It has long been reported that hydrogen gas can achieve therapeutic effects through anti-oxidation. In 1975, Dole M found that high-pressure hydrogen gas could effectively treat malignant skin tumors . HRW has also shown positive therapeutic effects in many studies [20–22]. When H2 enters the body and takes part in the reaction, only water is produced, and the excess H2 is discharged from the body, implying that HRW can be safely used to treat diseases without any side-effects. This is the first study in veterinary-related research to purposefully assess the effects of HRW on simple cutaneous wound healing in dogs. In this study, the positive effect of HRW treatment on skin wound healing in dogs was determined, which was primarily attributed to the its antioxidant potential.
The gold-standard assessment of skin wound healing involves the visual inspection of the wound for surface epithelization and reduction in the skin wound size with time . Recent studies have shown that estrogen might be involved in the wound-healing process at the molecular level [25–27]. Thus, the dogs used in this study were all intact males to avoid differing levels of estrogen. In this established skin wound healing model, we measured the area of skin wounds using Image J software with the polygon selection tool. The HRW treatment regimen exhibited substantial effects between days 7 and 28; the area of skin wounds was always less than that in the DW group. HRW was found to facilitate the rapid formation of granulation tissue to shorten the healing time than the DW group, which was probably due to the fact that HRW promoted the formation of blood vessels in the early stage of wound healing as well as relatively rapid re-epithelialization. Here, HRW upregulated the expression of antioxidant genes and growth factor genes, which promoted wound healing. Additionally, the wound length in the HRW group was also significantly shorter than that in the DW group (Fig. 4) between days 7 and 14.
Collagen fibers is the main extracellular matrix component, which acts as a structural scaffold in tissues, regulating cell proliferation and migration during skin wound healing . Thus, the content of collagen fibers is an important parameter to examine skin wound healing . The HRW group showed that the collagen fibers within the skin wound site were observed to be more regular and higher in content compared to the DW group (from days 7 and 21, Fig. 6). Upregulated gene expression in PDGF stimulated collagen synthesis and the release of collagenase  (Fig. 8B). Thus, elevated gene expression of PDGF resulted in more collagen fibers in the HRW group than the DW group. It also explained why collagen fibers increased in skin tissue after treatment with HRW in skin wound healing and was probably one of the major principles involved in HRW-mediated skin wound healing.
During the early stages of skin wound healing, an inflammatory response followed by re-epithelialization of the wound area occurs along with the establishment of granulation tissue accompanied by neovascularization . Among them, neovascularization is indispensable for wound healing and tissue regeneration . Neovascularization provides nutrients and oxygen to the wound, supports keratinocyte migration, transports mesenchymal stem cells to the skin, and then supports wound regeneration . Angiogenesis is a process orchestrated by multiple angiogenic factors, among which VEGF is an essential growth factor regulating the critical steps of angiogenic processes . At the same time, PDGF regulates angiogenesis by recruiting and primes the pericytes , promoting skin wound healing. In our experiment, the gene expression of VEGF (Fig. 8A) and PDGF (Fig. 8B) in both groups showed that the HRW group exhibited an enhanced ability for neovascularization at an early stage of skin wound healing (0–7 days), and complied with the results of the number of neovascular in the tissue section (Fig. 5A). Therefore, it was plausible that the effect of HRW on angiogenesis in skin wounds was regulated by increasing the expression of VEGF and PDGF, promoting the formation of granulation tissue. Studies have shown that VEGF increases cartilage formation during the early stages of endochondral bone formation, leading to a significant enhancement in bone formation and bone healing . Fan et al. found that in endometrial repair, VEGF blockade dramatically inhibited re-epithelialization . Elevated VEGF expression could drive re-epithelialization of the skin wound. Therefore, elevated gene expression of VEGF and angiogenesis were the positive signals during the skin wound healing process.
Nrf-2 is a critical regulator of antioxidant response , which can avoid the oxidative trauma as well as the development of oxidative stress. MDA is a metabolite of lipid peroxidation of unsaturated fatty acids by oxygen free radicals and indirectly reflects the severity of damage induced by free radicals [9, 28]. The changes in MDA levels reflect the changes in oxygen-free radical content in the tissues. As a scavenger of free radicals, the SOD activity also reflects the number of free radicals . The antioxidant roles of HRW were central to promoting wound healing. In the present study, we found that HRW activated the Nrf-2 antioxidant pathway and influenced SOD activity and MDA levels in skin tissue of dogs.
The NQO-1 and HO-1 genes are the downstream targets of Nrf-2 and these two genes are regulated by Nrf-2 expression, which is regarded as one of the most important intracellular antioxidant mechanisms . Research has shown that hydrogen molecules can activate the expression of Nrf-2 in lung tissue, thereby promoting the expression of HO-1 and NQO-1 . In our study, it was demonstrated that HRW treatment increased the expression of Nrf-2 (Fig. 7C, from days 7 to 21) and then elevated the expression of its downstream phase II metabolic enzymes HO-1 (Fig. 7D, from days 7 to 21) and NQO-1 (Fig. 7E, from days 7 to 21). Nrf-2 can trigger transcription of SOD, reducing the MDA content in the skin tissue . In our experiment, from days 7 to 14, the MDA content in the DW group was significantly higher than that in the HRW group (P < 0.01), and the SOD activity in the HRW group was significantly higher than that in the DW group (P < 0.05) on days 7, 21, and 28. As one of the reactive intermediates from oxidation, MDA is a marker of fatty acid oxidation; its elevated levels might lead to impaired and delayed skin wound healing in rats [41, 42]. Li et al. found that wound lipid peroxidation and enhancement of SOD expression promoted the wound healing of chronic ulcers . In the diabetic model, increased proinflammatory cytokines and oxidative stress in the wound microenvironment resulted in the delay of wound healing . This suggested that the activity of SOD reflected the oxidation levels of skin tissue, and lower oxidation level could avoid oxidative damage, causing faster healing. Ohsawa et al. reported that 2% hydrogen gas inhaled by animals could scavenge hydroxyl radical (OH•) and peroxynitrite anion (ONOO−), thereby improving oxidative stress-induced cell injury . SOD can catalyze the dismutation of superoxide anions into oxygen and hydrogen peroxide, and this step also requires the participation of hydrogen. HRW provides more hydrogen for SOD to participate in the scavenging of oxygen radicals; therefore, HRW scavenges the hydroxyl radicals and superoxide radicals by enhancing the SOD vitality, thereby reducing MDA content, promoting skin wound healing.