GIORNALE ITALIANO DI•

• DERMATOLOGE VENEREOL0(

OFFICIAL JOURNAL OF THE SOCIETÀ ITALIANA DI DERMATOLOGIACHIRURGICA, ESTETICA E DELLE MALATTIE SESSUALMENTE TRASMESS

THE ZEBRAFISH EMBRY0 DERIVATIVE AFFECTS CELL VIA]OF EPIDERMAL CELLS:

A POSSIBLE ROLE IN THE TREATMENT OF PSORIASI

G. NORMA. P. M. RIAVA. E DI N E M )

VOLUME 148 - No. 5 PAGES 479-483- o c r o m 2 0 13

In patients affected hy psoriasis, use of a topical formula con-taining a derivative of zebratish ernbryos was as,sociated withreduced skin inf iammation a n d derma' turnover, as well asa generally better outcome. In an attempt tu understand themolecola r mechanistns lying beyond these findings, we inves-ligated the anti-prol i ferative effects of the zebrafish embryosderivative hy a(ldressing the mitochondrial funct ion (1VITTassay) and celi nuclei distr ibut ion dloestch staining I n celicuitures stimulated vvith fetal cal i serum (FCS) or epidermalgrowth factor (-E G F ) , t h e z e b r af i s h d e r iv a t iv e s i g ni f i ca n t ly

inhibitcd celi pro l i ferat ion induced by either approach, al-though the effect was stronger in cells stimulated w i th l i r &These results suggest tha t the zebrafish embryos derivativemay dampen increased cel i pro l i ferat ion: th is observationmay be relevant to cutaneous pathologies related to alteredproliferative mechanisms, including psoriasis.KEY WORDS: Psoriasis - Zebrafish C e l i proliferation - Epider-unti growth factors.

filasotiasis i s a relapsing, non-infective, autoirn-mune, chronic fornì o f dermatitis (affecting the

scalp, elbows, knees, lumbosacral region, genitalskin, soles o f the feet and palms) that is extremelycommon worldwide. The most usual dermatologicevents are papules and clearly defined erythematouspapules that can develop erythematous-desquama-tive plaques)

From a histological point of view, psoriatic lesionsappear as skin hyper-proliferation areas — whose epi-dermal turnover is 5 lo M times faster than that ofthe norma! skin — accompanied by incomplete matu-ration of keratinocytes and the retention of nuclei in

Corresponding author: G. Norata, Department of Biomolecular Sei-enees, University of Milan, Milan, Italy. E-mail: danilo.norata@unimi.it

Cl ITAL. DERMAMI., VENERI

The zebra ish embryo defivative arects ceo epideE

a possa* role in the treatment

G. NORATA 13, P . M . B I A VA

'Department of BiomoUniversity o f M

Barts and The London School of MedQueen Mary Univet

3 Istituto di Ricovero e Cura a GaiMultime,

4Velleja Rese4

the stratum comeum (parakeratosis),larisation, increased blood fiow, proliami l imited capabilities o f the l y nAlso noticeable is the infiarnmator3ported by the infiltration of polymoriin the skin.2 The causes of psoriasis are stili unavailable data seems Lo hint at a multithat certainly involves genetics and alany case, most authors agree that thecause of the problem is an altered Thmune response, consequent to a pro,tory insidi. Whatever the triggeringof triggered T lymphocytes causesdi fferent active substances (gamma-iphokines, growth factors).

However, these do not seem tostances responsible f o r the reactiotof fact, psoriatic skin shows highp-endorphin and other angiogenic fa<a loc:d reactivity with reduced celi al

NORATA T H E ZEBRAFISH EMBRY0 DERIVATIV E A l21 1 3 C T S C E L I , V I A B I L I T Y 0 1

A preparation for topical use was recently devel-oped and successfully tested in clinica] t r ia ls;7, 8 l tcontains some active ingredients wel l known f o rtheir anti-inflammatory ( i 8-beta glycyrrhetic acidphytosome, boswellic acids, Zanthoxyllum bungea-num extract) and anti-psoriatic properties (7-clehy-drocholesterol), together wi th a partieular peptidederivative obtained f r o m zebrafish (Brachydaniorerio) embryos.9-16 T h i s d e r i v a ti v e ,1 7 c o n ta i n in g i ow -

molecular-weight protein factors (15 KDa) — ob-tained through homogenisation and subsequent f i l -tration and purification — seems Io be able to contro]some phases of the celi cycle with the stabilisationof the differentiation processes.18, 1 9 T h i s a c t i o n w a s

demonstrated in turo in tests intended to assay themitotic proliferation of keratinocyte clones. This ac-tion appeared to be partieularly beneficial in patientsaffeeted by psoriasis, vvhere the reduced capability ofceli clifferentiation of the basai lamina causes plaqueformation to be reduced owing to a lower prolifera-tive index of keratinocytes.20The study was thus aimed al verifying whetherthe effect that had previously been observed vvith thepeptide derivative obtained from zebrafish embryos7, 8 might be also linked to the anti-proliferative ac-tion o f the compound. For this reason the derivativeobtained from zebrafish embryos was tested in celicultures fol lowing induction o f celi proliferationthrough administration o f fetal calf serum (FCS) orepidermal growth factor (EGF).

Materials and methods

The study o f the anti-proliferative effect o f thezebrafish embryo derivative on epidermal cel ls(Keratinocytes, L i t e Technologies, U S A , ma in -tained in medium 154) was conducted using a mi-tochondrial function assay with methyl tetrazolium(MTT assay) purchased f rom Sigma (USA). The12-well culture plates (6x104 c e l l s p e r w e l l ) u s e d

for the test contained FCS or EGF in the presenceor absence of the zebrafish derivative. Celi viabil-ity was determined as the ratio between the cellsincubated w i th the zebrafish derivative and thecells incubated without i t (control). The M T T as-say was conducted according to a model describedin a previous work .21Viable and actively proliferating cells retain animportant mitochondrial activity, and th is aspect

is exploited by incubating the celiabove different experimental condcompound in question, which is the]mitochondria and produces a colouter is solubilised in isopropanol anca plate reader. The signal intensitythe amount of viable cells found in ting lo the adopted experimental patEuroclone, Italy), or EGF (I ng/mL;the vehiele alone (RPMI 1640) wererative stimuli_

Since the zebrafish derivative coreroi and 5% ethanol, the same quartiwas added lo final volume of the "e<

The zebrafish derivative concentr,experiments amounted to I Opg/mLtreatment with FCS as the proliferilluorescence analysis was also perto highlight the celi nuclei by Hoestfiuorescence analysis was per fo r i-nemethod described in a previous worl

The information provided by theis expressed as the mean value ± seand refers to 4 different experimentanalysis was performed using the Alowed by the Bonferroni analysis usoftware.

Results

The increase in celi viability folleulation (Figure I ) was signiticantlypresence of the compound (obtainelfour chief stages of the cmbryo develwhich are generally linkecl Io differtsion proftles), while the reductionthe cells were incubated with the ind

When looking at Figure I i t can Iviability decreases at 96 hours, andthe sign of some exhaustion of the cetial in the presence of FCS.

The detailed data concerning 72(Figure 2) shows a large and statisti,reduction due Io treatment with the 2tive containing all the four embryo s•

In the case of treatment with FCStive stimulus, a fluorescence analysiformed in order lo highlight the celiHoestch staining.

THE ZEBRANSH EMBRY0 D M v A n A F i q u i e r s CELI, O F EPIDERMAI CELLS

FCS

O l •I• I -2•• I , . • I t i o

Figure 3.—Immunolluorescence through Hoestch

Figure 3 clearly shows a reduction in the cells o fthe samples treated w i th the zebrafish derivative,partieularly in those treated with the derivative con-taining ali the embryo stages.

Treatment with EGF alone was found to be lessefficient in indueing celi proliferation, but even inthis experimental condition (Figure 4) the zebrafish

dio 1 • ( S

% ceti via bility

h • s

Figure 1.—Celi viabil i ty in the presence a FCS and zebrafish F i g u r e 2—Cel i v iab i l l y test at.72 hours folembryo derivative. Mean±standard error is shown; *P<0.05 vs. w i t h FCS. Meanistandard error is shown;FCS; **P<0.01 vs. FC.S. * * P < 0 . 0 vs . FeS.

7F1 Z F 4 Z f

derivative containing all the four stagithe only remedy capable o f slowingli feration•

The detailed data concerning 72 htive stimulus (Figure 5) shows a stati!eant reduction due to treatment wi t tderivative containing ali the four stagi

N ORAFA

Discussimi

Ti IF ZEIM E M B R V O D E R I V A-I P / L A r F E C T S V i A i l l t J TV 0 1

•

Figure v i a b i l i t y in the presence of EGF and zebrafish em-bryo derivative. Mean±standard error is shown; * 13< 0 . 0 5 v s . E G E

The aim of this work was Lo investigate the effectof the derivative obtained from zebrafish ernbryos oaceli viability in preliferating keratinocytes.

On the basis o f the rcI l l t ing experimental data itis possible to observe how the compound shows agood anti-proliferative action highlighted bythe M T T assay and nuclear analysis through fluo-rescence. Both the extract prepared during the firststage (1) and i hai [ l ' e v o-A d u r i n g t h e l a s t s t a ge ( 4 )

of the embryo eitiwiIi process demonstrated to pos-sess some antiproliterative capability; neverthelessthe derivative containing all the four stages (mix)proved to be more effective with a statistically sig-nificant activity•

This result was observed fol lowing stimulationwith either FCS or EGE It may be hypothesised thatthe higher elficacy recorded with FCS is due to thepresence of dilferent growth factors and the posi 1)11-ity that the extract containing the foui di fferent stag-es may have a more homogeneous composition thatis capable o f interfere wi th different rnechanismsassociated with «• Il proliferation. Future studies arewarranted to investigate the zebrafish ernbryo de-rivative effects in cells under psoriatic pathlogicalconditi ons.

These results agree with the observation that thezebrafish embryo derivative contained in the topicaiformulation i s effective in counteracting psoriasissymptoms;7, 8 w h i le a l so h i nt i ng at the p os si bi l it y

celi viability

i

Figure v i a b i l i t y test ai 72 hours widerivative. Meanistandard e r i-o r i s s h o w n ;that, in addition t o the antiphlogiserved in the preparation tinti ascribto its content in glycyruhetic acid aids, the preparation may re lieve ,k iI hrough n lodo lation of thu cui I turn

The preliminary indications o fscientific rationale for the use o fbryo derivative i n preparations wtarget is the control o f the skin ciparticular reference to possible deplications.

Riassunto

Il derivato di embrioni da zebrafishtà cellulare delle cellule epidermiche: •trattamento della psoriasi

L 'u t i l i zzo topic() di una preparazionerivato di embrioni da zebrafish ha dirtipsoriasici, di poter ridurre la sintornatczione cutanea e il turnover epidermico.sii meccanismi potessero essere legaticrescita cellulare, si è deciso di investiproliferativo di tale derivato sul i,'mica attraverso l'analisi della vitalitàla valutazione della funzionalità mito(della distribuzioi i a t leare (colorazicderivato embrionale d i zebratish è ri.•contrastare la proliferazione cellularestimolate con fetal calf serum (FCS) cfactor (EGF). I l dato è apparso particte nei saggi con FCS, in cui l 'effetto r

11112 ZILIMA1tS11 Etvit3RYO 11F R A F F E C TS C E L I, V I A B IL I TY OF E P I DE R M AL

essere la conseguenza della stimolazionc da parte di di f-ferenti fattori di crescita. I risultati ottenuti suggerisconoun possibile ruolo del derivato da zebratish nel controllarel'aumentata risposta prolifcrativa tipica dell'epidermoidedi pazienti psoriasici•PAROLE CHIAVE: Psoriasi - Zebratish - Cellule, prolifera-zione - Fattore di crescita epidermica•

References

i. Conway P. Currie Ci, Descriptive epidemiology of hospitallsationfor psoriasis Curr Med Rea Opin 2008:24:3487-91

2. leen M. Crowson CS, McEvoy MT, Danti Fi, Gabriel SE, Mara-dit Kreuners H. Trends in incidence of aduit-onset psoriasis overthree decades: a population-bascd study i Arn Acad Dermatol2009;60:394-401.

3. Blauvelt A , Bickenbach HZ. Kulesz-Martin MF, Bowcock AM.Montagna symposium 2008: the biologic basis of psoriasis. J InvestDermatol 2009:129:259-60,

4. Schmid-Ott G, Jaeger B, Boehm T, Langer K. Stephan M, Raapet al. Infintinological effects o f stress in psoriasis. Br i Dermatol2009;160:782-5,

5. Tsuruta D. NF-kappaB links keratinocytes and lymphocytes in thepathogcnesis of psorlasis. Recent Pat Inflamm Allergy Drug Discov2009;3:40-8.

6. Jain S, Kaur IR, Das S, Bhattacharya SN, Singh A. T helper I toT helper 2 shift in cytokine expression: an autoregulatory processin superantigen-associated psoriasis progression? .1 Med Microbiol2009;58(Pt 2):180-4.

7. D i Pierro F, Negri M. Bollero C. Terapia della Psoriasi: EfficaciaClinica di un Preparato Multicomponente. Cosmetic Technology2009:12:25-31.

8. Calzavara Pinton P, Rossi M. Una preparazione topica in concomi-tanza ala fototerapia. Valutazione dell'efficacia in pazienti affetti dapsoriasi di grado moderato. hi.techdermo 2012:7:29-35.

9. Mainini G. Rotondi M, Scaffa C. A new approach in the first-linetreatment of bacterial and mycotic vulvovaginitis with topical l i-pohydroperoxides and glycyrrhetic acid: a comparative study. ClinExp Obstet Gynecol .2011:38:243-6.

IO. Puglia C, Rizza L, Drechsler M. Bonina E Nanoemulsions as vehi-

cies for topical administration of glycyrrhelicand in vitro and in vivo eviduation. Drug Deli

1 , Veraldi S, De Micheli P, Schianchi R, Lunapruritus in mild-to-inoderate atopic dermatisteroidal agent. i Drugs Dermatol 2009;8:537

12. Pellati D, Fiore C. Armanini D, Rassu M, Befects of glycyrrhetinic acid on the growth of cidida albicans. Phytother Res 2009:23:572-4,

13. Calzavara-Pinton P, Zane C, Facchinetti E, CaTopical Boswellic acids fra treatment of photTher 2010;23 Suppi l S28-32,

14. Pedretti A, Capezzcra R, Zane C, FacchinettP. Effects of topical boswellic acid on photo aclinica', biophysical, and cchographie evaluatirandomized, split-face study, Planta Med 200

15. Singh S. Khaiutia A, Tancja SC, John RK, Singiic acids: A leukotriene inhibitor also effective ti-tion in inflammatory disorders. Phytomedicine

16. Artaria C, Maramaldi G. Bonfigli A. Rigano Iing properties of the alkamide fraction from 11thoxylum bungeanutm Int J COSITiet Sci 2011;

17. Blava PM, Produci with anti-psoriatic activity05101687.118, Biava PM, Carluccio A. Activation of anti-on,

by embryonic extracts in Vita) minor cells. i1997:4:9-15,

19. Biava PM, Post-transiational modification oprotein (pRb) induccd by in vitro administratbryonic extracts on human kidney adenocarci

r Marker Onc 2002; l 7:59-64.20, Webb AE, Driever W. Kimelman D. Psoriasú

development in nbratish. Dev Dyn 2008;237:21. Norata GD, Piriti() A, Callegari E. Hamsten

iksson P. Grate expression and intracellularendothelial dysfunction induced by VLDL 2Cardiovasc Res 2003:59: i 69-80.

Conllicts of interest.—Dr. Biava owns azebrafish embryo. He is also the president o f thCompany, which owns the "Starniblo" brand,

Received on November 13, 2012.Acceptcd for publication on May 13, 2013.