molecules Article
Gallic Acid Promotes Wound Healing in Normal and Hyperglucidic Conditions Dong Joo Yang 1,2,† , Sang Hyun Moh 3,† , Dong Hwee Son 1,4 , Seunghoon You 1 , Ann W. Kinyua 1,2 , Chang Mann Ko 1 , Miyoung Song 3 , Jinhee Yeo 5 , Yun-Hee Choi 3, * and Ki Woo Kim 1,2, * 1
2 3 4 5
* †
Department of Pharmacology, Wonju College of Medicine, Yonsei University, Wonju 26426, Korea;
[email protected] (D.J.Y.);
[email protected] (D.H.S.);
[email protected] (S.Y.);
[email protected] (A.W.K.);
[email protected] (C.M.K.) Department of Global Medical Science, Wonju College of Medicine, Yonsei University, Wonju 26426, Korea Anti-Aging Research Institute of BIO-FD & C Co. Ltd., Incheon 21990, Korea;
[email protected] (S.H.M.);
[email protected] (M.S.) Department of Wellness & Healthy Aging, Wonju College of Medicine, Yonsei University, Wonju 26426, Korea Jeongseon Agricultural Extension Center, Jeongseon 26103, Korea;
[email protected] Correspondence:
[email protected] (Y.-H.C.);
[email protected] (K.W.K.); Tel./Fax:+82-32-811-2027 (Y.-H.C.); Tel.: +82-33-741-0302 (K.W.K.); Fax: +82-33-742-4966 (K.W.K.) These authors contributed equally to this work.
Academic Editor: Pedro Mena Received: 19 March 2016; Accepted: 1 July 2016; Published: 8 July 2016
Abstract: Skin is the outermost layer of the human body that is constantly exposed to environmental stressors, such as UV radiation and toxic chemicals, and is susceptible to mechanical wounding and injury. The ability of the skin to repair injuries is paramount for survival and it is disrupted in a spectrum of disorders leading to skin pathologies. Diabetic patients often suffer from chronic, impaired wound healing, which facilitate bacterial infections and necessitate amputation. Here, we studied the effects of gallic acid (GA, 3,4,5-trihydroxybenzoic acid; a plant-derived polyphenolic compound) on would healing in normal and hyperglucidic conditions, to mimic diabetes, in human keratinocytes and fibroblasts. Our study reveals that GA is a potential antioxidant that directly upregulates the expression of antioxidant genes. In addition, GA accelerated cell migration of keratinocytes and fibroblasts in both normal and hyperglucidic conditions. Further, GA treatment activated factors known to be hallmarks of wound healing, such as focal adhesion kinases (FAK), c-Jun N-terminal kinases (JNK), and extracellular signal-regulated kinases (Erk), underpinning the beneficial role of GA in wound repair. Therefore, our results demonstrate that GA might be a viable wound healing agent and a potential intervention to treat wounds resulting from metabolic complications. Keywords: gallic acid; wound healing; cell migration; hyperglucidic condition
1. Introduction Gallic acid (GA) is a 3,4,5-trihydoxybenzoic acid, a phenolic acid found in almost all plants including fruits, leaves, and wild flowers [1,2], and has been reported to possess powerful health benefits, such as antioxidant, anti-inflammatory, analgesic, neuroprotective, anticancer, and anti-diabetic properties [3]. The skin comprising of outer epidermis, underlying connective tissue, and dermis functions as a barrier that protects the body from environmental stressors, such as pathogens, excessive water loss, temperature, and physical stress [4]. Skin injuries, such as abrasions and incisions, may cause Molecules 2016, 21, 899; doi:10.3390/molecules21070899
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damage to the epidermis and/or dermal layers, necessitating repair through a wound healing process comprised of four sequential phases; homeostasis, inflammation, proliferation/re-epithelialization, and maturation [5]. When this well-ordered process is disrupted by factors, such as age, hyperglycemia, poor circulation, repeated trauma, continuous pressure, infections, or systemic illnesses, the wound fails to close in an expected time frame developing into a chronic wound [6]. Both acute and chronic wounds represent a major public healthcare burden affecting a relatively large population [7]. Proper wound healing is essential not only for the restoration of disrupted anatomical continuity and the barrier function of the skin, but also for reducing the risk of infection and further complications [5]. The use of natural products from plants for treatment of wounds has been practiced since ancient times. Due Molecules to the 2016, fact21,that plant-extracted treatments are easily accessible and relatively safe, there is 899 2 of 14 a steadily growing interest in using natural compounds to prevent and combat skin pathologies [8]. [5]. When this well-ordered process is disrupted such as age, hyperglycemia, poor several circulation,chronic skin Moreover, polyphenols have been widely used by infactors, traditional medicines to treat repeated trauma, continuous pressure, infections, or systemic illnesses, the wound fails to close in an diseases, such as psoriasis vitiligo, they are [6]. also known to chronic be therapeutically expected time frame and developing into aand chronic wound Both acute and wounds representbeneficial in wound healing and show anti-inflammatory when topically [9,10]. a major public healthcare burden affectingeffects a relatively largeapplied population [7]. Proper woundDespite healing isthe fact that notbeneficial only for the properties restoration of including disrupted anatomical continuity and the barrier function of anti-tumor GA exhibitsessential several antioxidant, anti-inflammatory, and the skin, but also for reducing the risk of infection and further complications [5]. activities, thereThe is only little evidence on the role of GA in skin and skin pathology. Here, we studied use of natural products from plants for treatment of wounds has been practiced since ancient the effect of times. GA on healing in human keratinocytes. Previously, ourrelatively group safe, studied Duewould to the fact that plant-extracted treatments are easily accessible and there the is beneficial a steadily growing interest using naturaland compounds prevent and combat skin pathologies [8]. effects of GA on metabolic stressinconditions foundtoimprovement in whole body metabolism by Moreover, polyphenols have been widely used in traditional medicines to treat several chronic skin GA treatment [11]. In addition, GA has shown strong protective effects on disease progression in diseases, such as psoriasis and vitiligo, and they are also known to be therapeutically beneficial in type 1- andwound type 2-diabetes animal models [12]. Diabetes mellitus (DM) characterized healing and show anti-inflammatory effects when applied topically [9,10].isDespite the fact that by delayed GA exhibits several beneficial properties including antioxidant, anti-inflammatory, and anti-tumor of DM [13]. and impaired skin wound healing, leading to chronic ulcers, a common complication activities, there is only little evidence on the role of GA in skin and skin pathology. Here, we studied the We examined the effect of GA on DM-induced delayed wound healing in both keratinocytes and effect of GA on would healing in human keratinocytes. Previously, our group studied the beneficial fibroblasts using high glucose-containing medium to mimic diabetes. In the current study, we found effects of GA on metabolic stress conditions and found improvement in whole body metabolism by that GA hasGA antioxidant properties, as cellstrong migration effects normal and inhigh glucose treatment [11]. In addition,as GAwell has shown protective effectsin onboth disease progression 1- and type 2-diabetes animal models [12]. Diabetesfor mellitus (DM)wounds is characterized by delayed conditions, type suggesting that GA has a curative potential chronic in DM. and impaired skin wound healing, leading to chronic ulcers, a common complication of DM [13]. 2. Results We examined the effect of GA on DM-induced delayed wound healing in both keratinocytes and fibroblasts using high glucose-containing medium to mimic diabetes. In the current study, we found that GA has antioxidant properties, as well as cell migration effects in both normal and high glucose 2.1. GA Protects Skin suggesting Cells fromthat Oxidative conditions, GA has a Stress curative potential for chronic wounds in DM.
Polyphenols 2. Resultsare known to have a positive impact on cell viability by modulating oxidative stresses. To determine if GA has any effect on cell viability and oxidative stress, we first examined 2.1. GA Protects Skin Cells from Oxidative Stress the cytotoxicity of GA in human keratinocytes, HaCaT cells using 3-(4,5-dimethylthiazol-2-yl)Polyphenols are known(MTT) to haveassay a positive impact on cell viability GA by modulating oxidativecell viability 2,5-diphenyltetrazoliumbromide (Figure 1A). Although altered HaCaT stresses. To determine if GA has any effect on cell viability and oxidative stress, we first examined at high concentration (200 µM), cell viability was not affected at lower concentrations (10, 20, or 50 µM). the cytotoxicity of GA in human keratinocytes, HaCaT cells using 3-(4,5-dimethylthiazol-2-yl)-2,5As such, low concentration of GA (MTT) was used for subsequent experiments. MTT methylene blue diphenyltetrazoliumbromide assay (Figure 1A). Although GA altered HaCaT cell and viability at high concentration (200 μM), cell viability was not affected at lower concentrations (10, 20, or 50 μM). staining assays were performed to analyze cell viability following H2 O2 treatment. N-acetylcysteine As such, low concentration of GA was used for subsequent experiments. MTT and methylene blue (NAC), a strong antioxidant, was used as an antioxidant control [14]. Pretreatment with GA and NAC staining assays were performed to analyze cell viability following H2O2 treatment. N-acetylcysteine significantly(NAC), protected from was H2 O oxidative (Figure 1B) protective effect of 2 -induced a strongcells antioxidant, used as an antioxidant controlstress [14]. Pretreatment withand GA and NAC significantly protected cells fromwas H2O2observed -induced oxidative stress (Figure and protective of demonstrating GA GA from ROS-induced cytotoxicity at indicated time1B) points (Figureeffect 1C), from ROS-induced observed at indicated time points (Figure 1C), demonstrating that GA protects cells fromcytotoxicity oxidativewas stress in human keratinocytes. that GA protects cells from oxidative stress in human keratinocytes.
Figure 1. Cont.
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Figure 1. GA protects cells from oxidative human keratinocytes. (A)cells HaCaT Figure 1. GA protects cells from oxidative stress stress ininhuman keratinocytes. (A) HaCaT were cells were Figure GA protects cells from oxidative human keratinocytes. (A)MTT HaCaT cells assay. were treated with different concentrations GAstress for 24 hhand cellcell viability was assessed by assay. treated1.with different concentrations of ofGA for 24in and viability was assessed by MTT The values represent mean ± SEMof (* GA p < 0.05, one-way ANOVA followed bywas Bonferroni’s post by hoc MTT assay. treated with different concentrations for 24 h and cell viability assessed The values represent mean ± SEM (* p < 0.05, one-way ANOVA followed by Bonferroni’s post hoc test); (B) HaCaT cells were pretreated with either vehicle or two different GA concentrations (10 and The values represent mean ˘pretreated SEM (* p < 0.05, either one-way ANOVA followed by Bonferroni’s post hoc test); (B) HaCaT vehicle or two different (10test); and 20 μM) cells in thewere presence or absence with of H2O2 (1.8 mM) and then incubated for 4 h.GA Cell concentrations viability was (B) HaCaT cells were pretreated with either vehicle or two different GA concentrations (10 and 20 µM) shown by methylene blue staining; cells were and treated withincubated vehicle or GA in theviability was 20 μM) in the presence or absence of Hand 2O2(C) (1.8 mM) then for(104 μM) h. Cell inshown the presence or absence ofstaining; H2 Oand mM) then incubated for 4 h.vehicle Cell viability shown of H2O 2 (1.8 mM) cell viability was assessed intreated a time-dependent manner. TheGA datawas are μM) 2 (1.8 by presence methylene blue and (C)and cells were with or (10 in by the expressed as mean ± SEM (*** p < 0.05, two-way ANOVA followed by Bonferroni’s post hoc test). methylene blue staining; and (C) cells were treated with vehicle or GA (10 µM) in the presence of H O 2 2 presence ofScale H2Obar 2 (1.8 mM) and cell viability was assessed in a time-dependent manner. The data are = 200 μm. (1.8 mM) and cell viability was assessed in a time-dependent manner. The data are expressed as mean expressed as mean ± SEM (*** p < 0.05, two-way ANOVA followed by Bonferroni’s post hoc test). ˘Scale SEMbar (*** p200 < 0.05, ANOVA followed by Bonferroni’s post hoc test). Scale bar = 200 µm. 2.2.=Antioxidant Effects of GA in Human Keratinocytes μm. two-way Free radicals are known to cause tissue injuries, hence contributing to the pathology of many
2.2. Effects ofofGA human diseases [15]. To investigate the protective role of GA against oxidative stress in skin cells, 2.2.Antioxidant Antioxidant Effects GAin inHuman HumanKeratinocytes Keratinocytes the free radical scavenging activity of GA was examined. GA showed increased radical scavenging
Free are known tissue injuries, to the of activity in a dose-dependent manner (Figure 2A). In hence addition, GA significantly the Freeradicals radicals are knowntotocause cause tissue injuries, hencecontributing contributing toupregulated thepathology pathology ofmany many expression ofTo antioxidant genes,the including superoxide dismutase 2 (SOD2), catalase (CAT), and human diseases [15]. investigate protective role of GA against oxidative stress in skin human diseases [15]. To investigate the protective role of GA against oxidative stress in skin cells, cells, glutathione peroxidase 1 (Gpx1), in skin cells (Figure 2B). These results highly indicate that GA the scavenging activity ofofGA was examined. GA showed increased radical scavenging thefree freeradical radical scavenging activity GA was examined. GA showed increased radical scavenging exerts its antioxidant function by directly upregulating the expression of these antioxidant genes. activity activity in inaadose-dependent dose-dependent manner manner (Figure (Figure 2A). 2A).In Inaddition, addition,GA GAsignificantly significantlyupregulated upregulated the the expression of antioxidant genes, including superoxide dismutase 2 (SOD2), catalase (CAT), and expression of antioxidant genes, including superoxide dismutase 2 (SOD2), catalase (CAT), and glutathione in skin cellscells (Figure 2B). These results highlyhighly indicate that GAthat exerts glutathioneperoxidase peroxidase1 (Gpx1), 1 (Gpx1), in skin (Figure 2B). These results indicate GA its antioxidant function by directly upregulating the expression of these antioxidant genes. exerts its antioxidant function by directly upregulating the expression of these antioxidant genes.
Figure 2. Cont.
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Figure Figure 2. 2. Antioxidant Antioxidanteffects effects of of GA GA in in human human keratinocytes. keratinocytes. (A) (A)Scavenging Scavengingeffects effects of of GA GA on on 2,2-diphenyl-1-picrylhydrazyl 2,2-diphenyl-1-picrylhydrazyl(DPPH) (DPPH)radical. radical.HaCaT HaCaTcells cellswere weretreated treatedwith withGA GA(5, (5,10, 10,and and20 20μM) µM) or or vitamin vitamin CC (50 (50 μM) µM) for for 30 30 min min and and assayed assayed for for antioxidant antioxidantactivity activityby byDPPH DPPHscavenging scavengingassay. assay. Vitamin Vitamin C C was was used used as as aa positive positive control control for for radical radical scavenging scavenging activity. activity. Data Data are are presented presented as as the the means independent experiments experiments(*(*pp
