EGCG inhibits hypertrophic scar formation in a rabbit ear model

Hypertrophic scarring is a common skin fibro‐proliferative disease, but currently there has no satisfactory drugs for anti‐scar treatments. Previous study showed that epigallocatechin gallate (EGCG), the main catechin in green tea, improved wound healing and tissue fibrosis in both rats and mice. In the present study, the therapeutic effects of EGCG on hypertrophic scar were analyzed using a rabbit ear hypertrophic scar model.

of surgical patients may have secondary hypertrophic scar. 1,2 In developed countries, about 100 million people have scar after surgery every year. 3 In the United States, about 12 billion dollars are paid for treating skin scars every year. 3,4 Therefore, it is of great significance to explore a suitable drug for the prevention and treatment of hypertrophic scar.
The most typical features of hypertrophic scar tissue are the differentiation of fibroblasts into myofibroblasts. During this process, it will produce a large amount of extracellular matrix (ECM) partially resulting from the upregulation of transforming growth factor β1 (TGF-β1), collagen I (Col I), collagen III (Col III), and α-smooth muscle actin (α-SMA). 5,6 Drugs with the abilities to inhibit the expression of the above genes may assist in improving hypertrophic scarring.
Epigallocatechin gallate (EGCG, Figure 1A), an active compound isolated from green tea, 7 exhibits numerous biological properties, including anti-oxidative, anti-inflammatory, and anti-apoptotic. 8 Moreover, EGCG has been shown to have the anti-fibrosis effect on myocardial fibrosis, 9 lung fibrosis, 10 renal fibrosis, 11 and skin fibrosis. 12 The antifibrotic activities of EGCG on skin were also proved by many researchers. Park et al. found that EGCG suppressed keloids growth and collagen production using a keloid nude mice model 13 ; Syed et al. showed that EGCG inhibited the keloid tissues growth and induced human keloid tissue shrinkage in a human keloid OC model. 12 Moreover, both zonal priming and direct EGCG application showed a beneficial role for scar therapy in a double-blind trial. 14 These suggest its potential for improving hypertrophic scar and deserve further investigations.
In the present study, the role of EGCG in the prevention of hypertrophic scar formation were investigated using a rabbit ear hy-

| Rabbit ear hypertrophic scarring model establishment
The rabbit ear scarring model was built based on the procedures described in our previous study. 15 Briefly, 1.5% (15 g/L) pentobarbital sodium at 2 ml/kg and xoylazine hydrochloride (2%) at 0.5 ml/ kg were intravenously injection or intramuscularly injected to anesthetize the New Zealand white rabbits. Then, the rabbit ears were sterilized with iodophor, and four round wounds with a diameter of 1 cm were created on the ventral surface of each rabbit ear with a biopsy punch. Tissues, including epidermis, dermis, and perichondrium, were totally removed. Then, each wound was exposed to air and examined for infections for 3 days. All the wounds showing signs of infection or necrosis were excluded from this study.

| Treatment of hypertrophic scars with EGCG
Twenty three days after operation, all rabbit ear wounds were completely re-epithelialized, then 12 rabbits with 96 wounds were randomly divided into four groups (24 wounds in each group). Thirty one days after treatment, scar tissues were collected from each group. Half of the specimens were fixed in 4% formaldehyde solution and embedded in paraffin, and half of the specimens were frozen in liquid nitrogen and stored at −80°C.

| Histological quantification of the rabbit ear scars
Hematoxylin and Eosin (HE) staining and Masson' s trichrome staining images of hypertrophic scar tissues were used for scar elevation assessment and collagen fibers analysis, respectively. Scar elevation was obtained by calculating the scar elevation index (SEI) as previously reported. 15 The SEI is a ratio of total scar and wound height to the height of nearby normal dermis ( Figure 2A). SEI was measured using Image J software 1.52a.
The arrangement of the collagen fiber was examined by Masson's trichrome staining images. Eight images of dermis were taken at 100 × magnification in each scar. The mean collagen area fraction was measured using Image J software 1.52a.

| Immunohistochemistry
4μm thick scar tissue sections were used for immunohistochemical staining. After dewaxing, citrate buffer at pH 6.0 was applied for antigen retrieval for 20 min. Then, 5% bovine serum albumin (BSA) in phosphate buffer saline (PBS) was utilized for blockage. 3% hydrogen peroxide was used to quench endogenous peroxidase activ- Images were taken under a Nikon Eclipse E400 photomicroscope at a 100 × magnification. The numbers of capillaries in the scar sections were analyzed using the Image-J software 1.52a.

| qRT-PCR
To determine the mRNA levels in the scar tissues, total RNA was isolated with TRIzol (Invitrogen) according to the manufacturer's instructions and reversely transcribed into cDNA using a HiFiScript cDNA Synthesis Kit (cw2569M, CWBIOTECH). qRT-PCR experiments were conducted with UltraSYBR Mixture (CW0957M, CWBIOTECH). Data were analyzed using the 2 −ΔΔCt method. The results from three independent reactions were used to calculate the gene expression of chosen genes, which were normalized to the expression of the GAPDH. The primers were synthesized by Sangon Biotech Co., Ltd., and the sequences are shown in Table 1.

| Statistical analysis
Results from each experiment were presented as the mean ± SE of mean (SEM). Differences between groups were determined by analyzing variance (ANOVA) followed by Dunnett's multiple comparisons test using GraphPad Prism 7. p < 0.05 was considered to indicate statistical significance.

| Appearance changes of EGCG-treated scar
In the present study, the impacts of EGCG of different doses on rabbit ear hypertrophic scar formation were analyzed. The scar tissues in control group were convex, showing dark-red color and a moderately firm texture ( Figure 1B). While the scar tissues in either triamcinolone or 0.5 mg and 1.0 mg EGCG groups were flatter and softer.
Scars in triamcinolone group showed almost no bulge compared with the surrounding skin and displayed pale pink color.

| Pathological evaluation
To assess the influence of EGCG of different doses on rabbit ear hypertrophic scar, HE images scanned under NanoZoomer-SQ Digital slide scanner were used. The height of scars was quantified based on the SEI of each scar specimen ( Figure 2B). Significant differences were observed between groups (F = 16.25, DF = 31, p < 0.0001).

F I G U R E 2
The SEI of the scar tissues after EGCG treatment. (A) The diagram of Scar elevation index (SEI). The SEI is a ratio of total scar and wound height to the height of nearby normal dermis. (B) The HE staining images of hypertrophic scar tissues in control group, 0.5 mg EGCG group, 1.0 mg EGCG group, and triamcinolone group. The dotted line represents the measurement area of scar index. (C) Scar elevation index (SEI). A significantly decreased SEI was observed in the 1.0 mg EGCG group and triamcinolone group relative to that in the control group (p < 0.001), while there was no significant difference between the 0.5 mg EGCG group and control group.
No significant difference in SEI was observed between 0.5 mg EGCG group and control group (2.74 ± 0.16 vs. 3.13 ± 0.15, p = 0.25) ( Figure 2C). Histologically, the HE images revealed that the control scars were obviously elevated, presenting highly thicker and more tangled collagen fibers in dermis. In contrast, the scar tissues in 0.5 mg, 1.0 mg EGCG groups, and triamcinolone group were thinner and flatter at the scar site (Figure 3, magnification, ×100, scale bar = 250 μm).
The collagen fibers in the control group were denser, thicker, disorganized, and displayed in parallel arrangement, while the cells arranged in a circular or threaded manner. In comparison, collagen fibers in both the 0.5 mg and 1.0 mg EGCG groups and the triamcinolone group were relatively looser, thinner, and more regularly arranged ( Figure 4A, magnification, ×100, scale bar = 100 μm).
Then, the mean collagen area fraction in all Masson's trichrome staining images was measured by using Image J software 1.52a ( Figure 4B).

| EGCG reduced the number of capillaries in hypertrophic scars
Images for CD31 staining were applied to study the influence of EGCG on scar angiogenesis ( Figure 5A

| DISCUSS ION
Hypertrophic scar is a common skin fibrotic disease secondary to skin injury, which often causes adverse effects to patients. Due to the unclear pathogenesis of hypertrophic scar, the effects of currently used medications on hypertrophic scar is not ideal. Therefore, there is an urgent need to explore safe, feasible, and effective drugs to prevent and treat hypertrophic scar. In this study, we revealed the inhibitory effects of EGCG on hypertrophic scar formation using a rabbit ear hypertrophic scarring model.
Using this rabbit ear scarring model, we found that EGCG inhibited fibroblast activation and lowered collagen density through decreasing TGF-β1, Col I, Col III, and α-SMA gene expression. Previous studies confirmed that EGCG significantly inhibited Col I expression in mast cell-stimulated keloid fibroblasts. 16 Besides, EGCG also showed strong anti-fibrotic functions by suppressing ECM accumulation and keloids growth in a keloid nude mice model and a human keloid OC model. 12,13 These findings were consistent with our study.

F I G U R E 4
Masson's trichrome staining of the scar tissues after EGCG treatment. (A) Masson's trichrome staining of scars. Collagen fibers were dense and irregularly arrange in the control group. In triamcinolone group and both EGCG groups, the collagen fibers were regularly arranged. (B) The mean collagen area fraction analyzed by Image J software1.52a. Magnification, ×100, scale bar = 100 μm.*** p < 0.001, * p < 0.05, compared with the control group.
In the present study, the anti-scar effects of EGCG were confirmed using a rabbit ear scarring model, which is not reported previously. Among the existed scar research models, the rabbit ear hypertrophic scarring model reproduces many characteristics of human hypertrophic scar and is widely used for exploring anti- angiogenesis. 39 Ren et al. found that endostatin suppressed hypertrophic scar formation in a rabbit-ear scarring model through reducing scar vascularization and angiogenesis. 40 Previously, we also found the anti-angiogenic effect of UA (usnic acid) using the rabbit ear scarring model. 41  EGCG in a human keloid organ culture model. 12 In this study, qRT-PCR results confirmed that the expressions of these genes were also inhibited in the EGCG groups.
In this study, we selected triamcinolone, which is a commonly In summary, our results reveal the inhibitory effects of EGCG on the rabbit ear hypertrophic scarring, this may largely attribute to its anti-angiogenic and anti-fibrotic effects. Therefore, this study presents supportive evidences that EGCG can significantly inhibit hypertrophic scar formation.

CO N FLI C T O F I NTE R E S T
No potential conflict of interest was reported by the authors.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.