universidade federal do cearÁ - repositorio.ufc.br · “porque deus amou o mundo de tal forma,...

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UNIVERSIDADE FEDERAL DO CEARÁ

CAMPUS DE SOBRAL

SHIRLEY MOREIRA ALVES

LECTINA DE ABELMOSCHUS ESCULENTUS REDUZ HIPERNOCICEPÇÃO

INFLAMATÓRIA NA ARTICULAÇÃO TEMPOROMANDIBULAR DE RATOS

DEPENDENTE DE RECEPTORES OPIOIDES CENTRAIS

SOBRAL-CE

2016

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Dissertação de Mestrado apresentada ao Programa de Pós-Graduação – Curso Ciências da Saúde, da Universidade Federal do Ceará – Campus Sobral, como requisito parcial para obtenção do Título de Mestre em Ciências da Saúde. Área de concentração: Farmacologia.

Orientadora: Profª. Drª. Hellíada Vasconcelos Chaves

SOBRAL-CE

2016

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Dados Internacionais de Catalogação na Publicação Universidade Federal do Ceará

Biblioteca Universitária Gerada automaticamente pelo módulo Catalog, mediante os dados fornecidos pelo(a) autor(a)

M839l Moreira Alves, Shirley.


INFLAMATÓRIA NA ARTICULAÇÃO TEMPOROMANDIBULAR DE RATOS DEPENDENTE DE RECEPTORES OPIOIDES CENTRAIS / Shirley Moreira Alves. – 2017. 65 f. : il. color.

Dissertação (mestrado) – Universidade Federal do Ceará, Campus de Sobral, Programa de Pós-Graduação em Ciências da Saúde, Sobral, 2017. Orientação: Profa. Dra. Hellíada Vasconcelos Chaves.

1. Abelmoschus esculentus. 2. Articulação temporomandibular. 3. TNF-alfa. 4. Hipernocicepção. 5. Lectina. I. Título.

CDD 61

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Dissertação de Mestrado apresentada ao Programa de Pós-Graduação – Curso Ciências da Saúde, da Universidade Federal do Ceará – Campus Sobral, como requisito parcial para obtenção do Título de Mestre em Ciências da Saúde. Área de concentração: Farmacologia.

Orientadora: Profª. Drª. Hellíada Vasconcelos Chaves

Aprovado em:13/12/2016.

BANCA EXAMINADORA

__________________________________________________ Profª. Drª. Hellíada Vasconcelos Chaves (Orientadora)

Universidade Federal do Ceará (UFC) – Campus Sobral

_____________________________________________ Prof. Dr. Vicente de Paulo Teixeira Pinto

Universidade Federal do Ceará (UFC) – Campus Sobral

____________________________________________ Profª. Drª. Theodora Thaís Arruda Cavalcante

Instituto Superior de Tecnologia Aplicada (INTA) – Campus Sobral

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A Deus, por todo zelo e cuidado para

comigo. À minha família, alicerce firme

e norteador: Ana Lúcia Moreira, José

Alves da Costa, Sheila Moreira Alves,

Ernando Rodrigues Batista, Antônio

Dias Lima Filho.

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AGRADECIMENTOS

Ao Senhor Jesus, que é bom em todo o tempo. Tem coração manso e nos

reserva sempre o melhor. Obrigada Senhor!!!

À minha mãe, Ana Lúcia Moreira, por ter escolhido a família muitas vezes

em detrimento de si própria. Mãe, sem você jamais seria quem sou hoje.

Obrigada por você existir, te amo!

Ao meu pai José Alves da Costa, por ter me dado vida e discernimento.

À minha “contraparte clara”: minha irmã Sheila Moreira Alves que nas mais

diversas situações sempre esteve ao meu lado. Essa vitória também é sua.

Ao meu cunhado-irmão Ernando Rodrigues Batista, por se fazer porto

seguro sempre que necessário. Você é presente do Senhor.

Ao meu esposo Antônio Dias Lima Filho, pela paciência e serenidade nos

momentos mais turbulentos. Obrigada pelo apoio e carinho.

A todos os meus professores, desde os primeiros anos de ensino aos dias

de hoje. Sem eles essa conquista não seria possível.

Ao Prof. Dr. Paulo Roberto Santos, pela extrema humildade e sabedoria.

Exemplo a ser seguido.

A Todos que compõem o Laboratório de Farmacologia da UFC – Campus

Sobral na pessoa da Profª Ms. Danielle Rocha do Val. Agradeço cada dia de

dedicação, envolvimento e conhecimento empenhados. Aprendi muito com vocês.

À Profª. Drª. Mirna Marques Bezerra, por deixar transparecer amor em seu

trabalho. Deus é seu escudo e nos presenteia com sua presença.

À minha orientadora, Profª. Drª. Hellíada Vasconcelos Chaves, por toda a

compreensão, paciência e parceria nesse tempo de formação. Sua competência é

inquestionável.

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À Universidade Federal do Ceará (UFC); ao Laboratório de Farmacologia

de Sobral (LAFS – UFC); ao Programa de Pós-Graduação em Bioquímica; à

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES); ao

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq); à

Fundação Cearense de Amparo à Pesquisa (FUNCAP), e ao Instituto de

Biomedicina do Semi-Árido Brasileiro (INCT-IBISAB), pelo apoio e custeio do

presente projeto.

E a todos que de alguma forma contribuíram para a realização deste

trabalho.

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“Porque Deus amou o mundo de tal

forma, que deu seu único filho, para

todo aquele que nele crê não pereça,

mas tenha vida eterna.” (João, 3:16)

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RESUMO

Relevância etnofarmacológica: Abelmoschus esculentus é amplamente

cultivada no Nordeste do Brasil para fins medicinais, no tratamento de pneumonia,

bronquite e tuberculose pulmonar, apresentando também atividade anti-

inflamatória. Objetivo do estudo: Avaliar a atividade antinociceptiva e anti-

inflamatória da lectina de Abelmoschus esculentus (AEL) no modelo inflamatório

de hiperalgesia induzida por formalina na articulação temporomandibular (ATM)

de ratos. Materiais e métodos: Os experimentos comportamentais (CEUA nº

02/15) foram realizados em ratos Wistar machos (180-240 g). Os ratos foram pré-

tratados (i.v.) com salina ou AEL (0,001, 0,01 ou 0,1 mg/kg). Depois de 30 min.

receberam uma injeção intra-articular (i.art) de formalina (1,5 %/50 µl) ou solução

salina (controle) na ATM esquerda e foram monitorados durante 45 min. para

observar a resposta comportamental nociceptiva quantificada, em segundos,

pelos atos de coçar da região injetada e erguer a cabeça reflexivamente. Em

seguida, os animais foram anestesiados e submetidos a eutanásia e os tecidos

periarticulares, gânglio trigeminal e subnúcleo caudal foram removidos e

processados para dosagem de TNF-α pelo método ELISA. Para investigar a

permeabilidade vascular, os animais receberam 50 mg/kg (i.v) de corante azul de

Evans 30 min. antes da administração de AEL e, após 30 min., receberam uma

injeção intra-articular de formalina (1,5 %/50 uL). Após 45 min., os animais foram

eutanasiados e as ATMs removidas para análise. Ademais, foi estudada a

participação da via opioide na resposta antinociceptiva de AEL, utilizando o

antagonista opioide Naloxona (15 µg/10 µl) ou os antagonistas dos receptores

opióides Kappa (ҡ), Nor-Binaltorfimina (15 ou 45 µg/10 µl), ou Delta (δ),

Naltrindole (10 ou 30 µg/10 µl), ou mu (µ), CTOP (10 µg/10 µl) por via intra-tecal

15 min. antes da aplicação de AEL. Resultados: AEL (0,01 mg/kg) foi eficaz na

redução da nocicepção induzida por formalina (p<0,05) e extravasamento de azul

de Evans. Houve uma redução dos níveis de TNF-α (p<0,05) no tecido

periarticular, gânglio trigeminal e subnúcleo caudal de ratos pré-tratados com

AEL. Além disso, o efeito antinociceptivo de AEL é dependente dos receptores

opioides ҡ e δ, porém não de µ. Conclusão: AEL apresenta efeitos anti-

inflamatório e antinociceptivo, que podem resultar da inibição de citocinas, da

diminuição do extravasamento plasmático e da ativação de receptores opioides ҡ

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e δ.Palavras-chave: Abelmoschus esculentus, articulação temporomandibular,

TNF-alfa, hipernocicepção, lectina

Abstract:

Ethnopharmacological relevance: Abelmoschus esculentus is largely cultivated

in Northeastern Brazil for medicinal purposes, like in cases of pneumonia,

bronchitis, pulmonary tuberculosis and inflammation. Aim of the study: To

evaluate the Abelmoschus esculentus (AEL) in reducing formalin-induced

temporomandibular joint inflammatory hypernociception in rats. Materials and

Methods: The behavioral experiments (CEUA nº 02\15) were performed on male

Wistar rats (180–240 g). Rats were pre-treated (i.v.) with AEL (0.001, 0.01 or 0.1

mg/kg) thirty minutes before 1.5% formalin injection in the TMJ. Further, to analyze

the possible effect of opioid pathways on AEL efficacy, animals were pre-treated

via intrathecal injection of naloxone or CTOP (the antagonist of Mu (µ) opioid

receptor), naltrindole (antagonist of Delta (δ) opioid receptor) or Nor-

Binaltorphimine (antagonist of Kappa () opioid receptor) 15 minutes before AEL

followed by intra-TMJ injection of 1.5% formalin. Behavioral analysis were

perfomed, animals were monitored for a 45 min observation period to quantify the

nociceptive response. TMJ tissue, trigeminal ganglion and caudal subnucleus

collection was performed for TNF-α dosage (ELISA). In addition, vascular

permeability was evaluated by Evans Blue extravasation. Results: AEL

significantly reduced formalin-induced TMJ inflammatory hypernociception and

decreased Evans blue extravasation. It also decreased TNF-α levels in TMJ

tissue, trigeminal ganglion and caudal subnucleus. AEL antinociceptive effects,

however, were not observed in the presence of naltrindole or Nor-Binaltorphimine.

Conclusions: These findings suggest that AEL efficacy depends on TNF-α

inhibition and the activation of δ and opioid receptors.

Keywords: Abelmoschus esculentus; temporomandibular joint, TNF-α,

hypernociception, lectin

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LISTA DE ILUSTRAÇÕES

Quadro 1 - Classificação das Disfunções Temporomandibulares…………….. 19

Figura 1A - Anatomia do Complexo Trigeminal do Tronco Encefálico………… 17

Figura 1 - Eficácia da AEL na hipernocicepção inflamatória induzida por

formalina na ATM de ratos……………………………………………

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Figura 2 - Efeito de AEL sobre a permeabilidade vascular na

hipernocicepção inflamatória induzida por formalina na ATM de

ratos…………………………………….............................................

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Figura 3 - Efeito de AEL sobre os níveis de TNF-α na hipernocicepção

inflamatória induzida por formalina na ATM de

ratos…………..…………………………………………………………

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Figura 4 - Efeito do antagonista não seletivo (naloxona) e seletivos (µ,

eδ) na atividade antinociceptiva de AEL na hipernocicepção

inflamatória induzida por formalina na ATM de

ratos……………………………….…………………………………….

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LISTA DE SIGLAS E ABREVIATURAS

AEL: Lectina de Abelmoschus esculentus

ANOVA: Análise de Variância

ATM: Articulação Temporomandibular

ATP: Adenosina Trifosfato

BioGeR: Laboratório de Genética Bioquímica e Radiobiologia

b2-AR: Adrenoreceptor β2

CAPES: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior

CEUA: Comissão de Ética no Uso de Animais

COMT: catecol-O-metiltransferase

CNPq: Conselho Nacional de Pesquisa

CTOP: antagonista do receptor opióide µ

DAINES: Drogas anti-inflamatórias não-esteroidas

DEAE – Sephacel: permutador iônico

DTM: Disfunção temporomandibular

DBCA: Diretriz Brasileira para o Cuidado e a Utilização de Animais Para Fins

Científicos E Didáticos

δ: Receptor opioide delta

ELISA: Enzyme-Linked Immunosorbent Assay

FITC: fluorescein isothiocyanate

FUNCAP: Fundação Cearense de Apoio ao Desenvolvimento Científico e

Tecnológico

g: Grama

GRK: Cinase do receptor acoplado à Proteína-G

H2SO4: Solução de ácido sulfúrico

HO-1: Hemeoxigenase-1

i.art.: Intra articular

IL-1β: Interleucina-1beta

INCT- IBSAB: Instituto de Biomedicina do Semi -Árido Brasileiro

i.t.: Intra tecal

i.v.: Intra venoso

ҡ: Receptor opioide kappa

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LAFS: Laboratório de Farmacologia de Sobral

mg: Miligrama

mg/kg: Miligrama por quilo

µ: Receptor opioide mu

µg: Micrograma

µl: Microlitro

n: Número de animais

NaCl: Cloreto de Sódio

nm: Namômetro

TNF-α: Fator de necrose tumoral alfa

NT: Neuralgia do trigêmio

PGE2: Prostaglandina E2

pg/ml: picograma por mililitro

PZM21: Agonista seletivo do receptor opioide µ

p<0,05: Probabilidade de erro estatístico 5%

s: Segundos

SBCAL: Sociedade Brasileira de Ciência em Animais de Laboratório

s.c: Subcutânea

TJM: Temporomandibular Joint

º C: Grau Celsius

± EPM: Mais ou menos o erro padrão da média

5HTT: Recepor serotoninérgico

UFPB: Universidade Federal da Paraíba

Zy: Zymosam

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SUMÁRIO

1 INTRODUÇÃO……………………………….……………………...……….……. 15

1.1 Dor Orofacial......…………………………….………….………………………… 15

1.2 Processo inflamatório na região da articulação temporomandibular... 17

1.3 Disfunção temporomandibular………………….………….……...…………. 18

1.4 Lectinas …………………………….………….……………............................... 21

1.5 Propriedades biológicas de lectinas ………....……………..….………..…. 23

2 JUSTIFICATIVA…………………………….………….……………...………….. 25

3 OBJETIVOS…………………………….………….….…………………………... 26

3.1 Objetivo Geral…………………………….………….….……………………...… 26

3.2 Objetivos Específicos…………………………….………………….....……… 26

4 CAPÍTULO 1: LECTIN FROM ABELMOSCHUS ESCULENTUS

REDUCES RAT TEMPOROMANDIBULAR JOINT INFLAMMATORY

HYPERNOCICEPTION DEPENDENT FROM CENTRAL OPIOID

RECEPTORS…...............................................................................................

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REFERÊNCIAS…………………………….………….….……………………..… 51

APÊNDICE: Graphical Abstract Exigido pelo Periódico Journal of

Ethnopharmacology…………………………………………………………..….

58

ANEXO A: Declaração de Aceite do Comitê de Ética…………………….... 59

ANEXO B: Normas da Revista Journal of Ethnopharmacology………..... 60

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1 INTRODUÇÃO

1.1 Dor orofacial

A dor exerce papel fisiológico importante nos organimos no sentido de

alertar os sistemas biológicos para possíveis danos, o que torna a capacidade de

sentir dor um importante mecanismo de sobrevivência (SBED, 2010), e de

acorodo com a Associação Internacional para o Estudo da Dor (IASP, 2008) é

compreendida como uma experiência sensorial e emocional desagradável,

associada a um dano tecidual real ou potencial, ou ainda descrita nesses termos.

Os componentes da dor diferem da nocicepção, uma vez que a percepção

da dor envolve diversos fatores como estímulo dos nociceptores primários,

percepção emocional e cognitiva (componente subjetivo da dor) (Julius; Basbaum,

2001). Em contrapartida, a nocicepção (do latim nocere: nocivo; capere: captar,

receber) pode ser definida como a captação do nocivo, envolvendo apenas os

mecanismos de transmissão desse estímulo ao sistema nervoso central (SNC)

(Oliveira, 2001). Dessa forma, o termo dor deve ser aplicado aos estudos

envolvendo humanos, por possuírem a capacidade de identificar seu caráter

subjetivo, e nocicepção quando as pesquisas utilizarem animais, por estes serem

desprovidos da capacidade de captar o estímulo emocional da dor.

A dor orofacial tem se revelado como um grande problema de saúde

pública nas últimas décadas, comprometendo a funcionalidade articular e a

qualidade de vida dos indivíduos atingidos (Okeson, 1998; Hargreaves, 2011;

Monteiro et al., 2011). Adicionalmente, estudos tem demonstrado que os

processos dolorosos representam um custo financeiro elevado por conta do

grande número de horas disperdissadas durante o processo produtivo

(Macfarlane, 2002). Ademais, apesar de acometer indivíduos jovens, pesquisas

têm revelado que a dor orofacial possui alto grau de prevalência na população

mundial como um todo (Hargreaves, 2011). Obermann (2010) afirma que pelo

menos 10% da população adulta é acometida por dor orofacial, e que esse índice

aumenta em idosos em mais de 50%, com tendência de as mulheres serem mais

propensas às formas crônicas de dor orofacial, incluindo neuralgia do trigêmio e

as disfunções temporomandibulares (DTM).

Região comumente referida como dolorosa, a face abriga a mandíbula,

único osso móvel dessa região, que interage através de duas articulações

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temporomandibulares (ATM) reconhecidas como articulações especializadas que

realizam diversos tipos de movimentos a fim de cumprir suas funções fisiológicas.

Além disso, fazem parte do sistema estomatognático a ATM, os músculos faciais,

dentes, língua, glândulas, nervos, dentre outras estruturas (Okeson, 2008;

Barretto et al., 2013) que juntas permitem o desempenho harmônico de funções

como a respiração, mastigação, deglutição e fala. Estas estruturas estão sujeitas

a variações de pH (potencial de hidrogênio), temperatura, concentrações

moleculares e estímulos mecânicos que podem levar a lesões e inflamação,

provocando dor (Kitsoulis et al., 2011; Rando; Waldron, 2012; SBED, 2013).

Nesse contexto, a dor orofacial pode ser compreendida como qualquer dor

associada aos tecidos não mineralizados/moles (pele, vasos sanguineos,

glandulas ou musculos) e mineralizados (ossos e dentes) da cavidade oral e face,

estando normalmente relacionada aos eventos dolorosos que atingem a cabeça

e/ou região do pescoço, ou ainda à cervicalgia, odinofagia, cefaleias primárias e

doenças reumáticas tais como fibromialgia e artrite reumatoide (Leeuw, 2010).

Porreca et al. (2002) e Verri-Junior et al. (2006) relatam em seus estudos

que a dor, comum em diversas desordens clínicas, incluindo as que acometem a

região orofacial, se apresenta como um dos sinais clássicos da inflamação, sendo

iniciada pela sensibilização dos nociceptores aferentes primários. Assim, a

capacidade de reconhecer os possíveis agentes lesivos habilita a liberação de

mediadores da resposta inflamatória através do sistema imune (Robbins; Cotran,

2005). A percepção dolorosa da região orofacial está diretamente ligada ao

estímulo polongado de nociceptores periféricos (Reyes; Uyanik, 2014) que, por

sua vez, transmitem a informação nociva ao sistema nervoso central (SNC) e aos

centros superiores do tronco encefálico, comunicando-se com o complexo

trigeminal do tronco encefálico (tálamo e córtex somatosensorial) (Sessle, 2000).

Este possui, em sua maioria, os corpos celulares dos neurônios sensoriais

localizados no gânglio trigeminal, que mante´m de forma geral relação estreita

com o sistema límbico, responsável pelo processamento subjetivo da dor

(Matthews; Sessle, 2002).

É importante destacar que o complexo trigeminal (Figura 1A) compreende

o núcleo sensorial principal, responsável pela transmissão das sensações de

propriocecpção e tato; o núcleo motor, envolvido nas respostas motoras, e núcleo

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do trato espinal do trigêmio, dividido em três subnúcleos: oral, interpolar e caudal.

Sendo este ponto do tronco cerebral, também denominado como dorsal medular

por possuir semelhanças com o corno dorsal espinhal, reconhecido como o

principal sítio para a informação nociceptiva (Okeson, 2003; Takemura, 2006;

Ren, Dubner, 2011).

Figura 1A- Anatomia do Complexo Trigeminal no Tronco Encefálico. Gânglio de Gasser (gânglio trigeminal) (GG); núcleo motor do complexo trigeminal (NM do V); núcleo sensitivo principal do complexo trigeminal (NS do V); núcleo do trato espinhal do complexo trigeminal (NTE do V), subdividido em: subnúcleo oral (sno), subnúcleo interpolar (sni), e subnúcleo caudal (snc). Ta: tálamo.

Fonte: Adaptado de OKESON, 2003.

1.2 Processo inflamatório na região da articulação temporomandibular

A Inflamação possui sinais clássicos como dor, calor, rubor e tumefação

(edema), compreendendo uma fase aguda caracterizada pela vasoconstrição

transitória, vasodilatação e consequente aumento do fluxo sanguíneo, e uma fase

crônica entendida como aquela em que há uma lesão tecidual e células

mononucleares como macrófagos, linfócitos e plasmócitos são recrutadas

(Robbins; Cotran, 2005), assim como a liberação de mediadores químicos após a

lesão tecidual (Ellis; Bennett, 2013; Fong; Schug, 2014).

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Durante a resposta inflamatória mediadores lipídicos (prostaglandinas),

protéicos (citocinas), histamina, óxido nítrico, bradicinina e neuropeptídios são

responsáveis pela manutenção e amplificação do processo inflamatório.

Delineamentos de pesquisa envolvendo animais para a investigação do processo

inflamatório articular apontam os neutrófilos como sendo os primeiros a migrarem

para a artiulação afetada, além disso, a inflamação estimula a liberação de

mediadores pró-inflamatórios denominados citocinas, como as interleucinas IL-1β,

IL-6 e IL-8 e o fator de necrose tumoral alfa (TNF-α) (Venkatesha et al., 2011),

promovendo, dessa forma, a degradação da ATM (cartilagem articular, membrana

sinovial e parte óssea). Tais superfícies danificadas podem acarretar em dor,

inflamação e limitação da mobilidade local (Kostrzewa-Janicka et al., 2012).

Além disso, o TNF-α, produzido principalmente por macrofagos, é apontado

em diversos estudos como uma importante citocina mediadora do processo

inflamatório crônico e agudo observado durante o desenvolvimento de doenças

articulares degenerativas (Kostrzewa-Janicka et al., 2012). Outro mediador

inflamatório identificado em níveis elevados na sinóvia da ATM de pessoas que

apresentam dor inflamatória é a serotonina (5-HTT), encontrada no SNC e em

todos os tecidos periféricos (Oliveira-Fusaro et al., 2012). Contudo, mediadores

químicos que compõem a inflamação podem ser estimulados por células gliais

satélite que permeiam corpos neuronais, funcionando como um efetores da

resposta inflamatória, o que evidencia que a inflamação não é somente resultado

de mecanismos periféricos, mas também centrais (Ellis; Bennett, 2013).

1.3 Disfunção temporomandibular

As disfunções temporomandibulares (DTM) englobam um grupo de

condições musculoesqueléticas e neuromusculares envolvendo a ATM, os

músculos mastigatórios e todos os tecidos associados (Greene, 2010). Quanto

aos fatores etiológicos, hábitos parafuncionais podem desencadear DTM, como

bruxismo, onicofagia, ou mastigação não-funcional, assim como hábitos

ocupacionais, frouxidão ligamentar, traumatismos, iatrogenia (intubação) e

disfunção hormonal também fazem parte dos fatores predisponentes para as DTM

(Milam et al., 1987; Scrivani et al., 2008; Sharma et al., 2011; De Rossi et al.,

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2014). As DTM são classificadas em seis grandes grupos, e estes se ramificam

em outras disfunções, que podem ser observadas no Quadro 1.

Quadro 1: Classificação das Dinfunções Temporomandibulares

Disfunções Temporomandibulares

* Disfunções Articulares

* Dor articular - Artralgia - Artrite

* Disfunçoes articulares

- do complexo côndilo-disco - de hipomo-bilidade e hipermobilidade

* Doenças articulares

- Condilose - Osteocondrose dissecante - Osteonecrose - Condromatose sinovial - Articulares degenerativas

- Osteoartrite - Osteoartrose

* Disfunções congênitas ou de desenvolvimento

- artrite sistêmica - neoplasia

* Fraturas

Fonte: Adaptado de Leeuw; Klasser (2013).

Em um estudo realizado com 578 adolescentes chineses, a fim de

investigar a prevalência de sintomas de DTM e sua relação com a qualidade do

sono e distúrbios psíquicos, indicou que 61,4% da população estudada

apresentou pelo menos um sintoma de DTM, e que 1/3 dos indivíduos

experimentaram alteração do sono (não-reparador), depressão e estresse, e

ainda que 65,2% sofriam de ansiedade, dando robustez aos estudos que afirmam

a íntima relação entre distúrbios do sono e sofrimento de ordem psíquica com as

DTM (Lei et al., 2016). Sabe-se que condições dolorosas relacionadas à região

orofacial (principalmente a dor crônica) refletem negativamente na qualidade de

vida dos indivíduos afetados, gerando consequências graves como incapacidade

funcional e para o trabalho, prejuízos de ordem social, econômica e afetiva

(SBED, 2012; Greene, 2010).

Preditores sociodemográficos da incidência de DTM foram analisados em

um estudo de coorte prospectivo realizado com a população adulta dos Estados

Unidos (2.737 pessoas), sendo avaliados clinicamente os pacientes que relataram

sintomas. Foi constatado que 3,9% dos participantes desenvolveram DTM, e que

20

persistiram em 61% dos brancos contra 35% dos afro-americanos. Essas

variações podem estar relacionadas ao viés de incidência-prevalência, viés de

seleção que pode ocorrer em desenhos de pesquisas transversais (Slade et al.,

2016). Apesar das divergências encontradas na literatura acerca da prevalência

de DTM, com variações que giram em torno de 5-6% a 12% (De Rossi et al.,

2014), a dor associada à DTM foi relatada em 9-13% da população em geral (com

relação homem:mulher de 2:1), entretanto apenas 4-7% buscam tratamento (4

vezes mais mulheres). Já investigações realizadas por Steven; Kraus (2014) com

um grupo de 511 pessoas (8 hispânicos, 63 afro-americanos, 401 brancos e 39 de

outras etnias) que apresentavam quadro de DTM, com média de idade de 43,9

anos (44,9 entre as mulheres e 43,7 entre os homens), a proporção

homem:mulher foi de 5:1. Ademais, os sinais e sintomas atingem seu pico entre

20-40 anos de idade (Manfredini et al., 2011).

A DTM, portanto, é uma doença complexa que resulta da interação de

causas de domínios genéticos e ambientais (estresse, má qualidade no sono,

tabagismo, hábitos parafuncionais, doenças sistêmicas e etc.) (Slade et al., 2016).

No âmbito genético, pesquisas clínicas realizadas com genes da COMT (catecol-

O-metiltransferase), b2-AR (adrenoreceptor β2) e 5HTT (receptor serotoninérgico)

indicam que polimorfismos são relacionados com o processamento da dor e risco

de desenvolvimento de DTM (Maixner et al., 2011). Adicionalmente, estudos

realizados por Slade et al., 2015 e Slade et. al., 2016 demonstraram que a COMT,

participante da regulação do catabolismo de neurotransmissores de catecol,

possui papel importante na modificação da resposta ao estresse psicológico sobre

a dor, sendo aumentado em pessoas com DTM.

Os mecanismos periféricos envolvidos na dor relacionada às DTM

envolvem estimulação química e mecânica de nociceptores, liberação aumentada

de neuropeptídeos e mediadores inflamatórios como o óxido nítrico (NO),

citocinas, histamina, bradicinina, serotonina (5-hidroxitriptamina), adenosina

trifosfato (ATP), substância p, e eicosanoides, que por sua vez, possuem papel

relevante na artrite da ATM (Cairns, 2010; Chaves et al., 2011; Fusaro et al.,

2010). Já a via central da dor envolve células gliais (microglia e astrocitos),

neurônios nociceptivos do sistema trigeminal e vias aferentes que parecem estar

21

envolvidas na patogenia aguda e crônica de dor orofacial (Chiang et al., 2011;

Chiang et al., 2012).

O caráter multifatorial da DTM dificulta não só o diagnóstico como também

o seu tratamento. Neste sentido, intervenções terapêuticas farmacológicas, com

drogas anti-inflamatórias não-esteroidas (DAINES) tem sido a abordagem, muitas

vezes, de primeira escolha, para o alívio das dores e demais sintomas

associados. Contudo, o tratamento deve priorizar intervenções conservadoras

diante de quadros não-cirúrgicos e que não apresentem degeneração das partes

moles e nem óssea da ATM (Cairns, 2010). Outras modalidades terapêuticas

incluem placas oclusais, fisioterapia, exercícios madibulares, acupuntura,

laserterapia, toxina botulínica, dentre outros (Okeson, 2008; Fernandes et al.,

2009).

Nesse contexto, tem-se revelado promissora a pesquisa com recursos

naturais (como lectinas e polissacarídeos) na descoberta de ferramentas

farmacológicas que possam ser utilizadas para testes de novas substâncias a fim

de reduzir os efeitos pró-inflamatórios das DTM (do Val et al., 2014; Rivanor et al.,

2014; Rodrigues et al., 2014; Freitas et al., 2016).

1.4 Lectinas

A capacidade de se combinar específica e reversivelmente com várias

substâncias é uma característica da maioria das proteínas. Enzimas que se ligam

a seus substratos e inibidores ou anticorpos que se ligam ao antígeno, são

exemplos bem conhecidos (Sharon; Lis, 1989). Lectinas, entretanto, são

proteínas definidas inicialmente como moléculas que se ligam reversivelmente a

carboidratos, aglutinam células e/ou precipitam polissacarídeos e glicoproteínas.

A primeira definição de lectinas foi proposta por Boyd; Shapleigh (1954) que

utilizaram o termo “lectina”, oriundo da palavra latina legere, que significa

selecionar ou escolher, para definir uma classe de aglutininas de plantas

superiores, algumas das quais mostraram especificidade para grupos sangüíneos

humanos.

22

Goldstein et al. (1980) propuseram uma nova definição de lectinas, na qual

estas eram descritas como “proteinas de origem não imune, que se ligam a

carboidratos ou glicoproteínas, aglutinam células e/ou precipitam

glicoconjugados” definição modificada posteriormente por Kocourek; Horejsi

(1981), que sugeriram que lectinas “são proteinas ou glicoproteinas de natureza

não imune que se ligam a carboidratos, sem apresentar atividade enzimática

frente a esses açúcares e não requerem grupos hidroxilas livres para sua

ligação”.

Atualmente, a definição mais aceita para lectinas é a proposta por

Peumans; Van Damme (1995), que definem lectinas como proteínas de origem

não imune contendo pelo menos um domínio não-catalítico capaz de ligar-se

reversivelmente a mono ou oligossacarídeos específicos. Fundamentados no

conhecimento da estrutura das lectinas, Peumans; Van Damme (1995)

classificaram as lectinas em três grupos: merolectinas, hololectinas e

quimerolectinas. As merolectinas possuem um único sítio de ligação a

carboidratos sendo desprovidas de atividade hemaglutinante. As hololectinas são

semelhantes as merolectinas, entretanto, possuem dois ou mais sítios de ligação

a carboidratos podendo, desta forma aglutinar células ou precipitar

glicoconjugados. As quimerolectinas se diferenciam das duas outras classes por

possuírem além do sítio de ligação a carboidratos, um outro domínio não

relacionado que apresenta atividade biológica distinta e independente, podendo

ou não apresentar atividade hemaglutinante, dependendo do número de sítios de

ligação a açúcares.

As sequências de aminoácidos de uma grande quantidade de lectinas já

foram estabelecidas, e em geral as estruturas terciárias e quaternárias

encontradas são extremamente variáveis (Sharon; Lis, 2007), e podem ser

inativadas ou desnaturadas por processos como aumento de temperatura, pH

alterado (em relação ao seu pH ótimo) e tratamento com enzimas proteolíticas

tais como papaína ou tripsina (Gorakshakar; Ghosh, 2016).

A capacidade de reconhecimento entre proteinas e hidratos de carbono se

mostra fundamental em diversos processos biologicos como infecções virais,

bacterianas, a micoplasma e a parasitas e, ainda, na marcação de células e

componentes soluveis, fertilização, metástases cancerigenas e no crescimento e

23

ron, diferenciação celular (Pneumans; Van Damme, 1995; Beuth et al., 1995;

Sharon; Lis, 2004).

As Lectinas são empregadas em estudos diversos, sobretudo nos em que

há a necessidade de detectar, identificar e avaliar a funcionalidade de

carboidratos. Além disso, as lectinas podem ser utilizadas como ferramentas

para detecção antigenos nas células com base na sua estrutura superficial, e

suas interações com células e substâncias solúveis podem ser revertidas por

açúcares simples, sendo essa interação comumente utilizada como indicativo da

existência de hidratos de carbono (Sharon; Lis, 2007; Gorakshakar; Ghosh,

2016).

1.5 Propriedades biológicas de lectinas

Nas últimas décadas, pesquisadores têm voltado sua atenção à utilização

de moléculas de origem vegetal - proteínas e metabólitos secundários - na

perspectiva de avaliar a eficácia e segurança como agentes farmacológicos

(Cairns, 2010). A utilização de lectinas e polissacarídeos isolados de algas

marinhas no tratamento das condições inflamatórias das DTM já foi demonstrada

em estudos pré-clínicos (do Val et al.,Rivanor et al., 2014; Rodrigues et. al.,

2014). Ademais, atividade inflamatória, anti-inflamatória, anti-hipernociceptiva

(Alencar et al., 2007; Assreuy et al., 2009; Rangel et al., 2011; Figueiredo et al.,

2009), e ausência de citoxicidade aguda e crônica na utilização de lectinas

(Sabitha et al., 2011; Kumar et al., 2009) foram constatadas.

Diversas pesquisas têm descrito as atividades biológicas das lectinas,

aumentando o interesse e demonstrando segurança na utilização dessas

proteínas para fins terapêuticos como, por exemplo, na modulação da ligação de

insulina ao receptor de fator de crescimento 1, na síntese de óxido nítrico e até

mesmo na inibição do sistema nervoso central, através do mecanismo

dependente de GABA (Gadelha et al., 2005; Maniskosa et al., 2008; Vasconcelos

et al., 2009). Napimoga et al. (2007) em seus estudos apontaram a diminuição da

migração de leucócitos e da hipernocicepção mecânica por inibição na produção

de citocinas e quimiocinas. Em modelo de edema de pata induzido por

24

carragenina, também foi observada atividade anti-inflamatória de lectinas (Soares

et al., 2012).

Nesse contexto, destaca-se o potencial biológico da lectina de sementes de

Abelmoschus esculentus (AEL), planta originária da África, popularmente

conhecida como quiabeiro, e considerada de alto valor nutritivo (rico em cálcio,

vitaminas A, C e B1) (Panero et al., 2009). A AEL tem sido empregada no

controle glicêmico em pessoas com diabetes mellitus, além de inibir a absorção

de colesterol e reduzir o nível de lipídios e ácidos graxos no sangue (Khosrozadeh

et al., 2016), além de possuir efeito protetor gástrico e antioxidante (Ribeiro et al.,

2016). Adicionalmente, estudos envolvendo a AEL no tratamento da DTM tem

revelado seu potencial como agente anti-inflamatórios (Chaves et al., 2011;

Freitas et al., 2016).

25

2 JUSTIFICATIVA

É inegável que inúmeras conquistas ocorreram no âmbito da saúde bucal,

porém um número muito grande de pessoas sofrem com dor orofacial, e as DTM

estão entre as condições de maior queixa clínica (SBED, 2012). Uma abordagem

terapêutica eficaz pode fazer diferença no tratamento de sintomas como dor

crônica e incapacitante, contudo, o caráter multifatorial que envolve as DTM

dificulta essa escolha.

Portanto, esse cenário de alta prevalência, incidência e dificuldades na

identificação e tratamento que envolvem as DTM têm encorajado a comunidade

científica para o estudo de novas alternativas terapêuticas para o alívio de tais

condições através da utilização de recursos naturais como a AEL, dentre outras

(do Val et al., 2014; Rivanor et al., 2014; Rodrigues et al., 2014; Freitas et al.,

2016). O único registro de estudos utilizando a AEL em modelos de indução de

artrite na ATM de ratos por agentes pro-inflamatórios como zymosam foi

desenvolvido por nosso grupo de pesquisa, trazendo evidências da eficácia dessa

lectina na redução de eventos inflamatórios. Contudo, ainda não foi estudado o

papel anti-inflamatório e antinociceptivo da AEL produzidos na via central da dor

orofacial, tornando importante tal investigação. Ademais, o uso de substâncias

naturais pode se configurar como única forma de tratamento das DTM em muitas

comunidades em todo o mundo.

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3 OBJETIVOS

3.1 Objetivo Geral

Avaliar o uso da AEL como alternativa terapêutica para DTM verificando

sua ação antinociceptiva, anti-inflamatória e sobre receptores opioides em modelo

animal.

3.2 Objetivos Específicos

- Investigar o efeito antinociceptivo e anti-inflamatório promovido pela lectina de

Abelmoschus esculentus no modelo de hipernocicepção induzida por formalina na

ATM de ratos;

- Estudar o papel dos receptores opioides em seu mecanismos de ação no

modelo hipernocicepção inflamatória induzida pela formalina na ATM de ratos;

- Averiguar o envolvimento dos receptores opioides na resposta antinociceptiva

promovido pela lectina de Abelmoschus esculentus no modelo hipernocicepção

inflamatória induzida pela formalina na ATM de ratos.

27

4 CAPÍTULO 1: LECTIN FROM ABELMOSCHUS ESCULENTUS REDUCES

RAT TEMPOROMANDIBULAR JOINT INFLAMMATORY HYPERNOCICEPTION

DEPENDENT FROM CENTRAL OPIOID RECEPTORS

Shirley Moreira Alves1, Raul Sousa Freitas2, Danielle Rocha do Val3, Lorena

Vasconcelos Vieira4, Ellen Lima de Assis4, Carlos Alberto de Almeida Gadelha5,

Tatiane Santi Gadelha5, José Thalles Jocelino Gomes de Lacerda5, Juliana

Trindade Clemente-Napimoga6, Vicente de Paulo Teixeira Pinto7, Mirna Marques

Bezerra1,7 and Hellíada Vasconcelos Chaves1,7.

1Master of Healthy Sciences Degree Program, Federal University of Ceará,

Avenida Comandante Maurocélio Rocha Pontes, 100 Derby - CEP: 62.042-280

Sobral, Ceará, Brazil. [email protected]

2Department of Morphology Federal University of Ceará - UFC, Rua Delmiro de

Farias, s/n - Rodolfo Teófilo, CEP: 60.430-170, Fortaleza, Ceará, Brazil.

[email protected]

3Northeast Biotechnology Network (Renorbio), Federal University of Pernambuco -

UFPE, Avenida Prof. Moraes Rego, 1235 Cidade Universitária CEP: 50670-901,

Recife, Pernambuco, Brazil. [email protected]

4Faculty of Dentistry, Federal University of Ceará - UFC, Avenida Comandante

Maurocélio Rocha Pontes, 100 Derby - CEP: 62.042-280 Sobral, Ceará, Brazil.

[email protected]; [email protected]

5Department of Molecular Biology, Federal University of Paraíba - UFPB, Cidade

Universitária, CEP: 58059-900 João Pessoa, Paraíba, Brazil.

[email protected]; [email protected];

[email protected]

6Faculty of Dentistry, University of Campinas - UNICAMP, Avenida Limeira, 901,

Vila Rezende,CEP 13414-903, Piracicaba, São Paulo, Brazil.

[email protected]

7Faculty of Medicine, Federal University of Ceará - UFC, Avenida Comandante

Maurocélio Rocha Pontes, 100 -Derby - CEP: 62.042-280 Sobral, Ceará, Brazil.

[email protected]; [email protected];

[email protected]

28

*Corresponding author:

Profa. Dra. Hellíada V. Chaves

Faculty of Dentistry of Sobral - Federal University of Ceará

Avenida Comandante Maurocélio Rocha Pontes, 100

Derby - CEP: 62.042-280

Phone: 55 88-3611-2202 - Fax: 55 88-3611- 8000

Sobral - Ceará - Brazil

E-mail: [email protected]

Conflicts of Interest: The authors declare that there is no conflict of interests

regarding the publication of this paper.

Abstract:

Ethnopharmacological relevance: Abelmoschus esculentus is largely cultivated

in Northeastern Brazil for medicinal purposes, like in cases of pneumonia,

bronchitis, pulmonary tuberculosis and inflammation. Aim of the study: To

evaluate the Abelmoschus esculentus (AEL) in reducing formalin-induced

temporomandibular joint inflammatory hypernociception in rats. Materials and

Methods: The behavioral experiments (CEUA nº 02\15) were performed on male

Wistar rats (180–240 g). Rats were pre-treated (i.v.) with AEL (0.001, 0.01 or 0.1

mg/kg) thirty minutes before 1.5% formalin injection in the TMJ. Further, to analyze

the possible effect of opioid pathways on AEL efficacy, animals were pre-treated

via intrathecal injection of naloxone or CTOP (the antagonist of Mu (µ) opioid

receptor), naltrindole (antagonist of Delta (δ) opioid receptor) or Nor-

Binaltorphimine (antagonist of Kappa () opioid receptor) 15 minutes before AEL

followed by intra-TMJ injection of 1.5% formalin. Behavioral analysis were

perfomed, animals were monitored for a 45 min observation period to quantify the

nociceptive response. TMJ tissue, trigeminal ganglion and caudal subnucleus

collection was performed for TNF-α dosage (ELISA). In addition, vascular

permeability was evaluated by Evans Blue extravasation. Results: AEL

significantly reduced formalin-induced TMJ inflammatory hypernociception and

decreased Evans blue extravasation. It also decreased TNF-α levels in TMJ

tissue, trigeminal ganglion and caudal subnucleus. AEL antinociceptive effects,

29

however, were not observed in the presence of naltrindole or Nor-Binaltorphimine.

Conclusions: These findings suggest that AEL efficacy depends on TNF-α

inhibition and the activation of δ and opioid receptors.

Keywords: Abelmoschus esculentus; temporomandibular joint, TNFα,

hypernociception, lectin

1. Introduction

Temporomandibular joint (TMJ) arthritis presents as one of the differential

diagnoses in temporomandibular disorders (TMD), which, in turn, encompasses a

group of musculoskeletal and neuromuscular conditions involving the TMJ,

masticatory muscles and all associated tissues. TMDs are often associated to

acute or persistent pain, and the patients may suffer from other painful disorders.

Chronic forms of TMD may lead to withdrawal or disability at work or to social

activities, resulting to a impairment in the quality of life (Greene, 2010).

A study evaluating the prevalence of TMD symptoms and its relation to

sleep quality and psychic disorders, has shown that 61.4% of the studied

population showed less than one TMD symptom, and that 1/3 of the subjects

experienced altered sleep, depression and stress, and 65.2% had anxiety (Lei et

al., 2016). Socio-demographic predictors indicate a 3.9% incidence of TMD per

year with moderate or disabling pain (Slade et al., 2013). Despite the divergences

found in the literature about the prevalence of TMD (variations between 5%-6%

and 12%) (De Rossi et al., 2014), the pain related to TMD was reported in 9-13%

of the general population on proportion man: woman of 2:1). In addition, signs and

symptoms peaked around 20-40 years old (Manfredini et al., 2011).

TMD is, therefore, a complex disease that results from an interaction of

causes of genetic and environmental domains (Slade et al., 2016). The

multifactorial nature of TMD hinders not only the diagnosis but also its treatment.

In this way, pharmacological therapeutic interventions with non-steroidal anti-

inflammatory drugs (NSAIDs) have been an approach, often the first choice, for

the relief of the pain and associated symptoms.

30

In the recent decades, researchers have been growing interest in

alternative therapies and use of natural products to assessing their efficiency and

safety (Cairns, 2010; Rivanor et al., 2014; Freitas et al., 2016) in order to develop

potential tools for new therapies to ameliorate inflammatory pain, which have

encouraged scientific studies to search for new substances with therapeutic action

and to confirm the efficacy of medicines derived from plants.

Due to the high prevalence, incidence and difficulties in identifying and

treating the inflammatory conditions related to TMD, our group has demonstrated,

through preclinical studies, the use of natural products in the treatment of

inflammatory conditions of TMDs, especially those derived from plants such as

Abelmoschus esculentus lectin (AEL) (Freitas et al., 2016) and Tephrosia toxicaria

(Do Val et al., 2014), as well as lectins and polysaccharides derivated from marine

algae (Rivanor et al., 2014; Rodrigues et al., 2014) Abelmoschus esculentus

(Malvaceae) (popularly called okra) is originated from Africa and has spread

across a number of tropic countries, including northeastern Brazil. This species is

considered of high nutritional value (rich source of calcium and vitamins A, C and

B1), easy cultivation (tropical regions, temperatures between 18 and 35 °C), and

its commercialization has been used for medicinal purposes (treatment of

pneumonia, bronchitis, pulmonary tuberculosis, still acting as a laxative, and

inflammation) (Castro et al., 2008; Panero et al., 2009).

Monte et al. (2014) showed selective anti-tumor effects on human breast

cancer cells, and Soares et al. (2012) showed anti-inflammatory, antinociceptive

and hemagglutinating AEL activities. To our knowledge, the only study which

demonstrates that AEL, a plant-derived lectin, is able to exert antinociceptive and

anti-inflammatory effects on the TMJ pain, especially in the rat TMJ arthritis

models was developed by our group, bringing evidence of the efficacy of this lectin

in the reduction of inflammatory events in TMDs (Freitas et al., 2016). However,

there is still no study about the anti-inflammatory and antinociceptive role played

by AEL on the central and peripheral pathways of orofacial pain. Therefore, the

present study aimed to investigate the unexplored anti-nociceptive and anti-

inflammatory efficacy of AEL in the rat model of formalin-induced

temporomandibular joint inflammatory hypernociception. Additionally, we

31

investigated the role of opiod receptors and the putative involviment of TNF-α in

AEL efficacy.

2. MATERIALS AND METHODS

2.1. Animals

Male Wistar rats (160–220 g) (n=6) were housed in standard plastic cages

with food and water available ad libitum. They were maintained in a temperature

controlled room (23 ± 2 °C) with a 12/12- hour light-dark cycle. All experiments

were designed to minimise animal suffering and to use the minimum number of

animals required to achieve a valid statistical evaluation. The animal supplier for

this study was the Central Animal House of the Federal University of Ceará and

the experimental protocol was conducted in accordance with the Institutional

Animal Care and with the approval of the Ethics Committee of the Federal

University of Ceará, Fortaleza, Brazil (CEUA nº 02\15).

2.2. Source Material

A. esculentus seeds were collected in the municipality of Conde, Paraíba,

Brazil (geographical coordinates: S-7°17'629 "W-34°48'085") for botanical

identification. Professor Rita Balthazar de Lima (Department of Botany, Federal

University of Paraiba - UFPB, Brazil) identified species of the Malvaceae family to

which A. esculentus species belong. The specimen was deposited in the UFPB

herbarium under the identification number of 41,386. Lectin purification was

performed in BioGeR (Laboratory of Biochemical Genetics and Radiobiology).

2.3. Extraction of Lectin

Seeds were grounded to powder and its lipids removed with n-hexane. To

obtain the protein extract, the powder was placed in added in Tris-HCl buffer pH

7.4 with 0.1 M NaCl 0.15 M for 3 h and then centrifuged at 5.000 rpm at 4 °C for

20 min. The resulting precipitate was discarded, and the supernatant was

subjected to ammonium sulfate precipitation, obtaining a lectin fractionwithin the

range of 30–60% saturation. The lectin fraction was dialyzed exhaustively against

water, lyophilized, and then isolated by ion exchange chromatography on DEAE –

Sephacel (Ion exchanger) equilibrated with bibasic sodium phosphate 0.025 M pH

32

7.4. Lectin elution was prepared using the gradient of bibasic sodium phosphate

0.025 M and NaCl pH 7.4 1 M. Elution wasmonitored by spectrophotometer at a

wave length of 280 nm, being it dialyzed against water, frozen and lyophilized.

Furthermore, this lectin under study is endotoxin free which ultimately mean it

does not exert toxic effects on animals under investigation.

2.4. Efficacy of lectin from Abelmoschus esculentus on formalin-induced

temporomandibular joint inflammatory hypernociception in rats

2.4.1. TMJ injection

The animals were briefly anesthetized by inhalation of isoflurane and the

posteroinferior border of the zygomatic arch was palpated. Rats received an intra-

articular injection of 1.5% formalin (Roveroni et al., 2001). The needle was

inserted immediately inferior to this point and was advanced in an anterior

direction until reaching the posterolateral aspect of the condyle. TMJ injections

were performed via a 30-gauge needle introduced to the left TMJ at the moment of

the injection. A cannula consisting of a polyethylene tube was connected to the

needle and also to a 50 μL syringe (Hamilton, Reno, NV). Volume per injection

was 50 μL. Each animal regained consciousness approximately 30s after

discontinuing the anesthetic and was returned to the test chamber.

2.4.2. Experimental design

Thirty minutes before formalin injection rats were pre-treated (0.1 mL/100g

body weight) with AEL 0.001, 0.01 or 0.1 mg/kg) by intravenous (i.v.) injection or

0.9% sterile (sham group) fallowed by intra-TMJ injection of 1.5% formalin in a

final volume of 50 µl as above described. Immediately after the behavioral

analyzes, the animals were anesthetized and euthanized by decapitation, and the

periarticular tissues, trigeminal ganglion and caudal subnucleus were removed

and processed for biochemical analysis.

2.4.3. Behavioral tests for evaluation of nociceptive responses

Each animal was placed in a test chamber (30 X 30 X 30 cm mirrored-wood

chamber with a glass at the front side) for a 15-min habituation period to minimize

stress (Abbott et al., 1986). Each animal was used in only one experiment and

33

was sacrificed at the end of the experiment. Testing sessions took place during the

light phase (between 9:00 AM and 5:00 PM) in a quiet room maintained at 25 °C

and all animals were manipulated for 7 days before the experiment to be

habituated to the experimental manipulation. (Tjolsein, 1992). Each animal

immediately recovered from anesthesia after TMJ injection and was returned to

the test chamber for counting nociceptive responses during the following 45 min

observation period. The nociceptive response score was defined as the cumulative

total number of seconds that the animal spent rubbing the orofacial region

asymmetrically with the ipsilateral fore or hind paw plus the number of head

flinches counted during the observation period as described previously (Roveroni

et al., 2001). Results are expressed as the duration time of nociceptive behavior.

Rats did not have access to food or water during the test.

2.4.4. Collection of biological materials

2.4.4.1. TMJ tissue

The superficial tissues were dissected until reaching the left TMJ then the

TMJ soft tissues were collected. The samples were stored in a freezer -80º C.

2.4.4.2. Trigeminal ganglion

In order to access to the trigeminal ganglion, which is lodged at the base of

the skull in the trigeminal cavus region in the temporal bone, the skullcap and the

brain were removed, and the trigeminal ganglion was carefully identified and

collected. The samples were stored in a freezer -80 ºC.

2.4.4.3. Caudal subnucleus of the spinal tract of the trigeminal nerve

In order to have access to the caudal subnucleus, which is located in the

brainstem, the brain was removed, which was positioned in a specific matrix for

the neural structures of rats. The removal of the caudal subnucleus was performed

by cutting with a scalpel blade positioned 2 mm in the caudal direction to obex.

2.5. Evaluation of anti-inflammatory parameters

2.5.1. Evans blue extravasation measurement

34

In another sequence of experiments, AEL (0.01 mg/kg) was administered to

rats 30 min prior to formalin. Immediately after formalin injection (1.5% i.art) Evans

Blue dye 1% (5mg/kg, i.v.) was administered administered systemically to assess

plasma extravasation (Torres-Chavéz et al., 2012). After 45 minutes, the animals

were euthanized and the ATMs removed for analysis. Immediately after the

extraction, the periarticular tissue was weighed and placed in 1mL of formamide

overnight at 60 ºC (Fiorentino et al., 1999). The supernatant (100 μL) was

extracted, and the absorbance at 620 nm was determined in spectrophotometer.

The concentration was determined by comparison to a standard curve of known

amounts of Evans blue dye in the extraction solution, which was assessed within

the same assay. The amount of Evans blue dye (μg) was then calculated per mg

of exudates.

2.5.2. TMJ periarticular tissue, trigeminal ganglion and caudal subnucleus

TNF-α assay

TNF-α concentrations were determined in the TMJ periarticular tissue,

trigeminal ganglion and caudal subnucleus 45 min after formalin injection in rats

that received 0.01mg/kg AEL or vehicle (0.9% sterile saline). TMJ periarticular

tissue, trigeminal ganglion and caudal subnucleus were removed and stored at

−80 °C. The material was homogenized in a solution of RIPA Lysis Buffer System

(Santa Cruz Biotechnology, USA), and the supernatant was used to determine the

cytokine levels were quantified by the following kit: TNF-α–Rat TNF-

alpha/TNFSF1A Quantikine ELISA Kit (R&D Systems, catalog number RTA00) by

enzyme-linked immunosorbent assay (ELISA). All assays were carried out

according to the manufacturer's instructions. Briefly, microtiter plates were coated

overnight at room temperature (20–23 °C) with an antibody capture against rat

TNF-α (4.0 μg/mL). The plate was blocked by adding of Reagent Diluent to each

well, incubated at room temperature for a minimum of 1 h. After blocking the plate,

the samples and standard at various dilutions were added and incubated at room

temperature for 2 h. The plate was washed three times with buffer and of the

Detection Antibody, was added (100 μL/well). After further incubation at room

temperature for 2 h, the plate was washed and Streptavidin-HRP was added. The

colour reagent (H2O2 and Tetramethylbenzidine; 100 μL/well) was added 15 min

35

later and the plate was incubated in the dark at room temperature for 15–20 min.

The enzyme reaction was stopped with H2SO4 and absorbance was measured at

450 nm. TNF-α concentrations were expressed as pg/ml.

2.6. Evaluation of the involvement of the opioid pathway in the

antinociceptive AEL effect on formalin-induced temporomandibular joint

inflammatory hypernociception

2.6.1. Intrathecal drug administration

Rats were briefly anesthetized by the inhalation of isoflurane, and a small

area of the skin that covers a cervical region was trichotomized with an electric

appliance. The animals were positioned in the ventral decubitus position, in order

to the suboccipital space was easily found. A 30 gauge needle, connected to a 50

μL Hamilton syringe, by a polyethylene cannula, was used for the injection. First

the needle was inserted just below the occipital bone penetrating the skin over the

suboccipital space up to 4 mm deep and then to slightly inclined towards the

cranial. The needle was advanced plus 2 mm to puncture the atlanto-occipital

membrane and reach the bulbar subarachidoid space. This technique allows direct

administration of the drug in the cerebrospinal fluid in the proximity to the

trigeminal caudal subnucleus. The total volume of intrathecal injections was 10 μL

and administered in a rate of 1 μL/s, as previously standardized (Fischer et al.,

2009). Immediately after behavioral analysis, the animals were anesthetized and

euthanized by decapitation. The administered drugs were diluted in sterile saline

solution (0.9%).

2.6.2. Effect of the nonselective opioid receptor antagonist naloxone on AEL-

induced antinociception

Rats were pretreated (15 min) with an intrathecal injection of naloxone (Nlx;

15 µg/10 µl/intrathecal / n= 5; Eisenberg et al., 1996) followed by AEL (0.01

mg/kg/i.v.) 30 min prior 1.5 % formalin intra-TMJ injection (50 µl /TMJ). Behavioral

nociception response was evaluated for 45 min observation period. All animals

received a final volume of 50 µl of solutions into TMJ.

36

2.6.3. Effect of µ, δ and -opioid receptors on AEL-induced antinociception

Rats were divided in groups of five animals, and each group was pretreated (15 min) with

an intrathecal injection of a specific inhibitor of µ-opioid receptor CTOP (10 µg/10 µl

/intrathecal) the inhibitor of δ -opioid receptor Naltrindole (10 or 30 µg/10 µl/intrathecal)

or the selective -opioid receptor antagonist Nor-BNI (15 or 45 µg/10 µl/intrathecal);

followed by AEL (0.01 mg/kg /i.v.) 30 min prior 1.5% formalin intra-TMJ injection (50 µl

/TMJ). Behavioral nociception response was evaluated for 45 min period observation. All

animals received a final volume of 50 µl of solutions into TMJ (Picolo; Cury, 2004;

Clemente et al., 2004).

2.7. Statistical analysis

The data are presented in figures and text as the means±SEM. The number

(n) of animals per experimental group was at least 5. Differences between means

were compared using a one-way ANOVA followed by the Bonferroni test. A

probability level of less than 0.05 (P<0.05) was considered to indicate statistical

significance.

3. Results

3.1. AEL reduces formalin-induced temporomandibular joint inflammatory

hypernociception in rats

The formalin 1.5% injection (i.art.) resulted in a significant increase in the

behavioral nociceptive response compared to the saline group. Treatment with

AEL (0.001, 0.01 and 0.1 mg/kg; i.v.) significantly (p<0.05) reduced the behavioral

nociceptive response induced by the injection of formalin 1.5% into the TMJ when

compared to the formalin group (Fig. 1). Considering this result, the followed

experiments, in order to elucidate the mechanisms of action of this substance,

were performed using AEL 0.01 mg/kg.

37

Saline _ Mor 0.001 0.01 0.10

100

200

300

*

#

# #

Formalin 1.5% (50 L/art.)

AEL (mg/kg)

#

**

No

cice

pti

ve b

ehav

ior

(s)

Fig.1 Effects of AEL on formalin-induced temporomandibular joint inflammatory hypernociception in rats Pretreatment with AEL (0.001, 0.01 and 0.1 mg/kg; i.v.) and morphine (5 mg/kg; s.c.) reduced the the behavioral nociceptive response induced by the injection of formalin 1.5% (i.art.; 50μl). Saline (48.40 ± 2.24), formalin (246.0 ± 5.73), morphine (5.6 ± 3.09), AEL 0.001 mg/kg (100.5 ± 10.22), AEL 0.01 mg/Kg (70.13 ± 6.18) and AEL 0.1 mg/kg (83 ± 5.95). *p <0.05 compared to saline group; #p <0.05 compared to the formalin 1.5% group (ANOVA, Bonferroni). 3.2. AEL reduces Evans blue extravasation measurement on formalin-

induced TMJ inflammatory hypernociception in rats

The formalin 1.5% injection (i.art.) resulted in a significant increase in Evans

blue dye extravasation measurement in comparison with the saline group.

Pretreatment with AEL (0.01 mg/kg, i.v.) decreased (p<0.05) Evans Blue dye

extravasation compared with the formalin group (Fig. 2).

Saline _ AEL (0.01 mg/kg)0

10

20

30

40

50

Formalin 1.5% (50L/art.)

*

#

Evan

s b

lue (

µg

/mg

)

Fig. 2 Effects of AEL on Evans blue extravasation measurement on formalin-induced TMJ inflammatory hypernociception in rats Pretreatment with AEL 0.01 mg / kg (i.v.) significantly decreased the Evans blue extravasation measurement induced by the injection of formalin 1.5% compared with formalin

38

group. Saline (18.88 ± 4, 29), formalin (42.33 ± 5.13) and AEL 0.01 mg/kg (12.01 ± 1.14). *p <0.05 compared to saline group; #p <0.05 compared to the formalin group 1.5% (ANOVA, Bonferroni). 3.3. AEL decreases TNF-α levels in TMJ tissue, trigeminal ganglion and caudal subnucleus on formalin-induced TMJ inflammatory hypernociception in rats The formalin 1.5% injection (i.art.) resulted in a significant increase in TNF-α

levels in TMJ tissue, trigeminal ganglion and caudal subnucleus compared with

saline group. AEL 0.01 mg/kg (i.v.) also reduced TNF-α in TMJ tissue, trigeminal

ganglion and caudal subnucleus in comparison with formalin group.

Fig. 3 Effects of AEL on TNF-α levels in the TMJ periarticular tissue (A), trigeminal ganglion (B) and caudal subnucleus (C) on formalin-induced TMJ inflammatory hypernociception Pretreatment with AEL (0.01 mg/kg, i.v.)

Saline _ AEL 0.01 mg/kg0

50

100

150

200

250

300


*

#

A

TN

F-

(p

g/m

L T

MJ t

issu

e)

Saline _ AEL 0.01 mg/kg0

50

100

150

*

#

B


TN

F-

(p

g/m

L t

rig

em

inal

gan

gli

on

)

Saline _ AEL 0.01 mg/kg

0

50

100

150

200

250

*

#

C


TN

F-

(p

g/m

L c

au

dal

su

bn

ucle

us)

39

reduced levels of TNF-α in the TMJ tissue (Figure A), saline (61.29 ± 7.19), formalin (227.5 ± 15.5), and AEL 0.01 mg/Kg (131.8 ± 7.70); In the trigeminal ganglion (Figure B), saline (81 ± 4,16), formalin (113.8 ± 2.62), and AEL 0.01 mg/kg (84. 67 ± 0.88), and in the caudal subnucleus (Figure C), saline (97 ± 21.01), formalin (188.5 ± 27.18) and AEL 0.01 mg/kg (109.4 ± 10.51). * P <0.05 compared to saline group; #p <0.05 compared to the formalin group 1.5% (ANOVA, Bonferroni). 3.4 AEL inhibits formalin-induced temporomandibular joint inflammatory hypernociception through central opioids receptor activation In order to investigate whether the antinociceptive effect of AEL depends on

the central opioid activation, was tested the effect of the pretreatment with

naloxone, a non selective opioid receptor antagonist or the selective µ-opioid

receptor CTOP, δ-opioid receptor Naltrindole or the selective -opioid receptor

antagonist Nor-BNI 15 min before AEL treatment. The intrathecal administration of

naloxone (15 μl/10μl) significantly reversed (p <0.05) the antinociceptive effect of

AEL (0.01 mg/kg). This result suggests that the antinociceptive effect of AEL

depends on the central opioid receptors activation (Fig. 4A).

The Intrathecal administration of naltridole, a selective opioid receptor

antagonist delta (δ) (10 or 30 ug/10 uL), significantly reversed (p<0.05) the

antinociceptive effect of AEL (0.01 mg/kg). This result suggests that the

antinociceptive effect of AEL depends on the activation of the δ opioid receptor

(Figure 4B). The intrathecal administration of nor-binaltorfimine, a selective opioid

receptor antagonist kappa (ĸ) (15 or 45ug/10uL) 15 minutes prior AEL treatment

significantly abolishes (p<0.05) the antinociceptive effect of AEL 0.01 mg/Kg. This

fiding may suggests that the antinociceptive effect of AEL also depends on the

activation of the opioid receptor ĸ (Figure 4C). The intrathecal administration of

CTOP, a selective opioid receptor antagonist mu (μ) (10ug/10uL) 15 minutes

before intravenous injection of AEL does not reverse the antinociceptive effect of

AEL 0.01 mg/Kg what may suggests that the antinociceptive effect of AEL does

not depend on mu opioid receptor activation (μ) (Figure 4D).

40

Fig.4 Effect of the non selective (naloxone) and selective (μ, ĸ and δ) opioid receptor antagonists on the AEL antinociceptive efficacy on formalin-induced TMJ inflammatory hypernociception Naloxone (15ug/10uL/intrathecal) reversed the antinociceptive effect of AEL (0.01 mg/kg) (Figure A); Saline (48.4 ± 2.24), formalin (211.7 ± 7.82), morphine + naloxone (147.2 ± 4.60), AEL (0.01 mg/kg) (92.8 ± 15.66) and AEL (0.01 mg/kg) + naloxone (160 ± 8.51). Naltrindole (10 or 30ug/10uL) the delta (δ) opioid receptor antagonist reversed the antinociceptive effect of AEL (0.01 mg/kg) (Figure B); Saline (48.4 ± 2.24), formalin (211.7 ± 7.82), AEL (0.01 mg/kg) (82 ± 14.63), naltrindole 10 ug/10 uL + AEL (0.01 mg/Kg) (109.2 ± 5.59), naltrindole 30 ug/10 uL + AEL (0.01 mg/kg) (139.8 ± 7.34) and naltrindole 30 μg/10μl (203.8 ± 5,92). Nor-binaltorfimine (15 or 45μg/10μl) the opioid receptor antagonist kappa (ĸ) reversed the antinociceptive effect of AEL (0.01 mg/kg) (Figure C); Saline (47.57 ± 1.73), formalin (211.2 ± 8.36), AEL (0.01 mg/kg) (88.4 ± 8.88), nor-binaltorfimine 15μg/10μl + 1 mg/kg) (159.9 ± 12.34), nor-binaltorfimine 45μg/10μl + AEL (0.01 mg/kg) (174.5 ± 7.98) and nor-binaltorfimine (45μg/10μl) (235.2 ± 21.31). CTOP 10 (μg/10μl) the Opioid receptor antagonist mu (μ) did not reverse the antinociceptive effect of AEL (0.01 mg/kg) (Figure D); Saline (48.40 ± 2.24), formalin (165 ± 25.09), AEL (0.01 mg/kg) (71.4 ± 9.75), CTOP 10 μg/10μl + AEL (0.01 mg/Kg) (51.5 ± 14.82), CTOP 10μg/10μl (203.8 ± 5.92). * P <0.05 compared to saline group; #P <0.05 compared

Salina Nal Nal 0

100

200

300


*

#

+

AEL (0,01mg/kg)Morphine

#

&

A

No

cic

ep

tive b

eh

avio

r (s

)

Saline 10 30 300

100

200

300

AEL (0,01 mg/kg)

Naltrindole (µg/10 µL)


*

#

+

B

+

No

cic

ep

tive b

eh

avio

r (s

)

Salina 15 45 450

100

200

300

AEL (0,01 mg/kg)

Binaltorphirmine (µg/10 µL)


*

#

++

C

No

cic

ep

tive b

eh

avio

r (s

)

Saline 10 100

100

200

300

AEL (0,01 mg/kg)

CTOP (µg/10 µL)


*

#

D

No

cic

ep

tive b

eh

avio

r (s

)

41

to the formalin group 1.5%; P<0.05 compared to the morphine group and +P<0.05 compared to the AEL 0.01 mg/kg group (ANOVA, Bonferroni).

4. Discussion

We demonstrated the antinociceptive and anti-inflammatory effect of lectin

of Abelmoschus esculentus on formalin-induced temporomandibular joint

inflammatory hypernociception in rats and that its effects are mediated via central,

through opioid receptors activation. AEL effects depended in part on reduction of

inflammatory parameters, such as TNF-α levels, as there was a reduction of these

cytokines concentration in the TMJ tissue, trigeminal ganglion and caudal

subnucleus. Regarding inflammatory parameters, AEL administration also

decreased plasma extravasation in synovial exudates compared with formalin

group, being this parameter determined by Evans blue dye extravasation.

Alencar et al. (2007) demonstrated the effect of Vatairea macrocarpa lectin

on macrocytic activation and chemotaxis of inflammatory mediators. Assreuy et al.

(2009) demonstrated the relevance of vasodilation caused by diocletian lectin

(Canavalia genus) in the inflammatory process. The antinociceptive effect of

dioclenáceas lectins in the contortion model caused by acetic acid, in turn, was

demonstrated by Rangel et al. (2011). The Bolivian Canavalia lectin and its

antinociceptive effect, besides its toxicity, were studied by Figueiredo et al. (2009).

Additionally, our group demonstrated the anti-inflammatory and anti-nociceptive

effect of Caulerpa cupressóides lectin (CcL) on zymosam-induced TMJ arthritis in

rats (Rivanor et al., 2014). However, not only anti-inflammatory effects, are

demonstrated in plant lectins. Bauhinia bauhinioides and Dioclea wilsonii lectins

present proinflammatory effects by activation of proteolytic enzymes and induction

of neutrophil migration, respectively (Silva et al., 2011; Rangel et al., 2011).

The study of the properties of the lectin of Abelmoschus esculentus has

great relevance, since this species has been used to treat a variety of disorders,

such as microbial infection, hypoglycemia, constipation, urinary retention and

inflammation (Gürbüz et al., 2002; Kumar et al., 2009). Regarding the anti-

inflammatory effects, AEL was investigated in an experimental model of acute

inflammation, in which it showed an inhibitory effect on carrageenan-induced paw

edema but not on dextran-induced edema. This data suggests that the anti-

42

inflammatory effect of AEL occurs only in edema involving cell infiltrate, since

edema caused by carrageenin is due to intense neutrophil infiltration and it is

associated with the release of inflammatory mediators whereas dextran-induced

edema involves histamine, Serotonin and bradykinin (Masnikosa et al., 2008).

Similar results were found in other leguminous lectins (Assreuy et al., 2007).

Soares et al. 2012, in turn, demonstrated the anti-nociceptive effect of AEL, in the

same three doses tested in this work, on the model of the contortions induced by

acetic acid.

Recently, our group demonstrated the antinociceptive and anti-inflammatory

effect of AEL on zymosan-induced TMJ hypernociception in rats, we found

evidences that, at least in part, the antinociceptive and anti-inflammatory effects of

the AEL depend on the integrity of the HO-1 pathway, corroborating with other

works that show that the inhibition of the HO-1 pathway is associated with the

worsening of the inflammatory response (Freitas et al., 2016; Vicente et al., 2003).

The experimental use of formalin as a pro-inflammatory agent in rats, a

model used in this study, is considered quite representative of the pain clinically

observed in humans, and the similarity between clinical and experimental results

suggests that the formalin test in rat TMJ is an effective model for assessing the

mechanisms involved in TMD dysfunctions (Tjolsein et al., 1992; Roveroni et al.,

2001; Clemente et al., 2004). In addition to the nociceptive effects caused by

formalin, this substance also causes a local edema and plasma extravasation

induced directly and indirectly. Formalin promotes vascular effects by different

mechanisms that in common cause the stimulation of non-neuronal and neuronal

cells. In response to stimulation, both cells release inflammatory substances

causing intense edema and local plasma extravasation (Torres-Chávez et al.,

2012).

TNF-α, the cytokine investigated in this work, has a detrimental effect on

bone and cartilage (Gunson et al., 2012). In addition, a positive correlation was

found between cytokines in the synovial fluid and osteoarthritis. It has been

suggested that the presence of IL-1β and TNF-α in the synovial fluid of the TMJ

may affect the treatment outcome in patients with osteoarthritis (Hamada et al.,

2008). High TNF-α levels were found in symptomatic TMJs when compared to

normal joints (Shafer et al., 1994; Nordahl et al., 2000; Emshoff et al., 2000). The

43

anti-inflammatory effect of AEL was also observed by decreasing the plasma

extravasation and reducing of TNF-α levels in periarticular tissue, trigeminal

ganglion and caudal subnucleus.

We also demonstrated that the central antinociceptive response mediated

by AEL in the TMJ hypernociception results from the activation of the δ and

receptors, but not of μ opioid receptor. In addition, its anti-inflammatory effects

may also be related to opioid receptors. Nũnéz et al. (2007) provided the genetic,

proteomic and behavioral evidence for the involvement of peripheral opioid

receptors in relieving inflammatory pain from craniofacial muscle tissues and

suggested that all three subtypes of opioid receptors are involved in inflammatory

responses. Napimoga et al. (2007) demonstrated that the antinociceptive effects in

peripheral hypernociception of 15d-PGJ2, peroxisome proliferator-activated

endogenous protein (PPAR-ᵧ), recognized as a potent anti-inflammatory mediator,

promotes peripheral analgesia by endogenous opioid stimulation, suggesting that

this protein Can directly activate opioid receptors present in primary sensory

neurons. In addition, PPAR-ᵧ may stimulate the release of opioids that act to

control inflammatory pain by resident macrophages, supporting the understanding

that opioid receptors may be involved in the inflammatory response of orofacial

pain.

Pena-dos-Santos et al. (2009) showed that δ/ opioid receptors mediate

antinociception at the temporomandibular joint in rats, as also we demonstrated in

this study. Chicre-Alcântara et al. (2012) have provided evidence that the

activation of kappa opioid receptors located in the TMJ region reduces two

important parameters of inflammation, such as plasma extravasation and

neutrophil migration. Intra-articular administration of the selective kappa agonist

blocks plasma protein extravasation and neutrophil migration induced by formalin

in a dose-dependent way. Additionally, studies performed by Cunha et al. (2012)

evidenced that the peripheral activation of the kappa opioid receptor directly

blocks the inflammatory hyperalgesia induced by PGE2. The non-participation of

the μ opioid receptor in the induction of central antinociceptive response mediated

by AEL in the TMJ hypernociception can be explained by the fact that the opioid

receptors are sensitized in different ways.

44

The activation of μ receptor is related to the increasement of GRK

expression, a kinase coupled to a G-protein receptor, and the activation of beta-

arrestin, the protein responsible for the desensitization of receptors coupled to a

G-protein (Zang et al., 1998; Raehal et al., 2005). In other words, it is required a

higher phosphorylation GRK-mediated to activate the μ receptor than what is

required for δ and receptors, which suggests that the expression of this protein

in different cells and tissues may lead to distinct antinociceptive responses. In

addition, the action of morphine on the μ receptor, the opioid agent used in this

study, differs from other opioid agonists such as etorphine. It is noteworthy that

both morphine and etorphine effectively activate the μ receptor, but morphine is

not able to stimulate μ receptor phosphorylation by GRK in certain cell types,

indicating substantial differences in the agonist sites binding of this receptor (Zang

et al., 1998). Several studies have been carried out in order to elucidate the

molecular bases of the μ opioid receptor and to discover new ligands with

chemotypes for coupling of this receptor, such as the compound PZM21, a

selective and potent agonist of μ, However, it promotes analgesia only to the

affective component of the pain, what means it is more specific for reflexive spinal

responses (Manglik et al., 2016).

5. Conclusion

In conclusion, we demosntrated the antinociceptive and anti-inflammatory

activity of AEL in a model of formalin-induced TMJ inflammatory hypernociception

in rats. Additionally, our results strongly suggest that AEL efficacy involves TNF-α

inhibition and the activation of the δ and , but not of μ opioid receptor. Given the

well-demonstrated anti-nociceptive and anti-inflammatory efficacy of AEL, the

design of novel compounds is highly encouraged with the hope of defining new

pharmacological targets for the treatment of inflammatory TMJ pain.

6. Acknowledgments

This work was supported by Brazilian grants from Fundação Cearense de

Apoio ao Desenvolvimento Científico e Tecnológico (FUNCAP), Conselho

Nacional de Pesquisa (CNPq), Coordenação de Aperfeiçoamento de Pessoal de

45

Nível Superior (CAPES) and Instituto de Biomedicina do Semi-Árido Brasileiro

(INCT-IBSAB).

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APÊNDICE

Resumo Gráfico Exigido pelo Periódico Journal of Ethnopharmacology

59

ANEXO A

Declaração de Aceite do Comitê de Ética

60

ANEXO B

Normas da Revista Journal of Ethnopharmacology

Article structure

Subdivision - numbered sections

Divide your article into clearly defined and numbered sections. Subsections should

be numbered 1.1 (then 1.1.1, 1.1.2,), 1.2, etc. (the abstract is not included in

section numbering). Use this numbering also for internal cross-referencing: do not

just refer to 'the text'. Any subsection may be given a brief heading. Each heading

should appear on its own separate line.

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State the objectives of the work and provide an adequate background, avoiding a

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Calculation section represents a practical development from a theoretical basis.

Results

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Discussion

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combined Results and Discussion section is often appropriate. Avoid extensive

citations and discussion of published literature.

61

Conclusions

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If there is more than one appendix, they should be identified as A, B, etc.

Formulae and equations in appendices should be given separate numbering: Eq.

(A.1), Eq. (A.2), etc.; in a subsequent appendix, Eq. (B.1) and so on. Similarly for

tables and figures: Table A.1; Fig. A.1, etc.

Essential title page information Title

Concise and informative. Titles are often used in information-retrieval systems.

Avoid abbreviations and formulae where possible. • Author names and

affiliations. Please clearly indicate the given name(s) and family name(s) of each

author and check that all names are accurately spelled. Present the authors'

affiliation addresses (where the actual work was done) below the names. Indicate

all affiliations with a lower-case superscript letter immediately after the author's

name and in front of the appropriate address. Provide the full postal address of

each affiliation, including the country name and, if available, the e-mail address of

each author. • Corresponding author. Clearly indicate who will handle

correspondence at all stages of refereeing and publication, also post-publication.

Ensure that the e-mail address is given and that contact details are kept up

to date by the corresponding author. • Present/permanent address. If an

author has moved since the work described in the article was done, or was visiting

at the time, a 'Present address' (or 'Permanent address') may be indicated as a

footnote to that author's name. The address at which the author actually did the

work must be retained as the main, affiliation address. Superscript Arabic

numerals are used for such footnotes.

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Abstract

A concise and factual abstract is required. The abstract should state briefly the

purpose of the research, the principal results and major conclusions. An abstract is

often presented separately from the article, so it must be able to stand alone. For

this reason, References should be avoided, but if essential, then cite the author(s)

and year(s). Also, non-standard or uncommon abbreviations should be avoided,

but if essential they must be defined at their first mention in the abstract itself. The

author should divide the abstract with the headings Ethnopharmacological

relevance, Aim of the study, Materials and Methods, Results, and

Conclusions.

Graphical abstract

A Graphical abstract is mandatory for this journal. It should summarize the

contents of the article in a concise, pictorial form designed to capture the attention

of a wide readership online. Authors must provide images that clearly represent

the work described in the article. Graphical abstracts should be submitted as a

separate file in the online submission system. Image size: please provide an

image with a minimum of 531 × 1328 pixels (h × w) or proportionally more. The

image should be readable at a size of 5 × 13 cm using a regular screen resolution

of 96 dpi. Preferred file types: TIFF, EPS, PDF or MS Office files. You can view on

our information site. Authors can make use of Elsevier's Illustration and

Enhancement service to ensure the best presentation of their images also in

accordance with all technical requirements:

Keywords

After having selected a classification in the submission system, authors must in

the same step select 5 keywords. These keywords will help the Editors to

categorize your article accurately and process it more quickly. A list of the

classifications and set keywords can be found. In addition, you can provide a

maximum of 6 specific keywords, using American spelling and avoiding general

and plural terms and multiple concepts (avoid, for example, "and", "of"). Be sparing

with abbreviations: only abbreviations firmly established in the field may be

eligible. These keywords will be used for indexing purposes.

63

Chemical compounds

You can enrich your article by providing a list of chemical compounds studied in

the article. The list of compounds will be used to extract relevant information from

the NCBI PubChem Compound database and display it next to the online version

of the article on ScienceDirect. You can include up to 10 names of chemical

compounds in the article. For each compound, please provide the of the most

relevant record as in the following example: Glutamic acid (PubChem CID:611).

Please position the list of compounds immediately below the 'Keywords' section. It

is strongly recommended to follow the exact text formatting as in the example

below: Chemical compounds studied in this article Ethylene glycol (PubChem CID:

174); Plitidepsin (PubChem CID: 44152164); Benzalkonium chloride (PubChem

CID: 15865).

Plant names

In the Materials and Methods section there must be a separate heading for

describing the material used. That includes official name, local name, English

name (if known), GPS position in case of collection in the wild or cultivation, a

voucher specimen must be deposited in an official herbarium for possible future

comparison. In the text it should be stated that the plant name has been checked

with mentioning the data of accessing that website. In case of commercially

procured material should mention the source, batch number, quality control data.

Data on chemical characterization (metabolomics, chromatographic methods)

should also be presented, in case of known active compounds their quantitative

analysis should be presented.

Acknowledgements

Collate acknowledgements in a separate section at the end of the article before

the references and do not, therefore, include them on the title page, as a footnote

to the title or otherwise. List here those individuals who provided help during the

research (e.g., providing language help, writing assistance or proof reading the

article, etc.).

Formatting of funding sources

List funding sources in this standard way to facilitate compliance to funder's

64

requirements: Funding: This work was supported by the National Institutes of

Health [grant numbers xxxx, yyyy]; the Bill & Melinda Gates Foundation, Seattle,

WA [grant number zzzz]; and the United States Institutes of Peace [grant number

aaaa].

It is not necessary to include detailed descriptions on the program or type of grants

and awards. When funding is from a block grant or other resources available to a

university, college, or other research institution, submit the name of the institute or

organization that provided the funding.

If no funding has been provided for the research, please include the following

sentence: This research did not receive any specific grant from funding agencies

in the public, commercial, or not-for-profit sectors.

Math formulae

Please submit math equations as editable text and not as images. Present simple

formulae in line with normal text where possible and use the solidus (/) instead of a

horizontal line for small fractional terms, e.g., X/Y. In principle, variables are to be

presented in italics. Powers of e are often more conveniently denoted by exp.

Number consecutively any equations that have to be displayed separately from the

text (if referred to explicitly in the text).

Footnotes

Footnotes should be used sparingly. Number them consecutively throughout the

article. Many word processors can build footnotes into the text, and this feature

may be used. Otherwise, please indicate the position of footnotes in the text and

list the footnotes themselves separately at the end of the article. Do not include

footnotes in the Reference list.

References Citation in text

Please ensure that every reference cited in the text is also present in the reference

list (and vice versa). Any references cited in the abstract must be given in full.

Unpublished results and personal communications are not recommended in the

reference list, but may be mentioned in the text. If these references are included in

the reference list they should follow the standard reference style of the journal and

65

should include a substitution of the publication date with "Unpublished results".

"Personal communication" will not be accepted as a reference. Citation of a

reference as "in press" implies that the item has been accepted for publication.

Reference style

Text: All citations in the text should refer to:

1. Single author: the author's name (without initials, unless there is ambiguity) and

the year of publication;

2. Two authors: both authors' names and the year of publication;

3. Three or more authors: first author's name followed by 'et al.' and the year of

publication.

Citations may be made directly (or parenthetically). Groups of references should

be listed first alphabetically, then chronologically.

Examples: 'as demonstrated (Allan, 2000a, 2000b, 1999; Allan and Jones, 1999).

Kramer et al. (2010) have recently shown .…' List: References should be arranged

first alphabetically and then further sorted chronologically if necessary. More than

one reference from the same author(s) in the same year must be identified by the

letters 'a', 'b', 'c', etc., placed after the year of publication.

Examples:

Reference to a journal publication: Van der Geer, J., Hanraads, J.A.J., Lupton,

R.A., 2010. The art of writing a scientific article. J. Sci. Commun. 163, 51–59.

Reference to a book: Strunk Jr., W., White, E.B., 2000. The Elements of Style,

fourth ed. Longman, New York.

Reference to a chapter in an edited book: Mettam, G.R., Adams, L.B., 2009. How

to prepare an electronic version of your article, in: Jones, B.S., Smith, R.Z. (Eds.),

Introduction to the Electronic Age. E-Publishing Inc., New York, pp. 281–304.

Reference to a website:Cancer Research UK, 1975. Cancer statistics reports for

the UK. (accessed 11.11.16).

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