Mecanismos de ação envolvidos na atividade antinociceptiva e anti-inflamatória da (1- 3)(1_6) B-glucana isolada do Pleurotus Pulmonarius (Fr.)Quel

Orientadora: Prof. Dr. Adair Roberto Soares dos SantosCo-Orientadora : Profª Drª Maria Consuelo Andrade MarquesTese (doutorado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Farmacologia. Defesa: Curitiba, 06/04/2010Bibliografia: fls. 99-120Resumo: A b-glucana e um polissacarideo isolada do Pleurotus pulmonarius (Fr.) Quel., um cogumelo comestivel conhecido no Japao como gusuhiratake h. Uma variedade de efeitos biologicos tem sido atribuida a essas glucanas, como propriedades antitumoral, antioxidante, anti-inflamatoria e imunomodulatoria. O presente estudo avaliou a atividade antinociceptiva e anti-inflamatoria da (1 ¨3),(1 ¨6) b-glucana (GL), verificando seus possiveis mecanismos de acao. O GL administrado pela via oral (v.o.) e intraperitoneal (i.p.) reduziu de forma dependente da dose a nocicepcao induzida pelo acido acetico. Alem disso, o GL (i.p.) tambem reduziu a migracao de celulas e extravasamento plasmatico apos a injecao de acido acetico. A nocicepcao induzida pela formalina tambem foi diminuida com a administracao do GL (i.p.). O tratamento com o GL reduziu a migracao de leucocitos, neutrofilos e mononucleares induzidos pela carragenina, alem de aumentar os niveis de IL-10. Entretanto, a antinocicepcao provocada pela GL nao foi afetada pelo tratamento dos animais com o antagonista opioide (naloxona) e nem foi associada a uma acao sedativa ou relaxante muscular, observado no teste do campo aberto. A antinocicepcao causada pelo GL no teste do acido acetico foi parcialmente revertida pelo pre-tratamento dos animais com toxina pertussis (inativador da proteina Gi/o). O tratamento (i.p.) dos animais com o GL tambem preveniram a nocicepcao induzida pela injecao intraplantar (i.pl.) de capsaicina, cinamaldeido, mentol e salina acida, mas nao quando co-administrado na pata com esses agonistas. Alem disso, foi demonstrado que as fibras sensoriais do tipo C e AƒÂ sensiveis a capsaicina estao parcialmente envolvidas na atividade antinociceptiva promovida pela glucana. A nocicepcao induzida pelo glutamato i.pl., bem como, a nocicepcao induzida pela injecao intratecal (i.t.) de AEE para receptores ionotropicos e da citocina pro-inflamatoria, IL-1b foram reduzidas com a administracao do GL. O GL foi efetivo em inibir tanto a nocicepcao quanto a ativacao da PKCƒÃ (atraves da analise de Western blot) induzida pela injecao i.pl. de PMA (ativador da PKC). O tratamento dos animais com o GL tambem diminuiu a imunorreatividade para GFAP de astrocitos da medula espinhal apos a inducao da nocicepcao pela formalina. O GL inibiu a alodinia mecanica induzida pela constricao do nervo ciatico em camundongos. Coletivamente, o presente trabalho demonstrou que o GL possui um interessante efeito antinociceptivo quando avaliada em varios modelos de nocicepcao aguda quimica e cronica em camundongos. Alem disso, o GL apresentou marcada acao anti-inflamatoria no modelo de peritonite induzida por carragenina. Os mecanismos de acao antinociceptiva e anti-inflamatoria do GL ainda estao sob investigacao, contudo, neste trabalho pode-se demonstrar que a inibicao da PKC e da ativacao astrocitaria e o aumento dos niveis de IL-10 estao envolvidos na acao antinociceptiva e anti-inflamatoria do GL.Abstract: b-glucan is a polysaccharide isolated from Pleurotus pulmonarius (Fr.) Quel., an edible mushroom known as gusuhiratake h in Japan. A variety of biological effects have been ascribed to b-glucans, namely antitumoral, antioxidant, antiinflammatory and immunomodulatory properties. The present study assessed the possible antinociceptive and anti-inflammatory action of the (1 ¨3),(1 ¨6) b- glucana (GL), evaluating its possible mechanisms of action. The oral (p.o.) and intraperitoneal (i.p.) administration of GL reduced in a dose-dependent manner the nociception induced by acetic acid. Besides, GL (i.p.) also reduced the cellular migration and plasmatic extravasation after acetic acid injection. The formalininduced nociception was inhibited by GL administration (i.p.). GL treatment reduced the leukocyte migration, neutrophils and mononuclears induced by carrageenan, and increased the IL-10 levels. However, the antinociception caused by the GL was not affected by intraperitoneal treatment of mice with naloxone (a non-selective opioid receptor antagonist) and was not associated with nonspecific effects such as muscle relaxation or sedation, observed in the open-field test. The antinociception caused by GL in the acetic acid test was partially reverted by Gi/o protein inactivation (pertussis toxin treatment). I.p. treatment of animals with GL also prevented the nociception induced by intraplantar (i.pl.) injection of capsaicin, cinnamaldehyde, menthol and acidified saline, but not when these agonists were co-administered with GL. Therefore, the capsaicin-sensitive C-fibers seem partially play a critical role in the antinociception caused by the glucan. The nociception induced by glutamate (i.pl.) and by NMDA, AMPA, kainate and IL-1ƒÀ intrathecal (i.t.) injections were reduced GL administration. the administration of GL reduced the nociception induced by i.pl. injection of PMA. Western blot analysis revealed that GL treatment fully prevented PKCƒÃ activation by PMA in mice hindpaw. The treatment with GL also reduced the GFAP immunoreactivity of astrocytes from spinal cord after the formalin-induced nociception. Finally, the GL also reduced mechanical allodynia evaluated by von Frey monofilament fibers, induced by partial sciatic nerve injury neuropathy. Collectively, this present study showed that GL has an interesting antinociceptive and anti-inflammatory effect. The precise mechanisms through which GL exerts its action are currently under investigation, but the present study showed that the inhibition of PKC and astrocytes activation and the increasing IL-10 levels are involved in the antinociceptive and antiinflammatory action of GL

Plantas medicinais utilizadas pela Pastoral da criança de Almirante Tamandaré - Paraná - BR

Este trabalho está licenciado com a atribuição CC by-nc-ndUso adequado de Plantas Medicinais pela população assistida pela Pastoral da Criança de Almirante Tamandaré – PR coordenado por Maria Consuelo A. Marques no período de 2002-2005.Departamento de Farmacologia e Departamento de Botânica. Setor de Ciências Biológicas

Involvement of Interleukin-10 in the Anti-Inflammatory Effect of Sanyinjiao (SP6) Acupuncture in a Mouse Model of Peritonitis

In this study, we determined the anti-inflammatory effect of manual acupuncture at the Sanyinjiao or Spleen 6 (SP6) point on carrageenan-induced peritonitis in mice and investigated mechanisms that may underlie this effect. In the first set of experiments, male Swiss mice were allocated into five groups: the control (sterile saline), dexamethasone (DEXA), invasive sham-acupuncture (non-acupoint), SP6 acupuncture and carrageenan-treated groups. Ten minutes after needle retention or 30 min after DEXA treatment, mice received an intraperitoneal injection of carrageenan (750 μg/mouse). After 4 h, total leukocyte and differential cell counts (neutrophils and mononuclear), myeloperoxidase (MPO) activity, vascular permeability and cytokine levels were evaluated. In another set of experiments, adrenalectomized (ADX) mice were used to study the involvement of the adrenal gland on the therapeutic effects of acupuncture. Mice were allocated into two groups: the ADX and sham-operated animals (Sham ADX) that were subdivided into four subgroups each: the control (sterile saline), DEXA, SP6 acupuncture and carrageenan-treated groups. The SP6 and DEXA treatments inhibited the inflammatory cell infiltration, vascular permeability and MPO activity in carrageenan-injected mice. In addition, the SP6 treatment also increased interleukin (IL)-10 levels. In contrast, when the animals were adrenalectomized, the SP6 treatment failed to reduce total leukocyte and the plasma extravasation. In conclusion, this study clearly demonstrates the anti-inflammatory effect of SP6 acupuncture in a model of carrageenan-induced peritonitis. Our results demonstrated that SP6 acupuncture depends of the adrenal glands and increased IL-10 levels to produce its anti-inflammatory action

Original Article Involvement of Interleukin-10 in the Anti-Inflammatory Effect of Sanyinjiao (SP6) Acupuncture in a Mouse Model of Peritonitis

In this study, we determined the anti-inflammatory effect of manual acupuncture at the Sanyinjiao or Spleen 6 (SP6) point on carrageenan-induced peritonitis in mice and investigated mechanisms that may underlie this effect. In the first set of experiments, male Swiss mice were allocated into five groups: the control (sterile saline), dexamethasone (DEXA), invasive sham-acupuncture (non-acupoint), SP6 acupuncture and carrageenan-treated groups. Ten minutes after needle retention or 30 min after DEXA treatment, mice received an intraperitoneal injection of carrageenan (750 μg/mouse). After 4 h, total leukocyte and differential cell counts (neutrophils and mononuclear), myeloperoxidase (MPO) activity, vascular permeability and cytokine levels were evaluated. In another set of experiments, adrenalectomized (ADX) mice were used to study the involvement of the adrenal gland on the therapeutic effects of acupuncture. Mice were allocated into two groups: the ADX and sham-operated animals (Sham ADX) that were subdivided into four subgroups each: the control (sterile saline), DEXA, SP6 acupuncture and carrageenan-treated groups. The SP6 and DEXA treatments inhibited the inflammatory cell infiltration, vascular permeability and MPO activity in carrageenan-injected mice. In addition, the SP6 treatment also increased interleukin (IL)-10 levels. In contrast, when the animals were adrenalectomized, the SP6 treatment failed to reduce total leukocyte and the plasma extravasation. In conclusion, this study clearly demonstrates the anti-inflammatory effect of SP6 acupuncture in a model of carrageenan-induced peritonitis. Our results demonstrated that SP6 acupuncture depends of the adrenal glands and increased IL-10 levels to produce its anti-inflammatory action

Antinociceptive and gastroprotective actions of ethanolic extract from Pluchea sagittalis (Lam.) Cabrera

AbstractEthnopharmacological relevancePluchea sagittalis, an herbaceous plant widely distributed in South America, is used in folk medicine for the treatment of digestive diseases and inflammation.Aim of the studyThis study was designed to investigate the antinociceptive and gastroprotective effects of the ethanolic extract (EE) of aerial parts from Pluchea sagittalis in rodents.Materials and methodsThe antinociceptive effects of EE was evaluated in mice after oral administration in chemical tests (acetic-acid, glutamate and formalin) or by biting behavior following intrathecal administration of cytokines such as interleukin-1beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) in mice. Furthermore, rats were treated with EE and subsequently exposed to acute gastric lesions induced by 80% ethanol. Afterwards the gastric lesion extension and the mucus levels of gastric mucosa were measured.ResultsThe oral administration of EE showed a dose-dependent inhibition of acetic acid-induced abdominal constrictions and glutamate-induced pain in mice, with ID50 values of 624.0 (523.0–746.0)mg/kg and 368.0 (216.0–628.0)mg/kg, respectively. In the formalin test, the EE also produced significant inhibition of the inflammatory phase, with an ID50 value of 411.0 (183.0–721.0) mg/kg; however, it was ineffective in the neurogenic phase caused by formalin. In addition, oral treatment with EE caused a significant inhibition of biting behavior induced by i.t. injection of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α). The antinociception caused by the EE (300mg/kg, p.o.) was not reversed by naloxone (1mg/kg, i.p.) when assessed in the acetic acid writhing test. The EE (300–1000mg/kg, p.o.) did not affect the motor coordination of animals in an open-field model. Oral treatment with the EE protected rats against gastric lesions induced by ethanol, with an ID50 value of 55.0 (46.6–64.9)mg/kg, and increased the mucus levels of gastric mucosa to levels found in the non-lesioned group.ConclusionsThe mechanism by which the extract produced antinociception still remains unclear, but this effect seems to be primarily related to the modulation or inhibition of the action of pro-inflammatory mediators. Furthermore, these data support, at least in part, the ethnomedical use of Pluchea sagittalis

Antinociception of β-d-glucan from Pleurotus pulmonarius is possibly related to protein kinase C inhibition

Abstractβ-d-Glucan, a polysaccharide isolated from an edible mushroom Pleurotus pulmonarius (Fr.) Quel., presented antinociceptive activity in mice. This study evaluated the involvement of transient receptor potential (TRP) channels and protein kinase C (PKC) on antinociceptive effect of a (1→3),(1→6)-linked β-d-glucan (GL) in mice. Intraperitoneal administration of GL potently inhibited nociceptive responses induced by intraplantar injections of capsaicin, cinnamaldehyde, menthol, acidified saline and phorbol myristate acetate (PMA). Moreover, Western blot analysis revealed that GL treatment also prevented PMA-induced PKCɛ activation. Collectively, present results demonstrate that GL could constitute an attractive molecule of interest for the development of new analgesic drugs

Mecanismos envolvidos na atividade gastroprotetora do extrato aquoso das folhas de Achillea millefolium L.

Orientadora: Prof.ª Dr.ª Maria Consuelo Andrade MarquesCoorientadora: Dr.ª Sonia Mesia VelaDissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Curso de Pós-Graduação em FarmacologiaInclui referências: p. 97-109Resumo: A Achillea millefolium L. conhecida popularmente como mil folhas e pronto alívio é amplamente utilizada pela população mundial para uma variedade de doenças incluindo o tratamento de distúrbios gastrointestinais e dor. Estudos científicos já validaram alguns desses usos populares da espécie embora os efeitos da planta no trato gastrointestinal (TGI) permaneçam sem avaliação. Este trabalho teve por objetivo validar as ações do extrato bruto aquoso das folhas da A. millefolium, obtida de cultivo controlado e padronizado, no TGI como gastroprotetor. Também visamos determinar o(s) mecanismo(s) desta atividade assim como identificar a(s) provável(is) substâncias(s) ativa(s) responsável(is) por esta(s) ação(ões). Para a determinação da atividade gastroprotetora da A. millefolium, estudamos os efeitos do extrato bruto aquoso (EABA, 125-2000 mg/kg - vo) na lesão gástrica experimental assim como na secreção acida gástrica e na motilidade intestinal. Para isto, o extrato bruto aquoso das folhas de A. millefolium (EABA) foi preparado de acordo com a sua utilização popular (extração 10%, a 70 °C durante 30 minutos, três extrações sucessivas, rendimento 29%). A ação do EABA na úlcera gástrica experimental foi avaliada nos modelos de lesão aguda induzida por estresse (imobilização a 4 °C/3 h), por um antiinflamatório não-esteroidal (indometacina, 20 mg/kg - sc) e por um agente necrotizante etanol (70%, 0,5 ml/200 g - vo). O extrato da A. millefolium protegeu os animais contra lesões gástricas induzas pela indometacina e pelo etanol mas não aquelas induzidas por estresse. Como a proteção da mucosa gástrica pelo EABA foi mais potente contra lesões induzidas pelo etanol (DI50 = 936 mg/kg - vo) que pela indometacina (DI50 > 2000 mg/kg - vo), o modelo de etanol foi utilizado para o estudo dos mecanismos gastroprotetores do extrato. A ação do EABA sobre dois importantes fatores protetores da mucosa gástrica, como a concentração de muco gástrico e a concentração de grupamentos sulfidrílicos não-protéicos (GSH) assim como na atividade das enzimas antioxidantes gástricas (NADPH quinona oxidoredutase 1 - NQ01, glutationa S-transferase - GST e catalase - CAT), foram avaliadas neste modelo. O EABA diminuiu a depleção de GSH induzida pelo etanol sem estimular as atividades das enzimas antioxidantes, indicando que o efeito gastroprotetor da planta estaria relacionada com a ativação de fatores citoprotetores dependentes de GSH. No entanto, neste experimento de lesão aguda verificou-se que o EABA reduz a atividade da GST em 32%. Resultados similares foram obtidos após tratamento subcrônico (EABA, 1000 mg/kg - vo/7 dias) indicando que o extrato não possui uma ação antioxidante que contribua para o efeito gastroprotetor. O estudo dos efeitos do EABA na secreção ácida gástrica foi avaliada utilizando o modelo da ligadura pilórica (4 h em ratas), na presença de agonistas de receptores muscarínicos Mi e M3 (betanecol), receptores de gastrina CCKB (pentagastrina) e receptores histaminérgicos H2 (histamina). O EABA reduziu a secreção acida basal e a estimulada por histamina e betanecol sem alterar a secreção induzida por pentagastrina indicando uma possível atividade sobre a bomba de prótons. Complementamos então o estudo com a determinação das atividades do EABA sobre a atividade da enzima H+/K+-ATPase isolada de mucosa gástrica de cachorro, que foi inibida pelo EABA com CI50 = 382 [Mi]g/ml. Esses resultados mostram que há também participação de um efeito antisecretor por bloqueio da bomba de prótons no mecanismo de gastroproteção do extrato. Os efeitos do EABA na motilidade do trato gastrointestinal foi avaliado pelos métodos de esvaziamento gástrico, trânsito intestinal e diarréia induzida por óleo de rícino. O EABA não alterou nenhum desses parâmetros, permitindo-nos descartar efeitos colinérgicos e adrenérgicos na atividade gastroprotetora do extrato. Da grande variedade de compostos isolados da A. millefolium, obtivemos os flavonóides rutina e quercetina e o óleo volátil do Laboratório de Fitoquímica da UFPR, que trabalha com a mesma amostra da planta, e avaliamos seus efeitos no modelo de lesão por etanol e no modelo de hipersecreção por ligadura pilórica. A quercetina (50-200 mg/kg - vo) protegeu os animais contra as lesões gástricas induzidas pelo etanol (DI50 = 93 mg/kg - vo) mas não reduziu a secreção ácida gástrica nos animais com ligadura pilórica. No entanto, a rutina, nas doses testadas (50-200 mg/kg - vo) e o óleo volátil (5-50 mg/kg - vo) não alteraram a secreção ácida de animais com ligadura pilórica nem protegeram os animais de lesões gástricas induzidas por etanol. Ambos os flavonóides reduziram a atividade de hidrólise de ATP pela H+/K+-ATPase isolada de cachorro com CI50 = 231 e 339 [Mi]M, respectivamente. Em conjunto, nossos resultados permitem sugerir que o EABA possui efeito gastroprotetor contra lesões induzidas pelo etanol. O mecanismo da ação gastroprotetora parece ocorrer por mecanismos dependentes da ativação de fatores de proteção relacionados ao GSH e com participação do efeito inibitório da secreção acida gástrica. É evidente também que os flavonóides, rutina e quercetina, seriam dois possíveis mas não as únicas substâncias ativas da planta.Abstract: Achillea millefolium L. popularly known as "mil folhas" and "pronto alivio" is widely used for a variety of illnesses including gastrointestinal disturbances and pain. Scientific studies confirmed some of Achillea's folk uses although its effects on the gastrointestinal tract (GIT) remain without evaluation. The aim of the present study was to validate of the effects of aqueous crude extract (EABA) of A. millefolium leaves on the GIT as gastroprotective. The plant was obtained from a controlled and standardized germoplasm bank. We also try to identify the mechanism(s) as well as the active principles responsible for the gastroprotective action of the plant. For the determination of the gastroprotective activity of A. millefolium, we studied the effects of the EABA (125-1500 mg/kg - po) on experimental gastric lesion, gastric acid secretion and intestinal motility. For this, the EABA was prepared according to its popular use (10% extraction, 70 °C during 30 minutes, three extractions, yield of 29%). EABA action on experimental gastric ulcer was evaluated in models of acute lesion induced by stress (restraint and cold/3 h), nonsteroidal antiinflammatory (indomethacin, 20 mg/kg - sc) and necrotizing agent ethanol (70%, 0.5 ml/200 g). The extract of A. millefolium protected the animals against gastric lesions induced by indomethacin and ethanol but not against those induced by stress. Protection of gastric mucosa by EABA was more potent in the model induced by ethanol (ID50 = 936 mg/kg - po) that in the indomethacin (ID50 > 2000 mg/kg - po). Then, the model of ethanol was used for the study of gastroprotective mechanisms of the extract. EABA action on two important protective factors of gastric mucosa like the gastric mucus and non-proteic sulphydril groups (GSH) levels as well as the activity of gastric antioxidant enzymes (NADPH quinone oxidoreductase 1 - NQ01, glutathione S-transferase - GST and catalase - CAT) were also evaluated in this model. EABA reduced the GSH depletion induced by ethanol but did not alter the activity of antioxidants enzymes indicating that the gastroprotective effect of plant is related to the activation of cytoprotective factors depending of GSH and not to and antioxidant effect coming from tested enzymatic systems. However, in this experiment of acute lesion was verified that the EABA reduced the GST activity by 32%. Similar results were obtained after subchronic treatment (EABA, 1000 mg/kg - po/7 days). Although inexplicable with the present results, this effect deserves to be better studied in order to determine its full significance. The study of EABA effects on gastric acid secretion was evaluated using the pylorus ligature model (4 h in rats) with or without the presence of agonists of receptors mucarinics (bethanecol), CCKB (pentagastrin) and histaminergic (histamine). The EABA reduced both the basal and stimulated acid secretion by histamine and bethanecol without altering the one induced by pentagastrin, indicating a possible activity in a muscarinic and histaminergic receptors or in the proton pump, the final step of gastric acid secretion, common to both pathways. Thus, the study on gastric acid secretion was complemented with study of the activity of EABA on the hydrolysis of ATP by the H+/K+-ATPase isolated from dog gastric mucosa. The activity of the enzyme was inhibited by EABA with IC50 = 304 [Mi]g/ml. These results show that exist also participation of an anti-secretor mechanism which seems to be, at least in part, by blockade of the proton pump in the gastroprotective effect of the plant. EABA effects on GIT motility were evaluated through determination of its effects on gastric emptying, intestinal transit and diarrhea induced by castor oil. EABA did not alter any of these parameters allowing us to discard cholinergic effects on gastroprotective activity of the extract. From the large variety of isolated compounds of A. millefolium, we obtained the flavonoids rutin and quercetin and the volatile oil from Phytochemistry Laboratory of UFPR which works with the same lot of plant. We evaluated its effects on the ethanol induced gastric lesion and on hypersecretion model by pylorus ligature. Quercetin (50-200 mg/kg - po) protected the animals against gastric lesions induced by ethanol (ID50 = 93 mg/kg - po) but did not alter the gastric acid secretion of animals with pylorus ligature. In the other hand, rutin (50- 200 mg/kg - po) and the volatile oil (5-50 mg/kg - po) did not alter the acid secretion of animals with pylorus ligature neither protect the animals from gastric lesions induced by ethanol. However, both flavonoids reduced the hydrolysis of ATP by H+/K+-ATPase isolated of dog with IC50 = 231 and 307 [MI]M, respectively. Collectively, our results validate the popular use of A. millefolium as gastroprotective agent. The mechanism of gastroprotective action seems to occur by mechanisms depending of activation of cytoprotective factors related to GSH with participation of inhibitory effect of gastric acid secretion. It is also evident, that the flavonoids rutin and quercetin are two possible but not the only ones active principles of the plant

Action of crude aqueous extract of leaves of Achillea millefolium L. (Compositae) on gastrointestinal tract

Achillea millefolium L. (Compositeae) is used in folk medicine to treat gastric disturbances. Doses of 125, 1500 and 2000 mg/kg protected rats against ulcers induced by ethanol and restraint-in-cold-stress, but not against indomethacininduced ulcers. Injected into the duodenal lumen the extract inhibited the basal acid secretion. Data from studies indicate that the antiulcer activity of A. millefolium must been related to a inhibition of gastric secretion or to a increase of protective factors in gastric mucosa as mucus, bicarbonate and blood flow. In conclusion, this extract effectively protected the gastric mucosa and inhibited gastric acid secretion. Further studies should also be provided for the stimulation of receptors in the parietal cell to elucidate the route whereby the extract produce this action