Research Paper

Curine inhibits mast cell-dependent responses in mice

Jaime Ribeiro-Filho a, Fagner Carvalho Leite d, Hermann Ferreira Costa d,Andrea Surrage Calheiros a, Rafael Carvalho Torres b, Carolina Trindade de Azevedo b,Marco Aurélio Martins b, Celidarque da Silva Dias c, Patrícia T. Bozza a,n,1,Márcia Regina Piuvezamd,1

a Laboratório de Imunofarmacologia, Instituto Oswaldo Cruz, FIOCRUZ, Av. Brasil 4365, 21040-360 Rio de Janeiro, RJ, Brazilb Laboratório de Inflamação, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, Brazilc Laboratório de Fitoquímica, Departamento de Ciências Farmacêuticas, UFPB, João Pessoa, Paraíba, Brazild Laboratório de Imunofarmacologia, Departamento de Fisiologia e Patologia, UFPB, João Pessoa, Paraíba, Brazil

a r t i c l e i n f o

Article history:Received 18 March 2014Received in revised form29 May 2014Accepted 15 June 2014Available online 23 June 2014

Keywords:CurineChondrodentron platyphyllumAnti-allergicMast cellLipid mediators

a b s t r a c t

Ethnopharmacological relevance: Curine is a bisbenzylisoquinoline alkaloid and the major constituentisolated from Chondrodendron platyphyllum, a plant that is used to treat inflammatory diseases inBrazilian folk medicine. This study investigates the effectiveness of curine on mast cell-dependentresponses in mice.Materials and methods: To induce mast cell-dependent responses, Swiss mice were subcutaneouslysensitized with ovalbumin (OVA—12 μg/mouse) and Al(OH)3 in a 0.9% NaCl solution. Fifteen days later,the animals were challenged with OVA through different pathways. Alternatively, the animals wereinjected with compound 48/80 or histamine, and several parameters, including anaphylaxis, itching,edema and inflammatory mediator production, were analyzed. Promethazine, cromoglycate, andverapamil were used as control drugs, and all of the treatments were performed 1 h before thechallenges.Results: Curine pre-treatment significantly inhibited the scratching behavior and the paw edemainduced by either compound 48/80 or OVA, and this protective effect was comparable in magnitudewith those associated with treatment with either cromoglycate or verapamil. In contrast, curine wasa weak inhibitor of histamine-induced paw edema, which was completely inhibited by promethazine.Curine and verapamil significantly inhibited pleural protein extravasations and prostaglandin D2 (PGD2)and cysteinyl leukotrienes (CysLTs) production following allergen-induced pleurisy. Furthermore, likeverapamil, curine inhibited the anaphylactic shock caused by either compound 48/80 or an allergen.In in vitro settings, these treatments also inhibited degranulation as well as PGD2 and CysLT productionthrough IgE-dependent activation of the mast cell lineage RBL-2H3.Conclusion: Curine significantly inhibited immediate allergic reactions through mechanisms morerelated to mast cell stabilization and activation inhibition than interference with the pro-inflammatoryeffects of mast cell products. These findings are in line with the hypothesis that the alkaloid curine maybe beneficial for the treatment of allergic disorders.

& 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Allergic diseases are an important public health problem,affecting approximately 20% of the world population (Edwards et

al., 2009). The most common allergic diseases include asthma,rhinoconjunctivitis, sinusitis, food allergy, atopic dermatitis,angioedema, urticaria, anaphylaxis and allergy to drugs andinsects (Holgate and Polosa, 2008). The allergic reactions aretriggered by stimuli that lead to mast cell activation, causingdegranulation; the release of pre-formed mediators, such ashistamine and serotonin; and the synthesis of cytokines and lipidmediators, including prostaglandin D2 (PGD2) and cysteinil leuko-trienes (CysLTs) through specific signaling pathways (Cockcroft etal., 2007; Gould and Sutton, 2008). Under allergic conditions, mastcell activation is triggered by the IgE–FcεRI complex (Galli et al.,1999; Gould and Sutton, 2008). It has been demonstrated that

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Journal of Ethnopharmacology

http://dx.doi.org/10.1016/j.jep.2014.06.0410378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.

Abbreviations: AHR, airway hyper-responsiveness; BBA, bisbenzylisoquinolinealkaloid; β-hex, beta-hexosaminidase; COX, cyclooxygenase; CysLT, cysteinyl leu-kotriene; DNP, dinitrophenol; IgE, Immunoglobulin E; IL-(), interleukin-(); OVA,ovalbumin; PG (), prostaglandin

n Corresponding author. Tel.: þ5521 25621767.E-mail address: pbozza@ioc.fiocruz.br (P.T. Bozza).1 These senior authors contributed equally.

Journal of Ethnopharmacology 155 (2014) 1118–1124

mast cells can be activated by synthetic compounds, such ascalcium channel agonists and compound 48/80, which is animportant experimental tool in allergy research (Tatemoto et al.,2006). Because histamine is a major mediator in immediateallergic responses, histamine receptor antagonists and mast cellstabilizers have been used to treat many allergic symptoms,including edema and itch. However, there are numerous condi-tions for which they are not effective (Cook et al., 2002; Kim,2012), justifying the search for substances that can be used asmolecular templates in drug discovery for allergy.

Curine is a bisbenzylisoquinoline alkaloid (BBA) and the majorconstituent of the root bark of Chondrodendron platyphyllum(Menispermaceae). This plant is popularly known as “abútua”and has been used in Brazilian folk medicine to treat inflammatoryconditions (Correa, 1984; Gotfredsen, 2013). Earlier reportsdemonstrated that curine has vasodilator effects associated withthe inhibition of calcium influx, possibly through a direct blockadeof L-type Ca2þ channels (Dias et al., 2002; Medeiros et al., 2011).

We have demonstrated the anti-allergic properties of curineadministered orally using a mouse model of allergic asthma. Inthese experiments, curine significantly inhibited eosinophilicinflammation, eosinophil lipid body formation, cytokine produc-tion and airway hyper-responsiveness (AHR) in vivo. Verapamil,a calcium channel antagonist, had similar anti-allergic properties,and curine pre-treatment inhibited the calcium-induced trachealcontractile response ex-vivo, suggesting that the mechanism bywhich curine exerts its effects is through the inhibition of acalcium-dependent response. Importantly, oral treatment withcurine for 7 consecutive days in doses up to 10-fold higher thanits median effective dose (ED50) did not induce evident toxicity(Ribeiro-Filho et al., 2013).

Additionally, we have demonstrated that curine has anti-inflammatory and analgesic effects due to its inhibition of edemaand vascular permeability and its inhibition of the nociceptiveresponses associated with an anti-inflammatory but withoutneurogenic mechanisms. Importantly, curine inhibited PGE2 pro-duction in vitro, without affecting COX-2 expression. Moreover, theeffects of curine treatment were similar to those of indomethacin(a non-steroidal anti-inflammatory drug) but were different frommorphine (a central acting analgesic drug), suggesting that theanalgesic effects of curine do not result from a direct inhibitoryeffect on neuronal activation but that they depend on anti-inflammatory mechanisms that, at least in part, result from theinhibition of PGE2 production (Leite et al., 2014).

Despite the consistent anti-inflammatory and anti-allergicproperties of curine, the effects of this compound on mast cell-dependent responses remain to be elucidated. Therefore, this workaims to characterize the effects of curine on mast cell-dependentresponses in mice.

2. Methods

2.1. Curine purification

Chondrodendron platyphyllum Hill St. (Miers) was collected inSanta Rita, Paraíba, Brazil. The specimen voucher was deposited inthe Herbarium of Prof. Lauro Pires Xavier (UFPB - João Pessoa,Brazil), number 3631-P, and identified by Prof. Dr. Maria de FatimaAgra. The Chondrodendron platyphyllum bark was dried andpulverized in a HARLEY type grinder and then extracted underexhaustive percolation with 95% ethanol for 3–4 days. The extractwas concentrated under vacuum at temperatures ranging from501C to 601C to obtain the crude ethanol extract. This extract wasthen dissolved in 3% HCl, filtered through Celite (545 FischerScientific) and submitted to CHCl3 extraction alternated with

NH4OH (pH 8) basification. After washing with water and MgSO4,the solvent was evaporated, and this CHCl3 extract became thetotal tertiary alkaloid fraction (TTA). The TTA was separated usingcolumn chromatography (60�600 mm2) on aluminum oxideusing a step gradient of hexane, hexane-CH2Cl2 and CH2Cl2:MeOH.Fractions of 50 mL were collected and monitored using TLC with astep gradient of CHCl3–MeOH 100:0 (3 L) and 97:3 (2 L). Fractionsof 50 mL were collected for each system (hexane (100%), hexane:CH2Cl2 (1:1), CH2Cl2 (100%), CH2Cl2:MeOH (99:1), CH2Cl2:MeOH(8.5:1.5) and CH2Cl2:MeOH (2.5:7.5)), yielding 60 fractions. Frac-tions 52–60 monitored using TLC (system 6) contained curine(0.031% of dry plant; purity 498%) that achieved fusion at 215 1C[α]D–2251C (MeOH, c¼0.04). The structure was established usingspectroscopic NMR 13C and NMR 1H (CDCl3, 400 MHz) dataanalysis (Leite et al., 2014), which demonstrated that the productwas curine when compared to the literature data (Mambu et al.,2000). The curine solution was prepared using 1 mg of crystallinepowder, 50 μL of 1 N HCl and 500 μL of distilled water. The pH wasadjusted to 7–8 with 1 N NaOH. The volume was brought up to1 mL with phosphate buffered saline (PBS).

2.2. Animals

Male Swiss mice weighing 25–30 g were obtained from thebreeding units at the Oswaldo Cruz Foundation and FederalUniversity of Paraiba. The animals were maintained with foodand water ad libitum in a room with a temperature ranging from22 to 24 1C and a 12 h light/dark cycle. This study was carried outin accordance with the recommendations of the Brazilian NationalCouncil for the Control of Animal Experimentation (CONCEA). Theprotocols were approved by the Animal Welfare Committee of theOswaldo Cruz Foundation (CEUA/FIOCRUZ protocol # L-002/08)and by the Ethical Committee for Experimental Animal (CEPA No.0504/08, UFPB). Groups of 6–10 animals were used in eachexperiment.

2.3. Treatments

For the in vivo experiments, the animals were orally (p.o.) orsubcutaneously (s.c.) pre-treated with curine (2.5 mg/kg). Alter-natively, the groups of mice were treated with either an intraper-itoneal (i.p.) injection of sodium cromoglycate (10 mg/kg), anintramuscular (i.m.) injection of promethazine (10 mg/kg) or anoral (p.o.) administration of verapamil (2.5 mg/kg), all of whichwere used as reference drugs. PBS (p.o.) was given as a negativecontrol. All treatments were performed 1 h before the allergicchallenge. For the in vitro experiments, RBL-2H3 cells wereexposed to curine (1 or 10 μM) or vehicle 1 h before the stimulus.The dose of curine (2.5 mg/kg) was chosen based on the dataobtained from a prior study (Ribeiro-Filho et al., 2013). Of note, thecurine concentrations used in the in vivo experiments did notaffect cell viability (viability495%).

2.4. Induction and evaluation of the scratching behavior (itching)

This protocol was used to evaluate the potential of curine inmodulating allergic itching. For this purpose, Swiss mice weretreated subcutaneously (s.c.) with curine. One hour later, theanimals received an intradermal injection of compound 48/80(10 μg) dissolved in PBS (20 μL) in the rostral part of the back.Immediately after the injection, the animals were placed underobservation in individual boxes and their behavior was monitoredfor 60 min. The results are expressed as the number of times thatthe animals scratched the injection site with the hind paw over60 min (number of scratchings) (Inagaki et al., 2002).

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2.5. The anaphylactic shock reaction

The anaphylactic shock reaction was induced as previouslydescribed by Kim et al. (2004). Briefly, Swiss mice were orally pre-treated with curine, and 1 h later, they were intraperitoneally (i.p.)challenged with compound 48/80 (8 μg/g body weight). Alterna-tively, Swiss mice were subcutaneously (s.c.) sensitized withovalbumin (OVA—12 μg/mouse) and Al(OH)3 in a 0.9% NaCl solu-tion (saline 0.2 mL). Fifteen days later, the animals were intrave-nously (i.v.) challenged with OVA (200 μg/mouse) dissolved insaline (0.1 mL). In another set of experiments, the mice receiveda sub-lethal dose of OVA (60 μg/mouse, s.c.). Immediately after thechallenges, the animals were placed in individual boxes and theirmortality was monitored for 60 min. The results are expressed aspercent survival at 10, 30 and 60 min.

2.6. Paw edema induced by compound 48/80, histamine or OVA

One hour after the treatments, paw edema was induced aspreviously described by Henriques et al. (1987). Briefly, Swiss micewere injected in the left paw with a solution containing compound48/80 (100 ng/paw) dissolved in PBS (20 μL). The right pawreceived the same volume of PBS and was used as a control. Thediameter of each paw was measured using a caliper 30, 60 and120 min after stimulation with 48/80. In another series of experi-ments, Swiss mice were injected with histamine (100 μg/paw)dissolved in PBS (20 μL) in the left paw. Again, the right pawreceived the same volume of PBS and was used as a control. Thediameter of each paw was measured with a plethysmometer 30and 60 min after stimulation with histamine. Alternatively, OVA-sensitized mice were injected with OVA (12 μg/paw) dissolved inPBS (20 μL) in the left paw 15 days after allergic sensitization. In allof the experiments, the difference in the diameter of the left andthe right paw was expressed as edema.

2.7. Allergic pleurisy

Swiss mice were sensitized as described in Section 2.5. Fifteendays later, the orally pre-treated animals received an intrapleuralinjection of OVA (12 μg/mouse) in 0.1 mL saline. Four hours afterthe provocation, the pleural cavities were washed with 0.5 mL ofsaline using a sterile syringe. The pleural washes were centrifugedat 800g for 5 min, and the supernatants were collected. The totalprotein concentration of the samples was determined using a kitfor determination of total proteins (BCA, from Pierce, Rockford, IL)and was used as a parameter of pleural edema. The concentrationsof PGD2 and CysLT in the supernatants were analyzed usingDuoSet kits, according to the manufacturer's instructions (R & DSystems).

2.8. Cell culture

The RBL-2H3 cell line is a basophilic leukemia cell line isolatedfrom Wistar rat basophilic cells maintained as tumors. These cellshave high-affinity IgE receptors that can be activated to secretebeta-hexosaminidase (β-hex) and lipid mediators (Okuyama et al.,2005). The RBL-2H3 cells were maintained in complete (15% fetalbovine serum, penicillin 100 IU/ml, streptomycin 0.1 mg/ml) Dul-becco's modified Eagle's medium (DMEM) at 37 1C in a 5% CO2

atmosphere.

2.8.1. RBL-2H3 cells degranulation assayThe degranulation of the RBL-2H3 cells was evaluated by

measuring the release of β-hex (Han et al., 2009). Briefly, afterconfluence, cultured RBL-2H3 cells were dissociated with 0.25%

(w/v) Trypsin—0.53 mM EDTA solution and plated in a 48-wellplate (1.25�105 cells/well). The cells were sensitized with dini-trophenol (DNP)-specific IgE (1 μg/mL) for 20 h. After this period,the growth medium was replaced with Tyrode assay buffer(119 mM NaCl, 4.74 mM KCl, 2.5 mM CaCl2, 1.19 mM MgSO4,10 mM 4-2-hydroxyethyl-1-piperazineethane sulfonic acid (HEPES),5 mM glucose, and 0.1% (w/v) BSA, pH 7.3), and the cells were treatedwith curine or verapamil (1 or 10 μM) for 1 h. RBL-2H3 celltreatment was followed by stimulation with DNP–BSA (10 ng/mL)for 45 min, and the β-hex content was expressed using the percen-tage amount of the enzyme released into the supernatant. The β-hexreaction (37 1C, 50 min) with1 mM p-nitrophenyl N-acetyl-β-D-glu-cosamide in 0.1 M sodium citrate buffer (pH 4.5) was determinedusing a spectrophotometer at 405 nm after stopping the reactionwith 0.2 M glycine.

2.8.2. Determination of lipid mediator productionRBL-2H3 cells were plated in 24-well plates (1.25�105 cells/

well) and sensitized with dinitrophenol (DNP)-specific IgE (1 μg/mL)for 20 h in complete DMEM. After sensitization, the cells were treatedwith curine or verapamil (1 or 10 μM) for 1 h in DMEM withoutserum. Lipid mediator production was determined in supernatantscollected 1 h after stimulation with DNP–BSA (10 ng/mL). The con-centrations of PGD2 and CysLT in the supernatants were quantifiedusing DuoSet kits according to the manufacturer's instructions (R & DSystems).

2.9. Statistical analysis

The data were analyzed using a one-way ANOVA followed byTukey's test, and the survival data were analyzed using the Log-rank (Mantel-Cox) Test with GraphPad Prism software (GraphPad,San Diego, CA). The values are expressed as the means7S.E.M. Thedifferences with po0.05 were considered significant.

3. Results

3.1. Curine inhibits the scratching behavior induced bycompound 48/80

The intradermal injection of compound 48/80 significantlyinduced scratching behavior in non-treated mice. As shown inFig. 1, pre-treatment with curine significantly reduced the numberof scratches in the challenged mice compared with animals thatdid not receive treatment, indicating that curine has an inhibitoryeffect on mast cell-dependent itch.

3.2. The effects of curine on edema formation

Swiss mice challenged with compound 48/80 developed sig-nificant paw edema at 30, 60 and 120 min (Fig. 2). The group ofanimals pre-treated with curine had significantly reduced edemaformation compared with the non-treated group at all of the timesanalyzed (Fig. 2A). The groups of animals treated with cromogly-cate, a mast cell stabilizer drug, and promethazine, an antagonistof the histamine receptor 1 (H1), showed a significant inhibition ofthe paw edema induced by compound 48/80, and there were nodifferences between curine and cromoglycate treatments, demon-strating that mast cells participate predominantly in the mechan-ism of paw edema formation by releasing histamine andsuggesting that curine plays a modulator effect in this process.As shown in Fig. 2B, the injection of histamine into the pawinduced significant edema formation in untreated mice. The edemaformation was partially inhibited by the curine pretreatment,

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whereas the promethazine pretreatment completely inhibited thisresponse suggesting that curine might inhibit the allergic reaction bymodulating mast cell degranulation instead of blocking histamineaction at the allergic site. Because we have demonstrated that theeffects of curine are associated with calcium influx inhibition andthat curine and verapamil have similar anti-allergic effects, weanalyzed the effect of these drugs on allergic edema. As shown inFig. 2B, curine and verapamil significantly inhibited the edema andthere were no differences between the two treatments, suggestingthat the effects of curine on mast cell degranulation may result fromthe inhibition of a calcium-dependent response.

3.3. Curine and verapamil inhibit mediator production in a mousemodel of allergic pleurisy

An allergic challenge with OVA in actively sensitized miceinduced a significant increase in the total protein and the CysLT

and PGD2 concentrations in the supernatants of the pleuralwashes at 4 h (Fig. 3 A–C), and these increases were significantlydecreased by oral pretreatment with curine. Interestingly, verapa-mil administered with the same conditions (dose, time and path-way) had similar effects. Because, at the time point that theanalyses were performed, mast cell is the main source of PGD2

and CysLT, the data suggest that curine and verapamil inhibit theproduction of these mediators by modulating mast cell activation.

3.4. Curine and verapamil inhibit mast cell activation in vitro

Antigen-stimulated RBL-2H3 cells have been used as theprincipal model of mast cell activation in immediate allergicreactions. The level of activation of this cell lineage is directlyassociated with the level of cell degranulation, which can bedetermined by measuring hexosaminidase release, a well-established granule marker (Hwang et al., 2013), as well as byanalyzing the production of degranulation-independent media-tors, such as prostaglandin and leukotrienes. As shown in Fig. 4,RBL-2H3 cells sensitized with DNP–BSA and challenged with IgEanti-DNP produced a significant release of β-hex (A) and increasedconcentrations of CysLT (B) and PGD2 (C). Curine and verapamilsignificantly, but only partially, inhibited degranulation. However,the treatments produced significantly more expressive inhibitionof PGD2 and CysLT production indicating that curine inhibits mastcell activation by modulating both degranulation and mediatorproduction, through mechanisms that depend, at least in part, oncalcium influx inhibition.

3.5. The effect of curine on the anaphylactic shock reaction

The intraperitoneal administration of compound 48/80 in non-treated mice rapidly induced the development of an anaphylacticshock reaction followed by 40% and 100% mortality at 10 and30 min, respectively (Fig. 5A). Interestingly, mice orally pretreatedwith curine presented no mortality at 10 min, and the totalmortality at 60 min was 20% lower than that in mice challengedwith 48/80 but that did not receive treatment. Similarly, thesystemic administration of OVA (200 μg/mouse) in sensitized mice

Fig. 1. The effect of curine on scratching behavior. Swiss mice (n¼7–10) weresubcutaneously treated with curine (2.5 mg/kg) 1 h before an intradermal chal-lenge with compound 48/80. The animals were placed under observation inindividual boxes, and their behavior was monitored for 60 min. (A) The chemicalstructure of curine; (B) the number of scratchings. The data are expressed as thenumber of times that the animals scratched the injection site with its hind pawover 60 min (number of scratchings). These results are expressed as the mean7-SEM. þ Significantly different (po0.05) from the unchallenged group; n signifi-cantly different from the untreated, 48/80-challenged group.

Fig. 2. The effects of curine on allergic edema formation. Swiss mice (n¼7–10) were orally treated with curine (2.5 mg/kg), and 1 h later they were injected in the left hindpaw with compound 48/80 (100 ng/paw) or histamine (100 mg/paw) dissolved in PBS (20 μL). The allergen-sensitized mice were injected with OVA (12 μg/paw) 1 h after thetreatments. The right paw received the same volume of PBS and was used as a control. Cromoglycate (10 mg/kg), promethazine (10 mg/kg) and verapamil (2.5 mg/kg) wereused as control drugs. The diameter of each paw was measured using a plethysmometer 30 and 60 or 120 min after the challenge. The difference between the diameter of theleft and right paws was expressed as edema. (A) Compound 48/80-induced paw edema; (B) histamine-induced paw edema; (C) OVA-induced paw edema. These results areexpressed as the mean7SEM. þ Significantly different (po0.05) from the unchallenged group; n significantly different from the untreated, challenged groups.

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induced the development of an anaphylactic shock reactionfollowed by 60% and 100% mortality at 10 and 30 min, respectively(Fig. 5B). However, curine or verapamil treated mice presented nomortality at 10 min, and the total mortality at 60 min was 29%lower than in untreated mice. Additionally, when OVA was givensubcutaneously at a lower dose (60 μg/mouse), the untreatedgroup had 62.5% mortality, and the curine and verapamil treatedgroups had mortality rates of 37.5% and 28.5%, respectively, at both30 and 60 min (Fig. 5C), suggesting that these substances maymodulate the anaphylactic shock reaction in mice.

4. Discussion

Allergic reactions are associated with the activation of mastcells and the release of inflammatory mediators. In most cases, thisprocess relies on signaling through the FcεRI–IgE complex (Liew etal., 2010). However, it has been shown that other substances,including compound 48/80, can directly activate mast cells, thusinducing degranulation. Compound 48/80 is known to trigger mastcell activation by binding to G proteins in the cytoplasm andinitiating biochemical cascades that result in increased intracel-lular calcium and the release of inflammatory mediators (Tatemotoet al., 2006). The pathophysiological features of allergic diseasesare directly influenced by the environment of the organ in whichthey occur. Thus, itching is a key feature of most skin allergies

(Buddenkotte and Steinhoff, 2010) and, in this context, resultsprimarily from the stimulation of specific receptors in neurons bya wide range of mediators (Greaves, 2010; Garibyan et al., 2013).The severe itch is a major problem because it induces scratchingbehavior, thus resulting in the formation of skin lesions (Wahlgren,1991). Additionally, systemic allergic reactions may result in asevere clinical condition known as anaphylactic shock (Kemp andLockey, 2002), which is characterized by shortness of breath,hypotension, cardiac arrhythmia, vomiting, urticaria, headacheand unconsciousness and can result in death (Wade et al., 1989).

In the present study, we demonstrated the effectiveness ofcurine in inhibiting immediate allergic responses in mice. Oralcurine pre-treatment significantly inhibited the scratching beha-vior induced by compound 48/80. As previously described, it haslong been established that histamine is an important mediator ofitch (Lewis, 1927) acting mainly through receptor H1. However,some studies showed that the H4 receptor is also involved inhistamine-mediated itch (Bell et al., 2004). Although numerousstudies suggest that in allergic reactions, itch is mostly dependenton histamine release by mast cells, these cells can also releaseother mediators, such as adenosine triphosphate (ATP), tryptase,CysLTs, leukotriene B4 (LTB4), IL-31 and prostaglandins, whichdirectly activate nerve fibers. In turn, the activated nerve fibersmay release neuropeptides, such as substance P, which hasrecently been implicated in allergic responses, including modulat-ing scratching behavior (Hossen et al., 2006; Nakanishi and

Fig. 3. The effects of curine and verapamil on acute allergic pleurisy. OVA-sensitized Swiss mice (n¼6–8) were orally treated with curine (2.5 mg/kg) or verapamil (2.5 mg/kg)1 h before an allergic challenge. Four hours after the challenge, the pleural lavage was collected and the concentration of protein and lipid mediators in the supernatants wasanalyzed using BCA kits and ELISA, respectively. (A) The concentration of total proteins; (B) the concentration of CysLT; (C) the concentration of PGD2. These results are expressedas the mean7SEM of at least 6 animals. þ Significantly different (po0.05) from the unchallenged group; n significantly different from the untreated, OVA-challenged group.

Fig. 4. The effects of curine and verapamil on mast cell activation. RBL-2H3 cells (1.25�105 cells/well) were sensitized with IgE anti-DNP (1 μg/mL), incubated for 20 h,treated with curine (1–10 μM) or verapamil (1–10 μM) and stimulated with DNP–BSA (10 ng/ml) 45 min later. The β-hexosaminidase levels were quantified 45 min afterstimulus using a spectrophotometer at 405 nm. The mediator concentrations in the supernatants were analyzed 1 h after stimulus. (A) β-hexosaminidase release; (B) theconcentration of CysLT; (C) the concentrations of PGD2. These results are expressed as the mean7SEM. þ Significantly different (po0.05) from the unchallenged group;n significantly different from the untreated, DNP–BSA challenged group.

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Furuno, 2008). Therefore, the modulation of scratching behaviorby curine suggests that this alkaloid inhibits mast cell-dependentresponses in mice.

Edema is another important feature of allergic inflammation(Ash and Schild, 1997). Thus, to investigate the effects of curine onedema formation as well as to address the possible mechanismsinvolved in curine's effects in this model, we compared the effectsof curine treatment with cromoglycate, a mast cell stabilizer, andpromethazine, an antagonist of receptor H1. Using models ofmouse paw edema induced by compound 48/80 or histamineadministration, we demonstrated that curine had an anti-allergiceffect similar to cromoglycate but different from promethazine,suggesting that curine's effects may result from the inhibition ofmast cell degranulation, instead of the inhibition of histamine atallergic sites. Using a mouse model of allergic asthma, wepreviously demonstrated that curine and verapamil, a calciumchannel antagonist, had similar anti-allergic effects that areassociated with calcium influx inhibition (Ribeiro-Filho et al.,2013). Here, we demonstrated that curine and verapamil signifi-cantly inhibit allergen-triggered edema and that there were no

differences between these treatments, corroborating previouslydescribed data.

To better elucidate the effects of curine on immediate allergicresponses in mice, we used a model of acute allergic pleurisy,induced by sensitization and challenge with OVA. We demon-strated that curine and verapamil decreased the concentrations ofthe proteins PGD2 and CysLT in the pleural washes of OVA-stimulated mice. Because previous studies have shown that mastcells are a major source of CysLTs and PGD2 in the acute phase ofallergy (Boyce, 2003), these data suggest that curine and verapamilinhibited the production of these mediators, at least in part,through a direct effect on mast cell activation.

To confirm our hypothesis, we investigated the effects of curinepre-treatment in RBL-2H3 cells stimulated in vitro with IgE anti-DNP. This is a well-established model for evaluating the effect ofdrugs on mast cell activation (Paumet et al., 2000; Blank et al.,2002; Hemmerling et al., 2010). We demonstrated that curine andverapamil partially inhibited mast cell degranulation. However,the treatments produced significantly more expressive inhibitionof PGD2 and CysLT production, indicating that curine inhibits mastcell activation by modulating both degranulation and mediatorproduction.

Systemic anaphylaxis is a severe allergic reaction that resultsfrom a sudden release of mediators primarily from mast cells(Kemp and Lockey, 2002). Because anaphylaxis is one of the mostpotent immune reactions, it is a difficult condition to control and,as such, frequently causes death. We demonstrated that a singleoral pre-treatment with curine delayed death by anaphylacticshock in mice stimulated with either OVA or compound 48/80.Additionally, using a lower dose of OVA (of which the untreatedgroup presented mortality o100%), we found that curine reducedthe total mortality, and similar results were obtained with ver-apamil administered under the same conditions. These datasuggest that both curine and verapamil modulate the anaphylacticshock reaction in mice. Importantly, this is the first work todemonstrate the anti-anaphylactic effect of verapamil inthis model.

Previous studies have demonstrated the anti-allergic effects ofnatural products in mast cell-dependent responses both in vivoand in vitro. Hossen et al. (2006) demonstrated that treatmentwith caffeic acid, a phenolic compound widely distributed inplants such as coffee, inhibited scratching behavior, decreasedvascular permeability and reduced histamine release after chal-lenge with compound 48/80. Similar results were obtained withpropolis extract, which is rich in phenolic compounds, includingcaffeic acid and flavonoids (Shinmei et al., 2004). The ethanolextracts of Gleditsia sinensis (Leguminosae) and Plumbago zeylanica(Plumbaginaceae) inhibited the anaphylactic shock reaction andaffected mast cell activation (Dai et al., 2002), and the compoundpaeonol inhibited the anaphylactic shock reaction throughmechanisms dependent on the inhibition of histamine releaseand tumor necrosis factor (TNF)-α production (Kim et al., 2004).Interestingly, Bezerra-Santos et al. (2005) demonstrated that anethanol extract of Cyssampelos sympodialis (Menispermaceae)significantly inhibited the OVA-induced but not the 48/80-inducedanaphylactic shock reaction in mice. Finally, Costa et al. (2008)demonstrated that warifteine, a BBA isolated from Cyssampelossympodialis, significantly inhibited the scratching behaviorinduced by compound 48/80 in addition to reducing allergen-triggered mast cell activation in vitro corroborating the role of BBAin modulating immediate allergic responses.

As previously described, using a mouse model of allergicasthma, we demonstrated that oral pretreatment with curinesignificantly inhibited eosinophilic inflammation and airwayhyper-responsiveness through mechanisms that involve the inhi-bition of IL-13 and eotaxin production and the inhibition of

Fig. 5. The effect of curine on the anaphylactic shock. Swiss mice (n¼7–10) wereorally treated with curine (2.5 mg/kg) or verapamil (2.5 mg/kg) 1 h before thechallenge. Their mortality was monitored for 60 min. (A) Compound 48/80-inducedanaphylactic shock; (B) anaphylactic shock induced by systemically administered-OVA (200 μg/mouse); (C) anaphylactic shock induced by subcutaneously adminis-tered-OVA (60 μg/mouse). The data were analyzed using the Log-rank (Mantel-Cox)Test and expressed as percent survival after the challenge. þ Significantly different(po0.05) from the unchallenged group; n significantly different from the untreatedand challenged group.

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calcium influx (Ribeiro-Filho et al., 2013). Additionally, using thesame model, we demonstrated that curine and verapamil signifi-cantly inhibited the production of CysLT in the BAL (not shown),corroborating the data presented in this work. Because mast cellsproduce mediators that play key roles in asthma pathogenesis,including CysLT, eotaxin and IL-13 (Shakoory et al., 2004; Hong etal., 2013), the new data provided in this work suggest that theinhibitory effect that curine plays on mast cell activation maydirectly contribute to the anti-asthmatic effects of this alkaloid.

In conclusion, our results demonstrate that curine inhibits mastcell-dependent responses by modulating mast cell activationthrough mechanisms that may involve the inhibition of a calciumdependent response. This finding supports previous data suggest-ing that curine has the potential to be developed as a novel anti-allergic drug.

Acknowledgments

This work was supported by PRONEX/MCT, CNPq, FAPERJ andINCT-Cancer. The authors thank Edson Fernandes de Assis andJosenilson Feitosa de Lima for their technical assistance.

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