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Acta Histochemica 116 (2014) 1169–1177

Contents lists available at ScienceDirect

Acta Histochemica

jo ur nal homepage: www.elsev ier .de /ac th is

eed oil of Joannesia princeps improves cutaneous wound closure inxperimental mice

line Donato-Trancosoa, Lenicio Gonc alvesa, Andréa Monte-Alto-Costab,rancisco de Assis da Silvac, Bruna Romana-Souzab,∗

Department of Animal Biology, Rural Federal University of Rio de Janeiro, Seropédica 23890-000, BrazilDepartment of Histology and Embryology, State University of Rio de Janeiro, Rio de Janeiro 20950-003, BrazilDepartment of Chemistry, Rural Federal University of Rio de Janeiro, Seropédica 23890-000, Brazil

r t i c l e i n f o

rticle history:eceived 10 April 2014eceived in revised form 11 June 2014ccepted 16 June 2014

eywords:ngiogenesis

nflammationibroblast cultureound healing

a b s t r a c t

Joannesia princeps (Cotieira) is a well known medicinal plant in Brazil, however, the therapeutic effects ofoil obtained from its seeds have still not been demonstrated. The beneficial effects of J. princeps seed oilon cutaneous wound healing on the back of experimental mice were investigated. An excisional lesion inmale Swiss mice (n = 20 per group) was topically treated with mineral oil or J. princeps seed oil once a daybeginning on the day of lesion until the third day after wounding. Animals were killed and lesions collectedafter 14 days. Murine skin fibroblast cultures were treated with J. princeps seed oil and fibroblast activitywas evaluated. In the in vivo assay, J. princeps seed oil increased wound contraction and migratory tonguelength, but reduced neutrophil and macrophage number when compared with the control group. Bloodvessel number, collagen deposition, and VEGF levels were increased in treated lesions when compared

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with control lesions. However, J. princeps seed oil reduced myofibroblast density and carbonyl proteinlevels when compared with the control group. In the in vitro assay, treatment with J. princeps seed oilincreased fibroblast migration and proliferation, but reduced myofibroblastic differentiation in vitro. Inconclusion, J. princeps seed oil accelerates wound closure increasing angiogenesis, keratinocyte migration,and fibroblast activity while reducing inflammatory response and oxidative damage.

© 2014 Elsevier GmbH. All rights reserved.

ntroduction

Joannesia princeps Vellozo, known as “cotieira”, is a tree belong-ng to the Euphorbiaceae family native to the Atlantic Forests ofhe Southern Region in Brazil and which is also cultivated in trop-cal regions of Africa and Asia (Achenbach and Benirschke, 1997).he seeds of this tree contain oil which is used in anti-helminthicreatment in popular medicine owing to its induction of intesti-al motility (Freise, 1929; Achenbach and Benirschke, 1997). Theil obtained from J. princeps seeds consists of two units of linoleiccid and one unit of oleic acid (Berty et al., 2011). This compo-ition indicates that J. princeps seed oil may modulate cutaneous

ound healing, since linoleic and oleic acids are known to play a

ole in cutaneous wound healing (Cardoso et al., 2004; Rodriguest al., 2012). However, no study has so far been undertaken

∗ Corresponding author at: Department of Histology and Embryology, State Uni-ersity of Rio de Janeiro (UERJ), Rua Marechal Rondom, 381, 2◦ andar, 20950-003io de Janeiro, RJ, Brazil.

E-mail address: romanabio@yahoo.com.br (B. Romana-Souza).

ttp://dx.doi.org/10.1016/j.acthis.2014.06.005065-1281/© 2014 Elsevier GmbH. All rights reserved.

showing the effects of oil from J. princeps seeds on cutaneous woundhealing.

Linoleic acid (C18:2 cis 9.12) is an n-6 polyunsaturated fatty acidfound in the oils of safflower, sunflower, and soybean (McCuskerand Grant-Kels, 2010). Oleic acid (C18:1 cis 9) is an n-9 monoun-saturated fatty acid, which is a component of oil extracted fromolives (Piscopo, 2009). In skin, linoleic acid is the most abundantfatty acid in the epidermis, forming the permeability barrier of theStratum corneum and reducing trans-epidermal water loss (Hansenand Jensen, 1985; McCusker and Grant-Kels, 2010). The adminis-tration of natural oils rich in both fatty acids may be beneficialin cutaneous wound healing (Rojo et al., 2010; de Oliveira et al.,2013). Topical application of avocado oil composed mainly of oleicoil (67%) and linoleic acid (23%) may reduce inflammatory cell num-ber and increase collagen deposition in rat cutaneous lesions (deOliveira et al., 2013). An in vitro study showed that treatment withlucuma nut oil, composed mainly of linoleic acid (39%) and oleic

acid (28%), may promote migration of human fibroblasts (Rojo et al.,2010). Moreover, administration of these isolated fatty acids alsoproves their regenerative effect on cutaneous wound healing. Ithas been reported that topical treatment of oleic acid or linoleic

1170 A. Donato-Trancoso et al. / Acta Histoc

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ig. 1. Chemical structure of Joannesia princeps seed oil composed by two moleculesf linoleic acid connected to one molecule of oleic acid (C57H100O6 – 880 g/mol).

cid reduced the time for cutaneous wound closure (Cardoso et al.,004). More recently, it has been demonstrated that administrationf oleic acid or linoleic acid may modify neutrophil and macrophageunctions indicating that theses acids may affect the course ofnflammation (Rodrigues et al., 2010; Magdalon et al., 2012). Inddition, topical application of oleic acid or conjugated linoleiccid supplementation may modulate the inflammatory response ofutaneous wound healing (Park et al., 2010; Cardoso et al., 2011).ral administration of linoleic or oleic acids accelerates the early

nflammatory phase of wound healing leading to improved woundlosure (Rodrigues et al., 2012). The aim of this study was to investi-ate the therapeutic effects of topical application of J. princeps seedil on cutaneous wound healing in mice.

aterials and methods

eed materials

Seeds of J. princeps Vellozo (Euphorbiaceae) were collected inebruary 2010 from Jardim Peró, Cabo Frio, Rio de Janeiro, Brazil.lant materials were identified by Professor Dr. Pedro Germanoilho from the Department of Botany, Institute of Biology, Rural Fed-ral University of Rio de Janeiro. Voucher specimens (No. 34.630)ere deposited in the Herbarium of Department of Botany at theural Federal University of Rio de Janeiro. Collection of plant sam-les was undertaken in concordance with Brazilian Legislation.

xtraction, purification and characterization of the oil from. princeps seeds

The oil from J. princeps seeds was obtained by Soxhlet extrac-ion with hexane solvent (Vetec, Duque de Caxias, Brazil) from1.3 g of triturated seeds (Tang et al., 2014). After hexane extrac-ion, 3.7 g of light yellow oil were obtained. The composition andtructure of this oil was analyzed by NMR 1H and 13C (AC 500 of therucker), infra-red spectroscopy (Nicolet-FTIR Model 6700), andC/MS technique (GC/MS-QP2010 Plus) indicating only one triglyc-ride with 95–97% of purity formed by two units of linoleic acidnd one unit of oleic acid (Fig. 1), molecular mass of 880 g/mol andormula C57H100O6 (Berty et al., 2011). Spectroscopic comparisonsonfirmed the structure (Berty et al., 2011).

ound model and J. princeps seed oil treatment

Male Swiss mice weighing about 25–30 g (2–3 months of age)ere housed in plastic cages (30 cm × 20 cm × 13 cm) with a stain-

ess steel grid cover (Insight; Ribeirão Preto, Brazil) containing

hemica 116 (2014) 1169–1177

groups of five animals per cage in a room with controlled humid-ity (50%) and temperature (22 ◦C) on a 12-h light/dark cycle andan air exhaustion cycle (15 min/h). All procedures were carriedout in accordance with the Brazilian legislation regarding animalexperimentation (no. 11.794, from October 8, 2008). This study wasapproved by the Commission of Ethics in Research of the Rural Fed-eral University of Rio de Janeiro (no. 102/2011). Mice (n = 40) wereintraperitoneally anesthetized with ketamine (150 mg/kg b.wt) andxylazine (15 mg/kg b.wt). The hair of the back of the mice wasshaved and a full-thickness excisional wound (1 cm2) was createdwith excision of the epidermis, dermis and hypodermis exposingthe panniculus carnosus.

Animals were divided into a control group treated with mineraloil (Johnson & Johnson do Brasil Ltda.; São José dos Campos, Brazil);and an experimental group treated with J. princeps seed oil. In bothgroups, bed wounds were topically treated with 80 �l of oils begin-ning on the day of the lesion and maintained until three days later,and covered with an occlusive dressing (Cremer S.A.; Santa Cata-rina, Brazil). On the next day, the wound was cleaned with 0.9%saline, and oil and occlusive dressing applied again. From day fourafter wounding, wounds were left uncovered in both groups. Thisperiod of treatment was chosen because it has been demonstratedthat linoleic and oleic acid administration accelerates the initialinflammatory phase of wound healing resulting in faster woundclosure (Rodrigues et al., 2010, 2012; Cardoso et al., 2011).

To evaluate the effect of J. princeps seed oil on wound con-traction, a set of experiments was performed evaluating woundcontraction 7, 14, and 21 days after wounding (n = 10 in each group).After this, the time point of 14 days after wounding was cho-sen, another set of experiments was performed and the materialcollected for histological and biochemical analysis (n = 20 in eachgroup).

Macroscopic analyses

To evaluate wound contraction, a transparent plastic sheet wasplaced over the wound and its margins were traced soon afterwounding, and 7, 14 and 21 days later. After digitalization, thewound area was measured using ImageJ software (National Insti-tutes of Health, Bethesda, MD, USA). The results were expressed asa percentage of the original wound area.

Tissue harvesting and microscopic analyses

Fourteen days after wounding, mice were intraperitoneallyanesthetized with ketamine (150 mg/kg b.wt) and xylazine(15 mg/kg b.wt) and killed by CO2 exposure. Thereafter, five lesionsand adjacent normal skin per group were collected, fixed in forma-lin (pH 7.2) and embedded in paraffin wax. Fifteen lesions per groupwere collected and frozen at −80 ◦C. The remaining frozen lesionswere destined for assay of hydroxyproline (n = 5 each group), car-bonyl protein levels (n = 5 per group), and immunoblotting (n = 5per group).

Paraffin-embedded sections were stained with hematoxylin–eosin to analyze wound area and to evaluate blood vessel andmigratory tongue length. Blood vessel density was evaluatedusing point counting (Gundersen et al., 1988). For this, 20 ran-dom fields per animal were analyzed using a 40× objectivelens and videomicroscope system (TNB41 Opton microscope, aTecVoz CCD Sony video-camera, and CCE monitor) (Zeiss Opton;Oberkochen, Germany; Sony Corporation, Tokyo, Japan; Manaus,Brazil). Results were presented as volume density of blood vessels.

To measure migratory tongue length, the images of wound edgewere digitalized using 10× objective lens and videomicroscopesystem (Axiostar plus microscope and AxioCam Cc1 video-camera; Zeiss-Vision, Oberkochen, Germany) and the length of the

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igratory tongue was measured using ImageJ software (Tscharntket al., 2007). Migratory tongue length was defined as the distancein micrometers) from the wound edge to the tip of the tongue. Theound edge was defined through the different characters of normal

kin and newly formed dermal tissue; the proliferating keratino-yte lump on wound edge and delicate collagen fibers in newlyormed dermal tissue were two main factors to be considered toefine the wound edge (Chen et al., 2012; Assis de Brito et al., 2014).ections were also stained with Sirius red and observed underolarization microscopy to evaluate the organization of collagenbers and to quantify collagen deposition. For this, five random

mages observed under polarization optics per animal were digi-alized from wound edge to another using 40× objective lens.irefringent collagen fibers were measured using ImageJ softwareNIH). All quantifications were done blindly and repeated withoutignificant differences among them.

mmunohistochemistry and quantification

For quantification of myeloperoxidase-positive neutrophils and4/80-positive macrophages, sections were immunolabeled withat anti-myeloperoxidase (Santa Cruz Biotechnology, Santa Cruz,A, USA; 1:500) or anti-F4/80 antibodies (Serotec; Raleigh, NC,SA; 1:500), respectively. Subsequently, endogenous peroxidaseas inhibited. After washing, primary antibodies were detectedsing biotinylated anti-rat secondary antibody (Dako; Carpinteria,A, USA; 1:800) followed by incubation with streptavidin (Dako).or quantification of myofibroblasts and cell proliferation, sectionsere incubated with EnVision anti-mouse system (Dako; 1:60)

nd 3% H2O2 in methanol. After washing, sections were incubatedith a mixture of mouse anti-�-smooth muscle actin (SMA) anti-

ody (Dako; 1:600) plus EnVision anti-mouse (Dako; 1:20) or aixture of mouse anti-proliferating cell nuclear antigen (PCNA)

ntibody (Dako; 1:800) plus EnVision anti-mouse (Dako; 1:20)vernight. Diaminobenzidine (DAB) was used as a chromogen andections were counterstained with hematoxylin. No labeling wasbserved on sections where primary antibody was omitted. Inhe immunohistochemistry to detect PCNA, myeloperoxidase, and4/80, sections were incubated before labeling with citrate bufferpH 6.0) for antigen retrieval.

To quantify neutrophils and macrophages, 10 random fields pernimal (14,544 �m2) were analyzed using a 20× objective lens andideomicroscope system (Olympus BH-2 microscope, a SamsungDS-415 video-camera, and Samsung monitor) (Olympus Corp.,okyo, Japan; Samsung, Manaus, Brazil). Results were presenteds cells per mm2. Volume density of myofibroblasts was evaluatedsing point counting as described for blood vessels. Results wereresented as volume density of myofibroblasts. Cellular prolifera-ion was evaluated in neo-epidermis and in the granulation tissue.he percentage of PCNA-positive epithelial cells was calculated byounting 140 cells starting on wound margin (in neo-epidermis).uantification of PCNA-positive connective cells was performed asreviously described for F4/80-positive macrophages.

iochemical analyses

Collagen deposition in the wound area was quantified usingydroxyproline assay in five frozen lesions per group. Dry andefatted tissue samples were hydrolyzed in hydrochloric acid. Sam-les were then diluted, neutralized and centrifuged (Woessner,961). Diluted aliquots of hydrolysate were mixed with 40 �lhloramin-T (0.05 M) (Merck, Whitehouse Station, NJ, USA) and

ncubated for 20 min at 25 ◦C. Then, 40 �l perchloric acid (3.17 M)Vetec) and 40 �l 4-dimethylamino benzaldehyde (Merck) weredded, plates were again incubated for 20 min at 60 ◦C. Theeveloped color was read spectrophotometrically at 550 nm.

hemica 116 (2014) 1169–1177 1171

Hydroxyproline concentrations were determined from a standardcurve generated by different concentrations of l-4-hydroxyproline(Sigma–Aldrich, St. Louis, MO, USA). Data are expressed as nghydroxyproline per mg tissue.

Previous studies have demonstrated that conjugated linoleicacid supplementation and oleic acid administration may reducelipid peroxidation and hydrogen peroxide production (Park et al.,2010; Rodrigues et al., 2012). Thus, to determine if seed oil of J.princeps modulates oxidative damage in cutaneous lesions, car-bonylated protein content was measured (Levine et al., 1990).Carbonyl groups into proteins (aldehydes and ketones) are formedwhen proteins are oxidized by oxidative stress-induced free radi-cals (Levine, 2002). Thus, carbonyl protein content can be a methodto detect oxidative modification of proteins caused by oxidativestress (Levine et al., 1990). For this, five frozen lesions per groupwere macerated into potassium phosphate buffer (pH 7.5), aftercentrifugation, total protein concentration was estimated usingBradford method. Thereafter, these samples were mixed withtrichloroacetic acid (Sigma–Aldrich), incubated and centrifuged.Pellets were dissolved in sodium hydroxide. Hydrochloric acid or2,4-dinitrophenylhydrazine was added in each of sample and incu-bated. Later, trichloroacetic acid was added and pellet washed withmixture of ethanol–ethylacetate. Finally, pellets were dissolved inguanidine in water-baths. Carbonyl protein levels were read in370 nm and were expressed as �Mol carbonyl per �g total protein.

Immunoblotting

Five frozen lesions per group were macerated into lysis buffer(20 mM Tris–HCl pH 7.5, 138 mM sodium chloride, 10% glyc-erol, 1% Triton X-100, 2 mM ethylenediaminetetraacetic acid(EDTA), 10 �g/ml leupeptin, 0.025% phenylmethylsulfonyl fluo-ride) (Sigma–Aldrich), after centrifugation, total protein concen-tration was determined using the Bradford assay. Proteins (15 �g)were resolved by 8% or 12% sodium dodecylsulfate–polyacrylamidegels and were transferred to polyvinylidene fluoride (PVDF) mem-branes. Protein molecular weight standard (Bio-Rad, Hercules, CA,USA) was included. Membranes were blocked with 5% non-fatmilk in powder form (Nestlé Ind. Com. Ltda., São Paulo, Brazil)and probed with a mouse anti-VEGF antibody (45 kDa) (SantaCruz Biotechnology, Santa Cruz, CA, USA; 1:500) or a rat anti-platelet/endothelial cell adhesion molecule-1 (PECAM-1) antibody(130 kDa) (Santa Cruz; 1:500). Membranes were then washed andincubated with appropriated secondary antibodies conjugated withperoxidase (Dako). Bound antibodies were detected by enhancedchemiluminescence (Millipore, Hayward, CA, USA) and densitom-etry analysis was performed using Adobe Photoshop version 7.01(Adobe Systems Inc., San Jose, CA, USA). Results were expressedas arbitrary units. Subsequently, membranes were stripped witha stripping buffer plus �-mercaptoethanol (Sigma–Aldrich) andreprobed with anti-�-actin antibody (43 kDa) (Santa Cruz, CA;1:400).

In vitro assays

Primary fibroblasts were isolated from the skin of adult Swissmice by the standard procedure of explant technique (Freshney,2010). Skin fragments from the back of Swiss mice (1–2 months)were placed in culture plate with Dulbecco’s modified Eagle’smedium (DMEM) (Sigma–Aldrich) with 20% (v/v) fetal bovineserum (FBS) (Cultilab Ltda; Campinas, Brazil) containing antibiotics(100 UI/ml penicillin, 50 �g/ml kanamycin, 100 �g/ml strepto-

mycin, and 6 �g/ml amphotericin B) (Sigma–Aldrich) for 7 daysat 37 ◦C in 5% CO2. After cell separation from the dermis, cellswere transferred to a culture dish (75 cm2; Techno Plastic ProductsAG, Trasadingen, Switzerland) containing DMEM supplemented

1 Histochemica 116 (2014) 1169–1177

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Fig. 2. Macroscopic evaluation of wounds in control and treated mice. (A) Pho-tographs of wounds in control and treated mice 7, 14, and 21 days after wounding.(B) Percentage of original wound area in control and treated animals 7, 14 and21 days after wounding. (C) Representative images of migratory tongue in con-trol and treated groups 14 days after wounding. Black dotted lines show thelength of the migratory tongue, NS shows normal skin and sections stained withhematoxylin–eosin. (D) Length of migratory tongue in control and treated groups14 days after wounding. Data (n = 10) are expressed as mean ± SEM. *p < 0.05 vs.

172 A. Donato-Trancoso et al. / Acta

ith 10% FBS and antibiotics. For in vitro assays, J. princeps seedil was dissolved in serum-free DMEM containing 2.5% Tween-0 (Sigma–Aldrich) obtaining a stock solution of 200 mg/ml. In allxperiments, fibroblasts were cultured in DMEM with 10% FBS for4 h. After 80–100% confluence, the medium was changed to DMEMith 2% FBS for 12 h. Then, cells were treated with 0.001 mg/ml,

.01 mg/ml or 0.1 mg/ml of oil stock solution diluted in DMEM with% FBS. Control cells were treated with DMEM with 2% FBS alone.MEM with 10% FBS was used as control positive in all experi-ents. Final Tween-20 concentrations did not exceed 0.001% and

id not affect experiments. Cells from passages 3–7 were used inll experiments. Experiments were performed at least three times,n triplicate.

Cell migration was assessed by the ability of the cells to movento an acellular area in a two-dimensional scratch assay (Morellinit al., 2008). For that, fibroblasts (2.5 × 105 cells/well) were seededn a 24-well plate and treated with J. princeps seed oil and mit-mycin C (10 �g/ml) (Sigma–Aldrich) to block cell proliferation.oon after, cells were scratched in the center of the well with aellow pipette tip (100 �l) to create a cell-free area. The denudedreas were photographed using a microscopic system (Nikonclipse TS100 microscope and Nikon Coolpix 4500 camera) (Nikon,elville, NY, USA) soon after scratching and 1 and 2 days later. The

enuded areas were measured using ImageJ software (NIH) andata were expressed as percentage of the initial denuded area.

Fibroblast proliferation was determined using the 3-(4,5-imethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assayFierro et al., 2002). Fibroblasts (3 × 104 cells/well) were seeded in

96-well plate and, after 1 day of treatment, 10 �l of MTT solutionSigma–Aldrich) at 5 mg/ml were added to each well. After 4 h, theormazan product was solubilized with isopropyl alcohol (Vetec).he developed color was spectrophotometrically read at 570 nm.ata were expressed as percentage of the control (medium only)roup.

To evaluate the �-SMA protein expression, fibroblasts5 × 105 cells/well) were seeded in a 24-well plate. After treatment,ells were washed with ice-cold PBS and scraped in lysis buffer20 mM Tris–HCl, pH 7.5, 138 mM sodium chloride, 10% glycerol,% Triton X-100, 2 mM EDTA, 10 �g/ml leupeptin, 1 mM phenyl-ethylsulfonyl fluoride, 1 mM sodium orthovanadate). Proteins

20 �g) were resolved by 10% SDS–polyacrylamide gel and wereransferred to PVDF membranes. Membranes were blocked with 5%on-fat milk in powder form (Nestlé, São Paulo, Brazil) and probedith anti-�-SMA (Dako; 1:200) overnight at 4 ◦C. Membranes were

hen washed and incubated with the appropriate secondary anti-odies. Bound antibodies were detected by chemiluminescenceSanta Cruz Biotechnology) and densitometry analysis was per-ormed using ImageJ software (NIH). Subsequently, membranesere stripped with a stripping buffer plus �-mercaptoethanol and

eprobed with anti-�-actin (Sigma–Aldrich; 1:1000).

tatistical analysis

Data (mean ± standard error of mean) were analyzed byann–Whitney test or Student’s t test with Welch’s correction

sing the GraphPad Prism software (GraphPad Prism version 5.0,an Diego, CA, USA). Values of p < 0.05 were considered significant.

esults

n vivo assays

Topical application of J. princeps seed oil accelerated woundontraction over the course of the experiment (Fig. 2A and B). Inddition, the length of migratory tongue length was greater in the

control group. Scale bar = 50 �m.

treated group than in the control group 14 days after wounding(Fig. 2C and D).

The control group presented a high number of inflammatorycells on granulation tissue 14 days after wounding, while thetreated group presented a predominance of ‘fibroblast-like’ cellsand fewer inflammatory cells on granulation tissue (Fig. 3A andB). To measure inflammatory infiltrate, the number of neutrophilsand macrophages were counted 14 days after wounding. The num-ber of MPO-positive neutrophils and F4/80-positive macrophageswas smaller in the treated group than in the control group 14days after wounding (Fig. 4A and B). Cell proliferation on granu-

lation tissue was greater in the treated group than in the controlgroup 14 days after wounding (Fig. 4C). However, keratinocyte pro-liferation on neo-epidermis was similar to control (62 ± 7%) and

A. Donato-Trancoso et al. / Acta Histochemica 116 (2014) 1169–1177 1173

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ig. 3. Microscopic analysis of wound area in control (A, C) and treated groups (B, Dresents a high number of inflammatory cells, while in the treated group ‘fibroblastellow-reddish, thick, and arranged parallel to surface 14 days after wounding in co

reated (47 ± 6%) groups 14 days after wounding. Volume den-ity of myofibroblasts was smaller in the treated group than inhe control group 14 days after wounding (Fig. 4D). In both stud-ed groups, collagen fibers were mainly yellow-reddish, thick, andrranged parallel to surface 14 days after wounding (Fig. 3C and D).

morphometric analysis using image analysis software demon-trated that collagen fiber density was greater in the treated grouphan in the control group 14 days after wounding (Fig. 4E). Toonfirm this observation, hydroxyproline levels were measuredn lesions 14 days after wounding. Hydroxyproline levels werereater in the treated group (4.05 ± 0.04 ng/mg tissue) than in theontrol group (2.97 ± 0.17 ng/mg tissue) (p < 0.05). Volume den-ity of blood vessels was greater in the treated group than inhe control group 14 days after wounding (Fig. 4F). VEGF proteinxpression was greater in the treated group than in the con-rol group 14 days after wounding (Fig. 4G). Results of PECAM-1rotein expression confirmed the increase in blood vessel den-ity (Fig. 4H). The levels of carbonyl protein were smaller inhe treated group (1.96 ± 0.20 �mol/�g total protein) than in theontrol group (2.57 ± 0.11 �mol/�g total protein) 14 days afterounding (p < 0.05).

n vitro assays

In the scratch assay, the denuded area (fibroblast-free area) wasmaller in the cells treated with 0.1 mg/ml of J. princeps seed oilompared with control cells one day after scratching (Fig. 5A and). The denuded area in control positive (DMEM with 10% FBS)as completely covered by fibroblasts 1 day after treatment. In theTT assay, the rate of fibroblast proliferation was greater in the

ells treated with 0.1 mg/ml of J. princeps seed oil than in controlells after one day of treatment (Fig. 5C). �-SMA protein expressionas smaller in the cells treated with J. princeps seed oil than in the

ontrol cells 1 day after treatment (Fig. 5D).

ays after wounding. (A, B) In hematoxylin–eosin stained sections, the control groupcells predominate. (C, D) In Sirius red-stained sections, collagen fibers were mainly

and treated groups. Scale bar = 50 �m.

Discussion

The use of medicinal plants has been increasing throughout theworld for maintenance and improvement of health and for treatingvarious human conditions and diseases. Topical administration ofoils or fatty acids can be a low-cost alternative for treating cuta-neous lesions when compared to conventional synthetic drugs.Several studies show that oil administration rich in linoleic oroleic acids has therapeutic effects on skin tissue repair (Otrantoet al., 2010; Cardoso et al., 2011; Kim et al., 2009; Magdalon et al.,2012; Rodrigues et al., 2012). Recently, it has been reported that oilobtained from J. princeps seeds is composed of two units of linoleicacid and one unit of oleic acid (Berty et al., 2011). Therefore, wehypothesized that oil from J. princeps seeds may improve cutaneouswounding healing.

Inflammation, which requires the migration of neutrophils andmacrophages to wound areas, should occur rapidly to allow thedevelopment of subsequent phases. Several studies propose thatconjugated linoleic acid supplementation or oleic acid admin-istration accelerates the inflammatory phase of wound healingimproving cutaneous wound healing (Linz et al., 1994; Parket al., 2010; Cardoso et al., 2011; Rodrigues et al., 2012). Ini-tially, it has been shown that a diet rich in linoleic decreasesmononuclear cell number and TNF-� levels in wound fluid (Linzet al., 1994). Conjugated linoleic acid supplementation or oleicacid administration decreases nuclear factor �B (NF�B) activa-tion and downregulates cyclooxygenase-2 expression reducinginflammatory response (Park et al., 2010; Cardoso et al., 2011).In addition, leukocyte migration, neutrophil chemoattractant-2��(CINC-2��), and TNF-� secretion and NF�B activation are greaterin wound area of rats treated with linoleic or oleic acids 1 h after

wounding, whereas they are smaller after 24 h (Rodrigues et al.,2012). Similar results are observed in neutrophil culture wherelinoleic or oleic acid increases production of interleukin-1� (IL-1�)and CINC-2�� after 4 h in culture and linoleic or oleic acid decreases

1174 A. Donato-Trancoso et al. / Acta Histochemica 116 (2014) 1169–1177

Fig. 4. Microscopic and biochemical evaluation of wounds in control and treated mice. Number of myeloperoxidase (MPO)-positive neutrophils (A), F4/80-positivemacrophages (B), and PCNA-positive connective tissue cells (C) on wound area of control and treated groups 14 days after wounding. Stereological analysis showingvolume density for myofibroblasts (Vv[myofibroblasts]%) (D) in control and treated groups 14 days after wounding. Collagen density (in pixels) (E) on wound area of controland treated groups 14 days after wounding. Stereological analysis showing volume density for blood vessels (Vv[blood vessels]%) (F) in control and treated groups 14 days afterwounding. Densitometry expressed as arbitrary units (a.u.) for immunoblotting of VEGF (45 kDa) (G) and PECAM-1 (130 kDa) (H) in wound lysate 14 days after wounding.�-Actin (43 kDa) was used as a loading control protein. Data (n = 5) are expressed as mean ± SEM. *p < 0.05 vs. control group.

A. Donato-Trancoso et al. / Acta Histochemica 116 (2014) 1169–1177 1175

Fig. 5. Effects of Joannesia princeps (JP) seed oil on the activity of murine dermal fibroblasts in vitro. Representative images (A) and quantification (B) of the denuded area(fibroblast-free area) in murine dermal fibroblast cultures 1 (1d) and 2 days (2d) after scratching. Cell proliferation (C) in murine dermal fibroblast cultures 1 day aftert ensita s usedc

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reatment. DMEM with 10% serum fetal bovine (SFB) was used as positive control. Dctin (�-SMA) (42 kDa) (D) in cell lysate 1 day after treatment. �-Actin (43 kDa) waontrol group (0 mg/ml).

he release of the same cytokines after 18 h (Rodrigues et al., 2010).n macrophage culture, linoleic or oleic acid decreases produc-ion of TNF-�, IL-1�, and CINC-2�� (Yaqoob and Calder, 1995;

agdalon et al., 2012). Topical administration of J. princeps seed oilecreased neutrophil and macrophage number in the wound area.hese results indicate that linoleic and oleic acids present in the oilrom J. princeps seeds may decrease inflammatory phase acceler-ting the subsequent phases of healing process and, as a cascade,ccelerating wound closure.

During the inflammatory phase, neutrophils and macrophageselease reactive oxygen species (ROS) as hydrogen peroxide that killicroorganisms and degrade damaged tissue structures (Bickers

nd Athar, 2006). Toxic concentrations of ROS cause severe damageo cellular components such as protein, lipids and DNA impair-ng wound healing (Wlaschek and Scharffetter-Kochanek, 2005).igh levels of hydrogen peroxide cross cellular membranes and inssociation with metal ions induce lipid peroxidation and proteinxidation (Wlaschek and Scharffetter-Kochanek, 2005). Conju-ated linoleic acid supplementation or oleic acid administrationay have antioxidant effects and decrease oxidative damage (Park

t al., 2010; Rodrigues et al., 2012). Conjugated linoleic acid supple-entation reduces lipid peroxidation in mouse liver (Park et al.,

010). Oral administration of linoleic or oleic acid increases hydro-en peroxide production 1 h after wounding and decreases theelease of this ROS after 24 h (Rodrigues et al., 2012). Thus, linoleicr oleic acid administration may accelerate the production of ROS

n wound site leading to accelerated migration of inflammatoryells (Rodrigues et al., 2012). The oil from J. princeps seeds reducedarbonyl protein levels on wound area 14 days after wounding. Thisesult suggest that linoleic and oleic acids present in J. princeps seed

ometry expressed as arbitrary units (a.u.) for immunoblotting of �-smooth muscle as a loading control protein. Data (n = 9) are expressed as mean ± SEM. *p < 0.05 vs.

oil may decrease oxidative damage induced by ROS contributing toa reduction of inflammatory cell migration and an improvement ofwound healing.

Angiogenesis refers to new vessel growth by the sprouting ofpre-existing vessels adjacent to the wound. New blood vessels areimportant to sustain the development of granulation tissue. Growthof new blood vessels is mainly stimulated by VEGF (Barrientoset al., 2008). Topical administration of linoleic or oleic acids, or oraladministration of sunflower oil does not alter blood vessel num-ber and VEGF protein expression in rat wounds (Cardoso et al.,2004; Otranto et al., 2010; Rodrigues et al., 2012). However, a dose-dependent increase in VEGF expression is observed in neutrophilculture incubated in the presence of oleic or linoleic acids (Pereiraet al., 2008). In addition, topical application of ozonated olive oilincreases VEGF expression in the epidermal keratinocytes of pigwound (Kim et al., 2009). Wounds topically treated with J. princepsseed oil presented an increase in the blood vessel number and VEGFprotein expression. Thus, we propose that the increase in VEGFexpression induced by linoleic and oleic acids in J. princeps seed oilstimulates blood vessel formation promoting the wound healing.

Dermal reconstruction is characterized by the formationof granulation tissue, which includes angiogenesis, fibroblastsproliferation and differentiation, and extracellular matrix (ECM)deposition. Topical treatment with J. princeps seed oil increasedconnective tissue cell proliferation and collagen deposition in micewound developing a more vascularized and organized collagenous

granulation tissue. Dermal fibroblasts are essential for granulationtissue formation, because they permit the dermal reconstruc-tion. These cells proliferate and migrate to lesion area anddeposit a collagen-rich matrix. Some fibroblasts differentiate into

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yofibroblasts (�-SMA-positive cells) that are responsible foround contraction. We observed that oil from J. princeps seedsirectly enhanced cell migration and proliferation and reduced-SMA protein expression in murine skin fibroblast cultures. These

esults indicate that linoleic and oleic acids in J. princeps seed oilromote granulation tissue formation due to the increase in theermal fibroblast proliferation and migration.

Closure of cutaneous lesions is a result of the two mainvents: contraction and re-epithelialization. Interaction betweenyofibroblasts and its surrounding extracellular matrix plays an

mportant role in wound contraction (Desmoulière et al., 2005). Re-pithelialization is a process of restoring the epidermis and consistsf proliferation and migration of keratinocytes. In this study, the oilrom J. princeps seeds accelerated wound closure by contraction ande-epithelialization. However, J. princeps seed oil reduced myofi-roblast differentiation in vivo and in vitro assays. Recent studiesave shown that �-SMA expression is not the main event whichtimulates wound contraction, since �-SMA knockout mice presentormal contraction of cutaneous lesions (Tomasek et al., 2013). Inddition, the reduction of myofibroblastic differentiation may have

therapeutic role in treatment of excessive scar as hypertrophiccar and keloids. It has been demonstrated that the treatment withleic or linoleic acid might increase collagen deposition and woundontraction during early phase of wound healing (Cardoso et al.,004, 2011). In addition, J. princeps seed oil increased the lengthf the migratory tongue, but did not alter keratinocyte prolifera-ion 14 days after wounding; indicating that linoleic and oleic acidsn J. princeps seed oil stimulates re-epithelialization through thencrease in the keratinocyte migration.

Some studies have demonstrated that neutrophils isolated fromats supplemented with oleic and linoleic acid present an increasen the lipid content (either in the membrane or in droplets) of oleicnd linoleic acid (Rodrigues et al., 2010). In human umbilical veinndothelial cells, the incorporation of oleic acid by cell membranesay protect against lipid peroxidation (Vossen et al., 1995; Sola

t al., 1997). In addition, treatment with n-3 PUFA may decrease-6:n-3 PUFA ratio in erythrocyte membranes leading to rever-al of the vascular oxidative stress in ovariectomized rats (Gortanappellari et al., 2013). However, the mechanism by which fattycids are incorporated by cell membranes is still not understood.

In conclusion, topical application of the oil from J. princeps seedsmproves cutaneous wound closure of mice due to an increasen angiogenesis, collagen deposition, keratinocyte migration, andbroblast activity and a reduction in the local inflammatoryesponse and oxidative damage.

cknowledgements

This study was partially supported by the National Councilor Scientific and Technological Development (CNPq) (grant num-er: 472898/2010-8) and the Carlos Chagas Filho Foundation foresearch Support in the State of Rio de Janeiro (FAPERJ) (grantumber: E-26/110.668/2011).

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