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Braz. j. biol ; 84: e250936, 2024. graf
Article in English | LILACS, VETINDEX | ID: biblio-1345557


Abstract This study was carried out to evaluate the effect of Glutamine, as a dipeptide or a free amino acid form, on the progression of burn injuries in rats. Thirty male Wistar rats were burned with a comb metal plate heated in boiling water (98 °C) for three minutes, creating four rectangular full-thickness burn areas separated by three unburned interspaces (zone of stasis) in both dorsum sides. The animals were randomized into three groups (n=10): saline solution (G1-Control) and treated groups that orally received Glutamine as dipeptide (G2-Dip) or free amino acid (G3-FreeAA). Two and seven days after burn injury, lesions were photographed for unburned interspaces necrosis evolution assessment. Seven days after injury, glutathione seric was measured and histopathological analysis was performed. By photographs, there was a significant reduction in necrosis progression in G3-Free-AA between days two and seven. Histopathological analysis at day 7 showed a significantly higher stasis zone without necrosis and a higher number of fibroblasts in G2-Dip and G3-FreeAA compared with G1-Control. Also, glutathione serum dosage was higher in G2-Dip. The plasmatic glutathione levels were higher in the G2-Dip than the G1-Control, and there was a trend to higher levels in G3-FreeAA. The reduction in histological lesions, greater production of fibroblasts, and greater amounts of glutathione may have benefited the evolution of burn necrosis, which showed greater preservation of interspaces.

Resumo Este estudo foi realizado para avaliar o efeito da Glutamina, como um dipeptídeo ou forma de aminoácido livre, na progressão de queimaduras em ratos. Trinta ratos Wistar machos foram queimados com um pente de metal aquecido em água fervente (98 °C) por três minutos, criando quatro áreas retangulares queimadas separadas por três interesespaços não queimados (zona de estase) em ambos os lados do dorso. Os animais foram randomizados em três grupos (n = 10): solução salina (G1-Controle) e grupos tratados que receberam glutamina via oral como dipeptídeo (G2-Dip) ou aminoácido livre (G3-FreeAA). Dois e sete dias após a queimadura, as lesões foram fotografadas para avaliação da evolução da necrose entre os espaços não queimados. Sete dias após a lesão, foi dosada a glutationa sérica e realizada análise histopatológica. Pelas fotografias, houve uma redução significativa na progressão da necrose no G3-Free-AA entre os dias dois e sete. A análise histopatológica no dia 7 mostrou uma zona de estase significativamente maior sem necrose e número mais elevado de fibroblastos em G2-Dip e G3-FreeAA em comparação com G1-Controle. Os níveis plasmáticos de glutationa foram maiores no G2-Dip em relação ao G1-Controle, e houve tendência a níveis mais elevados no G3-FreeAA. A redução das lesões histológicas, maior produção de fibroblastos, maior quantidade de glutationa podem ter beneficiado a evolução da necrose da queimadura, que mostrou maior preservação dos interespaços.

Animals , Male , Rats , Burns/drug therapy , Glutamine , Rats, Wistar , Dipeptides , Disease Models, Animal , Amino Acids
Braz. j. med. biol. res ; 47(10): 876-885, 10/2014. tab, graf
Article in English | LILACS | ID: lil-722165


The aim of the present study was to determine the mechanisms underlying the relaxant effect of adrenomedullin (AM) in rat cavernosal smooth muscle (CSM) and the expression of AM system components in this tissue. Functional assays using standard muscle bath procedures were performed in CSM isolated from male Wistar rats. Protein and mRNA levels of pre-pro-AM, calcitonin receptor-like receptor (CRLR), and Subtypes 1, 2 and 3 of the receptor activity-modifying protein (RAMP) family were assessed by Western immunoblotting and quantitative real-time polymerase chain reaction, respectively. Nitrate and 6-keto-prostaglandin F1α (6-keto-PGF1α; a stable product of prostacyclin) levels were determined using commercially available kits. Protein and mRNA of AM, CRLR, and RAMP 1, -2, and -3 were detected in rat CSM. Immunohistochemical assays demonstrated that AM and CRLR were expressed in rat CSM. AM relaxed CSM strips in a concentration-dependent manner. AM22-52, a selective antagonist for AM receptors, reduced the relaxation induced by AM. Conversely, CGRP8-37, a selective antagonist for calcitonin gene-related peptide receptors, did not affect AM-induced relaxation. Preincubation of CSM strips with NG-nitro-L-arginine-methyl-ester (L-NAME, nitric oxide synthase inhibitor), 1H-(1,2,4)oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, quanylyl cyclase inhibitor), Rp-8-Br-PET-cGMPS (cGMP-dependent protein kinase inhibitor), SC560 [5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethyl pyrazole, selective cyclooxygenase-1 inhibitor], and 4-aminopyridine (voltage-dependent K+ channel blocker) reduced AM-induced relaxation. On the other hand, 7-nitroindazole (selective neuronal nitric oxide synthase inhibitor), wortmannin (phosphatidylinositol 3-kinase inhibitor), H89 (protein kinase A inhibitor), SQ22536 [9-(tetrahydro-2-furanyl)-9H-purin-6-amine, adenylate cyclase inhibitor], glibenclamide (selective blocker of ATP-sensitive K+ channels), and apamin (Ca2+-activated channel blocker) did not affect AM-induced relaxation. AM increased nitrate levels and 6-keto-PGF1α in rat CSM. The major new contribution of this research is that it demonstrated expression of AM and its receptor in rat CSM. Moreover, we provided evidence that AM-induced relaxation in this tissue is mediated by AM receptors by a mechanism that involves the nitric oxide-cGMP pathway, a vasodilator prostanoid, and the opening of voltage-dependent K+ channels.

Animals , Male , Adrenomedullin/pharmacology , Calcitonin Receptor-Like Protein/analysis , Muscle, Smooth/drug effects , Parasympatholytics/pharmacology , Penis/drug effects , Vasodilator Agents/pharmacology , /pharmacology , /analysis , Adrenomedullin/genetics , Adrenomedullin/metabolism , Blotting, Western , Calcitonin Receptor-Like Protein/antagonists & inhibitors , Cyclic GMP-Dependent Protein Kinases/antagonists & inhibitors , Cyclooxygenase Inhibitors/pharmacology , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , Immunohistochemistry , Indazoles/pharmacology , Muscle Relaxation , Muscle, Smooth/metabolism , Nitric Oxide Synthase/antagonists & inhibitors , Nitric Oxide/analysis , Nitric Oxide/analogs & derivatives , Penis/metabolism , Potassium Channels, Voltage-Gated/metabolism , Rats, Wistar , Real-Time Polymerase Chain Reaction , RNA, Messenger/metabolism , Receptor Activity-Modifying Protein 1/genetics , Receptor Activity-Modifying Protein 1/metabolism , /metabolism , /genetics , /metabolism , Receptors, Calcitonin Gene-Related Peptide/metabolism
Braz. j. med. biol. res ; 46(1): 71-80, 11/jan. 2013. graf
Article in English | LILACS | ID: lil-665802


Gliomas are the most common and malignant primary brain tumors in humans. Studies have shown that classes of kaurene diterpene have anti-tumor activity related to their ability to induce apoptosis. We investigated the response of the human glioblastoma cell line U87 to treatment with ent-kaur-16-en-19-oic acid (kaurenoic acid, KA). We analyzed cell survival and the induction of apoptosis using flow cytometry and annexin V staining. Additionally, the expression of anti-apoptotic (c-FLIP and miR-21) and apoptotic (Fas, caspase-3 and caspase-8) genes was analyzed by relative quantification (real-time PCR) of mRNA levels in U87 cells that were either untreated or treated with KA (30, 50, or 70 µM) for 24, 48, and 72 h. U87 cells treated with KA demonstrated reduced viability, and an increase in annexin V- and annexin V/PI-positive cells was observed. The percentage of apoptotic cells was 9% for control cells, 26% for cells submitted to 48 h of treatment with 50 µM KA, and 31% for cells submitted to 48 h of treatment with 70 µM KA. Similarly, in U87 cells treated with KA for 48 h, we observed an increase in the expression of apoptotic genes (caspase-8, -3) and a decrease in the expression of anti-apoptotic genes (miR-21 and c-FLIP). KA possesses several interesting properties and induces apoptosis through a unique mechanism. Further experiments will be necessary to determine if KA may be used as a lead compound for the development of new chemotherapeutic drugs for the treatment of primary brain tumors.

Humans , Apoptosis/drug effects , Cell Survival/drug effects , Diterpenes/pharmacology , Glioblastoma/drug therapy , Mikania/chemistry , Cell Line, Tumor , /drug effects , /drug effects , Diterpenes/isolation & purification , Fas Ligand Protein , Flow Cytometry , Glioblastoma/enzymology , Glioblastoma/pathology , Real-Time Polymerase Chain Reaction , Signal Transduction , Time Factors
Int. j. morphol ; 30(3): 979-985, Sept. 2012. ilus
Article in English | LILACS | ID: lil-665512


NMDAR (N-methyl-D-aspartate receptor) is one subtype of ionotrophic glutamate receptor which is extensively distributed in the central nervous system (CNS). In the mammalian CNS, NMDAR serves prominent roles in the pathophysiologic process of cerebral ischemia. This study aimed to investigate the pattern of expression of protein and gene of the excitatory neurotransmitter NMDAR in experimental focal cerebral ischemia and the hole of neuroprotection with hypothermia and ketoprofen. 120 rats were randomly divided into 6 groups (20 animals each): control - no surgery; sham - simulation of surgery; ischemic - focal ischemia for 1 hour, without reperfusion; ischemic + intraischemic hypothermia; ischemic + previous intravenous ketoprofen, and ischemic + hypothermia and ketoprofen. Ten animals from each experimental group were used to establish the volume of infarct. Transient focal cerebral ischemia was obtained in rats by occlusion of the middle cerebral artery with an intraluminal suture. The infarct volume was measured using morphometric analysis of infarct areas defined by triphenyl tetrazolium chloride and the patterns of expression of the protein and gene NMDA were evaluated by immunohistochemistry and quantitative real-time PCR, respectively. Increases in the protein and gene NMDA receptor in the ischemics areas were observed and these increases were reduced by hypothermia and ketoprofen. The increase in the NMDA receptor protein and gene expression observed in the ischemic animals was reduced by neuroprotection (hypothermia and ketoprofen). The NMDA receptor increases in the ischemic area suggests that the NMDA mediated neuroexcitotoxicity plays an important role in cell death and that the neuroprotective effect of both, hypothermia and ketoprofen is directly involved with the NMDA...

NMDAR (N-metil-D-aspartato) es un tipo de receptor de glutamato ionotrópico y está ampliamente distribuido en el sistema nervioso central (SNC). En el SNC de mamíferos, NMDAR se destaca de manera importante en el proceso fisiopatológico de la isquemia cerebral. Este estudio tuvo como objetivo investigar el patrón de expresión de proteínas y genes para el NMDA neurotransmisor excitatorio experimental de la isquemia cerebral focal y el vacío en la neuroprotección con hipotermia y ketoprofeno. Se dividieron 120 ratas aleatoriamente en grupos de 6 animales cada uno (20): Control - sin cirugía; Sham - simulación de cirugía; isquémicas - isquemia focal durante 1 hora, sin reperfusión isquémica; hipotermia intra-isquémica; isquemia; previa aplicación de ketoprofeno intravenoso, e hipotermia isquémica y ketoprofeno. Diez animales de cada grupo experimental fueron utilizados para establecer el volumen de infarto.La isquemia cerebral focal transitoria fue obtenida en ratas mediante oclusión de la arteria cerebral media con una sutura intraluminal. El volumen de infarto fue medido mediante análisis morfométrico de las áreas de infarto definidas por cloruro de trifenil tetrazolio y patrones de expresión de la proteína y el gen de NMDA, fueron evaluados por inmunohistoquímica y PCR cuantitativa en tiempo real, respectivamente. Se observaron aumentos en la proteína y en el gen del receptor de NMDA en las áreas isquémicas y estos aumentos fueron reducidos por la hipotermia y ketoprofeno. El aumento de la proteína del receptor de NMDA y la expresión génica observada en los animales isquémicos fue reducido mediante hipotermia y ketoprofeno. Los aumentos del receptor de NMDA en el área isquémica sugiere que la neuro excitotoxicidad mediada por NMDA desempeña un papel importante en la muerte celular y que el efecto neuroprotector de ambos, hipotermia y ketoprofeno está directamente relacionado al NMDA...

Animals , Rats , Brain Ischemia/metabolism , Brain Ischemia/pathology , Receptors, N-Methyl-D-Aspartate/metabolism , Ketoprofen/metabolism , Neuroprotective Agents/metabolism , Gene Expression , Hypothermia , Immunohistochemistry , Real-Time Polymerase Chain Reaction , Receptors, N-Methyl-D-Aspartate/genetics
Int. j. morphol ; 30(3): 1029-1034, Sept. 2012. ilus
Article in English | LILACS | ID: lil-665520


The aim was to analyze the protein expression of apoptotic genes caspase-3, caspase-8 and bcl-2 with the immunohistochemistry technique, correlating with tumor grade (I, II and III) and with the patient survival in order to understand the basic mechanism of tumoral transformation. The immunohistochemistry reactions on 50 samples of squamous cell carcinoma were carried out with the avidin-biotin immunoperoxidase method and antigen recovery. The analyses were made using the graduation method "in crosses" (0 to 4 crosses - no stain to more than 75 percent of positives cells) and in categories (low, intermediate, high) of the cytoplasm immunoreactivity of the epidermoid penile carcinoma cells. It was observed a statistically significant difference when the expression of caspase-3 were compared with the grades I and II of the tumor (p=0.0010) and when comparing the patient survival with the grades I and II of the tumor (p=0.0212). The protein bcl-2 was more expressed than caspase-3 and caspase-8 proteins, suggesting that the apoptotic rate in this carcinoma is low. The higher expression of the anti-apoptotic protein bcl-2 suggests a higher preservation of the tumoral cells...

El objetivo fue analizar la expresión de las proteínas de genes de apoptosis caspasa-3, caspasa-8 y Bcl-2-con la técnica de inmunohistoquímica, en correlación con el grado tumoral (I, II y III) y la supervivencia del paciente con el fin de comprender el mecanismo básico de la transformación tumoral. Se analizaron las reacciones inmunohistoquímicas sobre 50 muestras de carcinoma de células escamosas mediante el método de la inmunoperoxidasa avidina-biotina y la recuperación de antígeno. Los análisis se realizaron utilizando el método de graduación "en cruces" (0 a 4 cruces - no tinción a más del 75 por ciento de las células positivas) y en categorías (baja, media, alta) de la inmunorreactividad citoplasmática de las células de carcinoma epidermoide de pene. Se observó una diferencia estadísticamente significativa cuando la expresión de la caspasa-3 se comparó con los grados I y II del tumor (p = 0,0010) y cuando se comparan la supervivencia de los pacientes con los grados I y II del tumor (p = 0,0212). La proteína bcl-2 se expresa más que la caspasa-3 y caspasa-8, lo que sugiere que la tasa de apoptosis en este carcinoma es baja. La mayor expresión de la proteína anti-apoptótica bcl-2 sugiere una mayor preservación de las células tumorales...

Humans , Male , Carcinoma, Squamous Cell/metabolism , Carcinoma, Squamous Cell/pathology , Penile Neoplasms/metabolism , Penile Neoplasms/pathology , Apoptosis , /metabolism , /metabolism , Immunohistochemistry , Predictive Value of Tests , /metabolism , Survival Analysis
Rev. chil. anat ; 19(3): 263-269, 2001. ilus, tab
Article in English | LILACS | ID: lil-310235


Ha sido reconocido que la ingesta crónica de etanol causa alteraciones morfológicas en varios tejidos. En el presente trabajo fue realizado un análisis morfométrico de los acinos seromucosos y de los conductos granulosos de las glándulas submandibulares de ratones sometidos a alcoholismo crónico experimental. Ratones Wistar machos fueron sometidos a dieta alcohólica con etanol al 6 por ciento (v/v). A los 5, 10 y 15 meses tras el inicio del tratamiento, fueron recolectadas muestras de las glándulas submandibulares para analizar el área de los ácinos seromucosos y conductos granulosos. Los resultados indicaron que el consumo crónico de etanol reduce significativamente el área de las células de los acinos seromucosos y de las células de los conductos granulosos. Además, hubo un ensanchamiento en el área de los acinos seromucosos y de los conductos granulosos tras el consumo crónico de etanol. Concluimos que los efectos del alcohol fueron más graves mientras mayor fue el periodo de tratamiento

Animals , Rats , Alcoholism , Ethanol , Submandibular Gland , Submandibular Gland/pathology , Rats, Wistar
Rev. chil. anat ; 16(2): 229-35, 1998. ilus
Article in English | LILACS | ID: lil-243967


Cuarenta ratas machos adultas se dividieron en dos grupos de 20 animales cada uno (grupos control y experimentales). El grupo experimental recibió alcohol de caña de azúcar comercial, marca "51 Pirassununga", diluida a 30º g/L como dieta líquida por un período de 240 días y agua potable por los siguientes 60 días. Los animales se sacrificaron después de 60, 120, 180, 240 y 300 días de tratamiento. Se observaron varios cambios morfológicos en el grupo experimental de animales de hasta 240 días de tratamiento, encontrándose dilatación de las cisternas del retículo endoplasmático rugoso, espacios intercelulares aumentados, espacios entre las interdigitaciones basolaterales de las células, inflamación del tejido conjuntivo y presencia de una gran cantidad de células pinocíticas, de vacuolas digestivas y de vesículas con contenido electrolúcido a lo largo del citoplasma. Después de 300 días de tratamiento, el grupo experimental de animales presentó una regresión importante de los cambios descritos. No se observó cambios en el grupo de animales control

Animals , Rats , Alcoholism/etiology , Choroid Plexus/ultrastructure , Choroid Plexus/anatomy & histology , Microscopy, Electron , Cerebral Ventricles/ultrastructure
Rev. chil. anat ; 16(1): 59-66, 1998. ilus
Article in English | LILACS | ID: lil-242633


En el mono Cebus apella, las glándulas salivales submandibulares se localizan en la área cervical, lateralmente a tráquea y están constituidas morfológicamente por cuatro tipos de células parenquimatosas que forman diferentes estructuras: 1. Acinos: que presentan células secretoras seromucosas, mucosas y mioepiteliales. Las células secretoras tienen retículo endoplásmico rugoso y complejo de Golgi desarrollados lo que permite a éstas una gran capacidad de síntesis, además presentan una acumulación considerable de gránulos de secreción. 2. Ductos intercalares: se caracterizan por tener células epiteliales cuboidales simples y células mioepiteliales. Los ductos conectan las células secretoras a los ductos granulosos y presentan escencialmente una función excretora. 3. Ductos estriados: consisten en células columnares altas con pliegues extensos en su membrana en las zonas basal y lateral. Presentan una cantidad pequeña de retículo endoplásmico rugoso y de gránulos, lo que sugiere una función de transporte de iones y agua. 4. Ductos excretores: constituidos por células columnares altas y forman la porción final de la vía excretora

Humans , Submandibular Gland/ultrastructure , Haplorhini/anatomy & histology , Submandibular Gland/cytology , Submandibular Gland , Microscopy, Electron/methods