotes collagen deposition in vitro in MSCs, fibroblasts and keratinocytes. A. Three positive markers in MSCs phenotype. B. Five negative markers in MSCs phenotype. C. α-SMA, Vimintin, and Col-1 expression in the HUCPF (fibroblast) between 66% H2 and control groups 24 h after treatment. D and E. Col-1 deposit in the HaCat (keratinocyte) and HPF (fibroblast) cells between 66% H2 and control groups 24 h after treatment. Supplemental Figure S8. 24-, 48-, and 72-h time points tissue cytokine profiling and four-color Flow CytoMetry (FCM) of different targets showing quantification of CD3+, CD4+, and CD8+ T cells, as well as of Th1, Th2, Th17 and Treg subgroups. A. FCM quantification of CD4+ and CD8+ cell distribution in all three groups. B. T-bet+/CD4+ T-cell distribution, double checked against IFN-γ+/CD4+ T-cell distribution. C. GATA3+/CD4+ T-cell distribution, double-checked against IL-4+/CD4+ T-cell distribution. D. IL-17A+/CD4+ T-cell distribution. E. CD25high/CD127low T-cell distribution. F. Example figures of gating strategy used in cytometry flow test, and the first four plots showed procedure of gating strategy from one of the typical samples in Th1 subgroup cells analysis, the form showed the cell abundance during the gating strategy. G. Pro-inflammatory cytokine profile (TNF-α, IL-1β, and IL-6), Th17-related cytokine (IL-17a and IFN-γ), and Anti-inflammatory cytokine IL-10 expression profile in all of the three groups at three time points. Data in A-C and G processed Two-way ANOVA test, and data in D and E processed unpaired t test. All data were plotted as Mean±SEM. * P-value < 0.05; ** P-value <0.01; *** P-value < 0.001; no stars for P-value > 0.05. Supplemental Figure S9. High concentration of H2 promoted early mussel and nerve repair. A. Left: Scheme of a mouse gastrocnemius strike model. Right: Timeline of animal experiments and daily H2 treatment. B. Capture of the gastrocnemius strike area and blood perfusion 0-72 hours post-wounding in the 66% H2 Control groups. C. & D. Quantification of diameter and blood perfusion of the gastrocnemius strike area between two groups. E. & F. Representative immunofluorescence images for NFH (green) nerve injury repair and axons maturation in dermal wounds of at day 3 and 11 post wounding. Data in C & D were processed unpaired T test. All of the data are plotted as Mean±SEM. * P-value < 0.05; ** P-value <0.01; *** P-value < 0.001; no stars for P-value > 0.05; Scale bar = 100 μm. White dotted line in E & F indicates the boundary between the epithelium and dermis. Arrow in E & F indicates NFH+ cells. Table S1. List of primary and secondary antibodies used in experiments. IHC indicates immunochemistry, IF indicates immunofluorescence; FC indicates flow cytometry. Table S2. Gradient conditions for reversed phase C18 separation. Table S3. Gradient conditions for HILIC separation of polar metabolites. Table S4. DEGs annotation of all the 6 groups. Table S5. DEGs annotation of D1H vs D1C. Table S6. DEGs annotation of D2H vs D2C. Table S7. DEGs annotation of D3H vs D3C. Table S8. Raw data of nontargeted metabolomic analysis between D3H and D3C. Table S9. 12 metabolites identified through hydrophilic-product test between 66% H2 and Control group. Table S10. Top 20 significant pathways discovered by nontargeted metabolomic analysis between the D3H and D3C groups.