| Background and ObjectionDiabetes mellitus(DM)is reaching epidemic proportions worldwide.A global health reports that the number of DM patient dramatically increased during the past 2 decades.If the situation keeps developing without any interruption,the patient number will reach more than 360 million people by 2030.Up to 25%of DM patients will develop a foot ulceration,with half developing infection and requiring hospitalization,and 1 in 5 requiring amputation.Wound healing is a dynamic and complex process that can be divided into four partly overlapping phases:hemostasis,inflammation,proliferative and remolding.DM induces impaired wound healing by affecting one or more segments in the process.Re-epithelialization and fibroblast-mediated centripetal contraction play important roles in wound closure.Keratinocytes in diabetic wounds have been found to have abnormal characteristics,as decreased differentiation and impaired migration ability.Fibroblasts are also found to exhibit decreased proliferation,increased apoptosis and decreased migration.Although there were some topical treatments for non-healing diabetic wounds have been reported to be effective,it need to be noticed that there is only one single recombinant growth factor,recombinant human platelet-derived growth factor-BB(rhPDGF-BB)has been approved by the USA Food and Drug Administration.But because of the easy degradation character,the application of growth factors is quite complicated and instable.So far as we know,there is no ointment therapy that can stimulate keratinocyte and fibroblast activity for diabetic wounds has been reported.Thus an ointment agent which is easy to use and effective by stimulating proliferation and migration of keratinocyte and fibroblast to heal intractable ulcers are needed.Recently,a new agent in ointment form,which is a synthetic agonist of BLT2,for diabetic wound healing was discovered.BLT2 is a receptor for leukotriene B4(LTB4).There are two distinct G protein-coupled receptors(GPCR)for LTB4 defined as high-affinity receptor BLT1 and the low-affinity receptor BLT2.LTB4 is a potent inflammatory lipid mediator derived from arachidonic acid.It is a chemotactic factor and activator of phagocytic cells,differentiated T cells,and dendritic cells,involved in inflammation and immune responses.BLT2 is primarily expressed in epidermal keratinocytes and small intestinal tissue in mouse.But,in human,BLT2 was found to be expressed mostly in spleen,followed by liver,ovary and leukocytes,with weak signals in some other organs,such as lung,thymus and brain et al.,Liu M.et al.investigated the BLT2 expression in mouse and human skin and found that BLT2 mRNA is prominently detected in both mouse and human epidermal keratinocyte,but not in dermal fibroblast.They also uncovered that wound healing process was delayed in BLT2-knockout mice and both of the endogenous and synthetic BLT2 agonist promoted diabetic and non-diabetic wound healing by enhancing keratinocytes migration in wild type mice.BLT2 agonists accelerates keratinocyte migration by stimulating NF-?B signaling,which then induced the expression of TNF and MMPs.(17)12(S)-Hydroxyheptadeca-5Z,8E,10E-trienoic acid(12-HHT)which was considered as the by-product of Thromboxane A2(TXA2)biosynthesis,has been identified as a endogenous ligand for BLT2.(18)Iizuka Y.et al.reported a synthetic BLT2-specific agonist 4-{[pentanoyl(phenyl)amino]meth-yl}-1,1-biphenyl-2-carboxylic acid(CAY10583)that activates both human and mouse BLT2 at low concentration than LTB4,and induces Ca2+ mobilization and phosphorylation of extracellular signal-regulated kinase through BLT2(19).The effect of synthetic BLT2 agonist on wound healing has only been evaluated in mouse,and investigated on epidermal keratinocyte,there is no study about its effect on dermal fibroblasts has been reported yet.In current study,we investigated the efficiency of synthetic BLT2 agonist on fibroblast.Also,prior to verifying its therapeutic potential on promoting wound healing in human,we applied synthetic BLT2 agonist on wounds in larger animals,such as rat,as preclinical study for future practical use.Materials and MethodsAnimalsLewis rats(6-9weeks of age,180-220g of weight)purchased from Japan SLC Inc.(Tokyo,Japan)were used for this study and maintained under a 12hour light/12 hours dark cycle.Food and water were available ad libitum.Animals were induced diabetic by a single intraperitoneal injection of 65mg/kg streptozotocin(STZ)(Sigma-Aldrich Co.Ltd.,St.Louis,M.O.)dissolved in 0.01M sodium citrate buffer pH4.5.24hours and 48 hours after injection,nonfasting blood glucose was measured with a glucometer(Sanwa Kagaku Kenkyusho Co.Ltd.Nagoya,Japan)in the blood obtained from tail vein,and the rats with consecutive glycemia above 350mg/dl were kept.4 weeks later,the blood glucose of selected rats was measured,the rats maintained hyperglycemia were considered diabetic and used for wound healing models.All animal procedures were performed in accordance with the guidelines of the Juntendo University Animal Care and Use Committee.Wound healing modelThe procedure were performed on anesthetized animals having dorsal hair removed with shaver(THRIVE(?),Daito Electric Machine Industry Co.Ltd.,Osaka,Japan)and NairTM hair remove cream(Church&Dwight Co.Inc.,Ewing,N.J.).Two 1.5cm-diameter round full-thickness skin defects were made in dorsal skin cleansed with 10%popiyodon solution(Yoshida Pharmaceutical Co.Ltd.Tokyo,Japan)and 75%alcohol on each rat.Four Indian ink tattoos were intradermally made at a distance of 4mm to the border of each wound to evaluate skin contracture.A silicone ring with 2cm inner diameter and 3.6cm outer diameter was placed with glue(Daiichi-Sankyo Co.Ltd.,Tokyo,Japan)around each wound and sutured with 4-0 nylon(Akiyama-seisakusyo Co.Ltd.,Tokyo,Japan)to minimize skin contracture.The synthetic BLT2 agonist,CAY10583(CAY)(Caymen Chemical Co.Ltd.,Ann Arbor,Mich.),was dissolved in vehicle dimethyl sufoxide(DMSO)(Sigma-Aldrich Co.Ltd.,St.Louis,Mo.)to make a CAY solution(100?M).CAY solution mixed with white Vaseline(Wako Pure Chemical Industries Ltd.,Osaka,Japan)was applied to the wounds(n=6)in CAY group(final concentration of CAY =10?M).The wounds(n=6)in control group were topically treated with white Vaseline mixed with only DMSO of the same concentration as CAY group.All wounds were covered with surgical dressing(Alcare Co.Ltd.,Tokyo,Japan)and Tegaderm film(3M,Maplewood,Minn.).Dressing and medication were changed daily.Digital photographs of wounds were gained every two days with a digital camera(Canon Inc.,Tokyo,Japan).Wound area was measured with computer-assist image analysis software(BZ-? Analyzer;Keyence Co.Ltd.,Osaka,Japan)and was calibrated by calculating the percentage of the wound area to the inner area of silicone ring to correct for magnification,perspective,or parallax.Wound closure ratio was calculated on days 0,2,4,6,8,10,12 and 14 with following formula:Hematoxylin and Eosin(H-E)StainingAnimals were sacrificed on post-operative day 14.Wound tissue samples were collected,fixed in 10%formalin solution(Wako Pure Chemical Industries Ltd.,Osaka,Japan)and paraffin embedded.4 micro-meter sections from central area of wounds were used for H-E staining.In H-E staining slides,photographs were gained with BZ-9000 microscopy(Keyence Co.Ltd.,Osaka,Japan),re-epithelialization and granulation tissue formation were assessed by measuring epithelium gap and thickness of granulation respectively.In every slide,the thickest and thinnest of the thickness of granulation were measured,and then the average of the thickness was calculated as the thickness of the granulation tissue by using computer-assist image analysis software(BZ-? Analyzer;Keyence Co.Ltd.,Osaka,Japan).Epithelium gap distance was obtained by measuring the distance of the two ends of epithelium tongue.Keratinocyte and fibroblast culturesPrimary rat keratinocyte and fibroblast were isolated from Lewis new-born rats within 3 days.Rat keratinocytes were isolated and cultured in rat keratinocyte culture medium(Cell Application Inc.,San Diego,Calif.)by following the protocol previously published.Rat fibroblasts were cultured in Dulbecco's Modified Eagle Medium(DMEM)(Thermo Fisher Scientific Inc.,Waltham,Mass.)containing 10%fetal bovine serum(FBS)(Thermo Fisher Scientific Inc.,Waltham,Mass.),100U/ml penicillin and 100mg/ml streptomycin in a humidified incubator at 37? with 5%CO2 atmosphere.For experiments,the cells were used between two and three passages.Reverse transcription-polymerase chain reaction(RT-PCR)Total RNA was prepared from cultured rat keratinocytes or fibroblasts by using TRIzol reagent(Thermo Fisher Scientific Inc.,Waltham,Mass.).Collected RNA was used for the RT reaction with high capacity RNA-to-cDNA kit(Applied Biosystem,Waltham,Mass.).A BLT2 expression vector was used as a positive control.After the RT reaction,PCR was performed by using Ex Taq(Takara Bio Inc.,Shiga,Japan)and a pair of primers to target rat BLT2(forward:5'-CGT CTT TAC TGC GGG TGATT-3',reverse:5'-TGC CCT GAC CCA CTA CTT TC-3,)or rat GAPDH(forward:5'-GGC AAG TTC AAT GGC ACA GT-3',reverse:5'-TGG TGA AGA CGC CAG TAG ACT C-3').The PCR mixtures were incubated at 95?/5m(initial denaturation),followed by 35 amplification cycles of 95?/3s(denaturation),55?/30s(annealing)and 72?/30s(extension).The reaction products were analyzed by electrophoresis(100V/30m)on 2%agarose gels,stained with ethidium bromide(Bio-Rad Laboratories Inc.,Hercules,Calif.),and visualized under UV light.Molecular size of the amplified fragments were determined by compared with the 100bp DNA ladder(Takara Bio Inc.,Shiga,Japan).The expected product sizes for BLT2 and GAPDH were 155bp and 260bp.GAPDH served as a internal quantitative control.Conditioned medium of keratinocytesRat keratinocytes(3×106)were seeded on a 100mm dish coated with Type I collagen(Corning Inc.,Corning,N.Y.)and cultured in 10ml keratinocyte medium.After 48 hours,the medium was replaced with 10ml CAY+ keratinocyte medium(keratinocyte medium + 100nM CAY10583 + 0.02%DMSO)or CAY-keratinocyte medium(keratinocyte medium + 0.02%DMSO).After cultured for another 48 hours,the CAY+ keratinocyte supernatant and CAY-keratinocyte supernatant were collected,centrifuged at 200×g,4? for 5 minutes to remove cell debris and stored at-80? until it was used for the following fibroblast scratch assay and ELISA.Scratch assayRat keratinocytes and fibroblasts were seeded on 96-well ImageLock tissue culture plate(Essen BioScience Inc.,Ann Arbor,Mich.)and incubated in a standard CO2 incubator to form cell monolayers.The 96-well-plates for keratinocytes were coated with Type I Collagen(Corning Inc.,Corning,N.Y.).Fibroblast monolayers were starved by being cultured in 1%FBS medium for at least 12 hours before the scratches were made.Wounds were made with 96-well wound maker(Essen BioScience Inc.,Ann Arbor,Mich.).Each wound was washed twice with culture medium to remove the unattached cells.In keratinocyte scratch assay,scratches were divided into two groups and applied with:Group 1:keratinocyte medium + CAY(100nM)+ 0.02%DMSO.Group2:keratinocyte medium + 0.02%DMSO.To investigated the indirect application of CAY on fibroblast by scratch assay,we separated fibroblast scratches into four groups and treated with:Group:KC CAY+ sup:CAY+ keratinocyte supernatant + DMEM + FBS(1%)Group2:KC CAY-sup:CAY-keratinocyte supernatant + DMSO + DMEM +FBS(1%)Group3:KC med:keratinocyte medium + DMSO + DMEM + FBS(1%)Group4:KC med + CAY:keratinocyte medium + CAY(100nM)+DMSO + DMEM +FBS(1%)The photographs of wounds were automatically taken by IncuCyte zoom software(Essen BioScience Inc.,Ann Arbor,Mich.).In fibroblast scratch assay for investigating the direct application of CAY on fibroblast.We seeded rat fibroblast on 6-well plate(Corning Inc.,Corning,N.Y.)and incubated in a standard CO2 incubator to form cell monolayer.The cell monolayers were starved in 1%FBS medium for at least 12hours before scratching.The scratches were made with P1000 tips.After moved the unattached cells,we applied 1%FBS medium w/o different concentration of CAY(1nM,10nM,100nM,1000nM)to scratches.Wound closure ratio was expressed as migration area by using computer-assist image analysis software(BZ-? Analyzer;Keyence Co.Ltd.,Osaka,Japan).Cell proliferation assayThe proliferation of keratinocyte and fibroblast was evaluated by performing 3-(4,5-Dimethylthiazol-2-yl)-5-(3-Carboxymethoxyphenyl)-2-((4-Sulfophenyl)-2H-T etrazolium)Chloride(MTS)Assays according to the manufacture's protocol using Cell Tilter 96 AQueous One Solution Cell Proliferation Assay(Promega Co.Ltd.,Fitchburg,Wis.).2×103 fibroblast or keratinocyte were seeded per well in 96-well plate,cultured for another 24 hours and allowed to attach.For keratinocyte,96-well plates were coated with collagen type I.Then keratinocytes were treated with keratinocyte culture medium containing CAY at different concentrations(1nM,10nM,100nM,1000nM).In control group cells were treated with keratinocyte culture medium containing same concentration of DMSO.After 96 hours' incubation,20 mL of One Solution Reagent was added into each well of the 96-well assay plate containing the samples in 100 mL of culture medium.The cells were incubated for an additional 2h at 37? in a humidified,5%CO2 atmosphere.The production of formazan produced by viable cells was measured at an absorbance of 490 nm using a 96-well plate reader.cell proliferation was analyzed..Fibroblasts were cultured with KC CAY+ sup,KC CAY-sup,KC med or KC med + CAY for 24 hours,then cell proliferation was evaluated.Enzyme-linked immunosorbent assay(ELISA)Transforming Growth Factor-?1(TGF-p 1),basic Fibroblast Growth Factor(bFGF),Vascular Growth Factor(VEGF)and Hepatocyte Growth Factor(HGF)content was determined on the keratinocyte conditioned medium by using Quantikine ELISA kit(R&D Systems Inc.,Minneapolis,Minn.)according to manufacturer's protocol.Colorimetric reactions were read at 450nm in a microplate reader(Molecular Devices LLC,Sunnuvale,Calif.).The samples for detecting the content of TGF-?1 was concentrated six-fold using an ultra-o.5 Contrifugal Filter with a molecular weight cutoff(MWCO)of 30K(Merk Millipore Co.Ltd.,Darmstadt,Germany)..Statistical analysisData are all presented as the mean ± SD.All statistical analyses were performed using unpaired Student's t test(two groups)or one-way ANOVA with post hoc tests(greater than two groups).Significant was considered at P<0.05.All statistics were performed by Prism 5 software(GraphPad Software Inc.,La Jolla,Calif.).ResultsTopical application of CAY accelerated in vivo wound healingThe STZ-induced rats were confirmed to be diabetic after four weeks of blood sugar level higher than 3 50mg/dl before wounding.Application of CAY(10?M)to silicone ring stented skin defects demonstrated accelerated wound closure compared to control group at day4(22.57±3.3vs.16.01±5.99%,;p=0.0407),day6(54.74±12.75 vs.39.45±7.84%;p=0.0313),day8(78.05±12.22vs.59.84±11.09%;p=0.0222),day10(90.82±9.32vs.71.04±9.68%;p=0.0048),day12(97.89±2.05vs.82.73±4.84%;p<0.0001),and day 14(100±0vs.90.44±15.34%;p=0.0071)BLT2 was expressed only in keratinocyte,not in fibroblastThe gene expression of BLT2 receptor was investigated in epidermal keratinocyte and dermal fibroblast which play a main role in the process of re-epithelialization and granulation formation respectively.RT-PCR was performed on cultured neonatal rat primary keratinocytes and fibroblasts.The result showed that BLT2 mRNA was only detected in rat keratinocyte,not in fibroblast.The data suggested that BLT2 agonist CAY can only be functional to rat keratinocyte.CAY promoted re-epithelialization and granulation formationOn the post-operation 14th day,wound tissue samples were harvested for H-E staining to measure the width of epithelium gap and granulation thickness.It was found that the epithelium gaps of the wounds in control group were significantly wider compared to the controls(0±0 vs.3411±2025 im;p=0.0021).(figure 3B)Surprisingly,the thicknesses of the granulation tissue of the wounds in CAY group were significantly increased.(968.3±198.1 vs.526.1±203.4?m;p=0.0034).(Figure.3C)Direct application promoted rat keratinocyte scratch closure by promoting its migrationWe performed rat keratinocyte scratch assay to assess the in vitro effect of CAY on rat keratinocyte.When the keratinocyte scratches were directly applied with CAY,the cell-free area was significantly reduced in 96th hour after the scratches were made compared to control group(90.63±11.23 vs.40.08± 12.21%;p<0.0001)(figure 4).Different concentration of CAY was also directly applied to keratinocyte when performing MTS assay.There was not significant difference found in each group.(figure 7A)This indicated that keratinocyte scratch closure accelerated by CAY was due to the increased keratinocyte migration.Direct application of CAY did not promote rat fibroblast scratch closure.As BLT2 mRNA was only detected in rat keratinocyte,not in rat fibroblast,it indicates that CAY does not make effect on rat fibroblasts directly.When we directly applied different concentraions of CAY(1nM,10nM,100nM or 1000nM)to rat fibroblast in scratch assay,no significant difference of cell-free area was observed between control group and the other different CAY concentration groups.(figure 5)This confirmed that CAY does not affect rat fibroblast directly.Indirect application of CAY promoted rat fibroblast scratch closure by promoting its proliferationCAY has been demonstrated to be not effective to rat fibroblast.However,the thickness of granulation tissue was found to be increased in CAY-treated wounds.Therefore we hypothesize that,after CAY-treatment,the dermal fibroblast was promoted by some cytokines released from the CAY-stimulated epidermal keratinocytes.To test this hypothesis,we added the supernatant of keratinocytes stimulated with CAY(100nM)to fibroblast in scratch assay.We found that the closure was significantly accelerated in the scratches,which were cultured with CAY-stimulated keratinocyte supernatant for 24 hours.(75.95±4.09 vs.49.69±4.49 vs.47.78±8.34 vs.47.24±4.39%;p<0.0001)Under the same condition as scratch assay,in MTS assay,we found that the fibroblast proliferation was significantly enhanced when treated with the CAY+ keratinocyte supernatant.The levels of TGF-?1 and bFGF increased in CAY+ keratinocyte supernatantAs the previous results demonstrated only indirect application of CAY enhanced fibroblast proliferation and in vitro fibroblast scratch closure.We performed ELISA to measure the level of possible fibroblast stimulating factors in CAY+ keratinocyte supernatants.The level of TGF-?1 and bFGF were found to significantly increase in the CAY+ keratinocyte supernatant.(TGF-?1,15.79±3.16 vs.9.29±1.4pg/ml;p=0.001.bFGF,38.84±10.7 vs.24.79±5.1pg/ml;p=0.0158)There was no significant difference found in the VEGF or HGF content.(VEGF,60.96±15.12 vs.63.88±27.48pg/ml;p=0.8243.HGF,74.02±9.153 vs.73.84±3.191pg/ml;p=0.9651)DiscussionIn 2000,during the course of analysis of the genomic structure of human and mouse BLT1,a putative seven-transmembrane-type receptor with sequence similarities to BLT1 was designated as BLT2 by Yokomizo et al..Nowadays,in contrast to the well-defined BLT1,little was known about the role of BLT2.Until recently,BLT2 has been reported to be involved in wound healing process.The endogenous BLT2 agonist(12-HHT)and synthetic BLT2 agonist(CAY)were found to accelerate the wound healing by promoting keratinocyte migration in mice.It suggests that BLT2 agonist would be potential on promoting normal or diabetic wound healing.Moreover,12-HHT is one of lipid mediators(LM),which are considered to be involved in various processes including host defense,inflammation,ischemia reperfusion,hemostasis,and also wound healing.On wound healing,many studies have addressed the effects of growth factors and cytokines,but little was learned about the role of LM.The exploitation of the potential of LM on promoting wound healing would provide a new method on the management of wounds.In current study,our data demonstrated that the synthetic BLT2 agonist,CAY10583,promoted the healing of impaired wounds in diabetic rats with enhanced re-epithelialization and increased granulation tissue.CAY directly promoted only the migration of rat keratinocyte,which contributed to the enhanced re-epithelialization of the wounds.Consistent with the result of RT-PCR that BLT2 mRNA was only detected in rat keratinocyte,CAY did not directly promote the closure of rat fibroblast scratches.When applied with the supernatant of rat keratinocyte cultured of CAY to rat fibroblast,the scratch closure and the proliferation of rat fibroblast were both promoted.This indirect and positive stimulation of CAY on rat fibroblast leads to the increased granulation tissue formation.And furthermore,elevated content of TGF-?1 and bFGF was found in the supernatant of CAY-stimulated rat keratinocyte.Wound healing is a sequence of complex biological and molecular events including cell migration,cell proliferation and extracellular matrix deposition.Non-healing diabetic wounds is an intractable problem needed to be solved for patients and doctors.To restore an intact epidermal barrier is considered to be essential for the optimal wound closure.Re-epithelialization begins within several hours after skin injury.Without complete re-epithelialization,a wound can not be considered healed.The keratinocyte in chronic wounds,such as diabetic wound,have been found to be lack of migration ability which leads to the absence of the migration of the keratinocyte at the edge of the diabetic non-healing wound.Here we used a diabetic wound model to demonstrate that CAY promoted impaired diabetic wound healing with enhanced re-epithelialization.In vitro keratinocyte scratch assay and proliferation assay,it revealed that CAY promoted rat keratinocyte's migration,not proliferation.We established an anti-contracture wound model in diabetic rats and looked into the formation of granulation tissue.The granulation formation plays an important role in wound healing process,however the effect of CAY on granulation has not been yet investigated.Our data shows that,although there was no BLT2 mRNA detected in rat dermal fibroblast,the granulation formation was still promoted.So we hypothesized that CAY stimulated rat fibroblast indirectly via the cell mediator(s)produced and released from CAY-stimulated keratinocyte.In order to verify our hypothesis,we applied the supernatant of CAY stimulated keratinocyte to fibroblast.It is found that the scratches closure and the proliferation of fibroblast were promoted.Complicated interaction between keratinocyte and fibroblast throughout the healing process is considered critical for the wound closure and repair.It showed that the level of TGF-?1 and bFGF was increased in the supernatant of the keratinocyte after the CAY stimulation.In wound healing,TGF-?1 involves in inflammation,angiogenesis,re-epithelialization and connective tissue regeneration.The most well-known function of TGF-?1 on dermal fibroblast is to induce the fibroblast-to-myofibroblast differentiation.Myofibroblast plays an key role in the contraction of collagen matrix and wound closure.Except for TGF-?1,bFGF content in keratinocyte supernatant was also found to be increased after the stimulation of CAY.Although epidermal keratinocyte is not the major resource of bFGF,it reported that the expression or protein level of bFGF in keratinocyte were increased in different kinds of wounds and species,including human burnr wound,rat burn wound,mouse excisional wound and pocrine excisional wound.bFGF is widely considered to be an important factor of promoting the proliferation and migration of fibroblast.Take together with our data mentioned above,we conclude that synthetic BLT2 agonist stimulates epidermal keratinocyte to release TGF-?1 and bFGF to promote fibroblast.There is a limitation of current study includes not knowing the exact mechanism of how CAY promotes the secretion of TGF-?1 and bFGF in epidermal keratinocyte.The condition of the keratinocyte or fibroblast culture in this study was not simulated as in vitro high blood glucose environment.It did not completely morphologically or functionally represent the diabetic-impaired keratinocyte or fibroblast.It is a limitation of this experiment.But we have assessed the function of CAY on rat diabetic in vivo wounds.Taking together,it suggest CAY could be effective on both wound healing for both diabetic and non-diabetic patients.CAY has been examined by Iizuka Y.et al.that it can induce calcium mobilization and ERK phosphorylation only through human and mouse BLT2,not through BLT1.Also,in the mice deficient in BLT1,the wound closure was found to be normal.These indicate the BLT1 is not involved in wound closure and current study.When mentioned about BLT2,the role BLT2 is not thoroughly known.As mentioned above,BLT2 mRNA expression is found abundantly in human leukocyte.In details,high expression of BLT2 was found in human CD8+ cytotoxic T-,CD4+helper T-,and CD 19+ B-cells.These suggest that BLT2 may involve in immune system and chemotaxis.Mathis et al.found that BLT2 plays a role in murine autoantibody-induced severe inflammatory arthritis.But BLT2 was also reported to own a protective function of enhancing the epithelial cell barrier in the murine inflammatory colitis.Wang et al.investigated the effect of CAY on NK cells,and revealed that CAY has no effect on NK cell cytotoxicity.More investigations for the risk of CAY inducing local or systemic inflammation are needed.Some studies suggest the relation of BLT2 and cancer.BLT2 was found to expressed in some pancreatic cancer and stimulate the tumor cell proliferatio.However,the mechanism of BLT2 in cancers is not completely known yet.Pro-inflammation and enhancing cancer could be the potential side effects of the synthetic BLT2 agonist treatment for wounds.If its application could be used on clinic,these potential side effects need to be on the alert.In current study,the BLT2 agonist applied on diabetic wound are in ointment form.So far as we known,this is the first report about a ointment topical treatment for diabetic wounds.Compared with current existing treatments for diabetic wounds,it is more stable and convenient to use.In summary,we have demonstrated that the synthetic BLT2 agonist,CAY promotes impaired wound healing in diabetic rats by directly promoting epidermal keratinocyte migration and indirectly enhancing dermal fibroblast.CAY enhances fibroblast via the increased secretion of TGF-?1 and bFGF from keratinocyte.ConclusionCAY promotes keratinocyte migration directly and enhances fibroblast proliferation via the TGF-?1 and bFGF released from CAY-stimulated keratinocytes to accelerate rat diabetic wound healing.CAY will be an alternative pharmaceutical agent for diabetic wounds. |
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