| 1. IntroductionClinical experiments have shown that flushing with ozonated water promotes the healing of different types of wounds, including scratches, contusions, cuts, burns, crushing, cervical erosion, refractory ulcers caused by diabetes, and gangrenous pyoderma[1-4]. Early clinical practice has also confirmed that the treatment of gastric duodenal ulcers caused by Helicobacter pylori with ozone produced satisfactory results[5]. Another basic experiment also demonstrated that ozone effectively promotes the regeneration of mouse liver[6].The infection of skin wounds can be prevented and controlled effectively by flushing the wound with ozonated water, bathing it with ozone gas, flushing the wound with ozonated water, or fumigation, and wound healing occurs more rapidly than after conventional sterilization. Throughout the world, animal experiments have confirmed that flushing with ozone water promotes wound healing. Several principles underlying this effect, other than the antibacterial mechanism, have been proposed:(1) ozone opens capillaries and improves the capacity of red blood cells to carry oxygen, thus promoting the repair function; (2) ozone induces cells to generate hydroxyl deoxyguanosine (8-OHdG) and express hemeoxygenase-1, increasing oxidation within the body[7]; (3) ozone activates immune cells, stimulating the release of the cytokines interferon, interleukin (1L), tumor necrosis factor (TNF) and granulocyte macrophage colony-stimulating factor, and thus improves the local immune functions[8]; (4) ozone stimulates local epidermal growth factor expression, reduces TNF-α expression, and thus accelerates cell regeneration; (5) ozone increases the expression of platelet-derived growth factor, transforming growth factor, vascular endothelial growth factor, and cell-cycle D1 protein to accelerate wound healing, but does not increase fibroblast growth factor (FGF) expression[8,9]. Research has shown that ozone promotes tissue regeneration, with no obvious tissue specificity.The infection control and innate immune responses of the zebrafish are highly conserved throughout the vertebrates, which makes the zebrafish an ideal animal model of inflammation and wound healing[10-17]. For the purposes of this study, the most favorable characteristic of the zebrafish is that many of its organs and tissues can be regenerated. The caudal fin regeneration model is especially easy to establish, is structurally simple, and provides rapid tissue regeneration. Research has shown that after 95% resection, the regeneration of the zebrafish caudal fin is complete within two weeks. Complete regeneration is achieved in five days after the resection of the caudal fins of young fish[18].In our experiment, we established a new animal model of ozone treatment in the zebrafish, first determining the safe concentration of ozone that can be administered. With this model, we confirmed that ozone stimulates tissue regeneration after tissue damage. We discuss this effect in terms of the organization of the early inflammatory response. We measured the expression of IL-10 and STAT3, which regulate the expression of TNF-α, and measured the expression of IL-1β,which is also regulated by IL-10 and STAT3, to confirm the reliability of the experimental results. Therefore, we conclude that ozone promotes tissue regeneration by influencing tissue inflammation.2. Materials and Methods2.1. Zebrafish strains and maintenanceThe zebrafish strains AB (wild type) and Tg(mpo:GFP)[19] were obtained from the Key Laboratory of Zebrafish Modeling and Drug Screening for Human Diseases Institute, Southern Medical University, Guangzhou, China. Zebrafish embryos were maintained and raised according to the protocol described by Westerfield[20]. The zebrafish were kept at 28.5℃ with a 14 h:10 h ligh:dark cycle in a recirculating tank system with local tap water (pH 7.2-7.6, salinity 0.03%-0.04%). Fertilized embryos, collected after natural spawning, were cultured at 28.5℃ in clean Petri dishes in egg water. To inhibit melanin formation on AB (used for the for acridine orange test) and Tg(mpo:GFP) strains,0.003%1-phenyl-2-thiourea was added to the egg water after 10-12 h. The Tg(mpo:GFP) strain expresses green fluorescent protein (GFP) in its neutrophils under the control of the myeloperoxidase (MPO) promoter, which allows neutrophil migration to be tracked in vivo during the acute inflammatory reaction[19]. All containers were autoclaved before use[21,22].2.2. Ozonated water preparationOzone is a strong oxidizing gas and is more readily soluble in water than oxygen[23,24]. Ozonated water was produced by injecting ozone gas into water for five minutes (high oxygen liquid preparation equipment;Forefront Medical Technology company,Shandong,China). The ions in ozonated water affect the stability of ozone and can accelerate its degradation. However, because distilled deionized (dd)H2O greatly affects the growth and survival of young fish, we prepared the ozonated water with egg water, which contains some ions.2.3. Determining the concentration of ozonated waterTo determine the concentration of ozonated water, we made a working liquid solution by mixing fresh ozonated water with egg water in ratios of 1:0,2:1,1:1,1:2, 1:3, and 1:4. We measured the percentage concentration of ozonated water semiquantitatively with the DPD Ozone Test Kit (Hangzhou,China)2.4. Determining the safe concentration of ozone waterSurvival curves were constructed for AB (wild-type) larvae at 72 hours post fertilization (hpf) that had been treated with different concentrations of ozonated water. The 72 hpf AB larvae were divided into six groups to ascertain the suitable ozone concentration. The numbers of zebrafish deaths were checked every 5 min for 1 h under a microscope for each ozone concentration.2.5. Acridine orange staining for apoptosisAB zebrafish (72 hpf) were divided into six groups to ascertain the suitable concentration of acridine orange for 10 min to detect apoptosis. They were then incubated in 1 mL of 5 g/mL acridine orange for 30 min. After the larvae were washed three times with phosphate-buffered saline containing Tween 20, cell apoptosis was observed in the larval bodies under a fluorescence microscope[25].2.6. Morphological observation of caudal fin growthThe caudal fins of AB zebrafish (72 hpf) were resected with a needle of 1 mL syringe along the length of the vertical axis under a stereo microscope (Fig.2A) [26]. The zebrafish were then randomly divided into three groups (a, b, and c) and given egg water, single ozonated water (ozonated water given just once), of multiple ozonated water (replaced with fresh ozonated water every 12 h), respectively. The concentrations of ozone in the water were monitored. The caudal fin lengths in the three groups were measured and compared at 0,24,48,72, and 120 hpa under a microscope with a scale.2.7. Neutrophil migration after tail fin resectionThe caudal fins of 72 hpf Tg(mpo:GFP) zebrafish were removed as described above, and the zebrafish were randomly divided into two groups, which were given egg water or ozonated water. The numbers of neutrophils that migrated to the caudal incision were observed and recorded under a confocal microscope at 0.5,1,2, and 3 hpa[19].2.8. Fluorescent quantitative RT-PCR (RT-qPCR)The caudal fins of 72 hpf AB zebrafish were removed, as described above, and the zebrafish were randomly divided into two groups, and given egg water or ozonated water.the specimens were observed with a microscope at 0,6,12,24,48, and 72 hpa[21]. RNA was extracted from the samples with RNAiso Plus(Takara, Japan), and reverse transcribed with PrimeScriptTM RT reagent Kit with gDNA Eraser(Takara,Japan). The cDNA generated was used as the template for qPCR to monitor gene expression using the LightCycler Nano System (Roche, Switzerland) and SYBR Premix Ex TaqTM(Tli RNaseH Plus) (Takara, Japan). PCR was performed(95℃ for 5s,55℃ for 30s,72℃ for 30s; 40 Cycles)to detect the gene expression by using a set of primers.The mRNA levels were normalized according to the level of Elfla.Fold-differences in gene expression were calculated by the AACt equation:ACT= CTtarget gene-CTGAPDH, AACT= ACTexperiment-ACTcontrol, fold difference= 2-AACT.2.9. ImmunoblottingThe caudal fins of 72 hpf AB zebrafish were removed as described above. The zebrafish were randomly divided into two groups, one given egg water and the other multiple ozonated water. The specimens were observed with a neuromicroscope at 0, 6,12,24,48, and 72 hpa. TNF-a expression was determined after the fish proteins were extracted and analyzed with immunoblotting.The specimens were washed 2-3 times with ice-cold Tris-buffered saline (TBS). Ten volumes of TBS (to which protease inhibitors had been added a few minutes earlier) were added as the organization volume, and the samples were thoroughly homogenized. The lysates were incubated for 30 min on ice before centrifugation at 12,000 ×g for 5 min at 4℃. The proteins in the supernatants were denatured by boiling for 5 min in sodium dodecyl sulfate (SDS) sample buffer, and were then separated with SDS-polyacrylamide gel (5%) electrophoresis and transferred to nitrocellulose membranes. Following transfer, equal protein loading was verified with Ponceau stain. The membranes were blocked with 5%skim milk and incubated with the indicated antibodies:monoclonal rabbit anti-actin antibody (diluted 1:1000, SC-1616R,KPL,Santa cruz) or monoclonal rabbit anti-TNF-α antibody (1:1000, ab6671, abcam,England). The bound antibodies were visualized with horseradish-peroxidase-conjugated secondary antibody (1:3000, KPL, Santa cruz) and enhanced chemiluminescence. The AlphaEaseFC software(AIpha Innotech) was used to analyze the optical density values of the target proteins.2.10. Statistical methodsCaudal fin growth was investigated with analysis of covariance, and caudal fin neutrophil counts and qPCR results were analyzed with a t test.p values of< 0.05 were considered significant. 3. Results3.1 Preparation of ozonated waterOzonated water is unstable and degrades spontaneously[27,28]. The ion content, fluid temperature, fluidity, and pH of the water can affect the rate of degradation. Therefore, we first ascertained the survival rates of zebrafish maintained in ddH2O and egg water. The survival rate was much lower in dd H2O than in egg water. Because the sample loss was greater in dd H2O, we selected egg water as the basis of ozonated water, even though the NaHCO3 in egg water can accelerate the degradation of ozone. The water in which zebrafish are usually maintained is treated with bacteriostatic methylene blue, but ozone reacts with methylene blue, generating an unknown product. Therefore, we used no methylene blue in this experiment.3.2. Safe concentration of ozonated waterWe determined the safe concentration of ozonated water, so that the required concentration could be prepared simply and quickly. We mixed fresh ozonated water with egg water in proportions of 1:0,2:1,1:1,1:2,1:3, and 1:4 to produce ozone concentrations of 1.5,1.0,0.5,0.2,0.1, and 0.05 mg/L, respectively (temperature, 28.5℃).3.3. Survival times in different concentrations of ozonated waterBecause it is a strong oxidant, ozone in water can injure the tissues and organs of zebrafish, and different concentrations produce different levels of damage. Therefore, we determined the safe concentration of ozone water. The survival times were less than 20 min at ozonated water concentrations of 1.5,1.0, and 0.5 mg/L. At a concentration is 0.2 mg/L, the survival time clearly increased, with no obvious deaths after 1 h. No deaths were observed at concentrations of 0.1 and 0.05 mg/L. These results suggest that the safe concentration of ozonated water is 0.2 mg/L, and the survival time increased as the concentration decreased. At concentrations less than 0.2 mg/L, larval survival was not affected by the concentration of the ozonated water. The ozonated water was replaced with fresh ozonated water every 12 h, causing no cumulative damage to the zebrafish or change in the survival rate.These experimental results showed that ozonated water has no effect on the survival time of 72 hpf AB zebrafish at concentrations of 0.2 mg/L (Fig.1A).3.4. ApoptosisAccording to the results described above, the concentration of ozonated water should be 0.2mL/L, but fish survival does not mean that there is no damage to the tissues of the young fish (Fig.1C). When the ozonated water concentration was 1.5 mg/L, there were several signs of apoptosis, causing severe damage to the fins, respiratory system, and vascular system, and death. At concentrations of 1.0 and 0.5 mg/L, the apoptotic signal was less than at 1.5 mg/L, but still suggested serious injury to the respiratory and vascular systems and tail fins. When the concentration was 0.2 mg/L, there were a few apoptotic signals, and only minor damage to the circulatory system and the caudal fin. When the concentration of ozone water was 0.1mg/L, only basic metabolic apoptotic signals were observed. These results show that higher concentrations of ozonated water cause more serious damage to zebrafish. Thus, when the concentration of ozonated water was 0.2 mg/L, the circulatory system and tail fin were only slightly damaged, and survival was not affected. When the concentration was lower than 0.1 mg/L, the zebrafish were undamaged. Based on these experimental results, we used an ozonated water concentration of 0.1 mg/L.3.5. Comparison of caudal fin growth up after resectionThe zebrafish is a useful experimental model because it contains 70% of human protein-coding genes and 84% of the genes related to human diseases[45]. However, although clinical experiments and animal experiments have shown that ozone stimulates tissue regeneration in humans and mice, we cannot be completely sure that it promotes zebrafish caudal fin regeneration. Therefore, we first verified this phenomenon in vivo.After caudal fin resection, we recorded and compared the extent of caudal fin growth in the three groups (a, b, and c) every 12 h under a microscope (Fig.2B). In the first 12 hpa, there was no change in group a, but growth had commenced in groups b and c. At 24 hpa, caudal fin growth was apparent in all three groups, but the fins of group a were shorter than those of groups b and c. At 48 hpa, the fins of group a were still shorter than those of groups b and c, but there was no obvious difference between groups b and c. At 72 hpa, there was no obvious difference between groups a and b, but their caudal fins were shorter than those of group c, in which the outline and fin ray were almost completely reestablished. At 120 hpa, tail fin regeneration was complete in the three groups. Thus, within 24 h of excision of the tail fin, group a showed no obvious regenerative repair, but the regenerative repair of groups b and c had begun. Within 48 h of resection, the caudal fin length in groups b and c was greater than in group a. At 72 h after resection, the caudal fin outline and fin regeneration was basically complete group c, whereas the caudal fins of group a and b were completely regenerated at 120 hpa.3.6. Measured and recorded caudal fin growth after resection in three groupsThrough experimental observation, we confirmed that ozonated water promotes the regeneration of the zebrafish caudal fin. Next, we measured the growth of the caudal fin, and compared the differences in caudal fin growth in the different dose groups at each time point (Fig.2C-D).There was no difference in caudal fin length between groups a and b at 0,24, or 72 hpa. However, the two groups differed significantly at 48 hpa (p<0.01), but this difference disappeared before 72 hpa.At 0 hpa, there was no difference in the caudal fin growth in groups a and c. However, at 24,48, and 72 hpa, the caudal fin length was significantly shorter in group a than in group c at the same points (p<0.0001). Thus, the difference in caudal fin growth between groups a and c began after first 24 hpa (p<0.0001) and continued until 72 hpa.3.7. Monitoring the concentration of ozonated waterTo explain the differences in caudal fin growth in the single dose group and the multiple dose group, we tested the ozone concentrations in the water of the two groups. The ozone concentration in the single dose group decreased over time, reaching 0 mg/mL after 48 hpa. The concentration in the multiple dose group also decreased gradually over time, but because the ozonated water was refreshed every 12 hours, the concentration never reached 0 mg/mL (Fig.2E-F).These data demonstrate that ozone promotes the regeneration of the zebrafish caudal fin, and that this effect is associated with contact time.3.8.Neutrophil migration after resectionRelated research has shown that the early inflammatory response promotes tissue regeneration [43]. The migration of neutrophils (early inflammatory cells) to the wound began 3 h after resection. The number of neutrophils that migrate to a wound within a certain time reflects the extent of the tissue inflammatory reaction, and indirectly, the status of tissue regeneration. Conventional ozone is deemed an antiseptic because it kills germs and inhibits local inflammation[4]. Therefore, we determined the migration of neutrophils after the resection of the zebrafish caudal fin. At 0.5,1,2, and 3 hpa, photographs were taken to record the neutrophil migration in the caudal fins of 72 hpf Tg(mpo:GFP) zebrafish under a confocal microscope (Fig.3A). Visible results (Fig.3B) were apparent at 0.5,2, and 3 hpa, when the numbers of neutrophils migrating to the caudal fin wound were greater in the experimental groups than in the control group (p<0.01). At 1 hpa, the result was same, but the difference was smaller than at the other time points (p<0.05). Therefore, we conclude that soon after tissue damage, ozone does not inhibit tissue inflammation, but promotes tissue regeneration by promoting tissue inflammation.3.9. TNF-αAs the main factor in the inflammatory response, TNF-α has been the focus of many studies. An animal experiment confirmed that ozone inhibits the expression of TNF-α[9], whereas our results demonstrate that ozone does not simply inhibit the expression of TNF-α. Within 24 h of the excision of the tail fin, the expression of the inflammatory factor TNF-α was increased by ozone. After 24 hpa, tissue regeneration commenced after inflammation subsided, at which time ozone inhibited the expression of TNF-α(Fig.4). Thus, ozone promotes tissue regeneration by regulating the expression of TNF-a two-way. According to our immunoblotting results, the role of ozonated water in regulating TNF is not only reflected in the expression of the Tnf-a gene, but also at the protein level. However, the immunoblotting results are not completely consistent with the gene expression data, suggesting that, in addition to regulating gene expression, ozone also regulates protein translation, thus adjusting the amount of TNF to more accurately meet the requirements of tissue regeneration.3.10. Mechanism by which ozone regulates TNF-αHow does ozone regulate TNF-α? Does it directly affect TNF-areceptors or indirectly affect TNF-α by regulating other genes? We examined the expression of two genes upstream from TNF-α, IL-10 and STAT3, and the expression of IL-1β which is also regulated by IL-10 and STAT3. Our results show that STAT3 expression was induced by IL-10 after 12 hpa, and the expression of these two proteins tended to be similar. The trend in IL-1β expression show in Fig.5 was as previously reported[21].We can see when the IL-1β expression increase,the IL-1β expression of ozone group is higher than the control group.When the IL-1β expression decrase,the IL-1β expression of ozone group is lower than the control group/This experimental results are basically consistent with the known expression of TNF-α. TNF-α is regulated by the expression of STAT3, but this regulation acts two-way. The expression of these genes was related to the regeneration time, and 24 hpa was the dividing time (Fig.5).4. DiscussionOzone is a strong oxidizing gas with strong sterilization properties[29-32]. It has been used in clinical practice for a long time to kill germs in wounds, promote wound healing, and clean wounds. However, in recent years, it has been demonstrated that ozone treatment not only promotes wound healing, but does so more rapidly than other methods of disinfection[1-6]. Ozone treatment is often used to heal wounds that have failed to heal when treated with conventional disinfection. The results discussed above indicate that ozone promotes wound healing not only because it provides a sterile environment, but also because other factors are involved in the promotion of regeneration. Relevant clinical data and basic experiments have suggested a series of possible mechanisms, including antiseptic sterilization, the promotion of microcirculation, and the promotion of cell regeneration mechanisms[7-9]. Although many mechanisms may be involved in ozone-stimulated regeneration, the major mechanism is still unclear.Regeneration is a complex process, regulated by a variety of genetic pathways. At present, the main pathways known to control regeneration include the WNT, FGF, activin A, retinoic acid, hedgehog signaling, etc.[33-36,38-42]. Recent research suggests that although various regulatory pathways and inflammatory pathways are involved in the regulation of tissue regeneration after injury, the expression of inflammatory cytokines in the early stage of trauma affect regeneration. The increased expression of early inflammatory factors promotes tissue regeneration[43]. However, the persistent high expression of inflammatory factor inhibits regeneration.The effects of ozone on tissue regeneration were investigated with zebrafish as the model organism. First, we confirmed the injury and survival of 72 hpf zebrafish in different concentrations of ozonated water, to determine the safe concentration of ozonated water for use in the experiment. When the ozonated water concentration was 0.2 mg/L, the zebrafish body was mildly damaged, but the damage was reversible and did not affect the survival of 72 hpf zebrafish. However, tissue damage can exert other effects on experimental factors, such as gene expression, in the zebrafish, which may influence the experimental results to some extent. Therefore, although this concentration did not affect the larval survival rate, it could not be used as the experimental concentration. When the concentration of ozonated water was 0.1 mg/L, the apoptotic signals in the larval body were identical to those for normal physiological apoptosis (control group in an ozonated water concentration of 0 mg/L), and did not affect the results of the experiment. Thus,0.1 mg/L ozonated water not only ensured the accuracy of the experimental results, but maximized the ozone effect.Using the model described above, we compared the growth of the zebrafish caudal fin in the control group (normal egg water), single drug group (ozonated water given only once), and multiple drug group (ozonated water refreshed every 12 hours) at different time points after resection. The ozone concentrations in the single dose and multiple dose groups were>0 mg/L and their fins grew faster than in the control group. At 48 hpa, the ozone concentration in the single dose group was 0 mg/L, and the difference in the caudal fin growth rates between the control and single dose groups disappeared. The caudal fin growth rate in the multiple drug group remained higher than both. These results confirm that ozonated water promotes caudal fin regeneration in the zebrafish, and the effect disappears as the ozonated water is gradually depleted. Therefore, the differences in the rates of zebrafish caudal fin growth between the control group, single dose group, and multiple dose group were attributable to ozone. Thus, ozone promotes the regeneration of zebrafish caudal fin after resection.Related research has shown that the early inflammatory response promotes tissue regeneration [43]. The migration of neutrophils (an early inflammatory cell) to the wound began 3 hpa. The number of neutrophils that migrate to a wound within a certain time reflects the extent of the tissue inflammatory reaction, and indirectly affects tissue regeneration. Conventionally, ozone is considered an antiseptic agent that kills germs and inhibits local inflammation[4].On the contrary, in our experiment, ozone did not inhibit inflammation in the early stage of trauma. Using 72 hpf Tg(mpo:GFP) zebrafish, we confirmed that ozonated water promotes neutrophil migration to a wound. Thus, ozone promoted wound inflammation in the early stage of wound healing, accelerated the removal of dead cells from the caudal fin, and promoted tissue regeneration. These results suggest that ozonated water promotes caudal fin regeneration by promoting inflammation.TNF-α, the main inflammatory factor in the body, plays an important role in many physiological processes. As well as promoting inflammation, TNF-α directly regulates the cell cycle, accelerates cell apoptosis, and inhibits cell regeneration. Research has confirmed that ozone can inhibit the expression of TNF-α and promote tissue regeneration. However, continuous high-level expression of TNF-α and IL-1β delays wound healing and tissue regeneration. Our results show that ozone did not simply inhibit the expression of TNF-α. Within 24 hpa, ozone promoted the expression of TNF-α and tissue inflammation. Because of the powerful sterilization effect of ozone, this should be aseptic inflammation. According to the theory of inflammation, ozone promotes inflammation by promoting TNF-α in the early stage of trauma, thus promoting early tissue regeneration. After 24 hpa, the inflammation in the caudal fin had almost disappeared, and the regeneration process began, so the effect of ozone in promoting TNF-α weakened, and gradually changed into inhibition, thereby promoting tissue regeneration. This result confirms that ozone regulates TNF-α by adjusting it two-way, which are fully compliant with the requirements for tissue regeneration.TNF-α expression is regulated by many proteins in the body, including IL-10. When the expression of STAT3 is promoted, IL-10 inhibits the expression of TNF-α and IL-1β. Our experimental results are consistent with these regulatory pathways.This pathway was conventionally activated withing the first 24 hpa, whereas after 24 hpa, an alternative mechanism was activated that inhibited TNF-α expression. This suggests that the mechanism by which ozone regulates TNF-α is complex, and not simply dependent on IL-10. Even though STAT3 is directly regulated by IL-10, it is not fully regulated by IL-10. Thus, the regulation of gene expression by ozone in the body is complex and requires further research.A limitation of this study was that because the zebrafish is an aquatic animal, ozone cannot be applied specifically to the wound. In theory, this should not influence our experimental results, but there is as yet no appropriate way to validate this assumption or any theoretical support for it. Although we have confirmed that ozone promotes tissue regeneration, this was only a preliminary study of the mechanisms involved, and further studies of these mechanisms are required. |
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