Investigação do papel dos hormônios sexuais na atividade cicatrizante do extrato hidroalcoólico das folhas de Eugenia punicifolia na doença ulcerosa péptica experimental.
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Data
2018-03-06
Autores
Périco, Larissa Lucena
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Universidade Estadual Paulista (Unesp)
Resumo
As úlceras pépticas abrangem tanto as úlceras gástricas, quanto as úlceras duodenais e afetam cerca de 10% da população mundial, sendo 6,8% homens e 4,9% mulheres. A maior ocorrência de úlceras pépticas em homens do que em mulheres, provavelmente, está relacionada aos hormônios sexuais, pois existem relatos de que os hormônios femininos exercem um efeito protetor contra as ulcerações. A patogênese complexa e multifatorial desta doença ocorre devido a um desequilíbrio entre os fatores agressores [ácido clorídrico, pepsina, etanol, anti-inflamatórios não esteroidais (AINEs), estresse, Helicobacter pylori, entre outros] e os protetores (muco, bicarbonato, prostaglandinas e fluxo sanguíneo) da mucosa do estômago e/ou duodeno. Numa tentativa de proteger a mucosa gástrica do ácido clorídrico, melhorar a cicatrização da úlcera, e prevenir a sua recorrência, o controle farmacológico da secreção ácida tem sido uma das estratégias utilizadas ao longo dos anos. Dentre as opções disponíveis no mercado para o tratamento das úlceras pépticas existem os antagonistas de receptores H2, os inibidores da bomba de prótons e o uso de antibióticos para a erradicação da H. pylori. Entretanto, estes medicamentos podem promover não somente uma gama de efeitos colaterais, mas também uma menor eficácia no tratamento das úlceras, bem como o surgimento das recidivas logo após a interrupção do tratamento. Para tanto, se faz necessário as buscas de novas opções terapêuticas para tratamento adequado das ulceras pépticas a partir de produtos naturais. Eugenia punicifolia (Kunth) DC., uma espécie comumente encontrada na região Amazônica e no Cerrado, tem sido utilizada popularmente para o tratamento de feridas, infecções e inflamações. Em estudos realizados previamente em nosso laboratório, o extrato de E. punicifolia apresentou um efetivo efeito anti-inflamatório e gastroprotetor sobre a mucosa gástrica em modelos experimentais in vivo e in vitro. Porém, a presença de uma ação gastroprotetora de um extrato não assegura que o mesmo também possua ação cicatrizante. Portanto, o objetivo deste trabalho foi caracterizar os mecanismos envolvidos na ação cicatrizante do extrato hidroalcoólico das folhas de Eugenia punicifolia (HEEP) ao combater os efeitos lesivos promovidos pela ação do etanol, ácido acético, AINEs e as injúrias oriundas de um processo de isquemia-reperfusão e analisar a diferença entre o efeito do extrato na cicatrização de machos e fêmeas (intactas e ovariectomizadas). Para tanto, foram desenvolvidos modelos experimentais in vivo no qual, inicialmente foi implantada a lesão gástrica ou duodenal nos ratos Wistar e posteriormente foi realizado o tratamento com HEEP em períodos que variaram de 1 a 14 dias de tratamento. Foram analisados a cicatrização gástrica e duodenal nos diferentes grupos experimentais e para isso utilizou-se parâmetros macroscópicos como a área de lesão, parâmetros bioquímicos antioxidantes (superóxido dismutase, glutationa reduzida, catalase, mieloperoxidase e peroxidação lipídica), pró e anti-inflamatórios (TNF-α, IL-1β, IL-6, IFN-γ, IL-5 e IL-10), expressão proteica de fatores envolvidos no processo de cicatrização [fatores de crescimento endotelial vascular (VEGF) e epidermal (EGF), ciclooxigenase 1 e 2, marcadores pró-apoptóticos - caspase-3, caspase-8 e caspase-9 e antiapoptóticos, como a Bcl-2] e zimografia [metaloproteinases (MMP) 2 e 9]. A atividade de proliferação celular do HEEP em fibroblastos (células L929) também foi determinada por ensaios in vitro. Com a realização dos diferentes tratamentos foi possível avaliar os efeitos tóxicos através da evolução do peso corporal, letalidade, análises macroscópicas dos órgãos e alguns parâmetros bioquímicos no plasma. Adicionalmente, os efeitos do HEEP sobre a diarreia, a motilidade intestinal e o acúmulo de fluido intestinal in vivo assim como sua ação antimicrobiana in vitro, foram estudados. O HEEP foi capaz de cicatrizar a mucosa gástrica de machos, fêmeas intactas (4 dias) e fêmeas ovariectomizadas (6 dias) no modelo de etanol 80% devido à redução da atividade da mieloperoxidase e aumento nos níveis de glutationa reduzida. Nas lesões gástricas induzidas por AINEs, os ratos machos tratados com HEEP (2 dias) apresentaram uma efetiva cicatrização da mucosa gástrica com redução nos níveis da citocina pró-inflamatória IL-5. O mesmo resultado não foi obtido com as fêmeas tratadas com HEEP durante o mesmo período. Nas lesões gástricas e duodenais promovidas pela isquemia e reperfusão, o tratamento de ratos machos e fêmeas (intactas e ovariectomizadas) com HEEP (6 dias) foi capaz de cicatrizar a mucosa apresentando aumento nas atividades de catalase e superóxido dismutase e redução da atividade de mieloperoxidase e dos níveis de glutationa reduzida. Os animais que foram submetidos a lesão gástrica induzida por ácido acético e tratados com o HEEP (14 dias) apresentaram uma efetiva ação cicatrizante. Os resultados da análise de western blotting demonstraram que o tratamento com HEEP por 14 dias no modelo de indução de lesão gástrica por ácido acético não alterou a expressão proteica (EGF, VEGF, COX-1 e 2, Bcl-2 e as caspases-8, 9 e 3), porém, a cicatrização da mucosa gástrica pelo tratamento com HEEP nesse modelo experimental foi devido ao aumento dos níveis de prostaglandina E2, inibição da MMP-9 e manutenção constante da atividade da MMP-2 da mucosa gástrica. O efeito cicatrizante do HEEP apresenta um envolvimento com os hormônios sexuais femininos nos modelos de etanol 80%, isquemia-reperfusão e ácido acético. Podemos constatar que houve diferenças em relação ao tempo de tratamento, em que as fêmeas intactas apresentaram significativa ação cicatrizante durante um período menor de tratamento quando comparado com as fêmeas ovariectomizadas ou em relação ao grau de severidade da lesão, no qual as fêmeas intactas obtiveram uma área de lesão menor quando comparadas aos machos ou fêmeas ovariectomizadas. Além disso, houve diferença na expressão proteica de EGF, VEGF, Caspase-8 e Bcl-2, entre as fêmeas e os machos. Não foram observados efeitos tóxicos nos ratos machos e fêmeas com base na ausência de letalidade, alteração do peso corporal, análises macroscópicas dos órgãos e parâmetros bioquímicos do plasma pelo tratamento com HEEP. O efeito cicatrizante do HEEP também foi comprovado em estudos in vitro ao promover aumento da proliferação e cobertura de feridas riscadas em monocamadas de fibroblastos. Adicionalmente ao efeito antiulcerogênico, foi possível caracterizar que o extrato também possui ação antimicrobiana contra Staphyloccocus aureus e foi capaz de reduzir o trânsito intestinal e prevenir ocorrência da diarreia induzida por agente catártico. Desta forma, podemos concluir que o HEEP pode contribuir efetivamente para a cicatrização da lesão gástrica e duodenal promovidas por uma grande diversidade de agentes ulcerogênicos e que os mecanismos envolvidos nesta ação se mostram diversificados e abrangentes de acordo com o grau de severidade da lesão e do tempo de tratamento do extrato. Nossos resultados demonstram que existe uma clara interferência do hormônio sexual feminino no processo de cicatrização das úlceras pépticas. Aliado a ação cicatrizante, o extrato também exerce uma ação antidiarreica e antimicrobiana, inclusive no combate a um dos principais indutores de gastroenterite no homem, a S. aureus.
Peptic ulcers include both gastric and duodenal ulcers and affect about 10% of the world population, being 6.8% men and 4.9% women. The higher occurrence of peptic ulcers in men than in women is probably related to sex hormones, as there are reports that female hormones exert a protective effect against ulcerations. The complex and multifactorial pathogenesis of this disease occurs due to an imbalance between the aggressive (hydrochloric acid, pepsin, ethanol, nonsteroidal anti-inflammatory drugs (NSAIDs), stress, Helicobacter pylori, among others) and protective factors (mucus, bicarbonate, prostaglandins and blood flow) of the stomach and duodenum mucosa. In an attempt to protect gastric mucosa from the hydrochloric acid, improve ulcer healing, and prevent recurrence, pharmacological control of acid secretion has been one of the strategies used over the years. Among the options available in the market for the treatment of peptic ulcers are H2 receptor antagonists, proton pump inhibitors and the use of antibiotics for the eradication of H. pylori. However, these drugs may promote not only a range of side effects, but also a lower efficacy in the treatment of ulcers, as well as the occurrence of relapses soon after discontinuation of treatment. Therefore, it is necessary to search for new therapeutic options for appropriate treatment of peptic ulcers from natural products. Eugenia punicifolia (Kunth) DC., a specie commonly found in the Amazon region and in the Cerrado, has been popularly used for the treatment of wounds, infections and inflammation. In previous studies in our laboratory, the extract of E. punicifolia showed an effective anti-inflammatory and gastroprotective effect on the gastric mucosa in experimental models in vivo and in vitro. However, the presence of a gastroprotective action of an extract does not ensure that it also has healing action. Therefore, the objective of this work was to characterize the mechanisms involved in the healing action of the hydroalcoholic extract from the leaves of Eugenia punicifolia (HEEP) by combating the harmful effects promoted by ethanol, acetic acid, NSAIDs and injuries originating from a process of ischemia-reperfusion and analyzing the difference between the healing effect of the extract on male and female (intact and ovariectomized). For this, experimental models were developed in vivo in which the gastric or duodenal lesion was initially implanted in the Wistar rats and later the treatment with HEEP was performed in periods ranging from 1 to 14 days of treatment. The gastric and duodenal healing in the different experimental groups was analyzed using macroscopic parameters such as lesion area, antioxidant and biochemical parameters (superoxide dismutase, reduced glutathione, catalase, myeloperoxidase and lipid peroxidation), pro and anti-inflammatory agents (TNF, IL-6, IFN-γ, IL-5 and IL-10), protein expression of factors involved in the healing process [vascular endothelial (VEGF) and epidermal (EGF) growth factor, cyclooxygenase 1 and 2, pro-apoptotic markers - caspase-3, caspase-8 and caspase-9 and antiapoptotic markers such as Bcl-2] and zymography [MMPs 2 and 9]. With the accomplishment of the different treatments it was possible to evaluate the toxic effects through the evolution of the body weight, lethality, macroscopic analysis of the organs and some biochemical parameters in the plasma. In addition, the effects of HEEP on diarrhea, intestinal motility and intestinal fluid accumulation in vivo as well as its antimicrobial action in vitro were studied. HEEP was able to heal the gastric mucosa of males, intact females (4 days) and ovariectomized females (6 days) in the ethanol 80% model due to reduce of myeloperoxidase activity and increased levels of reduced glutathione. In the gastric lesions induced by NSAIDs, male rats treated with HEEP (2 days) showed an effective healing of the gastric mucosa with reduction in the levels of the pro-inflammatory cytokine IL-5. The same result was not obtained with the females treated with HEEP during the same period. In the gastric and duodenal lesions promoted by ischemia and reperfusion, the treatment of male and female (intact and ovariectomized) rats with HEEP (6 days) was able to heal the mucosa presenting an increase in the catalase and superoxide dismutase activities and reduction of myeloperoxidase activity and levels of reduced glutathione. Animals that underwent gastric injury induced by acetic acid and treated with HEEP (14 days) presented an effective curative action. The results of western blotting analysis demonstrated that 14-day HEEP treatment in the acetic acid gastric injury induction model did not alter protein expression (EGF, VEGF, COX-1 and 2, Bcl-2 and caspase-8, 9 and 3); however, healing of the gastric mucosa by treatment with HEEP in this experimental model was due to increased levels of prostaglandin E2, inhibition of MMP-9 and constant maintenance of MMP-2 activity of the gastric mucosa. The healing effect of HEEP shows an involvement with female sex hormones in the ethanol 80%, ischemia-reperfusion and acetic acid models. We observed that there were differences in treatment time, in which the intact females had a significant healing effect during a shorter treatment period when compared to the ovariectomized females or in relation to the degree of lesion severity, in which the intact females obtained a lower lesion area compared to males or ovariectomized females. In addition, there was a difference in the protein expression of EGF, VEGF, Caspase-8 and Bcl-2 between females and males. No toxic effects were observed in male and female rats based on the absence of lethality, body weight change, macroscopic organ analysis and biochemical parameters of plasma by treatment with HEEP. The healing effect of HEEP has also been demonstrated with in vitro studies in promoting increased proliferation and coverage of scratched wounds in fibroblast monolayers. In addition to antiulcerogenic effect, it was possible to characterize that the extract also has antimicrobial activity against Staphylococcus aureus and was able to reduce the intestinal transit and prevent occurrence of diarrhea induced by the cathartic agent. In this way, we can conclude that HEEP can effectively contribute to healing of the gastric and duodenal lesions promoted by a great diversity of ulcerogenic agents and that the mechanisms involved in this action are diversified and comprehensive according to the degree of severity of the lesion and the time of treatment of the extract. Our results demonstrate that there is a clear interference of the female sexual hormone in the healing process of peptic ulcers. Combined with healing action, the extract also exerts an anti-diarrheal and antimicrobial action, including in combating one of the major inducers of gastroenteritis in humans, S. aureus.
Peptic ulcers include both gastric and duodenal ulcers and affect about 10% of the world population, being 6.8% men and 4.9% women. The higher occurrence of peptic ulcers in men than in women is probably related to sex hormones, as there are reports that female hormones exert a protective effect against ulcerations. The complex and multifactorial pathogenesis of this disease occurs due to an imbalance between the aggressive (hydrochloric acid, pepsin, ethanol, nonsteroidal anti-inflammatory drugs (NSAIDs), stress, Helicobacter pylori, among others) and protective factors (mucus, bicarbonate, prostaglandins and blood flow) of the stomach and duodenum mucosa. In an attempt to protect gastric mucosa from the hydrochloric acid, improve ulcer healing, and prevent recurrence, pharmacological control of acid secretion has been one of the strategies used over the years. Among the options available in the market for the treatment of peptic ulcers are H2 receptor antagonists, proton pump inhibitors and the use of antibiotics for the eradication of H. pylori. However, these drugs may promote not only a range of side effects, but also a lower efficacy in the treatment of ulcers, as well as the occurrence of relapses soon after discontinuation of treatment. Therefore, it is necessary to search for new therapeutic options for appropriate treatment of peptic ulcers from natural products. Eugenia punicifolia (Kunth) DC., a specie commonly found in the Amazon region and in the Cerrado, has been popularly used for the treatment of wounds, infections and inflammation. In previous studies in our laboratory, the extract of E. punicifolia showed an effective anti-inflammatory and gastroprotective effect on the gastric mucosa in experimental models in vivo and in vitro. However, the presence of a gastroprotective action of an extract does not ensure that it also has healing action. Therefore, the objective of this work was to characterize the mechanisms involved in the healing action of the hydroalcoholic extract from the leaves of Eugenia punicifolia (HEEP) by combating the harmful effects promoted by ethanol, acetic acid, NSAIDs and injuries originating from a process of ischemia-reperfusion and analyzing the difference between the healing effect of the extract on male and female (intact and ovariectomized). For this, experimental models were developed in vivo in which the gastric or duodenal lesion was initially implanted in the Wistar rats and later the treatment with HEEP was performed in periods ranging from 1 to 14 days of treatment. The gastric and duodenal healing in the different experimental groups was analyzed using macroscopic parameters such as lesion area, antioxidant and biochemical parameters (superoxide dismutase, reduced glutathione, catalase, myeloperoxidase and lipid peroxidation), pro and anti-inflammatory agents (TNF, IL-6, IFN-γ, IL-5 and IL-10), protein expression of factors involved in the healing process [vascular endothelial (VEGF) and epidermal (EGF) growth factor, cyclooxygenase 1 and 2, pro-apoptotic markers - caspase-3, caspase-8 and caspase-9 and antiapoptotic markers such as Bcl-2] and zymography [MMPs 2 and 9]. With the accomplishment of the different treatments it was possible to evaluate the toxic effects through the evolution of the body weight, lethality, macroscopic analysis of the organs and some biochemical parameters in the plasma. In addition, the effects of HEEP on diarrhea, intestinal motility and intestinal fluid accumulation in vivo as well as its antimicrobial action in vitro were studied. HEEP was able to heal the gastric mucosa of males, intact females (4 days) and ovariectomized females (6 days) in the ethanol 80% model due to reduce of myeloperoxidase activity and increased levels of reduced glutathione. In the gastric lesions induced by NSAIDs, male rats treated with HEEP (2 days) showed an effective healing of the gastric mucosa with reduction in the levels of the pro-inflammatory cytokine IL-5. The same result was not obtained with the females treated with HEEP during the same period. In the gastric and duodenal lesions promoted by ischemia and reperfusion, the treatment of male and female (intact and ovariectomized) rats with HEEP (6 days) was able to heal the mucosa presenting an increase in the catalase and superoxide dismutase activities and reduction of myeloperoxidase activity and levels of reduced glutathione. Animals that underwent gastric injury induced by acetic acid and treated with HEEP (14 days) presented an effective curative action. The results of western blotting analysis demonstrated that 14-day HEEP treatment in the acetic acid gastric injury induction model did not alter protein expression (EGF, VEGF, COX-1 and 2, Bcl-2 and caspase-8, 9 and 3); however, healing of the gastric mucosa by treatment with HEEP in this experimental model was due to increased levels of prostaglandin E2, inhibition of MMP-9 and constant maintenance of MMP-2 activity of the gastric mucosa. The healing effect of HEEP shows an involvement with female sex hormones in the ethanol 80%, ischemia-reperfusion and acetic acid models. We observed that there were differences in treatment time, in which the intact females had a significant healing effect during a shorter treatment period when compared to the ovariectomized females or in relation to the degree of lesion severity, in which the intact females obtained a lower lesion area compared to males or ovariectomized females. In addition, there was a difference in the protein expression of EGF, VEGF, Caspase-8 and Bcl-2 between females and males. No toxic effects were observed in male and female rats based on the absence of lethality, body weight change, macroscopic organ analysis and biochemical parameters of plasma by treatment with HEEP. The healing effect of HEEP has also been demonstrated with in vitro studies in promoting increased proliferation and coverage of scratched wounds in fibroblast monolayers. In addition to antiulcerogenic effect, it was possible to characterize that the extract also has antimicrobial activity against Staphylococcus aureus and was able to reduce the intestinal transit and prevent occurrence of diarrhea induced by the cathartic agent. In this way, we can conclude that HEEP can effectively contribute to healing of the gastric and duodenal lesions promoted by a great diversity of ulcerogenic agents and that the mechanisms involved in this action are diversified and comprehensive according to the degree of severity of the lesion and the time of treatment of the extract. Our results demonstrate that there is a clear interference of the female sexual hormone in the healing process of peptic ulcers. Combined with healing action, the extract also exerts an anti-diarrheal and antimicrobial action, including in combating one of the major inducers of gastroenteritis in humans, S. aureus.
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Palavras-chave
Eugenia punicifolia, Ação cicatrizante, hormônios sexuais femininos, Úlceras pépticas, Eugenia punicifolia, Healing action, Female sex hormones, Peptic ulcers