The Effects of Hydrogen-Rich Water on Skin Wound Healing in Dogs

Background: Hydrogen-rich water (HRW) has been shown to be one of underlying therapeutic strategies regarding wound treatments. This study explored the effects of drinking HRW on skin wound healing in dogs. Eight circular wounds were analyzed in each dog. The experimental group was treated with HRW twice daily, while the control group was provided with distilled water (DW). The wound tissues of dogs examined histopathologically. The broblasts, inammatory cell inltration, the average number of new blood vessels, and the level of malondialdehyde (MDA) and superoxide dismutase (SOD) activity in the skin homogenate of the wound was measured using the corresponding kits. The expression of Nrf-2, HO-1, NQO-1, VEGF, and PDGF were measured by real-time uorescence quantitative method. Results: We observed that HRW wound had signicant average healing rate and faster average healing time. Histopathological results showed that the average number of blood vessels and the average thickness of epidermis were signicantly different from the DW group. The MDA levels were higher in the DW group than in the HRW group but the SOD levels were higher in the HRW group than in the DW group. The results of qRT-PCR showed that the expression of each gene was signicantly different between the two groups. Conclusions: The results showed that HRW treatment could promote skin wound healing in dogs, accelerate wound epithelization, reduce inammatory reaction, stimulate the expression of cytokines related to wound healing, and shorten wound healing time.


Background
As the largest organ of animals, the skin plays a critical role in preventing animals from pathogenic microorganisms and wounds [1]. Brie y, the skin wounds caused by diverse elements usually occur in clinical veterinary setting. The recovery of severe wounds associated with numerous factors, such as therapy duration and treatment strategies might lead to various therapeutic effects affecting the time and effects of wound healing [2]. wound healing involves a series of mechanisms, such as in ammatory response, oxidative stress, etc., which play vital roles in wound pathophysiology [3]. Numerous therapeutic approaches have been applied in clinical treatments, including photodynamic therapy, farinfrared ray therapy, and adipose tissue extraction therapy [4][5][6].
Hydrogen gas has the lowest molecular weight, along with a strong antioxidant capacity to remove excessive reactive oxygen species (oxygen-free radicals) from the body [7]. Clinical studies have shown that hydrogen therapy has therapeutic potential for the treatment of rheumatoid arthritis, diabetes, Parkinson's disease, and metabolic syndrome [8,9]. Tomohiro et al. found that hydrogen molecules could activate the NF-E2-related factor (Nrf-2) antioxidant defense pathway and ameliorate hyperoxic lung injury via the induction of Nrf-2 dependent genes, such as HO-1 [10]. Several studies have suggested that the inhalation of hydrogen-containing gas constitutes a novel guideline for practical clinical care, focusing on the treatment of skin wounds [11].
Hydrogen gas is a ammable and explosive gas, which is dangerous and di cult to store; however, hydrogen gas-saturated water, which is known as hydrogen-rich water (HRW), is economically safe and easy to absorb, and is frequently used in medicine [10]. HRW has been reported to exhibit antioxidant and anti-in ammatory effects, reducing the levels of pro-in ammatory cytokines, effectively protecting against organ damage [12]. Hydroxyl radical and peroxynitrite (ONOO-) ion have strong oxidation toxicity, and the potential to cause lipid peroxidation, DNA damage, and protein peroxidation, resulting in cell damage [13]. However, hydrogen dissolved in water can react with them, reducing the consequent damage. The four methods commonly used to infuse H2 gas into water involve high pressure, electrolyzing water to producing H2, and reacting magnesium metal or its hydride with water to produce HRW [14].
Previous studies on animal testing have used both oral ingestion of HRW and injection of hydrogen-rich saline (HRS) as major routes of administration of HRW [15]. Studies have shown that HRS can scavenge hydrogen peroxide and reactive oxygen species (ROS) [16,17] by upregulating the expression of heme oxygenase (HO-1) as well as the reduction of quinine (NQO-1) [18]. Additionally, recent studies have demonstrated the antifatigue effects of HRW in animals and humans [19]. HRW also induces antiapoptotic effects in myocardial cells [20] and inhibits the production of α, β-dicarbonyl compounds and ROS in the kidneys of rats [21]. The treatment principle of HRW is mainly re ected by its biological roles in anti-in ammation and antioxidation. Another study suggested that the use of HRW could enhance the rate of palatal wound recovery [22]. Thus, HRW has been shown to be one of underlying therapeutic strategies regarding wound treatments. Although HRW has been proven to accelerate the recovery of skin wounds [11], there is little information available on the use of HRW in the trauma associated with canine skin.
Here we aimed to explore whether HRW could play an essential role in the recovery of skin wounds in a canine model. The speci c objectives were (1) to compare the effect of the healing of open wounds in dogs treated with HRW and the ones treated with distilled water (DW); (2) to compare the level of the oxidative stress and in ammatory indicators; (3) to explore whether or not HRW had therapeutic value in the recovery of canine skin wound. We hypothesized that HRW promoted angiogenesis and elevated tissue antioxidant gene expression to promote wound healing and accelerate the rate of wound healing, making it a safe and effective clinical treatment.

Dogs
Twelve healthy male beagle dogs (age: 2-4 y; weight: 9.0-11.3 kg) were from Breeding Base of Harbin and housed in a standard environment and provided free access to standard dog food and water. Food and water were withdrawn 6 h prior to anesthesia. The Laboratory Animal Welfare and Ethics Committee of Northeast Agricultural University (#NEAU-2019-04-0245-13) approved the experimental design and animal surgery procedures in this study. The study was carried out in compliance with the ARRIVE guidelines. All methods were handled in accordance with good animal practices required by the Animal Ethics Procedures and Guidelines of the People's Republic of China. Make every effort to minimize animal suffering and reduce the number of animals used. No deaths were reported in this study; both mental status and diet of the dogs were normal. No animals were killed and all dogs were healthy and adopted by animal protection organization after the experiment was nished.

Surgical Operation
Dogs were randomly divided into control and experimental groups; each group contained six dogs. The hair was clipped on the dorsolateral trunk on day 0. Cephalic intravenous catheter was placed, and the dogs were anesthetized using propofol (0.65 mg/kg), followed by the inhalation of 3.5% iso urane via endotracheal intubation, and the physiological state of the experimental dogs was monitored in real time during the operation.
Four circular wounds with a diameter of 15 mm were created on each side of the midline of the shaved dorsum using a dermal biopsy punch. Each wound was 20 mm from the midline of the dorsum, and the adjacent wounds were 25 mm from each other. From the head to the tail side, and from left to right, the wounds were labeled as A1, A2, A3, A4, B1, B2, B3, B4 (Fig. 1). Post-operative pain was managed with meloxicam anesthetic (0.1 mg/kg, subcutaneously). Each dog was kept in single cage, wearing Elizabethan collars. The wound area was disinfected daily using iodophor. Hydrogen gas (99.99%, v/v) produced from a hydrogen gas generator (QL-500; Saikesaisi Hydrogen Energy Co., Ltd., Shandong, China) was bubbled into 400 mL of distilled water at the rate of 500 mL/min for 10 min to achieve saturation. All dogs had free access to standard food after postoperative fasting; the control group was provided DW (400 mL) twice a day and the experimental group was provided saturated HRW (400 mL) twice a day. Each dog had only one hour at a time to drink to keep the concentration of HRW constant.
Skin wound samples were collected at 7, 14, 21, and 28 day after surgical operation, intubation, analgosedation, and monitoring; the surgical procedures were identical to the skin wound model. The healthy skin around 1 cm of the wound was removed when skin wound sampling was performed. The skin wound at A1 and B1 was sampled on day 7; A2 and B2 were sampled on day 14, A3 and B3 were sampled on day 21, A4 and B4 were sampled on day 28. Following sampling, the wound was closed using a suture and sterilized with iodophor. A1, A2, A3, and A4 samples were xed in 4% paraformaldehyde (20g paraformaldehyde powder dissolved in 500 mL PBS buffer, followed by constant stirring using a magnetic stirrer. NaOH was used to adjust the solution pH.). B1, B2, B3, and B4 samples were stored in a refrigerator at -80℃.

Examination of the skin wound
The changes in the wounds in the HRW and the DW groups were observed on days 7, 14, 21, and 28 after the operation for bleeding, in ammatory exudate traits, wound healing, wound infection, etc. The wounds were photographed on days 3, 7, 14, and 21, and wound are was quanti ed using the Image J software.
where Sn, S0 (the wound area at 0 day). Dog skin wound healing was measured until granulation growth for 100% of the wound; the time of this process was the time of wound healing. Para n sections were prepared for histopathological examination. H&E staining was used for evaluating epithelial thickness and neovascularization and Masson staining for collagen ber. Brie y, ve sections were chosen from each dog, and ten visual elds were randomly selected from different areas in each section. The evaluation of section was performed by two independent and blinded observers.
Oxidative stress was assessed by detecting malondialdehyde (MDA) and super oxide dismutase (SOD) levels in skin tissues following the instructions on the detection kits (Jiancheng Biotech Nanjing).
Total RNA was isolated from skin tissue using TRIzol Reagent (Invitrogen). The RNA was reverse transcribed using Super Script First Strand cDNA System (Invitrogen), and qRT-PCR was performed using the Light Cycler 480 Real-Time PCR System (Roche Diagnostics, Germany). Table 1 provides the list of primer sequences. Statistical Analysis All data were statistically analyzed using independent-sample t-test using SPSS 22.0 statistical software.
All results are expressed as mean ± SD; P > 0.05 indicates no signi cant difference, P < 0.05 indicates a signi cant difference, and P < 0.01 indicates that the difference was extremely signi cant.

Wound Healing
Clinical Wound Evaluation Figure 2 shows the presence of a signi cant difference in skin wound area between the HRW and DW groups between days 3 and 21. On day 3, the skin wound showed obvious hemorrhage, edema, and in ammatory reaction in the DW group and was not obvious in the HRW group. On day 7, there were abundant granulation tissue, newborn epithelia, and wound surfaces were humid and glossy in the HRW group, whereas the DW group had fewer granulation tissues and newborn epithelia, and wound surfaces were dried and matte, with large necrotic tissues and with in ammatory exudate. On day 14, skin tissue defects were lled with granulation tissue in all dogs; the HRW group showed better re-epithelialization than the DW group with the appearance of scar tissue and hair growth. On day 21, wounds were almost fully re-epithelialized in the HRW group. On day 28, it showed complete wound closure and healing in both HRW and DW groups.

Wound Healing Rate and Average Healing Time
During the experimental period, the rate of skin wound healing in the DW and HRW groups increased gradually until complete healing was achieved (Fig. 3A)

Wound length
The results from histopathological analysis of skin showed that on day 7, the wound area was still large and the surface of wound was covered with the granulation tissue in DW and HRW groups. On day 14, the skin wound area in the HRW group was signi cantly smaller and showed better re-epithelialization than the DW group (P < 0.05).

Re-epithelialization
The formation of neoepithelium was observed along the wound surface margin in the HRW group, initiating keratinocyte differentiation, epidermal cell differentiation, epidermis development, skin development, and keratinization. On day 14, undifferentiated keratinocytes were found in the DW group.
On day 21, the HRW group showed marked reduction in epithelial attening, gland formation, and epithelium thickness, whereas the DW groups showed irregularly thick epithelium (Fig. 5A) Fig. 5C).

Collagen Fiber
The collagen bers were stained blue, and the muscle bers were stained red after Masson's trichrome staining. The results of the pathological section showed an increase in the collagen bers with time both in the DW and HRW groups (Fig. 6). The collagen bers in the HRW group were con gured regularly between days 7 and 21 and were similar to healthy skin until day 28. The DW group showed staggered and irregular collagen bers between days 7 and 21 and were fewer than those in the HRW group. On day 28, there was a dense deposition of irregular collagen bers in the DW group.

Oxidative Stress Detection
During the experimental period, the level of MDA in skin tissues (Fig. 7A)  Between days 21 and 28, the MDA levels in the skin tissues dropped sharply, and there was no signi cant difference between the two groups (P > 0.05).
During the experimental period, the SOD levels in the skin tissues (Fig. 7B) increased from day 7 to 28 in DW group, whereas they rst decreased (from day 7 to 14) and then increased (from day 14 to 28) in the HRW group. On day 7, compared with the SOD levels in the skin tissue in the DW group (12.36 ± 0.96U/mg), the same in the HRW group (22.82 ± 1.57 U/mg) was signi cantly higher (P < 0.05). On day 21, the SOD levels in the DW group (23.36 ± 4.04 U/mg) were signi cantly lower than that in the HRW group (31.23 ± 6.13 U/mg) (P < 0.05). On day 28, the SOD levels in the DW group (24.39 ± 2.87 U/mg) were signi cantly lower than that in the HRW group (33.78 ± 7.11 U/mg) (P < 0.05).
During the experimental period, the gene expression of Nrf-2 in HRW/DW ratio was extremely signi cantly different between days 7 and 21, and was signi cantly different on day 14 (Fig. 7C). The gene expression of HO-1 in HRW/DW ratio was extremely signi cantly different between days 7 and 21 (Fig. 7D). The gene expression of NQO-1 in HRW/DW ratio was extremely signi cantly difference between days 7 and 21, and was signi cantly different on day 14 (Fig. 7E).

Growth Factor Detection
During the experimental period, the gene expression of VEGF in HRW/DW ratio was extremely signi cantly different between days 7 and 14 (Fig. 8A). The gene expression of PDGF in HRW/DW ratio was also extremely signi cantly different between days 7 and 14 (Fig. 8B).

Discussion
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 [23]. HRW has also shown positive therapeutic effects in many studies [20][21][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 rst 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 [24]. Recent studies have shown that estrogen might be involved in the wound-healing process at the molecular level [25][26][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 signi cantly shorter than that in the DW group (Fig. 4) between days 7 and 14.
Collagen bers is the main extracellular matrix component, which acts as a structural scaffold in tissues, regulating cell proliferation and migration during skin wound healing [28]. Thus, the content of collagen bers is an important parameter to examine skin wound healing [23]. The HRW group showed that the collagen bers 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 [29] (Fig. 8B). Thus, elevated gene expression of PDGF resulted in more collagen bers in the HRW group than the DW group. It also explained why collagen bers 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 in ammatory response followed by re-epithelialization of the wound area occurs along with the establishment of granulation tissue accompanied by neovascularization [30]. Among them, neovascularization is indispensable for wound healing and tissue regeneration [31]. Neovascularization provides nutrients and oxygen to the wound, supports keratinocyte migration, transports mesenchymal stem cells to the skin, and then supports wound regeneration [32]. Angiogenesis is a process orchestrated by multiple angiogenic factors, among which VEGF is an essential growth factor regulating the critical steps of angiogenic processes [33]. At the same time, PDGF regulates angiogenesis by recruiting and primes the pericytes [34], 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 signi cant enhancement in bone formation and bone healing [35]. Fan et al. found that in endometrial repair, VEGF blockade dramatically inhibited re-epithelialization [36]. 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 [37], 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 re ects the severity of damage induced by free radicals [9,28]. The changes in MDA levels re ect the changes in oxygen-free radical content in the tissues. As a scavenger of free radicals, the SOD activity also re ects the number of free radicals [38]. 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 in uenced 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 [39]. 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 [22]. 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 [40]. In our experiment, from days 7 to 14, the MDA content in the DW group was signi cantly higher than that in the HRW group (P < 0.01), and the SOD activity in the HRW group was signi cantly 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 [43]. In the diabetic model, increased proin ammatory cytokines and oxidative stress in the wound microenvironment resulted in the delay of wound healing [44]. This suggested that the activity of SOD re ected 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 [45]. 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.

Conclusion
We demonstrated that HRW could increase the number of blood vessels in the granulation tissue, accelerating wound epithelization, reducing in ammatory reaction, stimulating the expression of cytokines related to wound healing, shortening wound healing time, thereby accelerating skin wound healing. The dogs showed no discomfort in following the treatment strategy. These results showed that HRW is a safe and effective veterinary clinical treatment for dog skin wound healing. The datasets generated and/or analysed during the current study are available from the corresponding author on request.

Funding
This study was funded by the by the National Natural Science Foundation of China (31402261), "Academic Backbone"Project of Northeast Agricultural University (18XG22).
The funding body did not have any in uence over the design, analysis and reporting of the study.     HRW (54.16 ± 2.53 μm) and DW (123.54 ± 46.14 μm) on day 21; HRW (50.12 ± 8.69 μm) and DW (86.50 ± 18.78 μm) on day 28, *P < 0.05, **P < 0.01. (C) Average number of blood vessels. The number of neovascular structures in skin tissue were assessed by H&E staining of the para n-embedded section.

Figure 6
Masson section of wound: Skin wound para n sections were subjected to Masson staining. The collagen bers were stained blue, and the muscle bers were stained red after Masson's trichrome staining.

Figure 7
Antioxidant indices: (A) MDA activity of sample was determined using test kits. The DW group had higher MDA levels than the HRW group on days 7 and 14, *P < 0.05, **P < 0.01. (B) SOD activity of sample was determined using test kits. The HRW group had higher SOD activity than the DW group on days 7, 21, and 28, *P < 0.05, **P < 0.01. (C) Nrf-2 gene content. Relative Nrf-2 mRNA levels were measured by TaqMan real-time PCR. The HRW group had higher mRNA expression than DW group on days 7, 14, and 21, *P < 0.05, **P < 0.01. (D) HO-1 gene content. Relative HO-1 mRNA levels were measured by TaqMan real-time PCR. The HRW group had higher mRNA levels than the DW group on days 7, 14, and 21, **P < 0.01. (E) NQO-1 gene content. Relative NQO-1 mRNA levels were measured by TaqMan real-time PCR. The HRW group had higher NQO-1 gene levels than the DW group on days 7, 14, and 21, *P < 0.05, **P < 0.01.

Figure 8
The gene expression levels of growth factor: (A) VEGF gene content. Relative VEGF mRNA levels were measured by TaqMan real-time PCR. The HRW group had higher mRNA levels than DW group on days 7 and 14, **P < 0.01. (B) PDGF gene content. Relative PDGF mRNA levels were measured by TaqMan realtime PCR. The HRW group had higher mRNA levels than DW group on days 7 and 14, **P < 0.01.