The heterogeneous and highly plastic cell populations of macrophages are important mediators of cellular responses during all stages of wound healing, especially in the inflammatory stage. Molecular hydrogen (H2), which has potent antioxidant and anti-inflammatory effects, has been shown to promote M2 polarization in injury and disease. However, more in vivo time series studies of the role of M1-to-M2 polarization in wound healing are needed. In the current study, we performed time series experiments on a dorsal full-thickness skin defect mouse model in the inflammatory stage to examine the effects of H2 inhalation. Our results revealed that H2 could promote very early M1-to-M2 polarization (on days 2-3 post wounding, 2-3 days earlier than in conventional wound healing), without disturbing the functions of the M1 phenotype. Time series analysis of the transcriptome, blood cell counts, and multiple cytokines further indicated that peripheral blood monocytes were a source of H2-induced M2 macrophages and that the functions of H2 in macrophage polarization were not only dependent on its antioxidant effects. Therefore, we believe that H2 could reduce inflammation in wound care by shifting early macrophage polarization in clinical settings.
Background: Despite progress in developing wound care strategies, there is currently no treatment that promotes the self-tissue repair capabilities. H2 has been shown to effectively protect cells and tissues from oxidative and inflammatory damage. While comprehensive effects and how H2 functions in wound healing remains unknown, especially for the link between H2 and extracellular matrix (ECM) deposition and epidermal stem cells (EpSCs) activation. Methods: Here, we established a cutaneous aseptic wound model and applied a high concentration of H2 (66% H2) in a treatment chamber. Molecular mechanisms and the effects of healing were evaluated by gene functional enrichment analysis, digital spatial profiler analysis, blood perfusion/oxygen detection assay, in vitro tube formation assay, enzyme-linked immunosorbent assay, immunofluorescent staining, non-targeted metabonomic analysis, flow cytometry, transmission electron microscope, and live-cell imaging. Results: We revealed that a high concentration of H2 (66% H2) greatly increased the healing rate (3 times higher than the control group) on day 11 post-wounding. The effect was not dependent on O2 or anti-reactive oxygen species functions. Histological and cellular experiments proved the fast re-epithelialization in the H2 group. ECM components early (3 days post-wounding) deposition were found in the H2 group of the proximal wound, especially for the dermal col-I, epidermal col-III, and dermis-epidermis-junction col-XVII. H2 accelerated early autologous EpSCs proliferation (1-2 days in advance) and then differentiation into myoepithelial cells. These epidermal myoepithelial cells could further contribute to ECM deposition. Other beneficial outcomes include sustained moist healing, greater vascularization, less T-helper-1 and T-helper-17 cell-related systemic inflammation, and better tissue remodelling. Conclusion: We have discovered a novel pattern of wound healing induced by molecular hydrogen treatment. This is the first time to reveal the direct link between H2 and ECM deposition and EpSCs activation. These H2-induced multiple advantages in healing may be related to the enhancement of cell viability in various cells and the maintenance of mitochondrial functions at a basic level in the biological processes of life.
Since Ohsawa et al. reported a biological antioxidant function of hydrogen in 2007, researchers have now shown it to exert protective effects in a wide range of human and animal disease models. Clinical observations and scientific arguments suggest that a selective scavenging property of H2 cannot adequately explain the beneficial effects of hydrogen. However, there is no experiment challenging the original published data, which suggested that molecular hydrogen dissolved in solution reacts with hydroxyl radicals in cell-free systems. Here we report that a hydrogen-saturated solution (0.6 mM) did not significantly reduce hydroxyl radicals in the Fenton system using 1 mM H2O2. We replicated the same condition as Ohsawa’s study (i.e. 5 μM H2O2), and observed a decrease in •OH radicals in both the H2-rich and N2-rich solutions, which may be caused by a decreased dissolved oxygen concentration. Finally, we determined the effect of hydrogen on a high-valence iron enzyme, horseradish peroxidase (HRP), and found that hydrogen could directly increase HRP activity in a dose-dependent manner. Overall, these results indicate that although H2 and •OH can react, the reaction rate is too low to have physiological function. The target of hydrogen is more complex, and its interaction with enzymes or other macro-molecules deserve more attention and in-depth study.