Angiogenesis activity of Jatropha curcas L. latex in cream formulation on wound healing in mice

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1 Veterinry Worl, EISSN: Aville t RESEARCH ARTICLE Open Aess Angiogenesis tivity of Jtroph urs L. ltex in rem formultion on woun heling in mie Ummu Blqis 1, Drmwi 2,3, Cut Dhli Isknr 4 n Muhmm Nur Slim 1,5 1. Lortory of Pthology, Fulty of Veterinry Meiine, Syih Kul University, Bn Aeh 23111, Inonesi; 2. Lortory of Miroiology, Fulty of Veterinry Meiine, Syih Kul University, Bn Aeh 23111, Inonesi; 3. Lortory of Reserh, Fulty of Veterinry Meiine, Syih Kul University, Drusslm, Bn Aeh 23111, Inonesi; 4. Lortory of Histology, Fulty of Veterinry Meiine, Syih Kul University, Bn Aeh 23111, Inonesi; 5. Stuy Progrm of Mthemtis n Applie Sienes, Syih Kul University, Bn Aeh 23111, Inonesi. Corresponing uthor: Muhmm Nur Slim, e-mil: mnurslim@unsyih..i Co-uthors: UB: ummu.lqis@unsyih..i, Drmwi: rmwi@unsyih..i, CDI: ut.ifn@gmil.om Reeive: , Aepte: , Pulishe online: oi: /vetworl How to ite this rtile: Blqis U, Drmwi, Isknr CD, Slim MN (2018) Angiogenesis tivity of Jtroph urs L. ltex in rem formultion on woun heling in mie, Veterinry Worl, 11(7): Astrt Aim: The im of this reserh ws to etermine the ngiogenesis tivity of Jtroph urs ltex in rem formultion on CD34 immune expression uring woun heling phse in mie skin. Mterils n Methos: Amount of 36 2-month-ol mle mie were use etween 30 n 40 g. To surgil proeures, woun skin inision ws performe 2.0 m in length until suutneous on the prverterl of eh niml. The tretment ws rrie uner lolly nesthetize with proine rem. All mie were ivie into four groups, nmely the se rem s ontrol group (A), sulfizine 0.1% rem (B), Jtroph urs ltex rem 10% (C), n J. urs ltex rem 15% (D). All groups were trete entire surfe of woun. All experiments were performe twie y for 10 ys. Experiments were terminte on ys 3, 7, n 10, respetively. The woun heling ws ssye in stine histologil setion in immunohistohemil of the wouns. The CD34 expression ws investigte uner mirosope. Results: The results showe tht the rem from 10% n 15% ltex J. urs revele moerte immune retion to CD34 on ys 3 n 7 in woun heling of mie skin. Conlusion: We onlue tht the rem from 10% n 15% ltex J. urs hs potentil s ngiogenesis tivity in woun heling of mie skin. Keywors: ngiogenesis, CD34, Jtroph urs ltex rem, woun heling. Introution The woun is efine s rw skin surfe n lost its surfe epithelil overge use y vrious types of injuries or iseses [1,2]. It n e mge or isruption to the norml ntomil struture n funtion of skin [3]. Wouns tht exhiit impire heling, inluing elye ute wouns n hroni wouns, generlly hve file to progress through the norml stges of heling [4]. Neovsulriztion or ngiogenesis is importnt for woun heling proess. Angiogenesis plys role in woun heling y forming new loo vessels from preexisting vessels y inving the woun lot n orgnizing into mirovsulr network throughout the grnultion tissue [5]. However, ngiogenesis lso plys role in the provision of nutrients n oxygen to the woun re n inreses the formtion of grnultion tissue with its ollgen n onnetive tissue proteins Copyright: Blqis, et l. Open Aess. This rtile is istriute uner the terms of the Cretive Commons Attriution 4.0 Interntionl Liense ( whih permits unrestrite use, istriution, n reproution in ny meium, provie you give pproprite reit to the originl uthor(s) n the soure, provie link to the Cretive Commons liense, n inite if hnges were me. The Cretive Commons Puli Domin Deition wiver ( puliomin/zero/1.0/) pplies to the t me ville in this rtile, unless otherwise stte. eposition [6]. A supopultion of hemtopoieti progenitor CD34 ells oul iretly ontriute to the neovsulriztion n woun repir proess [7]. CD34 is ell surfe mrker tht is expresse y ro rnge of ells inluing hemtopoieti, stroml, epithelil, n enothelil ells [8,9]. Woun heling is influene y mny ftors inluing the type of meition use. One is the use of tritionl meiine. The use of tritionl meiine is inresingly fvore euse of fewer sie effets suh s rugs from hemils [10], the ost is reltively low, n it is esily essile [11]. Seonry metolites from herl plnts tht re potentil s miroil gents my help overome the prolem of ntiioti resistne [12]. Jtroph urs L. (Euphoriee) is multiple purpose with potentil for ioiesel proution n meiinl uses [13]. Some of the enefits of J. urs re tht they n pt well to ry ln, re esy to ultivte, n their utiliztion oes not ompete with foo rops suh s ssv, mize, oonut, n plm oil [14]. J. urs is generlly grown s live fene for protetion of griulturl fiels from mge y livestok s it is not eten y ttle [15] n to prevent erosion [16]. All prts of J. urs hve lso een use in tritionl meiine n for veterinry purposes [17]. Veterinry Worl, EISSN:

The leves n ltex re use in heling of wouns, refrtory ulers, n septi gums, n stypti in uts n ruises [18]. J. urs ontins numer of iotive ompouns tht re flvonois, sponins, tnnins, n polivenol.

2 The leves n ltex re use in heling of wouns, refrtory ulers, n septi gums, n stypti in uts n ruises [18]. J. urs ontins numer of iotive ompouns tht re flvonois, sponins, tnnins, n polivenol. The ompouns ply role in ntiteril [19], ntioxint, nti-inflmmtory, ntiner [20], ntiieti tivities [21], n woun heling [22]. However, limite informtion is ville on the woun heling proess of this plnt, espeilly ngiogenesis tivity. The im of this reserh ws to etermine the ngiogenesis tivity of J. urs ltex in rem formultion on CD34 immune expression uring woun heling phse in mie skin. The results will then e ompre to tht of tretment with sulfizine in stnr woun therpy. Mterils n Methos Ethil pprovl This reserh ws performe ppropritely following the regultion of Animl Ethis Committee. This reserh ws pprove y the Animl Ethis Committee of Fulty of Veterinry Meiine, Syih Kul University, Bn Aeh, Inonesi (Approvl No. 004/KEPH-C/VII/2017). Mie Amount of 36 hes mle mie (Mus musulus) ge 2 months ol n weighing g were use in this stuy. All mie mintine iniviully in ges t the Lortory of Pthology, Fulty of Veterinry Meiine, Syih Kul University. Fee n wter were given liitum for 2 weeks. Preprtion of o/w rem formultion The ltex use in the present stuy quire rek of J. urs lef stems whih were proure from lol frm roun yr res of Bn Aeh. The ltex ws ollete in the morning with rek of lef stems, ltex t pity into sterile ottle. The ltex ws formulte in rem following the proeure s esrie y Muntih et l. [23]. J. urs ltex rem me with se of oil in wter (O/W) euse the formultion is homogeneity, spreility, simply to use, non-stiky, soothing on the skin, n esy to len. A rem se ws e little y little with the 10% n 15% ltex of J. urs in porelin ish ontining 100 g of rem n stirre until homogeneous t room temperture. The osges of 10% n 15% ltex of J. urs hve een proven to e effetive to use for topil for topil woun heling [24]. Formtion of exision woun moel The mie skin woun ws one y men of surgil proeures s expline y Slim et l. [24]. Mie were lolly nesthetize y topil rem with ontining lioine 25 mg n priloine 25 mg. Longituinl wouns of out 2.0 m were me on the prverterl region till to suutneous uner the septi onition n were oserve throughout the experimentl stuy. Grouping of mie for experimentl esign The mie were seprte into four groups ontine nine mie of eh. group A s negtive ontrol, the mie skin woun reeive se rem. group B s positive ontrol, the mie skin woun reeive stnr rug ontining sulfizine 0.1% rem. group C, the mie skin woun trete with rem ontining J. urs ltex 10%. group D the mie skin woun trete with rem ontining J. urs ltex 15%. The topil experimentlly pplie twie ily t m n pm strting from the y of wouning till to mesure on 3 r, 7 th, n 10 th post-wouning y. Histopthologil n immunohistohemil expression of CD34 Woun skin tissue smples were ollete in 10% uffere formlin for histopthologil exmintion [25,26]. The tissues were proesse y routine prffin emeing tehnique, n 5 µm setion ws stine with immunohistohemil stining y stnr methos s esrie y Drmwi et l. [27], using streptviin-iotin omplex. For ngiogenesis immunoretion, we use the CD34 monolonl mouse ntioy (Dko, 1:50 ilution) ilute in 1% BSA in PBS. We efine the sores of CD34 expression s negtive, 0 points (Gre 0), mil 1 point (Gre 1), moerte 2 points (Gre 2), n severe 3 points (Gre 3) [28]. The intensity of stining ws reore s wek, moerte, n strong [29]. Results The results of the present stuy showe tht immunohistohemil of CD34-positive expression is hrterize y rown olor on mirosopi slies. The CD34 monolonl mouse ntioy h strong retion with ntigen on the enothelil progenitor ells n enothelil ells s shown in Figures-1-3. On y 3, the CD34 positive ells were seen in enothelil ells of eh group. The miniml immunoretivity of CD34 positive ells ws oserve on group A (Gre 1). Menwhile, the moerte immunoretivity Figure-1: Photomirogrph of wouns skin on y 3 of tretment (streptviin-iotin 40 ). CD34-positive ells in enothelil ells eh group tht mrke the rown olor on Group A (Gre 1). (-) Moerte immunoretivity CD34-positive ells on Groups B, C, n D (Gre 2). Veterinry Worl, EISSN:

Figure-3: Photomirogrph of wouns skin on y 10 of tretment (streptviin-iotin 40 ). Immunoretivity CD34 ells eh group tht mrke the rown olor on Group A (Gre 1).

3 Groups B, C, n D s sign of the woun heling proess. Figure-2: Photomirogrph of wouns skin on y 7 of tretment (streptviin-iotin 40 ). CD34-positive ells in enothelil ells eh group tht mrke the rown olor on Group A (Gre 1). (-) Moerte immunoretivity CD34-positive ells on Groups B, C, n D (Gre 2). Figure-3: Photomirogrph of wouns skin on y 10 of tretment (streptviin-iotin 40 ). Immunoretivity CD34 ells eh group tht mrke the rown olor on Group A (Gre 1). (-) Negtive immunoretivity CD34 ells on Groups B, C, n D (Gre 0). of CD34 positive ells ws foun in group B, C, n D (Gre 2) s seen in Figure-1. In this phse, CD34- positive immunoretion is sign of ngiogenesis or new pillry formtion n the migrtion of enothelil progenitor ells from the vsulr. The results of the y 7 showe tht the migrtion of enothelil progenitor ells n new pillries more lerly seen. As seen in Figure-2, the miniml immunoretivity of CD34 positive ells in enothelil ells were oserve on group A (Gre 1), ut on group B, C, n D we foun the moerte immunoretivity of CD34 positive ells (Gre 2). The rem from 10% n 15% ltex of J. urs n sulfizine 0.1% rem revele moerte immune retion to CD34 on ys 3 n 7 in mie skin woun heling. The results show tht the numer of enothelil progenitor ells in migrtory n ifferentite tissue ereses with the enothelil formtion on y 10. As shown in Figure-3, the miniml immunoretivity of CD34 positive ells ws only foun on group A (Gre 1). However, the negtive immunoretivity of CD34 ell ws foun on group B, C, n D (Gre 0). In this phse, CD34 is negtive immune retion in Disussion The simplest interprettion of our fining on y 3 is shown in Figure-1 n y 7 is shown in Figure-2; it ws foun tht the ngiogenesis on Groups B, C, n D (Gre 2) is etter thn the Group A (Gre 1). Guo et l. [7] reporte lose orreltion etween n inrese in irulting CD34 + ells in response to trumti injury n ngiogenesis in trumti rin injury. A signifint inrese in the numer of irulting CD34+ ells etete in trumti rin injury. Inee, the high levels of CD34+ were lso oserve in 7 ys fter the injury. The results foun tht ngiogenesis involve in inflmmtion phse (y 3) n prolifertive phse (y 7). The levels of ngiogenesis in wouns often orrelte with the inflmmtory response, lrgely euse inflmmtory ells proue n unne of prongiogeni meitors [7]. In onfirmtion of our previous stuy, we foun tht the rem from the 10% n 15% ltex of J. urs revele moerte immune retion to CD68 on woun heling [24]. Mrophges s inflmmtory ells serete vsulr enothelil growth ftor (VEGF) tht inues ngiogenesis [30]. Here, we esrie tht the CD34 monolonl mouse ntioy h strong retion with ntigen on the enothelil progenitor ells n enothelil ells. Clinil n niml stuies provie eviene tht stem progenitor ells re ritil for neovsulriztion [31]. Angiogenesis is the growth of new loo vessels from existing enothelil ells of the woun s jent y mture stroml ells suh s firolsts [32]. The similr phenomenon esrie y Ieronimkis et l. [33] who notie tht the frtion of CD34 + ells remins stey uring homeostsis n injury regenertion. In this stuy, we gree with n support those of Kiry et l. [34] who expline tht most hemtopoieti stem ells express CD45, n suset of hemtopoieti stem ells express CD34. Bloo vessels re ompose of epithelil tues with epithelil ells tht trnsport ruil flui. During lumen formtion, enothelil ells in mture loo vessels represente s the first phenotype n seon phenotype, n enothelil ells polriztion often strts with the elivery through exoytosis, suh s CD34 [35]. The effet of J. urs espeilly on woun heling hs een stuie using niml moels. Mny previous reports exist out the effiy of J. urs on woun heling. Jsmi et l. [36] evlute tht the ointment from 10% ltex of J. urs hs the potentil to elerte the woun heling of urns on the skin of mie. Slim et l. [37] inite tht methnol extrt of J. urs leves ws potentil in elerting woun heling in mie. Our previous stuy inite tht ointment from 10% ltex of J. urs hs proven to elerte the epithelil perio [38,39], n remoeling perio on woun heling in mie skin [40]. This Veterinry Worl, EISSN:

4 uses tht the ointment from 10% ltex of J. urs effetive to e use for topil therpy on woun heling in mie skin. The queretine n routine flvonois t J. urs n improve the woun heling proess on the regultion of VEGF expression for the growth of new loo vessels (ngiogenesis) [18]. Angiogenesis involve in the prolifertive phse whih is prinipl step in epitheliliztion of woun heling [5]. This results stuy showe tht on y 10, CD34 is unetete on Groups B, C, n D (Gre 0). The erese in the numer of migrtory n ifferentite enothelil progenitor ells ws in line with enothelil ells formtion [41]. DiPietro [42] stte tht fter n injury, levels of prongiogeni ftors inrese, rehing pek slightly efore mximum pillry ontent ours, n then susie to nerly unetetle levels. Conlusion We onlue tht the rem from 10% n 15% ltex J. urs hs potentil s ngiogenesis tivity in woun heling of mie skin. Authors Contriutions Drmwi n MNS esigne the reserh; UB n CDI performe immunohistohemistry; UB, Drmwi, CDI, n MNS prepre the mnusript. All uthors re n pprove the finl mnusript. Aknowlegments We wish to thnk the Diretorte of Reserh n Community Servie, the Diretorte Generl of Strengthening Reserh n Development, Ministry of Reserh, Tehnology n Higher Eution of Repuli Inonesi for funing of the work grnt from the Hih Bersing with ontrt numer: 129/SP2H/ LT/DRPM/III/2016. Competing Interests The uthors elre tht they hve no ompeting interests. Referenes 1. Prsetyono, T.O.H. (2009) Generl onept of woun heling, revisite. Me. J. Inones., 18(3): Agyre, C., Bokye, Y.D., Bekoe, E.O., Hensel, A., Dph, S.O. n Appih T. (2016) Review: Afrin meiinl plnts with woun heling properties. J. Ethnophrmol., 177: Velnr, T., Biley, T. n Smrkolj, V. (2009) The woun heling proess: An overview of ellulr n moleulr mehnism. J. Int. Me. Res., 37: Guo, S. n DiPietro, L.A. (2010) Ftor ffeting woun heling. J. Den. 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(2014) Test effetivity Jtroph multifi L. to hel woun infetion use y Stphyloous ureus in rit (Orytolgus uniulus). Phrmon, 3(3): Slim, M.N., Msyith, D., Hrris, A., Blqis, U., Isknr, C.D., Hml, M. n Drmwi (2018) Anti inflmmtory tivity of Jtroph urs Linn. ltex in rem formultion on CD68 expression in mie skin woun. Vet. Worl, 11(2): Drmwi, D., Blqis, U., Hml, M., Tiuri, R., Frengki n Priosoerynto, B.P. (2013) Muosl mst ell response in jejunum of Asrii glli-infete lying hens. Mei Peternk., 36(2): Blqis, U., Hml, M., Rinir, Athillh, F., Ismil, Azhr, Vn, H. n Drmwi, D. (2017) Cutiulr surfe Veterinry Worl, EISSN:

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