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Investigation of anti-inflammatory and leishmanicidal activity of lupan in cutaneous leishmaniasis models
Brazil (IFMSA-Brazil) - Universidade Federal do Ceara, Fortaleza
Department of Pathology and Legal Medicin, Rua Monsenhor Furtado, s/n - Rodolfo Teófilo - CEP 60441-750 - Fortaleza - CE
Maria Jânia Teixeira
Maria Jânia Teixeira
English and Portuguese
Type of Research Project
- Basic science
What is the background of the project?
Leishmaniasis includes a complex of diseases caused by different Leishmania species whose clinical presentations vary according to species involved in the infection and with the immunological and genetic host. Leishmania braziliensis is the main species involved in it. Combretum leprosum is a native plant of the Caatinga, an important biome of the Brazilian semi-arid region. This species is commonly used in Brazil as a healing agent, as well as in the treatment of skin diseases. The bioactive components of C. leprosum are 3beta, 6beta, 16beta-trihydroxylup-20 (29) -ene, lupano-triperpene, isolated from its leaves, which has been reported as presenting leishmanicidal effect in vitro against L. amazonensis. The lupano group has been extensively used in studies of anti-cancer effects; however, its possible activity against protozoan parasites is still little known. This study will evaluate the inflammatory and leishmanicidal effects of lupan-triterpene isolated from Combretum leprosum, in mice infected with L.braziliensis.
What is the aim of the project?
Check the inflammatory and leishmanicidal effects of lupan- triterpene isolated from Combretum leprosum in mice infected with L. braziliensis
What techniques and methods are used?
Animals. We will use 36 mice at 8 weeks of life, females, from the vivarium of the Department of Pathology and Medicine Of the Faculty of Medicine of the Federal University of Ceará (UFC) Chemical constituent. The triterpene 3®, 6®, 16®- trihydroxylup-20 (29) -ene (Lupen- triterpene) will be provided by the Laboratory of Biomolecules of the Federal University of Ceará. The ointment, containing 100æg / ml lupus-triterpene, treatment of the animals, will be prepared in the laboratory of Profa. Luzia Kalyne Almeida Moreira Leal, Faculty of Pharmacy, UFC. Parasites . A strain of Leishmania (V.) braziliensis originally obtained from lesions of a patient with localized cutaneous leishmaniasis of the Ceará. The parasites are grown. For the experiments, will be used promastigotes to 5 passage of culture. The viability of the parasites will be analyzed by motility under optic microscopy. Infection of animals. The metacyclic promastigote forms in the stationary phase will be inoculated into the dermis of the direct ear, using insulin syringe and needle. Pre-treatment monitoring . The thickness of the lesion will be accompanied by 45 days, measure every 5 days, using a circular scale caliper and expressed in millimeters. The thickness of the lesion will be represented by the difference infected right ear and contralateral uninfected. Treatment. Treatment will begin after 5 weeks of infection, after the onset of ulcers. The animals will be separated into 03 groups: One group of infected animals (n= 12) will be part of the negative control group. Another group of animals (n = 12) will be treated with daily applications of the drug, lupano-triterpene (100 ug/ml) at the ulcer site for 15 consecutive days. positive patients will be treated with Glucantime (n = 12), 100mg/kg/day, intramuscular. Aspects macroscopic examination of the ears will be recorded by digital photomicrographs for healing. Euthanasia. There will be two moments of euthanasia with two groups of animals with 18 animals each. After 15 days of treatment, equivalent to 45 days after infection, 6 animals from each group (3 groups, n = 18) will be euthanized. After 30 days equivalent to 60 days post infection, the rest of the patients will be euthanized. animals from the 3 groups (n = 18). Euthanasia will occur according to the recommended method overdose of anesthetics, Xylazine/Ketamine. During euthanasia, the infected ears and retro-maxillary lymph nodes for subsequent loading studies inflammatory mediators and histopathological analysis. Evaluation of the parasitic load. The drainage lymph IFMSA International Secretariat, c/o IMCC, Nørre Allé 14, 2200 København N., Denmark nodes (retro-maxillaries) and the infected ears will be removed to quantify the number of parasites used to limiting dilution technique. The number of parasites present in the samples will be calculated. Dosage of inflammatory mediators. The production of cytokines will be determined in supernatants obtained from the culture of draining lymph node cells. For Nitric Oxide (NO) dosing, the supernatants from the cell cultures will be tested to detect the release of NO in the form of nitrite (NO2) by the reaction of Griess. Histopathological analysis. The histopathological analysis will be done with cut material and fixed in 10% buffered formalin. Subsequently this material will be processed, paraffin-embedded for lamination, with sections of 5mm thickness of the emblocated tissues. The staining will be with hematoxylin and eosin (H & E). At alterations will be analyzed under a common optical microscope with the aid of pathologist. Uninfected ears will be used as controls. Statistical analysis. To verify the statistical significance between the treated groups and the control will be applied the normality test and in case of normal distribution, t- Student test.
What is the role of the student?
- The student will observe the practical experiments but will be highly involved in the analysis of the results
- The tasks will be done under supervision
What are the tasks expected to be accomplished by the student?
The student will inoculate the parasite in the right ear of the animals, making use of needles. In the pre-treatment phase, it follows the measurement of the animal's ear thickening, while during treatment, it applies a drug in the test and glucantime groups, in the positive control. In addition, the student participates in the euthanasia of the animals, as well as the removal of infected ears and periauricular lymph nodes. Finally, the student is following the histopathological study and will help in the tabulation of data for statistical analysis, with the normal distribution test and the student t test.
Will there be any theoretical teaching provided (preliminary readings, lectures, courses, seminars etc)
No, just a brief explanation of how the study works and what is there to be done.
What is expected from the student at the end of the research exchange? What will be the general outcome of the student?
- The student will prepare a presentation - The student will prepare a scientific report
What skills are required of the student? Is there any special knowledge or a certain level of studies needed?
To be able to deal with In Vivo and In Vitro models (it can be learned during the exchange); To like working in the Lab
Are there any legal limitations in the student’s involvement
Type of students accepted
This project accepts: - Medical students - Pre-Medical students from the American-British system - Students in biomedical fields
- NASCIMENTO-NETO; LG; EVARISTO; FFV; ALVES; MFA; ALBUQUERQUE; MRJR; SANTOS; HS; FLAG; PN; ARRUDA; FVS; TEIXEIRA; EH; Effect of the triterpene 3b; 6B; 16B-trihydroxylup-20 (29) -ene isolated from the leaves of Combretum leprosum Mart. on cutaneous wounds in mice. J. Ethnopharmacol.; N. 171; p.116-120; 2015.
- TELES; CBG; MOREIRA-DILL; LS; SILVA; AA; FACUNDO; VA; ZULIANI; JP; STABELI; RG; et al. Activity of the lupane- triterpene isolated from Combretum leprosum against Leishmania amazonensis promastigotes. J. Braz. Chem. Soc.; Vol. 22; p.936-46; 2011. TELES; CBG; MOREIRA-DILL; LS; SILVA; AA; FACUNDO; VA; AZEVEDO JR; WF; SILVA; LHP; MOTTA; MC; STÁBELI; RG; SILVA-GARDIM; I. lupane-triterpene isolated from Combretum leprosum Mart. fruit extracts that interferes with the intracellular development of Leishmania (L.) amazonensis in vitro. BMC Complementary and Alternative Medicine; no. 15; vol. 165; p. 1-10; 2015.
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