Oxidative stress is characterized by an increase in endogenous reactive oxygen species such as hydrogen peroxide and superoxide. Low levels of hydrogen peroxide regulate physiological cellular events, while high levels of hydrogen peroxide observed in the inflammatory state generally cause loss of function and/or cytotoxicity in skin cells. In these conditions, hydrogen peroxide plays an indirect role by regulating metabolic pathways and intracellular signal cascades. Oxidative stress plays an important role in the pathogenesis of diseases such as: cardiovascular diseases, cancer, neurological diseases, diabetes, and aging. Defense mechanisms related to oxidative stress include: blocking and repair of these mechanisms, physical defense, and antioxidant defense. Enzymatic antioxidant defenses include: superoxide dismutase (SOD), glutathione peroxidase (GPxs), and catalase(Brito et al., 2006; Li et al., 2006; Pizzino et al., 2017; B. Wang et al., 2007).
Glutathione peroxidases (GPxs) effectively reduce hydroperoxidases to water and alcohol. These enzymes provide an antioxidant protective shield by removing reactive oxygen and nitrogen species from the body. In addition, GPxs are not only critical to this detoxification process, but also play a key role in regulating apoptosis, inflammation, and other processes(Brigelius-Flohé & Maiorino, 2013). Superoxide dismutases (SODs), a group of metalloenzymes, release hydrogen peroxide and molecular oxygen by splitting superoxide anion free radicals and reducing O2- levels (excessive accumulation leads to cell damage) (Younus, 2018). MDA is a lipid peroxidation product and has been reported in several studies to be an indicator of oxidative stress. When free radicals are formed in the body, they attack polyunsaturated fatty acids(Ayala et al., 2014).
GPX enzyme is a selenium-dependent enzyme and is known to have 8 subtypes in the human body. In previously conducted studies, a strong mRNA expression of the GPX1 enzyme in the inflammatory phase of wound healing provides a positive contribution to healing(Hariharan & Dharmaraj, 2020). In the study conducted by Iuchi et al in 2010, it was reported that GPX1 enzyme levels have decreased in the early phase of the wound in immunosuppressive rats which negatively affected the wound healing(Iuchi et al., 2010). A separate study, identified an increase of GPX1 within 3–7 days after skin damage(Woo et al., 2010). It becomes clear that low levels of GPX1 enzyme, which is known to play an important role in scavenging oxygen radicals, will adversely affect wound healing. This study found that GPX levels measured by ELISA in the RSV group were higher than in the control group. The excess amount of GPx indicates that the detoxification of reactive oxygen radicals is disproportionate, suggesting that it prevents cellular damage and delay in wound healing. We posit Resveratrol has a positive effect on wound healing due to its protective antioxidant effect.
Superoxide dismutase (SOD) is an important antioxidant that quickly cleanses reactive oxygen from the body and is known to have 3 subtypes. The more actively it can remove reactive oxygen radicals, the stronger its potency is considered. In other words, more SOD activity corresponds with the removal of various oxygen types from the body(Yoshida et al., 2020). In our study, no statistically significant difference was found in the amount of SOD between the RSV and control groups on the 14th day. The primary reason for this may be that the total amount of SOD was measured in our study, and no analysis was made of its subgroups. The authors of this study think that this situation can be overcome by isotyping in further studies. In addition, if this measurement had been made earlier, on the 7th day, perhaps different results could have been obtained since the inflammation was more intense in this period. The researchers of this study suggest that this situation should be taken into account in future studies.
Polyphenols are a family of antioxidants that include flavonoids, anthocyanins, phenolic acids, lignans, and stilbens. All are derivatives of phenyl alanine and contain aromatic ring with reactive hydroxyl group. Resveratrol (3,5,4-trihydroxystilbene), which is a subclass of stilbens, has trans and cis isomeric forms(Signorelli & Ghidoni, 2005).Resveratrol functions as an antioxidant in vivo and can capture peroxyl radicals in the skin; thus, it protects the skin from ischemia-reperfusion damage. Many studies have shown that Resveratrol has the ability to capture both superoxide and hydroxyl radicals. Resveratrol's in vivo antioxidant properties are strengthened by its ability to increase nitric oxide synthesis. Here, as an in vivo antioxidant, nitric oxide is capable of capturing superoxide. Resveratrol also maintains the intracellular concentrations of antioxidants found in biological systems. The measurement of oxidative stress in organisms can be made by evaluating MDA production. Cell and tissue damage caused by reactive oxygen radicals can be assessed with lipid peroxidation products such as MDA(Cherian et al., 2019). Resveratrol, a polyphenol found in grape, red wine, mulberry, and peanuts and is well-known for antioxidant properties. Its antioxidant capacity is due to the OH-group yielding electrons and its aromatic ring(Wu et al., 2019). The reason for this may be related to timing. In our study, biopsies were taken and evaluated on the 14th day. Perhaps lower MDA rates in the RSV group would have been detected if biopsies were taken during the period of higher inflammation. The authors of this study encourage further studies to confirm this theory.
Resveratrol induces anti-apoptotic signals to protect the skin. One study found that Resveratrol reduced protein tyrosine phosphorylation mediated by endothelin-1. Resveratrol has been shown to be anti-apoptotic effective(Das & Maulik, 2006). Although a parameter evaluating the anti-apopitic effect of RSV was not used in this study, it can be speculated that it shows this effect due to its healing effect on the wound, but studies designed accordingly are needed.
Blood flow to the damaged skin is related to the extent of vascularization. Laser Doppler flowmetry may be useful in measuring this. In this study, this parameter was evaluated with the PeriScan PIM 3 System Laser Doppler Blood Perfusion Imager. Microcirculation of the skin can be measured with the said device. While there was no difference between the groups on days 0, 7, and 14 in open wounds, the blood flow was found to be higher in the control group on day 21. The increase in perfusion in the open wound on the 21st day in control group was evaluated as a positive finding that RSV decreased the wound perfusion towards the last stage of healing. However, in histopathological studies, no statistical difference was found between the RSV and control groups on the 21st day. Examining the values of the 14th day, although neovascularization was higher in the RSV group, this could not be supported by Laser Doppler flowmetry. This can be attributed to the fact that the flow has not increased in immature vessels despite the increased neovascularization, but this must be proven by further studies. Ayrıca, The higher incidence of neovascularization in the RSV group on day 14 indicates the efficacy of RSV in the mid-stage of wound healing. Although there is no difference in perfusion, from the current situation, the effectiveness of carrier molecules in RSV may be responsible.In closed wounds, a statistically significant difference was not detected in the control and RSV groups on days 0, 7, 14, and 21. In histopathological studies, again, no significant difference was found in the control and RSV groups in terms of neovascularization. Accordingly, RSV may not be effective in the neovascularization phase of wound healing, but future controlled trials are needed to prove this.
It is known that vascular endothelial growth factor (VEGF) plays a specific and powerful role in the growth and migration of endothelial cells; thus having a central role in angiogenesis. Resveratrol plays a role in angiogenesis by providing a regulatory effect on VGEF expression with the help of polyphenols it contains. Previous studies have shown VEGF initiates the formation of granulation tissue(Afshar et al., 2017; H. Wang et al., 2010). In our study, the increased neovascularization (on day 14-open wounds) and granulation (on day 21-closed wounds) may have occurred due to Resveratrol causing VEGF secretion. However, VEGF measurement to prove this was not included in the design of this study. In our study, RSV caused significant histopathological changes in higher level of neovascularization and granulation rate compared with the control group.
This study adds to the existing literature by identifying the partial effects Resveratrol has on the wound healing. Researchers of this study recommend international community to plan and conduct further studies.
Study Limitations:
In this study, the fact that RSV was applied once a day instead of two, or more, may be the reason we observed minimal healing effect on the wounds. More robust parameters are required for future research.