Protection by Mikania laevigata (guaco) extract against the toxicity of Philodryas olfersii snake venom

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Toxicon 60 (2012) 614–622

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Protection by Mikania laevigata (guaco) extract against the toxicity ofPhilodryas olfersii snake venom

Rita de Cássia O. Collaço a,*, José Carlos Cogo b, Léa Rodrigues-Simioni c, Thalita Rocha d,Yoko Oshima-Franco e, Priscila Randazzo-Moura a

aDepartamento de Ciências Fisiológicas, Faculdade das Ciências Médicas e da Saúde, Pontifícia Universidade Católica de São Paulo (PUC/SP),Praça José Ermírio de Moraes 290, Sorocaba 18030-095, SP, Brazilb Serpentário do Centro de Estudos da Natureza, Universidade do Vale do Paraíba (UNIVAP), Av. Shishima Hifumi 2911, Urbanova,São José dos Campos 12244-000, SP, BrazilcDepartamento de Farmacologia, Faculdade de Ciências Médicas, Universidade Estadual de Campinas (UNICAMP), Rua Tessália Vieira de Camargo 126,Cidade Universitária “Zeferino Vaz”, Campinas 13083-881, SP, Brazild Laboratório Multidisciplinar de Pesquisa, Universidade São Francisco (USF), Av. São Francisco de Assis 281, Jd. São José, Bragança Paulista 12916-900, SP, BrazileMestrado em Ciências Farmacêuticas, Universidade de Sorocaba (UNISO), Rod. Raposo Tavares, Km 92.5, Sorocaba 18023-000, SP, Brazil

a r t i c l e i n f o

Article history:Received 29 February 2012Received in revised form 10 May 2012Accepted 23 May 2012Available online 4 June 2012

Keywords:AntiophidicAnti-inflammatoryGuacoNeuromuscular junctionPlant extract

* Corresponding author. Av. Antonino Vieira doSalete, Araçoiaba da Serra 18190-000, SP, Brazil. Telþ55 15 9657 9043 (mobile); fax: þ55 19 3289 2968

E-mail address: [email protected] (R.d

0041-0101/$ – see front matter � 2012 Elsevier Ltd10.1016/j.toxicon.2012.05.014

a b s t r a c t

Philodryas olfersii is responsible for most colubrid snakebites in Brazil. In this work, weexamined the ability of an ethanolic extract from Mikania laevigata (guaco) leaves to protectagainst the in vitro neuromuscular activity of P. olfersii venom in mouse phrenic nerve-diaphragm (PND) and chick biventer cervicis (BC) preparations. M. laevigata extract causedmoderate twitch-tension facilitation at low concentrations (107.4 � 6.2% with 20 ml/ml and118.9 � 9.3% with 40 ml/ml in PND, and 120.7 � 7.7% with 40 ml/ml and 114.5 � 4.4% with50ml/ml inBC after 120min; n¼ 4–6,mean� SEM). In PND, theethanol alone (40 ml/ml,n¼ 4)did not change the twitch-tensionwhen compared with control. However, in BC, the ethanolproduced a higher facilitation when compared to control. At higher concentrations(>50 ml/ml) the extract caused total and reversible blockade in both preparations. Venom(50 mg/ml) caused partial blockade in PND (58.5� 12%, n¼ 4) and almost total blockade in BC(93.5� 2.2%,n¼ 4). Pretreatment of thepreparationswith extract (40ml/ml) for 30minbeforeincubation with venom (50 mg/ml) completely protected PND from neuromuscular blockadeand delayed the blockade in BC. The extract alone caused onlymildmorphological alterations(12.5� 0.5% and10.9� 2.3%fiberdamage in PNDand BC, respectively, compared to2.3� 0.3%and 3 � 0 in controls; n ¼ 3), with no increase in expression of the inflammatory cytokinesTNFa and IFNg. The ethanol alone also caused slight muscle damage: 4.3 � 2.4% in PND and6.7� 3.3% in BC (both n¼ 3) and little or noTNFa and IFNg expression in both preparations asobserved in control. Venom (50 mg/ml) caused 53.5 � 8.5% and 55.8 � 4.3% fiber damage inPND and BC, respectively; (n¼ 3, p< 0.05 vs. controls) and enhanced expression of TNFa andIFNg. Pretreatment of the preparationswith extract protected against venom-inducedmuscledamage by 80.3 and 60.4 in PND and BC, respectively, and prevented TNFa and IFNgexpression. These results indicate that the M. laevigata extract protected nerve-musclepreparations against the myotoxic, neurotoxic and inflammatory effects of P. olfersii venom.

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Amaral 765, Jardim.: þ55 15 3281 1225,.eC.O. Collaço).

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1. Introduction

Philodryas olfersii (Dipsadidae) is an opisthoglyphouscolubrid responsible for most colubrid snakebites in Brazil.

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Bites by this species can result in local effects such asedema, erythema, pain, hemorrhage and necrosis, and,more rarely, systemic effects such as coagulopathy andinternal bleeding (Silva and Buononato, 1983/1984;Assakura et al., 1992; Salomão and Di-Bernardo, 1995;Ribeiro et al., 1999; Acosta de Pérez et al., 2003; Rocha et al.,2006). Prado-Franceschi et al. (1996) showed that P. olfersiivenom caused an in vitro irreversible neuromuscularblockade, in chick biventer cervicis preparations, witha direct myotoxic effect being caused by myotoxins in thisvenom (Prado-Franceschi et al., 1998). The related speciesPhilodryas patagoniensis also has neurotoxic and myotoxicactivities that may contribute to the clinical manifestationsof envenoming by this species (Carreiro-da-Costa et al.,2008). There is no specific antivenom for bites by Philo-dryas species and most cases of systemic envenoming havebeen treatedwith bothropic antivenom that neutralizes thetoxic effects but has little effect on the local tissue damage(Correia et al., 2010).

Many studies have examined the ability of extracts andpurified components from South American plants toprotect against the local damage and other activities asso-ciated with snakebite, particularly pitvipers of the generaBothrops, Crotalus and Lachesis (Martz, 1992; Maioranoet al., 2005). Some of the plants investigated so farinclude Renealmia alpinia, Mikania glomerata, Bixa orellanaL., Casearia sylvestris, Eclipta prostrata and Calendula offici-nalis (Mors et al., 1989; Alarcón et al., 2005, 2008; Oshima-Franco et al., 2005; Maiorano et al., 2005; Mise et al., 2009).Various species of the genus Mikania (Asteraceae) haveanti-edematogenic, antidiarrheal, anti-inflammatory, anti-malarial, anti-microbial, anti-mutagenic, anti-ulcerogenic,bronchodilator and hypoglycemic effects (Soares deMoura et al., 2002; Suyenaga et al., 2002; Fernandes andVargas, 2003; Barbosa-Filho et al., 2005; Bighetti et al.,2005; Salgado et al., 2005; Botsaris, 2007; Baratto et al.,2008). M. glomerata extract inhibits the biological activi-ties of Crotalus durissus spp. and Bothrops spp. venoms(Maiorano et al., 2005).

Few studies have examined the ability of plant extracts,particularly from Mikania species, to protect against thebiological activities of South American colubrid venoms. Inthis work, we examined the ability of an ethanolic extractof Mikania laevigata to protect against the toxic effects of P.olfersii venom using in vitro vertebrate neuromuscularpreparations.

2. Materials and methods

2.1. Venom, plant extract and reagents

P. olfersii venom was milked manually from snakes ofboth sexes maintained in the serpentarium of the Centrode Estudos da Natureza, UNIVAP. The ethanolic extract ofM. laevigata leaves was prepared and donated by Uni-versidade de Sorocaba. Acetylcholine chloride was fromSigma–Aldrich Chemical Co. (St. Louis, MO, USA), halo-thane was from Cristalia (Itapira, SP, Brazil), anti-TNFa andanti-IFNg antibodies and the ABC kit were from Santa CruzBiotechnology Inc. (Santa Cruz, CA, USA). Bovine serumalbumin (BSA), Canada synthetic balsam, eosin, ethanol,

hematoxylin, paraffin, paraformaldehyde, Tris–HCl, TritonX-100 and xylene were purchased from local suppliersand the salts for the physiological solutions were ofanalytical grade.

2.2. Animals

Male HY-LINE W36 chicks (4–8 days old) were suppliedby Granja Globo Aves Agrícola Ltda. (Campinas, SP, Brazil)and male Swiss mice (25–30 g) were supplied by theMultidisciplinary Center for Biological Research of the StateUniversity of Campinas (CEMIB/UNICAMP). The animalswere housed at 25 �C on a 12 h light/dark cycle and had freeaccess to food and water. All procedures were done inaccordance with the general guidelines of the BrazilianSociety of Laboratory Animal Science (SBCAL) and wereapproved by the Committee for Ethics in Research (CER/UNIVAP, protocol no. A025/CEP/2009).

2.3. Mouse phrenic nerve-diaphragm (PND) preparation

Mice were killed by exsanguination after halothaneanesthesia and the phrenic nerve-diaphragm preparationswere removed andmounted under a tension of 5 g in a 5mlorgan bath containing Tyrode solution (composition, inmM: NaCl 137, KCl 2.7, CaCl2 1.8, MgCl2 0.49, NaH2PO4 0.42,NaHCO3 11.9 and glucose 11.1; pH 7.4, 37 �C) aerated with95% O2 and 5% CO2 (adapted for mice from Bülbring, 1946).Muscle contractions were evoked by indirect stimulationwith supramaximal pulses (3 V, 0.1 Hz, 0.2 ms) deliveredfrom an ESF-15D stimulator and applied to the phrenicnerve by a bipolar electrode. The muscle twitches wererecorded using a force displacement transducer (Ugo Basilecat. no. 7003) coupled to a basic preamplifier (cat. no. 7080,Ugo Basile) linked to a two-channel Gemini flatbedrecorder (cat. no. 7070). The preparations were allowed tostabilize for at least 20 min before adding ethanol alone(40 ml/ml),M. laevigata extract (20, 40, 60 and 80 ml/ml) or P.olfersii venom (50 mg/ml). In some experiments, the prep-arations were pretreated with 40 ml/ml of M. laevigataextract for 30 min before adding venom (50 mg/ml). Allexperimental protocols were done at least four times withincubations lasting up to 120 min.

2.4. Chick biventer cervicis (BC) preparation

Male chicks were killed by halothane inhalation and thebiventer cervicis muscles were removed (Ginsborg andWarriner, 1960) and mounted under a tension of 1 g ina 5 ml organ bath containing Krebs solution (composition,in mM: NaCl 118.7, KCl 4.7, CaCl2 1.88, KH2PO4 1.17, MgSO41.17, NaHCO3 25.0 and glucose 11.65, pH 7.5, 37 �C) aeratedwith 95% O2 and 5% CO2. The preparations were allowed tostabilize for at least 20 min before adding a singleconcentration of venom (50 mg/ml). A bipolar platinum ringelectrode was placed around the muscle and coupled toa Grass S48 stimulator (5 V, 0.1 Hz, 0.2 ms). Isometricmuscle contractions and contractures were recorded viaa force displacement transducer (Load Cell BG-50) coupledto a Gould RS 3400 physiograph. Muscle responses toexogenous acetylcholine (ACh, 110 mM) and potassium

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chloride (KCl, 20 mM) were obtained in the absence of fieldstimulation before the addition of ethanol alone (40 ml/ml),M. laevigata extract (40, 50 and 60 ml/ml) or venom(50 mg/ml) or pretreatment with M. laevigata extract(40 ml/ml) for 30 min followed by venom (50 mg/ml). Allexperimental protocols were done at least four times withincubations lasting up to 120 min.

2.5. Morphological and morphometrical analyses

At the end of the experiments, diaphragm and biventercervicis muscles incubated with ethanol alone,M. laevigataextract, venom or M. laevigata extract þ venom asdescribed above were immediately fixed for 24 h in 10%paraformaldehyde, washed three times with saline solu-tion, dehydrated in increasing ethanol concentrations(70, 80, 95 and 100%, v/v), clarified in xylene and embeddedin paraffin. Sections (5 mm thick) obtained using a LeicaRM2245 microtome were stained with hematoxylin-eosin(H&E) and analyzed with a Nikon Eclipse E800 micro-scope coupled to a microcomputer (Dell Vostro 220S)equipped with image analysis software (NIS-ElementsAR30). Control preparations were prepared from muscleincubated with Tyrode or Krebs solution. The extent ofdamage in control and treated muscles was assessed bycounting 100 normal and damaged fibers (n ¼ 3 animals)and then expressing the number of damaged fibers asa percentage of the total number of fibers counted. Normalfibers were defined as those with a polygonal appearance,peripheral nucleus and evenly distributedmyofibers withinthe fascicle.

2.6. Immunohistochemistry for TNFa and IFNg

Paraffin sections were deparaffinized in xylene, rinsedin a graded ethanol series (100%, 95%, 90%, 80% and 70%),distilled water and 0.05 M phosphate-buffered saline, andpretreated with H2O2:50% ethanol (1:1, v/v) solution for15 min to quench endogenous peroxidase activity. The

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Fig. 1. Twitch-tension responses to M. laevigata extract in indirectly stimulated PND37 �C as described in Sections 2.3 and 2.4, respectively. The points represent the mea

sections were then washed in 0.05 M Tris-buffered saline(TBS), permeabilized with 0.3% Triton X-100 for 10 min,rinsed in TBS and incubated in 1% TBS/BSA for 2 h at roomtemperature. Rabbit anti-TNFa and anti-IFNg (sc 1350 andsc 1377, respectively) were applied as primary antibodies(diluted 1:50 in 1% TBS/BSA) overnight at 4 �C in a humid-ified chamber. The sections were subsequently rinsed in0.05 M TBS, incubated with biotinylated anti-rabbitsecondary antibody (sc 5491) and developed with DAB.Hematoxylin staining was used to assess the nuclearmorphology. Finally, the slides were rinsed, dehydrated,mounted in Canada synthetic balsam and analyzed by lightmicroscopy. Control reactions were done by omitting theprimary antibody.

2.7. Statistical analysis

The results were expressed as the mean � SEM. Statis-tical analyses were done using Student’s t-test(for comparison of two samples) and ANOVA followed bythe Tukey–Kramer test (for comparison of more than twosamples). P values < 0.05 were considered significant.

3. Results

3.1. Neuromuscular activity

The M. laevigata extract produced a concentration-dependent facilitation of twitch-tension at low concentra-tions [for PND: 107.4 � 6% after 70 min with 20 ml/ml and118.9 � 9% after 120 min with 40 ml/ml (p < 0.05 comparedwith control); for BC: 120.7 � 7.7% after 20 min with40 ml/ml and 114.5 � 4.4% after 80 min with 50 ml/ml;p< 0.05 for both compared to control]. High concentrationsof extract (>50 ml/ml) caused total, reversible neuromus-cular blockade after 120 min (n ¼ 4–6; p < 0.05 comparedto control preparations) (Fig. 1A, B).

In PND preparation, the ethanol alone (40 ml/ml) did notchange the twitch-tension when compared with control.

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(A) and BC (B). The preparations were mounted for electrical stimulation atn � SEM of 4–6 experiments. *p < 0.05 compared to Tyrode or Krebs control.

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Fig. 2. Neuromuscular blockade of PND (A) and BC (B) preparations incubated with Tyrode or Krebs solution (control), Ethanol alone (40 ml/ml), M. laevigataextract (40 ml/ml), P. olfersii venom (50 mg/ml) or M. laevigata extract (40 ml/ml) for 30 min followed by venom (50 mg/ml) for a further 90 min at 37 �C. The pointsrepresent the mean � SEM of n ¼ 4–6 experiments. *p < 0.05 compared to Tyrode or Krebs controls; #p < 0.05 compared to venom alone.

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However, in BC preparation, the ethanol did producea higher facilitation (increase of twitch-tension amplitude)when compared to the control (n ¼ 4; p < 0.05) (Fig. 2A, B).

Venom (50 mg/ml) caused partial blockade in PND(58.5 � 12.0%, n ¼ 4) and total blockade in BC (6.5 � 2.2%,n ¼ 4) (Fig. 2A, B), that was irreversible by washing in bothcases (data not shown). Pretreatment with M. laevigataextract (40 ml/ml) for 30 min followed by incubation withvenom (50 mg/ml) for an additional 90 min in both prepa-rations resulted in 100% protection in PND and delayed thevenom-induced neuromuscular blockade in BC (n ¼ 4–6)(Fig. 2A, B).

The contractures to exogenous ACh and KCl wererecorded before and after the addition of protocols. Incu-bation with venom significantly attenuated the contrac-tures to ACh and KCl (p < 0.05). In preparations pretreated,the contractures to KCl were w40% of the control whilethose to ACh were 15% of the control, indicating a slightprotection of these responses by the extract (data notshown).

3.2. Morphological and morphometrical analyses

The percentage of muscle damage, including vacuolatedand hypercontracted fibers, was considerably higher(p < 0.05) in venom-treated PND and BC preparations(53.5 � 8.5% and 55.8 � 4.3%, respectively; n ¼ 3) than incontrol preparations (3.0 � 0.1% and 2.2 � 0.3%, respec-tively; n ¼ 3), ethanol preparations (4.3 � 2.4% and6.7 � 3.3%, respectively; n ¼ 3) and in preparations treatedwith M. laevigata extract (12.5 � 0.5% and 10.9 � 2.3%,respectively; n ¼ 3) or extract þ venom (13.0 � 3.5% and26.4 � 4.5%, respectively; n ¼ 3). Pretreatment with M.laevigata extract reduced the muscle damage by 80.3% inPND and 60.4% in BC (Fig. 3).

3.3. Immunohistochemistry for TNFa and IFNg

Qualitative immunohistochemistry showed that TNFaand IFNg were expressed in damaged muscle fibers, espe-cially in vacuolated and hypercontracted fibers where the

myofilaments were condensed. TNFa expression was moreintense than that for IFNg in some fibers. The expression ofthese cytokines was qualitatively greater in venom-treatedpreparations than in those treated withM. laevigata extractor with M. laevigata extract þ venom. Little or no TNFa andIFNg expression was seen in control and ethanolic prepa-rations (Fig. 4).

4. Discussion

Plant extracts have been shown to have various effectson the neuromuscular junction. For example, Portulacaoleracea, C. sylvestris and Camellia sinensis (C. sinensis) causefacilitation of twitch-tension response (Habtemariam et al.,1993; Oshima-Franco et al., 2005; de Jesus Reis Rosa et al.,2010). In contrast, an extract from Piper sarmetosumcauses neuromuscular blockade, probably by preventingthe release of ACh from the presynaptic terminal (Ridtitidet al., 1998), and Cleisthanthus collinus causes neuromus-cular blockade by decreasing the excitability of nervesand membranes (Nandakumar et al., 1989). Kunzeaericoides reduces the contractile activity of BC preparationsbut has no activity in PND preparations (Lis-Balchin et al.,2000).

As shown here, the M. laevigata extract caused signifi-cant facilitation in PND preparations at low concentrations(20–50 ml/ml), indicating that the extract containscomponents that affect contractile activity in these prepa-rations. The increase in facilitation is also observed in BCpreparations, however the effect of ethanol alone, observedin BC ethanolic preparations displayed highest facilitationwhen compared to M. laevigata extract. Studies has shownthat ethanol have both presynaptic and post-synapticeffects on synaptic transmission. The neurotransmitterrelease is mediated by an inhibition of the delayed rectifierpotassium current (Anderson et al., 1988; Appel et al., 2003;Silinsky, 2004) that normally repolarized the nerve ending,enhancing Ca2þ entry into the nerve ending. These effectscause an increase in ACh release without changing thepresynaptic store of releasable neurotransmitter (Searl andSilinsky, 2010).

Fig. 3. Morphological alterations in diaphragm muscle incubated with Tyrode solution (control), Ethanol (40 ml/ml), M. laevigata extract (40 ml/ml), P. olfersiivenom (50 mg/ml) or M. laevigata extract for 30 min prior to addition of venom. Sections were stained with H&E and for TNFa and IFNg. Scale bar ¼ 20 mm. Noteedematous (e) and hypercontracted (h) fibers scattered amongst normal muscle fibers (n) with peripheral nuclei (arrow). *Immunohistochemical staining forcytokines.

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The facilitatory effect observed with ethanol did notaffect the results found in this study. Similar effect wasfound with polyethylene glycol 400 (PEG400) using thesame in vitro neuromuscular preparations in a similarexperimental model (Oshima et al., 2010). Besides, asethanol works as antidote against methanolic intoxication

(Ekins et al., 1985), as PEG400 has several clinical applica-tions (Oshima et al., 2010).

In contrast, M. laevigata extract at high concentrations(>50 ml/ml) induced a reversible neuromuscular blockade,as shown by washing the preparations (data not shown).The reversibility by washing indicated that the extract

Fig. 4. Morphological alterations in biventer cervicis muscle incubated with Krebs solution (control), Ethanol (40 ml/ml), M. laevigata extract (40 ml/ml), P. olfersiivenom (50 mg/ml) orM. laevigata extract for 30 min prior to addition of venom. Sections were stained with H&E and for TNFa and IFN. Scale bar ¼ 20 mm. Note theedematous (e) and hypercontracted (h) fibers scattered amongst. *Immunohistochemical staining for cytokines.

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components involved had low affinity for post-synapticnicotinic receptors (Nirthanan et al., 2003).

P. olfersii venom caused complete, irreversible neuro-muscular blockade in BC as previously observed by Prado-Franceschi et al. (1996). In contrast, only partial irreversible

blockade was seen in venom-treated PND. This greateractivity in avian preparations is characteristic of otherSouth America colubrid venoms (Prado-Franceschi et al.,1996) and several Bothrops spp. (Heluany et al., 1992;Cogo et al., 1993; Abreu et al., 2007) that show little activity

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in mammalian compared to avian preparations. Suchphenomenonmay reflect the dietary preference of P. olfersiifor birds (Leite et al., 2009). The absence of contractures toexogenous KCl suggested the presence of a myotoxiccomponent in the venom, a conclusion in agreement withthe previous identification of a myotoxin in this venom(Prado-Franceschi et al., 1998). The inhibition of ACh-induced contractures also agreed with the findings ofPrado-Franceschi et al. (1996) regarding a post-synapticaction of the venom.

The anti-snake venom activity of several Brazilian plantshas been tested against pitviper and elapid snake venoms(Borges et al., 2000; Otero et al., 2000; Núñez et al., 2004; deJesus Reis Rosa et al., 2010). However, few studies haveexamined such activities against the venoms of Philodryasspp. As shown here, pre-treatment with the M. laevigataextract totally protected PND fromvenom-induced damage.Interestingly, an extract of C. sinensis induced only 50%protection against muscle damage by Crotalus durissus ter-rificus venom (de Jesus Reis Rosa et al., 2010), whereas totalprotection against Bothrops jararacussu and its main toxin,bothropstoxin-I (Oshima-Franco et al., 2012), showing thatthe mechanism of action of each venom is determinant toevaluate the protection level against myotoxicity.

In BC, which is more sensitive to venom, there was onlyslight protection,with themost relevant effect being a delayin the ensuing blockade, as also observed for Dipteryx alataagainst B. jararacussu venom (Nazato et al., 2010).

Extracts of M. glomerata and M. laevigata have similarstructural, chemical and physical properties, although thatof M. laevigata contains a larger amount of coumarin(Acordi-da-Silva, 2008; Alves et al., 2009; Bolina et al.,2009) that may be related to its antiophidian activity. M.glomerata inhibits snake venom PLA2 activity (Maioranoet al., 2005; Floriano et al., 2009). Since P. olfersii venomis devoid of PLA2 (Assakura et al., 1992; Prado-Franceschiet al., 1998; Rocha et al., 2006; Rocha and Furtado, 2007)then the extract must act on other venom componentsinvolved in this response.

Histological analysis showed significant muscle damagein both preparations incubated with venom. The myonec-rosis observed with P. olfersii venom in vivo (Acosta dePérez et al., 2003) and with P. patagoniensis venomin vitro (Carreiro-da-Costa et al., 2008) results in edema-tous, vacuolated and hypercontracted fibers. These findingsmay be related to the ability of these venoms to degradefibrinogen without clot formation, thereby facilitating theaction of proteases, principally metalloproteases (Assakuraet al., 1992; Acosta de Pérez et al., 2003), and myotoxins(Prado-Franceschi et al., 1996, 1998).

M. laevigata extract caused slight but significant muscledamage when compared to control preparations. However,when used to pretreat preparations the extract protectedagainst venom-induced damage. Studies in other modelshave also shown thatM. laevigata can protect against tissuedamage (Nopimoga and Yatsuda, 2010). Edematous fiberswere less observed in pretreated preparations whencompared to venom. In addition, the oral or subcutaneousadministration ofM. laevigata extract decreases paw edemaand vascular permeability, as well as leucocyte rolling andadhesion in inflamed tissue (Suyenaga et al., 2002).

Prolonged TNFa activation has been associated witha loss of muscle fibers (Sharma and Anker, 2002). However,when released by infiltrating inflammatory cells orproduced by the myofibers themselves during myopathies(Kuru et al., 2003) or after muscle damage caused byvenom-derived cardiotoxin (Chen et al., 2005) or peptides(Rocha et al., 2010), TNFa actually contributes to improvemuscle regeneration. IFNg also influences skeletal musclehomeostasis and repair. IFNg expression is enhanced incultured muscle cells (Mantegazza et al., 1991) and duringmyoblast differentiation (Tomita and Hasegawa,1984; Kelicet al., 1993). As with TNFa, IFNg has been associated withskeletal muscle regeneration (Cheng et al., 2008; Rochaet al., 2010). Indeed, some studies have suggested theexistence of “cross-talk” between TNFa and IFNg expres-sion (Abbas and Lichtman, 2005; Rocha et al., 2010).

As shown here, incubation with venom increased theexpression of TNFa and IFNg in skeletal muscle. Thisenhanced expression was attenuated in preparations pre-treated with M. laevigata extract, which by itself alsoreduced the expression of these cytokines. These observa-tions for the extract agreed with the ability of M. laevigatato inhibit the production of pro-inflammatory cytokines(Alves et al., 2009), thereby contributing to the anti-inflammatory activity of this plant.

Based on the findings described here, we conclude thatan ethanolic extract of M. laevigata was able to protectvertebrate muscle against the neurotoxicity and myotox-icity caused by P. olfersii venom and that the extract alsoprotected against the inflammatory action of the venom.

Acknowledgments

This study was supported by PIBIC-CNPq. The authorsthank Gildo Bernardo Leite for technical assistance.

Conflicts of interest

The authors declare that there are no conflicts ofinterest.

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