Dermatologic Therapy - 2020 - Kaleci - Efficacy of Resveratrol in The Wound Healing Process by Reducing Oxidative Stress

Received: 22 July 2020 Revised: 14 September 2020 Accepted: 23 September 2020
DOI: 10.1111/dth.14357
ORIGINAL ARTICLE
Efficacy of resveratrol in the wound healing process by

reducing oxidative stress and promoting fibroblast cell
proliferation and migration
Belisa Kaleci | Meral Koyuturk
Department of Histology and Embryology,

Cerrahpasa Faculty of Medicine, Istanbul Abstract
University-Cerrahpasa, Istanbul, Turkey Oxidative stress (OS) is implicated in the pathogenesis of diabetic, chronic, and
Correspondence inflamed wounds. In these cases, wound healing is difficult and remains a big clinical
Belisa Kaleci, Department of Histology and challenge. As fibroblasts are crucial in wound healing by producing extracellular
Embryology, Cerrahpasa Faculty of Medicine,
Basic Science building, A block, Istanbul matrix and wound contraction, the researchers investigated the resveratrol (RES)
University-Cerrahpasa, Kocamustafapaşa, effects as a potential therapeutic alternative, on an in vitro OS and wound healing
Fatih Istanbul, Turkey.
Email: belisa.kaleci@istanbul.edu.tr model established by 3T3 Swiss Albino fibroblasts. OS was induced by H2O2, and
1-100 μM RES doses were applied on cells for up to 48 hours. The cell proliferation
Funding information
Bilimsel Araştirma Projeleri Birimi, Istanbul was assessed by the population doubling time and bromodeoxyuridine immunocyto-
Üniversitesi, Grant/Award Number: 21032 chemistry. The OS levels were determined by fluorescence staining and all values
were calculated by the ImageJ program. The collagen-I expression (COL1A2 mRNA)
was evaluated by a real-time PCR. Cell migration was evaluated by wound closure
assay and the ultrastructure was evaluated by electron microscopy. The cell prolifera-
tion index significantly decreased in H2O2 incubation and increased with RES applica-
tion. RES application reduced OS levels and stabilized cell proliferation, but no
differences were found in COL1A2 mRNA expression. RES-treated cells showed bet-
ter proliferation, migration rates, and ultrastructural preservation. RES administration
decreases OS levels and improves the healing process by increasing cell proliferation
and migration quality, suggesting it is a powerful candidate for the treatment of skin
wounds.
KEYWORDS
cell migration, cell proliferation, fibroblast, oxidative stress, resveratrol
1 | I N T RO DU CT I O N WH impairment and complications are common among diabetic-,2,3

immunosuppressed-,4,5 and radiotherapy-treated,6,7 patients. The man-
Wound healing (WH) is a complex process of cellular and molecular agement and treatment of WH are challenging and expensive. A recent
mechanisms that begins after injury and is essential to restoring skin study from the United States showed that total Medicare spending on
integrity and function. In normal physiological conditions, the WH all wound types ranged from $28.1 to $96.8 billion.8 Different theories
process involves four stages: hemostasis, inflammation, proliferation, about WH impairment in diabetes have been asserted, as diabetes is
and remodeling, which are superposed in time and space.1 Interrup- associated with a persistent low-grade inflammatory state, prolonged
tion of the process at different phases of these stages leads to compli- inflammatory response after injury, impaired angiogenesis and
cated and impaired WHs. vasculogenesis, microvascular and macrovascular dysfunction, impaired
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2 of 9 KALECI AND KOYUTURK
neuropeptide signaling, and hypoxia.3,9 Hypoxia is well-known as a free As a result of dose determination assays for H2O2 and RES, the
oxygen species (ROS) amplifier.2,9,10 The oxygen radicals are produced experimental groups were assigned as: control group, 500 μM H2O2
normally during the inflammatory phase by neutrophils, and they func- group, 1 μM RES group, 500 μM H2O2 + 1 μM RES group. The PDT,
tion as a defense against invader species. ROS levels are equilibrated BrdU immunostaining, ROS reactivity, cell migration test, Real Time-
between production and the scavenger process. Overproduction of ROS Polymerase Chain Reaction (RT-PCR), and transmission electron
causes cellular oxidative stress (OS). OS is associated with lipid peroxida- microscopic (TEM) analyses were performed for the final experimental
tion, protein modification, and DNA damage, which impair the WH pro- groups (Control, H2O2, RES, H2O2 + RES) with the defined doses and
cess via inducing cell apoptosis and/or senescence.10 Consequently, ROS time intervals.
also affects various cells of the connective tissue, such as fibroblasts.
These cells play a key role in the WH process5 by secreting extracellular
matrix components like collagen and fibronectin, and by remodeling. 2.3 | PDT analysis
Fibroblast contraction forces are essential for wound closure.11-13
Current therapies for WH involve the wound debridement, local The defined doses of H2O2 and RES were applied to 24-hour cultured
dressings, and conjugated (adjunctive) therapies.14 In addition to a cells for 48 hours. After the application, the cells were collected and
variety of treatments, some wound lesions certainly show high resis- counted in the Neubauer-Improved (Marienfield) hemocytometer.
tance to the common methods of treatment and therapeutic agents, PDT was calculated by following formulas18:
which is why discovering new therapeutic agents and treatment
methods continues to be the goal of recent research. Population doubling = log ðtotal cell number=cultured cell numberÞ × 3:33
0
Resveratrol (3,4 ,5-trihydroxy-trans-stilbene; RES) is a natural
polyphenolic compound found in more than 72 plant species, such as Population doubling time = Total time=population doubling
grapes, peanut products, and blueberries.14,15 Different studies have
shown that RES has antiinflammatory, antiaging, anticancer, anti-
diabetic, cardioprotective, and antioxidant properties. RES also pro- 2.4 | Immunocytochemistry staining
motes re-epithelialization of diabetic wounds in experimental
animals,16 and it is cost-effective and easy to administrate.14,15,17 At the last hour of experiments (H2O2 and RES exposure) BrdU
In this study, the researchers analyzed the effects of RES as a (20 mM) was added to the medium and incubated for 60 minutes at
potential therapeutic for improving WH with an in vitro WH model 37 C. In the end of 48th hour based on previously established
for fibroblasts under OS. protocols,20 the cells were fixed and routine immunocytochemical
procedures were applied.
2 | MATERIALS AND METHODS

2.5 | OS staining
2.1 | Cell culture
The OS levels of living cells were measured by using the CellROX
The 3T3 Swiss Albino Cell line was supplied by the American Type Deep Green Reagent (Invitrogen). Before the end of the experiment,
Culture Collection. Cells were cultured in a medium containing 1:1 the CellROX Deep Green Reagent was added with a final concentra-
DMEM-F12 + DMEM High-Glucose + 10% FBS (fetal bovine serum) tion of 5 μM and incubated for 30 minutes at 37 C.
+ antibiotics (100 μg/mL penicillin G + 100 μg/mL streptomycin), and The BrdU and OS levels were evaluated under the photo-

incubated at 37 C with 5% CO2 + 95% air mixture and moisture. microscope by using the ImageJ (ImageJ 151g NIH, The United States)
When confluence reached 80% density, cells were collected by using program to count positive cells.
trypsin and then centrifuged. Cell pellets were suspended, attached to
sterile coverslips, mediums were added and prepared for experiments.
2.6 | In vitro scratch assay
2.2 | Experimental design To test the cell migration performance of fibroblasts, an in vitro
scratch assay was performed by scratching a line in the middle of the
At the beginning of the study, to determine the effective doses, the 24-hour cultured cells.19 Then, RES and H2O2 were applied for 24 and
researchers applied 10, 25, 50, 100, 250, 500, 750, and 1000 μM 48 hours and examined under an inverted microscope. Cell migration
doses of H2O2 (Merck) and 1, 5, 10, 15, 25, 50, and 100 μM of RES or the distance of each scratch closure was calculated by the formula
(Resveratrol ≥99% HPCL, Sigma-Aldrich). The testing methods used ΔX (μm) = X1 − X2 (X1 is the distance between cells border at time
were population doubling time (PDT), bromodeoxyuridine (BrdU) 0 of generating the scratch, X2 is the distance between cells border at
immunostaining, and ROS reactivity was measured by the CellROX the end of 24th hour). After 48th hour, the data were not counted
fluorescence method. since the scratches were closed.
KALECI AND KOYUTURK 3 of 9
2.7 | Real time-polymerase chain reaction In vitro, preliminary tests showed that RES doses of more than
10 μM were toxic for the cells, in compliance with PDT indexes
In the first step, mRNA was isolated from the cells of all experimental (data not shown). To determine effective doses of RES, 1 μM RES,
groups. Afterward, cDNA synthesis was carried out by using a script 5 μM RES, and 10 μM RES were analyzed in combination with
cDNA synthesis kit (Jena Bioscience). Then real time-polymerase 500 μM H2O2.
chain reaction (RT-PCR) was performed by using the q-PCR Green On the one hand, the control and 1 μM RES groups showed sta-
Master with the Uracil-N-Glycosylase (UNG)/low ROX kit (Jena Bio- tistically similar results for the PDT index (Figure 1A). On the other
science, Cat no: PCR306). Ct values from the RT-PCR analysis were hand, incubation of the H2O2 group resulted in PDT indexes more
calculated with the 2−ΔΔCt algorithm method.20 than two times higher than the control group, while the indexes of
the H2O2 + RES group decreased significantly, compared with the
H2O2 group (P < .001) (Figure 1B).
2.8 | TEM analysis
The spheroids established by 3T3 fibroblasts from the final experimen- 3.2 | BrdU immunocytochemistry analysis
tal groups were prepared and selected for the TEM analysis, in accor-
dance with the literature.21 Araldite blocks were trimmed, semi-thin The highest proliferation rate was determined to be in the 1 μM RES
sections were analyzed, and finally, ultra-thin sections were contrasted group, compared with the other groups (P < .05) (Figure 2). Experi-
for evaluation and photographed under the TEM (Jeol: JEM-1011). mental group comparisons indicated that the H2O2 + 1 μM RES group
had a higher proliferation rate than the H2O2 only group (P < .001)
and a similar rate to the control group (Figure 2).
2.9 | Statistical analysis
All data were statistically evaluated with the SPSS program (IBM SPSS 3.3 | OS analysis
Advanced Statistics 20.0, 2011). Mean values were compared with a
one-way ANOVA, and variations between groups were compared The OS levels of the RES-applied groups showed no significant differ-
with the post-hoc Tukey-Kramer Multiple Comparison Test and the ences compared with the levels of the control group, but the best
Tukey HSD Multiple Comparison Test. P values <.05, <.01, and <.001 results were obtained by the 1 μM RES dose (Figure 3). The OS levels
were accepted as statistically significant. of the experimental groups (Figure 3), in comparison to the control
group, demonstrate that H2O2 application significantly increased the
level of OS (P < .001). RES addition decreased the levels of OS signifi-
3 | RESULTS cantly (P < .001) (Figure 3E1).
3.1 | PDT analysis

3.4 | In vitro scratch assay
According to the preliminary results of the dose assays, administration
of 500 μM H2O2 for 48 hours was enough to generate OS since the In the final experimental groups, cell migration was rated by an in vitro
PDT index doubled in the 500 μM H2O2 group, compared with the scratch assay; the results from the measurements are given in Figure 4.
control group (P < .001). The rest of the experiments were continued Compared with the control group, the shortest migration distance was
with this dose. detected in the H2O2 group, (P < .001). The longest migration distance
F I G U R E 1 PDT (%) results. RES dose groups: (1, 5, and 10 μM), (A). Experimental groups including Control, 500 μM H2O2, 1 μM RES, 500 μM
H2O2 + 1 μM RES on 3T3 fibroblasts, *P < .001 vs other groups, (B)
F I G U R E 2 Photomicrographs and graphics of BrdU proliferation assay. RES dose groups (A-E): A, Control; B, 1 μM; C, 5 μM; D, 10 μM,
×200. E, Graphical presentation of BrdU indexes. *P < .05 vs other groups. Experimental groups (A1-E1): A1, Control; B1, 500 μM H2O2; C1,
1 μM RES; D1, 500 μM H2O2 + 1 μM RES, ×200. E1, Graphical presentation of BrdU indexes. *P < .05 vs other groups, ***P < .001 vs other
groups
(even longer than the control group) was observed in the H2O2 + RES The control group showed normal ultrastructure of the cell mem-
group, but the difference was not statistically significant. brane, euchromatic nuclei with heterochromatin in small amounts, and
eccentrically located nucleoli. Intercellular spaces and junctions had a
regular appearance. Mitochondria had ordinary crista, and the cyto-
3.5 | Collagen expressions plasm had many free ribosomes (Figure 5A).
In H2O2-applied spheroids, fibroblasts were hypertrophic and
An RT-PCR assay of COL1A2 mRNA expression was performed to vacuolated. The mitochondria of the cells were locally degenerated
evaluate the effects of H2O2 and RES application on collagen produc- with remarkably damaged crista. In general, the intercellular distance
tion in 3T3 cells (Table 1). Comparing all groups in terms of COL1A2 increased, and some intercellular junctions were not preserved locally
mRNA expression levels, the lowest expression level was observed in (Figure 5B).
the H2O2 group while the highest level was seen in the RES group, The TEM images of the RES group indicated that the cells pre-
but the differences were not statistically significant. served their ultrastructure of the nucleus, nucleolus, mitochondria,
and normal intercellular junctions. The cytoplasm had many free ribo-
somes and showed less vacuolization (Figure 5C).
3.6 | Ultrastructural analysis The TEM images of the H2O2 + RES group also showed normal
structures of the nuclei, nucleoli, and cell membrane. A low number of
The TEM micrographs of the final experimental groups established by autophagosomes were detected. The cytoplasm had very few
500 μM H2O2 and 1 μM RES doses are given in Figure 5. degenerated mitochondria and even fewer vacuoles, compared with
F I G U R E 3 Photomicrographs and graphics of oxidative stress (OS) levels (%). RES dose groups (A-E): A, Control; B, 1 μM RES; C, 5 μM
RES; D, 10 μM RES, ×200. E, Graphical presentation of OS ratios. Experimental groups (A1-E1): A1, Control; B1, 500 μM H2O2; C1, 1 μM RES;
D1, 500 μM H2O2 + 1 μM RES, ×200. E1, Graphical presentation of OS ratios. ***P < .001 vs other groups
the H2O2 group. The cytoplasm showed preserved structures of Golgi, better antioxidative activities in an OS environment. Only RES treat-
many free ribosomes, and mitochondria with preserved crista. Space ment has an inductive effect in terms of cell proliferation while the
of intercellular distance and intercellular junctions were preserved combination with H2O2 may exhibit its protective effect against
(Figure 5D). OS. This study showed that an available antioxidant like RES has pro-
tective effects, the ability to improve biological cellular functions, and
potential as an adjuvant treatment in WH. However, further clinical
4 | DISCUSSION studies are required to elucidate this relationship.
Low concentrations of ROS stimulate the cell growth of gingival
Antioxidants have been used as supplementary and alternative treat- fibroblasts and epithelial cells in culture conditions, but high concen-
ments to conventional therapies for treating diseases associated by trations induce free radical chain reactions, inducing tissue
OS. In a study that examined the biological effects of antioxidants in damage.23,24
an H2O2-induced OS model on human gingival fibroblasts, Orihuela- Various RES effects on tissues and cells depend on the time and
Campos et al showed that a 50 μM concentration of RES increases dose administration.25 Varying concentrations of RES can affect OS
cell proliferation, inhibits ROS production, and stimulates mitochon- levels differently in different cell lines. RES has beneficial effects on
drial respiration in human gingival fibroblast cell cultures.22 In this health in medial doses, but high concentrations have shown
study, different doses of RES were used to investigate its effects on proapoptotic effects.26 In this manner, Nakagawa et al reported that
cellular functions, PDT index, proliferation ratio, and ROS levels in an an RES concentration ≤22 μM increased cell proliferation in human
in vitro OS model. The best PDT index and the highest cell ratio of mammary cancer cell lines, and concentrations ≥44 μM suppressed
the S phase were detected in the 1 μM RES group. Increasing doses of cell growth.27 In the literature, there are few studies about the effects
RES may lead to an increase in ROS levels. Additionally, RES shows of RES on proliferation rate, OS levels, and ultrastructural effects.28,29
F I G U R E 4 Migration assay findings of experimental groups (T0/ T24 hour). A/A1, Control group; B/B1, 500 μM H2O2 group; C/C1, 1 μM
RES group; D/D1, 500 μM H2O2 + 1 μM RES group, ×100. E, Graphical presentation of migration distances. ***P < .001 vs other groups
TABLE 1 COL1A2 mRNA RT-PCR findings of the experimental

groups the level of the control group, so RES may not be enough to totally
eliminate H2O2 effects.
Groups 2−ΔΔCt Pa Pb
In vitro studies showing the effects of RES on cellular morphology
Control 1 ± 0c – >.05 are limited and insufficient. Suwalsky et al investigated the protective
H 2O 2 0.75 ± 0.10 >.05 – effect of RES on human erythrocytes against oxidative damage and
RES 1.43 ± 0.36 >.05 >.05 their scanning electron microscope (SEM) findings showed that espe-
H2O2 + RES 1.33 ± 0.26 >.05 >.05 cially 1.0-10 mM doses of RES led to morphological changes and
affected cell membranes, driving erythrocytes to differentiate into
Notes: Values are given as mean ± SE mean.
a
vs control group. echinocytes.28 In another study, Opipari et al examined the effects of
b
vs 500 μM H2O2 group. a 24-hour incubation with low concentrations (50-200 μM) of RES on
c
Relative mRNA expression levels were normalized with actin gene. ovarian cancer cells, which showed that RES exposure resulted in
autophagocytic granule appearance, chromatin condensation, and
organelle degradation.29 In the present study, the TEM examination
According to the parameters used in this in vitro model of 3T3 fibro- has revealed that the cytoplasm of RES-treated cells had a low num-
blasts, RES significantly decreases the OS level (P < .001). The immu- ber of vacuoles. Therefore, RES cannot be considered precisely as a
nofluorescent results showed significantly increased ROS in all preserving agent under normal conditions but exhibits preserving
H2O2-treated groups, and the highest levels were detected in the behaviors under OS.
H2O2 only group. The selected dose of RES for this study did not The H2O2 + RES applied fibroblasts had a generally normal ultra-
stimulate OS production by itself. When RES was added to the H2O2 structural appearance but was associated with few mitophagic bodies
group, OS levels dramatically decreased, proving its benefits as an and vacuoles, suggesting that RES may not be sufficient to completely
antioxidant. But the OS level for the H2O2 + RES group did not reach protect the cellular ultrastructure against H2O2. Based on the
F I G U R E 5 TEM photomicrographs of experimental groups (A,C,D ×10 000 and B ×12 000). A, Control group; B, 500 μM H2O2 group; C,
1 μM RES group; and D, 500 μM H2O2 + 1 μM RES group. Small photomicrograph with white frame ×60 000. N, nucleus; Nu, nucleolus; Star,
mitochondria; Black arrows, cell membrane; Black circles, damaged mitochondria; White arrows, intercellular junctions; V, vacuoles
literature, ROS inhibits the mitochondrial fusion or stimulates the fis- and hydroxyproline (collagen) levels in fibroblasts that were obtained
sion.30,31 Mitochondrial fusion has claimed to be a saving or protec- from hypertrophic scar tissue, which demonstrated that RES concentra-
tive mechanism for cellular damage that increases under stress tions between 75 and 300 μM started to decrease the collagen levels
conditions.30 Robb El et al reported that RES leads to mitochondrial during the 24-hour incubation, and this decrease continued past
31
fusion in fibroblasts through mitofusin-2 protein. In correlation with 48 and 72 hours, depending on the dose.33 The study concluded that
these findings, the results of this study suggest that RES combination RES in appropriate doses can control the balance between collagen
may generate ultrastructural protective effects against H2O2 and may synthesis and accumulation and, thus, may be used in WH.33 In this
be used against degenerative effects of OS inducers. examination of H2O2 and RES applications on COL1A2 mRNA expres-
In the literature, few studies examined the effect of RES on cell sion in 3T3 fibroblast cells by RT-PCR, the increase in expression level
migration while in the presence of OS.25 Bosutti et al showed that was observed in H2O2 + RES groups but was statistically insignificant.
RES affected the plasticity of myoblasts, depending on the doses, and So, an increase in this level may be effective in rapid recovery without
that cell migration is stimulated with low doses of RES, whereas high causing hypertrophic tissue formation.
25
doses inhibit cell migration. In the same study OS at high concentra- The result of this study shows that RES has good protective
tions weakens cell migration. As a result, the shortest migration dis- effects on fibroblasts in the in vitro WH model under OS factors. It is
tance was observed in the H2O2 group. However, a remarkable important to remember that the effects of RES can change depending
improvement in migration distance was observed in the H2O2 + RES on doses and it may be preferred in limited doses as a therapeutic in
experimental group. Also, the migration distance was longer in the the WH process, especially with continuing OS. Low doses of RES
H2O2 + RES group than in the intact control group but was not statis- might be an adjuvant therapeutic agent in WH; however, further stud-
tically significant. ies are required to elucidate clinical trials.
In addition to the antioxidative effects, research has showed that
RES plays a role in the regulation of collagen production and profibrotic ACKNOWLEDG MENTS
factors in conditions such as WH.32,33 Zeng et al investigated the We acknowledge the Research Fund of Istanbul Üniversitesi for
effects of RES on Type I and Type III procollagen mRNA expression supporting the present work Project No. 21032.
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Received: 22 July 2020 Revised: 14 September 2020 Accepted: 23 September 2020

Efficacy of resveratrol in the wound healing process by

Belisa Kaleci | Meral Koyuturk

Department of Histology and Embryology,

cell migration, cell proliferation, fibroblast, oxidative stress, resveratrol

1 | I N T RO DU CT I O N WH impairment and complications are common among diabetic-,2,3

Dermatologic Therapy. 2020;33:e14357. wileyonlinelibrary.com/journal/dth © 2020 Wiley Periodicals LLC. 1 of 9

2 | MATERIALS AND METHODS

3.1 | PDT analysis

TABLE 1 COL1A2 mRNA RT-PCR findings of the experimental

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