Protective effect of melatonin against the inflammatory response elicited by crude venom from...

Protective effect of melatonin against the inflammatory responseelicited by crude venom from isolated nematocysts of Pelagianoctiluca (Cnidaria, Scyphozoa)

Introduction

Among Scyphozoa, Pelagia noctiluca is an off-shore jelly-fish (purple-stripped jelly) exclusively marine, which inhab-

its warm and temperate water as Mediterranean Sea,Adriatic Sea, off coast of California, Atlantic shores andNorthern Ireland, as recently reported. Pelagia noctiluca is

known for its bioluminescence being visible in the dark andwhen disturbed [1–4].Pelagia noctiluca has not caused human fatalities, but in

spite of this it can be a nuisance and a health and

economical problem when it appears in huge numbersduring outbreaks. Contact with all body portions – bell,tentacles, and oral arms – causes local pain, burning,

swelling, hyper pigmentation, and other local symptoms inhumans; repeated contacts with this jellyfish can producerecurrent skin eruptions, which are also observed without

further stings [5]. The four cardinal signs of inflammation,

recognized for centuries are: heat, rubor, tumor, and pain.Pain is one of the most prevalent conditions limiting

productivity and diminishing quality of life [6]. In thisregard, as highlighted by the effectiveness of the nonsteroi-dal anti-inflammatory agents, pain is part of the inflamma-

tory response. It is well appreciated that during tissue injuryand inflammation, hyperalgesia results from a persistentstate of peripheral afferent sensitization that subsequently

initiates spinal sensitization. The mechanism(s) are complexand involve peripherally and spinally formed inflammatorymediators including peptides, prostanoids, nitric oxide, aswell as spinal cord glial cell activation [6].

Edema formation in paw is a result of a synergismbetween various inflammatory mediators that increasevascular permeability and/or mediators that increase blood

flow [7]. Several experimental models of paw edema havebeen described. Rat paw edema has been characterized byan early phase caused by the release of histamine,

Abstract: Melatonin (N-acetyl-5-methoxytryptamine) is an efficient free

radical scavenger and antioxidant, both in vitro and in vivo. The role of

melatonin as an immunomodulator is, in some cases, contradictory. In this

study we have investigated the therapeutic efficacy of melatonin in rats

subjected to Pelagia noctiluca crude venom (of the familia Pelaguiidae; and

genus Pelagia) induced acute paw inflammation. In particular, injection of

the venom into the paw of rats elicited an acute inflammatory response

characterized by accumulation of fluid containing a large number of

polymorphonuclear neutrophils in the paw and subsequent lipid

peroxidation. Furthermore, the venom promoted an expression of iNOS,

nitrotyrosine and the activation of the nuclear enzyme poly (ADP-ribose)

polymerase as determined by immunohistochemical analysis of paw

tissues. Administration of melatonin 30 min, 1 and 6 hr after the challenge

with the venom, caused a significant reduction in all the parameters of

inflammation measured. Thus, based on these findings we propose that

melatonin may be useful a treatment of local acute inflammation induced by

P. noctiluca crude venom.

Angela Marino1,*, Rosanna DiPaola2,*, Concetta Crisafulli3,Emanuela Mazzon2, RossanaMorabito1, Irene Paterniti3, MariaGaluppo3, Tiziana Genovese3,Giuseppina La Spada1 andSalvatore Cuzzocrea2,3

1Department of Life Sciences ‘‘M. Malpighi’’,

Section of General Physiology and

Pharmacology, University of Messina,

Messina, Italy; 2IRCCS Centro Neurolesi

‘‘Bonino-Pulejo’’, Messina, Italy; 3Department

of Clinical and Experimental Medicine and

Pharmacology, School of Medicine, University

of Messina, Messina, Italy

Key words: inducible nitric oxide synthase,

inflammation, melatonin, NF-jB, nitric oxide,

oxidative stress, Pelagia noctiluca

Address reprint requests to Prof. Salvatore

Cuzzocrea, Department of Clinical and

Experimental Medicine and Pharmacology,

School of Medicine, University of Messina,

Torre Biologica – Policlinico Universitario Via

C. Valeria – Gazzi – 98100 Messina, Italy.

E-mail: [email protected]

*These authors contributed equally to this

work.

Received February 25, 2009;

accepted April 17, 2009.

J. Pineal Res. 2009; 47:56–69Doi:10.1111/j.1600-079X.2009.00688.x

� 2009 The AuthorsJournal compilation � 2009 John Wiley & Sons A/S

Journal of Pineal Research

56

Mo

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Bio

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5-hydroxytryptamine and bradykinin, followed by a latephase mainly sustained by prostaglandin release [8]. Var-ious studies have clearly demonstrated that the production

of reactive oxygen species such as hydrogen peroxide(H2O2), superoxide, and hydroxyl radicals, as well asperoxynitrite, also contribute to the tissue injury observedduring inflammation [9–11]. In addition, recently we have

clearly demonstrated that superoxide is a key player in painassociated with acute inflammation [12].

Melatonin, is an indole produced in and secreted from

the pineal gland nightly. It has been classically associatedwith circadian and circannual rhythm regulation, and withadjustments of physiology of animals to seasonal environ-

mental changes [13]. Melatonin production, however, is notconfined exclusively to the pineal gland, but other organsand tissues including retina, Harderian glands, gut, ovary,testes, bone marrow, and lens also have been reported to

produce it [14, 15]. In recent years it has become apparentthat melatonin�s actions transcended those of a hormonalmodulator. Melatonin had been shown to subtly influence

the function of a variety of tissues and cells not generallyconsidered in the endocrine category [13]. This led to asuspicion that melatonin had yet undefined actions at the

cellular level. Numerous data suggest a role for melatoninand its metabolites function in the antioxidative defense inall organisms that produce this indole [16–21], particularly

as melatonin crosses all morphophysiological barriers andenters all subcellular compartments. Several mechanismshave been proposed for the anti-inflammatory effect ofmelatonin.

Melatonin upregulates antioxidative defensive systems,including the activities of superoxide dismutase andglutathione peroxidase as well as levels of glutathione

[18–20]. Furthermore, melatonin and its metabolites scav-enge free radicals [16–19, 22, 23] and thus reduce theburden of oxidative stress. Recent studies show that

melatonin binds several metals, including iron [24, 25]and this effect may attenuate the Fenton reaction and thegeneration of the hydroxyl radical. In this study we

identified a possible mechanism by which P. noctilucavenom induced acute paw inflammation. Moreover, wealso found that melatonin exerts significant anti-inflam-matory effects against this venom. We propose that these

effects are related, at least in part, to antioxidant proper-ties of melatonin.

Materials and methods

Materials

Biotin blocking kit, biotin-conjugated goat anti-rabbit IgG,Primary anti-nitrotyrosine antibody and avidin–biotin per-oxidase complex were obtained from DBA (Milan, Italy).

All other reagents and compounds used were obtained fromSigma Chemical Company, Milan, Italy.

Animals

The study was carried out in Sprague–Dawley male rats

(200–230 g, Harlan, Nossan, Italy). The animals werehoused in a controlled environment and provided with

standard rodent chow and water. Animal care was incompliance with Italian regulations on protection ofanimals used for experimental and other scientific purposes

(D.M. 116192) as well as with the EEC regulations (O.J. ofE.C. L 358/1 12/18/1986).

Nematocysts isolation and crude venom extraction

The nematocysts isolation method has been previouslydescribed by Salleo et al. (1983). Briefly, specimens of

P. noctiluca were collected in the Strait of Messina. Theoral arms were excised and submerged in distilled water for2 hr at 4�C. After a complete detachment of the epidermis

the tissue was removed from the suspension containingboth epidermis and undischarged nematocysts derivingfrom the osmotic rupture of nematocytes. The nemat-ocysts, still attached to the epidermal tissue, were separated

by stirring. The nematocysts suspension was repeatedlywashed in distilled water and filtered through planktonnets (40, 60, and 100 lm mesh, respectively) to remove

most of the tissue debris, and then centrifuged at 4�C (ALCPK 120R, 4000 g for 5 min). Such obtained nematocystswere classified as holotrichous isorhizas according to

Mariscal (1974). Then, the suspension was frozen at)20�C until use. Before each experiment the nematocystswere defrosted, filtered, and washed again in distilled

water. This clean suspension of nematocysts was resus-pended in 0.01 m phosphate buffer containing 0.9% NaCl(pH 7.4; osmotic pressure = 300 mOsm/kgH20). Crudevenom was extracted by sonication on ice (Sonoplus,

70 mHz, 30 times, 20 s) of a population of isolateduntreated nematocysts (90 nematocysts/lL) and the crudeextract was then separated from crushed capsules by

refrigerated centrifugation at 4000 g for 10 min. Theobtained venom was kept at 4�C until use or stored at)20�C or )80�C, unless otherwise stated. Aliquots of crude

venom were employed to measure the protein content byBio-Rad Protein Assay (BioRad, Richmond, CA, USA).

Pelagia noctiluca venom-induced paw edema

Paw edema was induced in rats by subplantar injections of69, 115, 230 lg P. noctiluca crude venom protein (crude

venom). Control animals received the same volume ofvehicle. The volume of the paw was measured by plethys-mometry (model 7140; Ugo Basile, Varese, Italy) immedi-

ately after the injection as previously described [26].Subsequent readings of the volume of the same paw were

carried out at 60-min intervals and compared with the

initial readings. In a second group of experiments pawinflammation was induced in rats treated with melatonin(50 mg/kg) or with vehicle, both injected i.p. 30 min, 1 hr,and 6 hr after crude venom administration. The dose of

melatonin used in this study is in agreement with a previousstudy demonstrating the ability of melatonin to reduce theinflammatory process [27].

Myeloperoxidase activity

Myeloperoxidase (MPO) activity, an index of polymorpho-nuclear cells accumulation, was determined as previously

Melatonin reduces crude venom-induced oedema

57

described [28]. At the specified time following the intrapl-antar injection of crude venom, tissue from the pads of therat hind paw was removed with a scalpel and 5 mm pieces

were then obtained with a tissue punch. Each piece oftissues was homogenized in a solution containing 0.5%hexa-decyl-trimethyl-ammonium bromide dissolved in10 mm potassium phosphate buffer (pH 7) and centrifuged

for 30 min at 20,000 g at 4�C. An aliquot of the superna-tant was then allowed to react with a solution of tetra-methyl-benzidine (1.6 mm) and 0.1 mm H2O2. The rate of

change in absorbance was measured by spectrophotometerat 650 nm. Myeloperoxidase activity was defined as thequantity of enzyme degrading 1 lmol of peroxide per

minute at 37�C and was expressed in milliunits per gramweight of wet tissue.

Malonaldehyde (MDA) measurement

Levels ofMDA in the paw tissuewere determined as an indexof lipid peroxidation, as described by Okhawa et al. [29]. At

the specified time following the intraplantar injection ofcarrageenan, tissue from the pads of the rat hindpaw wasremoved with a scalpel and 5 mm pieces were then obtained

with a tissue punch. Each piece of tissue was homogenized in1.15%KCl solution. An aliquot (100 lL) of the homogenatewas added to a reaction mixture containing 200 lL of 8.1%

SDS, 1500 lL of 20% acetic acid (pH 3.5), 1500 lL of 0.8%thiobarbituric acid and 700 lL distilled water. Samples werethen boiled for 1 hr at 95�C and centrifuged at 3000 g for10 min. The absorbance of the supernatant was measured by

spectrophotometry at 650 nm.

Histological examination

For histopathological examination, biopsies of paws weretaken 5 hr following the intraplantar injection of crude

venom, tissue from the pads of the rats hindpaw wasremoved with a scalpel. The tissue slices were fixed inDietric solution (14.25% ethanol, 1.85% formaldehyde, 1%acetic acid) for 1 wk at room-temperature, dehydrated by

graded ethanol and embedded in Paraplast (SherwoodMedical). Section (thickness 7 lm) were deparaffinized withxylene, stained with Haematoxylin/Eosin (H&E), Masson

trichromic with blue aniline, and silver impregnation forreticulum studied using light microscopy (Dialux 22 Leitz;Leica Microsystems SpA, Milan, Italy).

Immunohistochemical localization of nitrotyrosine,PAR, iNOS, chymase, and tryptase

At the specified time following the crude venom injection,the tissues were fixed in 10% (w/v) PBS-buffered formalde-hyde and 8 mm sections were prepared from paraffin

embedded tissues. After deparaffinization, endogenous per-oxidase was quenched with 0.3% (v/v) hydrogen peroxide in60% (v/v) methanol for 30 min. The sections were permea-

bilized with 0.1% (w/v) Triton X-100 in PBS for 20 min.Nonspecific adsorption was minimized by incubating thesection in 2% (v/v) normal goat serum in PBS for 20 min.

Endogenous biotin or avidin binding sites were blocked bysequential incubation for 15 min with biotin and avidin

(DBA, Milan, Italy), respectively. Sections were incubatedovernight with anti-nitrotyrosine antibody (Upstate Bio-tech, Prodotti Gianni, Milan, Italy) (1:500 in PBS, v/v), or

anti-iNOS polyclonal antibody (1:500 in PBS, v/v), withanti-PAR polyclonal antibody (1:500 in PBS, v/v),or anti-chymase polyclonal antibody (1:1000), or anti-tryptase polyclonal antibody (1:1000). Sections were washed

with PBS, and incubated with secondary antibody. Specificlabeling was detected with a biotin-conjugated goat anti-rabbit IgG and avidin–biotin peroxidase complex (DBA,

Milan, Italy). To verify the binding specificity for nitroty-rosine, PAR, iNOS, Chymase and Tryptase, some sectionswere also incubated with only the primary antibody (no

secondary) or with only the secondary antibody (noprimary). In these situations no positive staining was foundin the sections indicating that the immunoreactions werepositive in all the experiments carried out. Immunocyto-

chemistry photographs (N = 5) were assessed by densi-tometry by using Imaging Densitometer (AxioVision, Zeiss,Milan, Italy) and a computer program.

Terminal deoxynucleotidyltransferase-mediated UTPend labeling (TUNEL) assay

TUNEL assay was conducted by using a TUNEL detectionkit according to the manufacturer�s instruction (Apotag,

HRP kit DBA, Milan, Italy). Briefly, sections were incu-bated with 15 lg/mL proteinase K for 15 min at roomtemperature and then washed with PBS. Endogenousperoxidase was inactivated by 3% H2O2 for 5 min at room

temperature and then washed with PBS. Sections wereimmersed in terminal deoxynucleotidyltransferase (TdT)buffer containing deoxynucleotidyl transferase and bio-

tinylated dUTP in TdT buffer, incubated in a humidatmosphere at 37�C for 90 min, and then washed with PBS.The sections were incubated at room temperature for

30 min with anti-horseradish peroxidase-conjugated anti-body, and the signals were visualized with diaminobenzi-dine. The number of TUNEL positive cells/high-power fieldwas counted in 5–10 fields for each coded slide.

Western blot analysis for IjB-a, NF-jB p65, iNOS,Bax, and Bcl-2

Cytosolic and nuclear extracts were prepared with slightmodifications. Briefly, paw tissues from each mouse were

suspended in extraction Buffer A containing Hepes 10 mm,KCl 10 mm, EDTA 0.1 mm, EGTA 0.1 mm, DTT 1 mm,PMSF 0.5 mm, pepstatin A 3 lg/mL, leupeptin 2 lg/mL,

Trypsin inhibitor 15 lg/mL, Benzamidina 40 lm, homog-enized at the highest setting for 2 min, and centrifuged at13,000 g for 3 min at 4�C. Supernatants represented thecytosolic fraction. The pellets, containing enriched nuclei,

were resuspended in Buffer B containing Hepes 20 mm,MgCl2 1.5 mm, NaCl 0.4 m, EGTA 1 mm, EDTA 1 mm,DTT 1 mm, PMSF 0.5 mm, pepstatin A 3 lg/mL, leupeptin

2 lg/mL, Trypsin inhibitor 15 lg/mL, Benzamidina 40 lm,NONIDET P40 1%, Glycerole 20%. After centrifugation10 min at 13,000 g at 4�C, the supernatants containing the

nuclear protein were stored at )80�C for further analysis.The levels of IjB-a, iNOS Bax, and Bcl-2 were quantified in

Marino et al.

58

cytosolic fraction from paw tissue collected at 5 hr aftercrude venom administration, while NF-jB p65 levels werequantified in nuclear fraction. The filters were blocked with

1 · TBS, 5% (w/v) nonfat dried milk (PM) for 1 hr at roomtemperature and subsequently probed with specific AbsIjB-a (1:1000; Santa Cruz Biotechnology, DBA, Milan,Italy), or anti-Bax (1:500; Santa Cruz Biotechnology), or

anti-Bcl-2 (1:500; Santa Cruz Biotechnology), or anti-iNOS(1:1000; Transduction) or anti-NF-jB p65 (1:1000; SantaCruz Biotechnology) in 1 · PBS, 5% w/v nonfat dried

milk, 0.1% Tween-20 (PMT) at 4�C, overnight. Membraneswere incubated with peroxidase-conjugated bovine anti-mouse IgG secondary antibody or peroxidase-conjugated

goat anti-rabbit IgG (1:2000; Jackson ImmunoResearch,West Grove, PA, USA) for 1 h at room temperature.

To ascertain that blots were loaded with equal amountsof proteic lysates, they were also incubated in the presence

of the antibody against b-actin protein (1:10,000 Sigma-Aldrich Corp.). The relative expression of the protein bandsof IjB-a (�37 kDa), NF-jB p65 (65 kDa), Bax (�23 kDa),

Bcl-2 (�29 kDa) iNOS (�130 kDa) was quantified bydensitometric scanning of the X-ray films with GS-700Imaging Densitometer (GS-700, Bio-Rad Laboratories)

and a computer program (Molecular Analyst, IBM), andstandardized for densitometric analysis to b-actin proteinlevels.

Data analysis

All values in the figures and text are expressed as

mean ± standard error of the mean (S.E.M.) of N obser-vations. For the in vivo studies N represents the number ofanimals studied. In the experiments involving histology or

immunohistochemistry, the figures shown are representa-tive of at least three experiments performed on differentexperimental days. The results were analyzed by one-way

ANOVA followed by a Bonferroni post hoc test for

multiple comparisons. A P-value of less than 0.05 wasconsidered significant.

Results

To study a possible mechanism by which crude venominduced the damage biomembranes and alter membrane

permeability, in a preliminary experiments we establishedthat injection into the rat paw of 69, 115, 230 lg of crudevenom protein evoked an inflammatory reaction in a dose

dependent manner that resulted in edema within 1 hr andwas maximal at 5 hr after administration (Fig. 1A). Basedon these findings, we have evaluated the effect of melato-

nin treatment in a further experiment, which was per-formed in rats subjected to paw inflammation induced bythe injection into the rat paw of 115 lg of crude venomprotein.

Therefore, the crude venom-induced paw edema wassignificantly reduced by treatment with melatonin at alltime points (Fig. 1B). Paw tissues were examined for the

measurement of MPO activity, the latter being indicative ofneutrophil infiltration. As shown in Fig. 1C, MPO activitywas significantly (P < 0.01) increased in the paw at 5 h

after crude venom injection when compared with sham rats.MPO activity was significantly (P < 0.01) reduced bymelatonin treatment (Fig. 1C).

At histological examination, the paws revealed patho-logic changes that correlated closely with the increases inMPO activity. Paw biopsies showed that after crude venomadministration, marked inflammatory changes were

observed including pronounced cellular infiltration (Fig-s 2Ab; see particles 2Ab1). Treatment with melatoninsignificantly reduced the pathological changes in the tissues

(Fig. 2Ac). No histological alteration was observed in thepaw tissues from sham-treated rats (Fig. 2Aa).Reticular and nervous fibers as well as connective

tissues structure were observed by Masson trichromic with

2.00(A)

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Time (hr)

Vehicle*Melatonin

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Crude venom (115 µg + vehicle)

Crude venom (230 µg + vehicle)Crude venom (115 µg + vehicle)Crude venom (65 µg + vehicle)

Paw

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T3 T4 T5

Fig. 1. Injection of the rat paw with 69,115, 230 lg of crude venom proteinevoked an inflammatory reaction in adose-dependent manner (A). Melatonin(100 mg/kg) significantly reduced thecrude venom-induced paw edema (115 lgprotein) at all time points (Fig. 1B). Pawstissue were examined for the measurementof MPO activity, the latter being indica-tive of neutrophil infiltration. MPOactivity was significantly increased in thepaw at 5 hr after crude venom injectionwhen compared with sham rats. MPOactivity was significantly reduced by mel-atonin treatment (C). The results in A andB are expressed as mean ± S.E.M. ofn = 5–6 rats and *P < 0.01 versus vehi-cle treated group at the indicated timepoints. The results in C are expressed asmeans ± S.E.M. of 12 rats for each groupand *P < 0.01 versus sham; �P < 0.01versus carrageenan.


59

blue aniline, and silver impregnation. In sham-treated rats

a normal presence of reticular and nervous fibers as wellas connective tissues was observed (Fig. 2Ba). On thecontrary a significant alteration of reticular and nervous

fibers as well as connective tissues associated with a

collagen reduction was observed in the paw tissuescollected at 5 hr after crude venom administration(Fig. 2Bb). Melatonin treatment significantly reduced the

(A)

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Fig. 2. Representative paw sections from(Aa) sham rats demonstrating the normalarchitecture of the paw. On the contrarypaw biopsies showed that after crudevenom administration, marked inflamma-tory changes were observed includingpronounced cellular infiltration (Ab seeparticles Ab1). Treatment with melatoninsignificantly reduced the pathologicalchanges in the tissues (Ac). Silverimpregnation: in sham-treated rats (Ba) anormal presence of reticular and nervousfibersas well as connective tissues wereobserved. On the contrary a significantalteration of reticular and nervous fibersaswell as connective tissues associated with acollagen reduction was observed in thepaw tissues collected at 5 hr after crudevenom administration (Bb). Melatonintreatment significantly reduced the alter-ation of reticular and nervousfibers as wellas connective tissues induced by crudevenom administration (Bc). Masson tri-chromic with blue aniline: No collagenalteration was observed in the paw tissuesfrom sham-treated rats (Ca). A significantalteration of collagen fiberswas observedin the paw tissues collected at 5 hr aftercrude venom administration (Cb). On thecontrary, after crude venom administra-tion a significant reduction in collagenalteration was observed in the paw tissuescollected from rats previously treated withmelatonin (Cc). Figure is representative ofat least three experiments performed ondifferent experimental days.

Marino et al.

60

alteration of reticular and nervous fibers as well asconnective tissues induced by crude venom administration(Fig. 2Bc). Moreover, to better study the collagen alter-

ation associated with crude venom administration the pawtissues were stained by Masson trichromic. In particular asignificant alteration of collagen fibers was observed in the

paw tissues collected at 5 hr after crude venom adminis-tration (Fig. 2Cb). On the contrary, significant less colla-gen alteration was observed in the paw tissues after crudevenom administration collected from rats, which have

been treated with melatonin (Fig. 2Cc). No collagenalteration was observed in the paw tissues from sham-treated rats (Fig. 2Ca).

To test whether melatonin may modulate the inflam-matory process induced by crude venom administrationthrough the regulation of the mast cell activation, we

analyzed the paw tissue expression of chymase andtryptase by immunohistochemistry. There was no stainingfor chymase and tryptase in the paw tissues obtained from

the sham rats (Figs 3Aa,Ba). A substantial increase inchymase and tryptase expression was found mainlylocalized in mast cell in the paw tissues collected at 5 hrafter crude venom administration (Figs 3Ab,Bb; also see

densitometry analysis C). Paw tissues expression ofchymase and tryptase were significantly attenuated in thepaw tissues collected from rats that have received mela-

8 Crude venom + vehicle

Sham + vehicle

Crude venom + Melatonin

Sham + Melatonin

7

6

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4

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% o

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0 ND

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ND

* *

* *

100 µm 100 µm

100 µm 100 µm

100 µm100 µma

(A)

(C)

(B)

a

b b

c c

Fig. 3. There was no staining for chymaseand tryptase in the paw tissues obtainedfrom the sham rats (Aa,Ba). A substantialincrease in chymase and tryptase expres-sion was found mainly localized in mastcell in the paw tissues collected at 5 hrafter Pelagia noctiluca crude venomadministration (Ab,Bb). Paw tissueexpression of chymase and tryptase wassignificantly attenuated in the pawtissues collected from melatonin-treatedrats (Ac,Bc). Densitometry analysis (C)of immunocytochemistry photographs(n = 5 photos from each sample collectedfrom all rats in each experimental group)for chymase and tryptase from paw wasassessed. The assay was carried out byusing Optilab Graftek software on aMacintosh personal computer (CPUG3-266). Data are expressed as % of totaltissue area. *P < 0.01 versus Sham;�P < 0.01 versus crude venom. ND: notdetectable.


61

tonin treatment. (Figs 3Ac,Bc; also see densitometryanalysis C).In addition, we have evaluated IjB-a degradation,

phosphorylation of Ser536 on the NF-jB subunit p65and nuclear NF-jB p65 by Western blot analysis toinvestigate the cellular mechanisms by which Melatonintreatment may attenuate the development of acute paw

inflammation induced by crude venom administration. Abasal level of IjB-a was detected in the paw tissues fromsham-treated animals, whereas IjB-a levels were substan-

tially reduced in the paw collected at 5 hr after crudevenom administration. Melatonin administration pre-vented the crude venom-induced IjB-a degradation (Figs

4A,5a1). In addition, NF-jB p65 levels in the nuclearfractions from paw tissue were also significantly increased

at 5 hr after crude venom administration compared withthe sham-treated rats (Figs 4B,5b1). Melatonin treatmentsignificantly reduced the levels of NF-jB p65 as shown in

Figs 4B,5b1. As it�s known the activation of NF-jB causesthe activation of various genes with subsequent expressionof some pro-inflammatory factors; in this study weevaluated iNOS expression, which is well known to play

an important role in acute paw inflammation [30]. TheiNOS expression was investigated by Western blot andimmunoistochemistry analysis in the paw tissues collected

at 5 hr after crude venom administration. A significantincrease in iNOS levels was observed in the paw fromcrude venom-treated rats (Figs 5Ac; also see densitometry

analysis 5a1,6E). The treatment of rats with melatoninsignificantly reduced the level of iNOS (Figs 5A,6D, seedensitometry analysis 5a1,6E).Moreover, the potential participation of nitrosative stress

in acute inflammation induced by crude venom wasevaluated by immunohistochemical detection of nitrotyro-sine. At 5 hr following the intraplantar injection of crude

venom, paw tissue sections, analyzed by immunohisto-chemistry using a specific anti-nitrotyrosine antibody,revealed a positive staining in paw tissues from crude

venom-treated rats, which was primarily localized ininflammatory cells (Fig. 6Ab; also see densitometry anal-ysis D). A marked reduction in nitrotyrosine staining

(Fig. 6Ac; also see densitometry analysis D) was found inthe paw tissues of the crude venom-treated rats treated withmelatonin. In addition, at 5 hr after crude venom admin-istration, thiobarbituric acid-reactant substances levels

were also measured in the paw tissues as an indicator oflipid peroxidation. A significant increase in thiobarbituricacid-reactant substances were observed in the paw tissues

collected at 5 hr after crude venom when compared withsham-treated rats (Fig. 6B). Thiobarbituric acid-reactantsubstances (Fig. 6B) were significantly attenuated by the

intraperitoneal injection of melatonin. At the same timepoint (5 hr after crude venom injection), tissue sectionswere taken to determine the immunohistological staining

for poly ADP-ribosylated proteins (an indicator of PARPactivation). A positive staining for the PAR (Fig. 6Cb; alsosee densitometry analysis D) was found primarily localizedin the inflammatory cells present in the paw tissue from

crude venom-treated rats. Melatonin treatment reduced thedegree of PARP activation (Fig. 6Cc). Please notethat there was no staining for either nitrotyrosine or

PAR in paw tissues obtained from sham-treated rats(Figs 6Aa,6Ca).Moreover, to test whether acute paw inflammation was

associated to cell death by apoptosis, we measuredTUNEL-like staining in the paw tissue. Almost noapoptotic cells were detected in the paw from sham-treated rats (Fig. 7A). At 5 hr after the crude venom

administration, tissues from vehicle-treated rats demon-strated a marked appearance of dark brown apoptotic cellsand intercellular apoptotic fragments (Fig. 7B,b1). In

contrast, tissues obtained from rats treated with melatonindemonstrated no apoptotic cells or fragments (Fig. 7C).Moreover, at 5 hr after crude venom administration, the

appearance of proapoptic protein, Bax, in paw homogen-ates was investigated by Western blot. Bax levels were

37 kDa

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(B)

(b1)

Fig. 4. With Western blot analysis, a basal level of IkB-a isdetected in the paw tissue homogenates of sham-operated animalsand it is decreased 5 hr after Pelagia noctiluca crude venomadministration, while in melatonin-treated rats IkB-a levels weresubstantially reduced (A, densitometric analysis a1). Moreover, thelevels of NF-jB p-65 subunit protein in the nuclear fractions of thepaw tissues were also significant increased by Pelagia noctilucacrude venom administration compared with the sham-operated rats(B, densitometric analysis b1). The levels of NF-jB p-65 proteinwere significantly reduced in the nuclear fractions of paw tissuesfrom animals that had received melatonin treatment (B, densito-metric analysis b1). b-actin (A) and laminin B1 (B) were used asinternal control. The results in a1 and b1 are expressed asmean ± S.E. mean from five blots. *P < 0.01 versus sham,�P < 0.01 versus crude venom + vehicle.

Marino et al.

62

appreciably increased in the paw tissues from rats at 5 hr

after crude venom administration (Fig. 7D; also seedensitometry analysis 7d1). On the contrary, melatonintreatment prevented the crude venom-induced Bax expres-

sion in the paw tissues (Fig. 7D; also see densitometryanalysis 7d1).

In addition, Bcl-2 expression in homogenates from paw

tissues of each rat was analyzed by Western blot. A basallevel of Bcl-2 expression was detected in paw tissue fromsham-treated rats (Fig. 7E; also see densitometry analysis7e1). Five hours after crude venom administration, the

Bcl-2 expression was significantly reduced in paw tissuesfrom vehicle-treated rats (Fig. 7E; also see densitometryanalysis 7e1). Treatment of rats with melatonin signifi-

cantly blunted the crude venom-induced inhibition ofanti-apoptotic protein expression (Fig. 7E; also seedensitometry analysis 7e1).

Discussion

One of the most relevant aspects of Cnidarian physiology is

related to the biologically active compounds of nemat-ocysts, organelles contained in specialized cells callednematocytes. The accidental touch with some Coelenterate

specimens can produce severe local and systemic patholo-gies, and, in some case, can lead to death.Human pathologies are mainly because of specimens

like Physalia physalis, Chironex fleckeri, and P. noctiluca.

However, the large variety of potentially toxic species andthe different extraction techniques employed to obtaincrude venom have prevented so far an exhaustive

description of nematocyte toxinological features. Never-theless, the notable impact of Cnidarian envenomation onpublic health has to be mentioned because of its

consequences on humans, so that investigations have

130 kDa

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0 Arb

itra

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100 µm

100 µm

100 µm

ND

*

o

Crude venom

7 Sham + VehicleSham + Melatonin Crude venom + Vehicle Crude venom + Melatonin

6

5

4

3

2

1

0 ND

*

* % o

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tal t

issu

e ar

ea

Crude venom + Mel

iNOS

b-actin

(A)

(a1)

(B)

(D) (E)

(C)

Fig. 5. At 5 hr after Pelagia noctilucacrude venom administration a significantincrease in the iNOS expression wasobserved in the paw tissue comparedwith the sham-treated rats (A, densito-metric analysis a1). Melatonin treatmentsignificantly reduced the iNOS expression(A, densitometric analysis a1). b-actinwas used as internal control. The resultsin a1 are expressed as mean ± S.E.mean from five blots. *P < 0.01 versussham, P < 0.01 versus crude venom.Moreover, immunohistochemical locali-zation of iNOS in paw tissue 5 hr follow-ing P. noctiluca crude venom injectionshows a positive staining (C), when com-pared with sham animals (B). In the pawtissue the expression of iNOS was signifi-cantly attenuated in samples collectedfrom rats that have received melatonintreatment (D). Densitometry analysis (E)of immunocytochemistry photographs(n = 5 photos from each sample collectedfrom all rats in each experimental group)for iNOS from paw was assessed. Theassay was carried out by using OptilabGraftek software on a Macintosh per-sonal computer (CPU G3-266). Data areexpressed as % of total tissue area.*P < 0.01 versus Sham; �P < 0.01 versuscrude venom. ND: not detectable.


63

been also performed with the aim of preventing orreducing pathological effects deriving from Cnidariantoxins. Immunological studies have been also carried outtrying to detect antibody titers against nematocysts

venom [31]. Bioactive compounds of nematocysts includeproteins (neurotoxins, pore-forming toxins or cytolysins,phospholipases, proteinase inhibitors) and secondary

metabolites with either toxic and/or biomedical proper-ties. Little is known on the mechanism of action ofnematocyst toxins. Many biological assays have been

carried out to evaluate toxicity of venoms and thehaemolytic one on red blood cells from different sourcesis the most used [32–35]. Neurotoxic [36], cardiotoxic [37]

or cytolytic tests [38] are also employed. Recently, anevaluation of cytotoxic activities has been performedon V79 cells with toxins of Rhizostoma pulmo [39],P. noctiluca [40] and of Anemonia sulcata and Aequorea

aequorea [41]. Moreover, we have also demonstrated thatcrude venom from nematocysts isolated from the Scy-phozoan P. noctiluca exerts an important haemolytic

effect that is slightly reduced by antioxidant compoundstreatment [1].

This study provides the first evidence that P. noctilucacrude venom administration induced: (i) the developmentof paw edema, (ii) the infiltration of the paw withpolymorphonuclear neutrophils, (iii) the degree of lipid

peroxidation in the paw, (iv) the expression of iNOS (byWestern blot analysis), (v) the nitration of tyrosine residues,(vi) NF-jB expression, (vii) apoptosis, (viii) Bax and Bcl-2

expression, and (ix) the degree of paw tissue injury.Moreover, in this study we have clearly demonstrated

that melatonin treatment caused a significant reduction in

all parameters related to inflammation evaluation. What,then, is the mechanism by which melatonin reduces acuteinflammation induced by crude venom administration?

The protective effects of melatonin have been demon-strated in various conditions of oxidant stress [42].Recently, a novel role of melatonin as an immunomodu-lator has also been proposed [43]. In particular, Maestroni

et al. [43] has clearly showed that melatonin antagonizesthe effects of corticosteroids on antibody production via theopioid system and subsequently described the effects of

melatonin on T-helper-2 lymphocytes. In addition, itsimmunomodulatory effects, melatonin has been shown to

100 µm 100 µm

50

MelatoninVehicle45

40 35 30 25 20 15 10 5 0

Sham Nitrotyrosine

1

2

3

4

5

6

7

8

0 ND ND

Sham + VehicleSham + Melatonin

Crude venom + MelatoninCrude venom + Vehicle

% o

f to

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PAR Crude venom

*

*

* *

*

o

Paw

MD

A le

vels

(µM

/mg

wet

tis

sue)

100 µm 100 µm

100 µm 100 µm

(A)

a a

b b

c c

(C)

(B) (D)

Fig. 6. There was no staining for nitro-tyrosine and PAR in the paw tissuesobtained from the sham rats (Aa,Ca).A positive nitrotyrosine and PAR stainingwas found in the paw tissues collected at5 hr after Pelagia noctiluca crude venomadministration (Ab,Cb). Paw tissueexpression of nitrotyrosine and PAR wassignificantly attenuated in the pawtissues collected from rats treated withmelatonin. (Ac,Cc). Densitometry analysis(D) of immunocytochemistry photographs(n = 5 photos from each sample collectedfrom all rats in each experimental group)for iNOS from paw was assessed. Theassay was carried out by using OptilabGraftek software on a Macintosh personalcomputer (CPU G3-266). Data areexpressed as % of total tissue area.*P < 0.01 versus Sham; �P < 0.01 versuscrude venom. ND: not detectable. Theassessment of MDA levels (B) shows anincrease of lipoperoxidation degree in thepaw tissues collected at 5 hr after P. noc-tiluca crude venom administration, whencompared with sham animals. The treat-ment with melatonin decreases the pawMDA levels (B).

Marino et al.

64

have oxygen radical scavenging effects, comparable withthat of vitamin E or glutathione [44, 45]. Because of the

recent interest in melatonin as an anti-inflammatory agent,in this study we have investigated the effect of melatonintreatment in the inflammatory responses associated with

administration of crude venom. There are a number of sites

where melatonin can interfere with the inflammatoryprocess.

There is some evidence that have clearly demonstratedthat melatonin reduces the activation of the transcriptionfactor NF-jB [30, 46]. NF-jB plays a central role in the

regulation of many genes responsible for the generation of

14,000

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Crude venom Crude venom + Mel



Sham

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Bax

b-actin

Bcl2

b-actin

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2000

0

(A)

(C)

(D)

(d1)

(E)

(e1)

(B)

(b1)

Fig. 7. At 5 h after Pelagia noctilucacrude venom administration, TUNEL-likestaining showed a marked appearance ofdark brown apoptotic cells and intercel-lular apoptotic fragments in paw tissue(B,b1), when compared with sham animals(A). On the contrary, the number of darkbrown cells was significantly reduced inpaw tissues by the treatment with mela-tonin (C). Moreover, Western blot analy-sis of Bax (D, analysis densitometric d1)and Bcl-2 (E, analysis densitometric e1)levels was realized in paw samples at 5 hrafter P. noctiluca crude venom-injection.A significant increase in the Bax expres-sion was observed in the paw tissue (D,densitometric analysis d1) compared withthe sham-treated animals, whereas inmelatonin-treated rats Bax levels weresubstantially reduce (D, densitometricanalysis d1). On the contrary, at 5 hr afterP. noctiluca crude venom administration,a decrease in the Bcl-2 expression wasobserved in the paw tissue (E, densito-metric analysis e1) compared with thesham-treated rats, while Bcl-2 expressionwas more evident in the paw tissue fromP. noctiluca crude venom-treated rats thatreceived melatonin treatment (E, densito-metric analysis e1).


65

mediators or proteins in inflammation. A recent evidencesuggests that the activation of NF-jB may also be underthe control of oxidant/antioxidant balance [47, 48]. Such

hypothesis is based primarily on the observation that lowdoses of peroxides, including H2O2 and tert-butyl hydro-peroxide, induce NF-jB activation whereas some antioxi-dants prevent it [49, 50].

The discovery in 1997 that inhibition of the activation ofNF-jB may be useful in conditions associated with local orsystemic inflammation [51] stimulated the search for agents

that prevent the activation of NF-jB. We report here forthe first time that P. noctiluca crude venom caused asignificant increase in the nuclear translocation of p65 in

the paw tissues at 5 hr, whereas treatment with themelatonin significantly reduced this activation. Moreover,we also demonstrate that melatonin treatment inhibitedIjB-a degradation. The observed effect of melatonin

treatment on NF-jB activation is in agreement withprevious studies [52].NF-jB plays a central role in the regulation of many

genes responsible for the generation of mediators orproteins in inflammation. These include the genes forTNF-a, IL-1b, iNOS, and COX-2, etc [53]. Moreover, we

demonstrate that melatonin attenuates the expression ofiNOS induced by crude venom. This inhibitory effect maybe related to the already known inhibitory effect of

melatonin, also demonstrated in this study, on the activa-tion of the transcription nuclear factor kappa B [30],involved, as known, in the process of iNOS expression [54–56]. In addition, an inhibition by melatonin of the expres-

sion of iNOS on the transcriptional level, melatonin mayalso directly inhibit the catalytic activity of NOS [57–59].Moreover, there is a large body of evidence showing that

the production of reactive oxygen and nitrogen species playkey roles in acute inflammation [60]. These species arecytotoxic agents, inducing lipid peroxidation and other

cellular oxidative stress by cross linking proteins, lipids, andnucleic acids, which then cause cellular dysfunction, dam-age, and eventually death [61–64]. In this study we

demonstrated that the treatment with melatonin caused asignificant reduction of the MDA levels, as an indicator oflipid peroxidation, in the injured paw tissue. In addition,nitrotyrosine formation, along with its detection by

immunostaining, was initially proposed as a relativelyspecific marker for the detection of the endogenousformation �footprint� of peroxynitrite [65]. There is, how-

ever, recent evidence that certain other reactions can alsoinduce tyrosine nitration; e.g. the reaction of nitrite withhypochlorous acid and the reaction of myeloperoxidase

with hydrogen peroxide can lead to the formation ofnitrotyrosine [66]. Increased nitrotyrosine staining is con-sidered, therefore, as an indication of �increased nitrosativestress� rather than a specific marker of the peroxynitrite

generation. In this regard, in this study we clearly demon-strated that melatonin at the highest dose used (50 mg/kg),fully inhibited the appearance of nitrotyrosine staining in

the inflamed tissue. This effect is likely related to a directscavenging effect of melatonin on peroxynitrite, as recentlydemonstrated in our in vitro studies [67]. Additional

protective effects of melatonin may lie within the abilityof this indole to reduce oxyradical-related oxidant processes

by either directly interfering with the oxidants [45, 68, 69] orup-regulating antioxidant systems such as superoxidedismutase [70, 71] or enhancing the catalytic activity of

glutathione peroxidase [72].Furthermore, various studies we clearly demonstrated

that acute inflammation induced the appearance of adhe-sion molecules on the damaged endothelial vascular wall,

which correlated with the reduction of leukocyte infiltra-tion. Activation and accumulation of leukocytes is one ofthe initial events of tissue injury due to release of oxygen

free radicals, arachidonic acid metabolites, and lysosomalproteases [60, 61].Recently, a novel pathway of inflammation has been

proposed in relation to ROS (hydroxyl radical and perox-ynitrite) induced strand breaks in DNA, which triggerenergy-consuming DNA repair mechanisms and activatesthe nuclear enzyme PARP resulting in the depletion of its

substrate NAD+ in vitro and a reduction in the rate ofglycolysis [73–75]. As NAD+ functions as a cofactor inglycolysis and the tricarboxylic acid cycle, NAD+ depletion

leads to a rapid fall in intracellular ATP. This process hasbeen termed �the PARP Suicide Hypothesis�.In this regard, in a previous study we clearly demon-

strated that melatonin treatment significantly abolishedPARP activation in various model of inflammation [76]. Wereport here for the first time that administration of venom

caused a significant increase in the PARP activation in thepaw tissues at 5 hr, whereas treatment with the melatoninsignificantly reduced this activation. In addition, oneconsequence of the increased oxidative stress is the activa-

tion and inactivation of redox-sensitive proteins [77].Generation of free radicals and nitric oxide by activated

macrophages has also been implicated in causing oligoden-

drocyte apoptosis. We have demonstrated in this study thatP. noctiluca crude venom induces apoptosis while melato-nin treatment attenuates the degree of apoptosis, measured

by TUNEL detection kit, in the paw tissue at 5 hr aftercrude venom administration.Therefore, we have also identified pro-apoptotic tran-

scriptional changes, including up-regulation of pro-apop-totic Bax and down-regulation of anti-apoptotic Bcl-2,using Western blot assay. We report in this study for thefirst time that the treatment with melatonin in acute

inflammation documents features of apoptotic cell deathafter crude venom administration, suggesting that protec-tion from apoptosis may be a prerequisite for anti-

inflammatory approaches. In particular, we demonstratedthat the treatment with melatonin lowers the signal forBax in treated groups when compared with paw sections

obtained from crude venom-treated rats, while on thecontrary, the signal is much more expressed for Bcl-2 inmelatonin-treated rats than in vehicle-treated rats. Thismeans that melatonin prevents the loss of the anti-

apoptotic way by inhibiting NF-jB and reduces the pro-apoptotic pathway activation with a mechanism still to bediscovered.

Taken together, the results arising from this studyenhance our understanding of the possible mechanism bywhich P. noctiluca venom induces acute paw inflammation

and support the view that melatonin exerts significant anti-inflammatory effects. Such results imply that melatonin

Marino et al.

66

may be useful also in the therapy of local acute inflamma-tion and provide a novel therapeutic strategy.

Acknowledgements

This study was supported by grant from a UniversityMinister grant. The authors would like to thank Carmelo

La Spada for his excellent technical assistance during thisstudy, Ms Caterina Cutrona for secretarial assistance andMs Valentina Malvagni for editorial assistance with the

manuscript.

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