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Investigation of the role of sex hormones in healing activity of the hydroalcoholic extract from the leaves of Eugenia punicifolia on experimental peptic ulcer disease.

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Larissa Lucena Périco
Total Authors: 1
Document type: Doctoral Thesis
Press: Botucatu. 2018-03-26.
Institution: Universidade Estadual Paulista (Unesp). Instituto de Biociências. Botucatu
Defense date:
Advisor: Clélia Akiko Hiruma Lima; Lúcia Regina Machado da Rocha

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. (AU)

FAPESP's process: 15/14797-3 - The role of the hydroalcoholic extract from the leaves of Eugenia punicifolia (Kunth) DC on experimental peptic ulcer disease: characterization of mechanisms of action anti-inflammatory, healing and antiapoptotic.
Grantee:Larissa Lucena Périco
Support Opportunities: Scholarships in Brazil - Doctorate