ABSTRACT
Green propolis produced by Apis melífera bees, having Baccharis dracunculifolia D.C. (Asteraceae) as the primary botanical source, has been used in traditional medicine to treat numerous disorders. However, studies evaluating propolis' potential in treating cardiovascular diseases via its effects on cholesterol metabolism are lacking. Therefore, this study investigated the effects of green propolis extracts on lipid metabolism in hypercholesterolemic guinea pigs. Chemical characterization of ethanolic extracts of green propolis samples was undertaken using HPLC. The in vitro characterization included an evaluation of the antioxidant capacity of the hydroalcoholic extract of green propolis (DPPH and FRAP assays) and its ability to act as an inhibitor of the HMG-CoA reductase enzyme. In vivo, we investigated the effect of the hydroalcoholic extract of green propolis on lipid metabolism in hypercholesterolemic guinea pigs. Results obtained validated previous reports of significant antioxidant activity. HPLC analysis confirmed that coumaric acid, artepillin C, and baccharin were the most common and abundant compounds in green propolis samples among the studied compounds. Furthermore, the compounds in these extracts acted as effective HMG-CoA reductase inhibitors in vitro. In vivo assays demonstrated that a hypercholesterolemic diet significantly reduced serum levels of the HDL cholesterol fraction. Simvastatin and propolis hydroalcoholic extracts promoted a significant increase in HDL cholesterol, suggesting that these extracts can improve the serum lipid profile of hypercholesterolemic guinea pigs. Results obtained in this study provide a perspective on the possible hypocholesterolemic effect of green propolis, suggesting that it can improve the serum lipid profile in hypercholesterolemic guinea pigs.
Subject(s)
Propolis , Guinea Pigs , Animals , Propolis/chemistry , Brazil , Cholesterol, HDL , Plant Extracts/pharmacology , Antioxidants , Lipid Metabolism , HomeostasisABSTRACT
Oxidative stress (OS) is a major disruption in the physiology of the lacrimal functional unit (LFU). Antioxidant enzymes have dual protective activities: antioxidant and antimicrobial activities. Peroxidases have been indistinctly used as markers of the secretory activity of the LFU and implicated in the pathophysiology, diagnosis and treatment of dry eye disease (DED), even though they comprise a large family of enzymes that includes lactoperoxidase (LPO) and glutathione peroxidase (GPO), among others. Assays to measure and correlate OS with other local LFU phenomena have methodological limitations. Studies implicate molecules and reactions involved in OS as markers of homeostasis, and other studies identify them as part of the physiopathology of diseases. Despite these conflicting concepts and observations, it is clear that OS is influential in the development of DED. Moreover, many antioxidant strategies have been proposed for its treatment, including calorie restriction to nutritional supplementation. This review offers a critical analysis of the biological mechanisms, diagnostic outcomes, drug use, dietary supplements, and life habits that implicate the influence of OS on DED.
ABSTRACT
ABSTRACT Purpose: This study aimed to compare the changes in the lacrimal functional unit in the following two models of neurogenic dry eye syndrome: sensory denervation of the cornea versus autonomic denervation of the lacrimal gland. Methods: The neural network supports the lacrimal functional unit. It can be divided into afferent (sensory) and efferent (autonomic) pathways and is affected by severe diseases that compromise the lacrimal functional unit. Male Wistar, 8-week-old rats were divided into the following three groups: 1) control naïve (n=16 animals); 2) autonomic denervation: where rats were subjected to right lacrimal gland nerve ablation and evaluated after 1 and 2 months (1M and 2M) after the procedure (n=7 animals per subgroup, autonomic denervation 1M and autonomic denervation 2M, respectively); 3) sensory denervation induced by 0.2% benzalkonium chloride eye drops, twice a day for 7 days in the right eye (n=10 animals). The corneal sensitivity was measured using the eye wipe test with capsaicin (10 µM). The quantitative real-time PCR was performed to compare the mRNA expressions of proinflammatory cytokines, such as Il-1β, Il-6, Tnf, Mmp9, in the cornea, trigeminal ganglion, and lacrimal gland. In addition, the mRNA of the promitotic factors in the lacrimal gland, such as Bmp7, Runx1, Runx3, Fgf10, and Smad1, was compared. Results: Sensory denervation induced corneal hyperalgesia (p=0.001). Sensory denervation and autonomic denervation increased the mRNA of proinflammatory cytokines in the cornea and lacrimal gland (p<0.05), but only sensory denervation increased the mRNA levels of Il-1β and Tnf in the trigeminal ganglion (p<0.05) compared with the control naïve. Conclusions: Autonomic denervation and sensory denervation models can have common features, such as inflammation of different parts of the lacrimal functional unit. However, hyperesthesia and inflammatory markers in the trigeminal ganglion because of sensory denervation and the expression of regenerative mediators in the lacrimal gland owing to autonomic denervation are the distinguishing features of these diseases that can be explored in future studies assessing dry eye syndrome secondary to neural damage of the lacrimal functional unit.
RESUMO Objetivo: O nosso objetivo neste estudo foi comparar as alterações na unidade funcional lacrimal em dois modelos de síndrome do olho seco neurogênica: desnervação sensorial da córnea versus desnervação autonômica da glândula lacrimal. Métodos: A rede neural é um importante suporte para a unidade funcional lacrimal. Pode ser dividido em vias aferentes (sensoriais) e eferentes (autonômicas), sujeitas a doenças graves que comprometem a unidade funcional lacrimal. Ratos Wistar machos, com 8 semanas de idade, foram divididos em três grupos: 1) Controle naïve (n=16 animais); 2) Desnervação autonômica: onde os ratos foram submetidos à ablação do nervo da glândula lacrimal direita e avaliados após um e dois meses (1 M a 2 M) do procedimento (n=7 animais por subgrupo, desnervação autonômica 1M e desnervação autonômica 2M, respectivamente); 3) Desnervação sensorial induzida por colírio a 0,2% de cloreto de benzalcônio, duas vezes ao dia por 7 dias no olho direito (n=10 animais). A sensibilidade da córnea foi medida pelo teste de movimento pata-olho com capsaicina (10 µM). A PCR quantitativa em tempo real foi aplicada para comparar a expressão relativa de mRNA de citocinas pró-inflamatórias: Il1b, Il6, Tnf, Mmp9, na córnea, gânglio trigêmio e glândula lacrimal. O mRNA dos agentes pró-mitóticos Bmp7, Runx1, Runx3, Fgf10 e Smad1 foram comparados na glândula lacrimal. Resultados: A desnervação sensorial induziu hiperalgesia da córnea (p=0,001). Desnervação sensorial e desnervação autonômica aumentaram o mRNA de citocinas pró-inflamatórias no córnea e glândula lacrimal (p<0,05), mas apenas desnervação sensorial aumentou o mRNA de Il1b e Tnf no gânglio trigêmio (p<0,05) quando comparado ao controle naïve. Conclusões: Os modelos de desnervação autonômica e desnervação sensorial podem ter características comuns, como inflamação de diferentes partes da unidade funcional lacrimal. No entanto, a hiperestesia e os marcadores inflamatórios no gânglio trigêmio de desnervação sensorial e a expressão de mediadores regenerativos na glândula lacrimal na desnervação autonômica são características que distinguem essas doenças, podendo ser investigadas em estudos futuros que abordam o olho seco secundário ao dano neural da unidade funcional lacrimal.
ABSTRACT
PURPOSE: This study aimed to compare the changes in the lacrimal functional unit in the following two models of neurogenic dry eye syndrome: sensory denervation of the cornea versus autonomic denervation of the lacrimal gland. METHODS: The neural network supports the lacrimal functional unit. It can be divided into afferent (sensory) and efferent (autonomic) pathways and is affected by severe diseases that compromise the lacrimal functional unit. Male Wistar, 8-week-old rats were divided into the following three groups: 1) control naïve (n=16 animals); 2) autonomic denervation: where rats were subjected to right lacrimal gland nerve ablation and evaluated after 1 and 2 months (1M and 2M) after the procedure (n=7 animals per subgroup, autonomic denervation 1M and autonomic denervation 2M, respectively); 3) sensory denervation induced by 0.2% benzalkonium chloride eye drops, twice a day for 7 days in the right eye (n=10 animals). The corneal sensitivity was measured using the eye wipe test with capsaicin (10 µM). The quantitative real-time PCR was performed to compare the mRNA expressions of proinflammatory cytokines, such as Il-1ß, Il-6, Tnf, Mmp9, in the cornea, trigeminal ganglion, and lacrimal gland. In addition, the mRNA of the promitotic factors in the lacrimal gland, such as Bmp7, Runx1, Runx3, Fgf10, and Smad1, was compared. RESULTS: Sensory denervation induced corneal hyperalgesia (p=0.001). Sensory denervation and autonomic denervation increased the mRNA of proinflammatory cytokines in the cornea and lacrimal gland (p<0.05), but only sensory denervation increased the mRNA levels of Il-1ß and Tnf in the trigeminal ganglion (p<0.05) compared with the control naïve. CONCLUSIONS: Autonomic denervation and sensory denervation models can have common features, such as inflammation of different parts of the lacrimal functional unit. However, hyperesthesia and inflammatory markers in the trigeminal ganglion because of sensory denervation and the expression of regenerative mediators in the lacrimal gland owing to autonomic denervation are the distinguishing features of these diseases that can be explored in future studies assessing dry eye syndrome secondary to neural damage of the lacrimal functional unit.
Subject(s)
Dry Eye Syndromes , Lacrimal Apparatus , Animals , Cornea/surgery , Denervation , Lacrimal Apparatus/surgery , Male , Rats , Rats, Wistar , TearsABSTRACT
Purpose: The aims of this work were a) to describe the histology of the lacrimal gland (LG) and cornea induced by an adenovirus (Ad) vector encoding the human erythropoietin (Epo) gene delivered to the LG and b) to evaluate the therapeutic potential of this strategy to prevent benzalkonium chloride (BAK) corneal toxicity.Methods: Structure and function of male Wistar rats LG were compared in the groups: 1) naïve control and 2) Ad-hEpo in the right LG (RLG). The protective response against BAK eye drops was compared among the groups 1) naïve control, 2) BAK in the right eye, 3) Ad-hEpo RLG + BAK and 4) Ad-hEpo in the right salivary gland (RSG)+BAK. Ad-hEpo groups received an injection of AdLTR2EF1a-hEPO (25 ul, 1010 particles/ml) in the right LG or SG (positive control). The BAK groups received 0.2% BAK in the right cornea twice a day. The tests applied after 7 days, included tear secretion, hEPO mRNA detection by qRT-PCR, LG and cornea histology, LG ELISA for cytokines and hematocrit.Results: hEPO mRNA was present in the Ad-hEpo RLG and RSG, but not kidney or liver samples (negative controls). TNF-α and IL-1ß increased in the LG exposed to Ad-hEpo compared to naïve control (p = .0115 and p = .0397, respectively). BAK reduced tear secretion, but this reduction was prevented by Ad-hEpo RLG+BAK and Ad-hEpo RSG+BAK (p = .017). The corneal epithelia were thinner in the BAK-treated groups independent of Ad-hEpo (p = .0009). Hematocrit increased only in the Ad-hEpo RSG group (p = .01).Conclusions: Ad-hEpo infection of rat LG and SG induces local, but only the SG infection induced systemic changes in rats. Importantly, Ad-hEpo attenuated the BAK-mediated toxic reduction in tear flow. Future studies must consider viral vector tissue tropism, biodistribution and effective therapeutic gene products for ocular surface diseases.
Subject(s)
Adenoviridae/genetics , Dry Eye Syndromes/therapy , Erythropoietin/genetics , Genetic Therapy/methods , Lacrimal Apparatus/diagnostic imaging , Animals , Benzalkonium Compounds/toxicity , Disease Models, Animal , Dry Eye Syndromes/chemically induced , Dry Eye Syndromes/diagnosis , Erythropoietin/metabolism , Genetic Vectors , Lacrimal Apparatus/drug effects , Lacrimal Apparatus/metabolism , Male , Rats , Rats, Wistar , Tears/metabolismABSTRACT
RESEARCH QUESTION: Is the profile of microRNA (miRNA) altered in cumulus cells of infertile women with early (EI/II) and advanced (EIII/IV) endometriosis? DESIGN: In this prospective case-control study, a miRNA profile including 754 targets was evaluated in samples of cumulus cells from infertile women with endometriosis (5 EI/II, 5 EIII/IV) and infertile controls (5, male and/or tubal factor) undergoing ovarian stimulation for intracytoplasmic sperm injection, using TaqMan® Array Human MicroRNA Cards A and B. The groups were compared with Kruskal-Wallis test, followed by Benjamini-Hochberg correction and Dunn's post hoc test. An in silico enrichment analysis was performed to list the possibly altered pathways in which the altered miRNA target genes are involved. RESULTS: Only the miRNA miR-532-3p showed significant differences among the analysed groups, being down-regulated in the EIII/IV group compared with the infertile control group, as well as compared with the EI/II group. The enrichment analysis showed that some genes regulated by this miRNA are involved in important pathways for the acquisition of oocyte competence, such as the oxytocin, calcium, Wnt, FoxO, ErbB and Ras signalling pathways, as well as the oocyte meiosis pathway. CONCLUSION: The present findings bring new perspectives to understanding the follicular microenvironment of infertile women with different stages of endometriosis. It is suggested that the dysregulation of miR-532-3p may be a potential mechanism involved in the aetiopathogenesis of endometriosis-related infertility. Further studies are needed to evaluate these pathways in cumulus cells of infertile women with the disease, as well as their impact on the acquisition of oocyte competence.
Subject(s)
Cumulus Cells/metabolism , Endometriosis/metabolism , Infertility, Female/metabolism , MicroRNAs/metabolism , Adult , Case-Control Studies , Endometriosis/complications , Female , Humans , Infertility, Female/etiology , Prospective StudiesABSTRACT
PURPOSE: Collagen deposition and myofibroblast differentiation are critical factors related to excessive scarring in ocular surgeries. This study evaluated the anti-fibrotic activity of rosmarinic acid on rabbit Tenon's capsule fibroblasts stimulated with transforming growth factor- ß2. METHODS: Primary cultures of rabbit Tenon's capsule fibroblasts were treated with various concentrations of rosmarinic acid for 12 h, in the presence and absence of transforming growth factor-ß2. After 48 h, the proliferation index of rabbit Tenon's capsule fibroblasts and the differentiation of myofibroblasts were investigated through immunofluorescence staining for proliferating cell nuclear antigen and alpha smooth muscle actin. An automated cell counter and colorimetric metabolic activity assay were used to evaluate cell number and viability. Collagen expression and production were determined by quantitative real-time polymerase chain reaction and hydroxyproline assay, respectively. RESULTS: Unstimulated rabbit Tenon's capsule fibroblasts treated with any concentration of rosmarinic acid exhibited diminished collagen expression (p<0.01) but showed no differences in proliferation index. Transforming growth factor-ß2 exposure induced myofibroblast differentiation and increased collagen production. Exposure to rosmarinic acid at 1.0 and 3.0 µM concentrations reduced the proliferation index (p<0.02), as well as the collagen expression and hydroxyproline content (p<0.05). Exposure to 3.0 µM rosmarinic acid reduced viability (p=0.035) in unstimulated rabbit Tenon's capsule fibroblasts and cell numbers (p=0.001) in both stimulated and unstimulated rabbit Tenon's capsule fibroblast cultures. CONCLUSIONS: Exposure to 1.0 µM rosmarinic acid was noncytotoxic and led to reduced collagen expression and proliferation of stimulated rabbit Tenon's capsule fibroblasts. These findings suggest that rosmarinic acid is a relatively non-injurious anti-fibrotic compound to rabbit Tenon's capsule fibroblasts, with potential application as an adjunctive agent in ocular procedures, particularly in glaucoma surgeries.
Subject(s)
Tenon Capsule , Animals , Cells, Cultured , Cinnamates , Depsides , Fibroblasts , Glaucoma , Rabbits , Rosmarinic AcidABSTRACT
ABSTRACT Purpose: Collagen deposition and myofibroblast differentiation are critical factors related to excessive scarring in ocular surgeries. This study evaluated the anti-fibrotic activity of rosmarinic acid on rabbit Tenon's capsule fibroblasts stimulated with transforming growth factor- β2. Methods: Primary cultures of rabbit Tenon's capsule fibroblasts were treated with various concentrations of rosmarinic acid for 12 h, in the presence and absence of transforming growth factor-β2. After 48 h, the proliferation index of rabbit Tenon's capsule fibroblasts and the differentiation of myofibroblasts were investigated through immunofluorescence staining for proliferating cell nuclear antigen and alpha smooth muscle actin. An automated cell counter and colorimetric metabolic activity assay were used to evaluate cell number and viability. Collagen expression and production were determined by quantitative real-time polymerase chain reaction and hydroxyproline assay, respectively. Results: Unstimulated rabbit Tenon's capsule fibroblasts treated with any concentration of rosmarinic acid exhibited diminished collagen expression (p<0.01) but showed no differences in proliferation index. Transforming growth factor-β2 exposure induced myofibroblast differentiation and increased collagen production. Exposure to rosmarinic acid at 1.0 and 3.0 µM concentrations reduced the proliferation index (p<0.02), as well as the collagen expression and hydroxyproline content (p<0.05). Exposure to 3.0 µM rosmarinic acid reduced viability (p=0.035) in unstimulated rabbit Tenon's capsule fibroblasts and cell numbers (p=0.001) in both stimulated and unstimulated rabbit Tenon's capsule fibroblast cultures. Conclusions: Exposure to 1.0 µM rosmarinic acid was noncytotoxic and led to reduced collagen expression and proliferation of stimulated rabbit Tenon's capsule fibroblasts. These findings suggest that rosmarinic acid is a relatively non-injurious anti-fibrotic compound to rabbit Tenon's capsule fibroblasts, with potential application as an adjunctive agent in ocular procedures, particularly in glaucoma surgeries.
RESUMO Objetivo: A deposição de colágeno e a diferenciação de miofibroblastos são fatores chaves relacionados à cicatrização excessiva em cirurgias oculares. Este estudo avaliou a atividade anti-fibrótica do ácido rosmarínico nos fibroblastos da cápsula de Tenon de coelhos estimulados com o fator de crescimento transformador-β2. Métodos: Culturas primárias de fibroblastos da cápsula de Tenon de coelhos foram tratadas com várias concentrações de ácido rosmarínico por 12h, na presença e na ausência do fator de crescimento transformador-β2. Após 48h, o índice de proliferação dos fibroblastos da cápsula de Tenon de coelhos e a diferenciação dos miofibroblastos foram investigados por coloração por imunofluorescência para proliferação de antígeno nuclear celular e α-actina de músculo liso, respectivamente. Um contador automático de células e um ensaio de atividade metabólica colorimétrica foram utilizados para avaliar o número e a viabilidade das células. A expressão e produção do colágeno foram determinadas por reação quantitativa em cadeia da polimerase em tempo real e ensaio de hidroxiprolina, respectivamente. Resultados: Fibroblastos da cápsula de Tenon de coelhos não estimulados tratados com qualquer concentração de ácido rosmarínico exibiram diminuição de colágeno (p<0,01), mas não mostraram diferenças no índice de proliferação. A exposição ao fator de crescimento transformador-β2 induziu a diferenciação de miofibroblastos e aumentou a produção de colágeno. A exposição ao ácido rosmarínico nas concentrações de 1,0 e 3,0 µM reduziu o índice de proliferação (p<0,02), bem como a expressão de colágeno e a quantificação de hidroxiprolina (p<0.05). A exposição a 3,0 µM de ácido rosmarínico reduziu a viabilidade (p=0,035) de fibroblastos da cápsula de Tenon de coelhos não estimulados e o número de células (p=0,001) em culturas de fibroblastos da cápsula de Tenon de coelhos estimuladas e não estimuladas. Conclusões: A exposição ao ácido rosmarínico 1,0 µM foi não citotóxica e levou à expressão reduzida de colágeno e menor proliferação de fibroblastos da cápsula de Tenon estimulados pelo fator de crescimento transformador-β2. Esses achados sugerem que o ácido rosmarínico é um composto antifibrótico relativamente não lesivo aos fibroblastos da cápsula de Tenon de coelhos, com potencial aplicação como agente adjuvante em procedimentos oculares, particularmente em cirurgias de glaucoma.
Subject(s)
Animals , Tenon Capsule , Rabbits , Cells, Cultured , Glaucoma , Cinnamates , Depsides , FibroblastsABSTRACT
PURPOSE: We aimed to determine the effects of different intensities of acute exercise on Hcy plasma levels, and the exercise-induced changes in Hcy liver metabolism. METHOD: First, thirty-two Wistar rats were randomly submitted to an acute bout of swimming exercise carrying a load of 2% (n=8), 4% (n=8) and 6% (n=8) of their total body weight attached in their tail. Control rats remained rested (n=8). Blood samples were taken from tail vein for plasma S-containing amino acids determination before (Rest) and post, 1, 2, 3, 4, 6, and 10h after acute swimming exercise. Second, 56 exercised rats (4% loads) were euthanized before (Rest) and1, 2, 3, 4, 6, and 10h after acute swimming exercise. Blood and liver samples were collected for amino acids and keys genes involved in the Hcy metabolism assay. RESULTS: Acute exercise increases (P<0.05) plasma Hcy concentration in an intensity-dependent manner (rest 7.7±0.8; 6% load 13.8±3.6; 4% load 12.2±2.9±and 2% load 10.1±2.6, µmol/L); this increase is transient and does not promote hyperhomocysteinemia (<15µmol/L).Exercise-induced increased plasma Hcywas accompanied by the decreased liver S-adenosylmethionine/S-adenosylhomocysteine ratio and elevated MAT1a mRNA content. Acute exercise also caused elevated mRNA of key enzymes of transsulfuration (CBS) and remethylation (BHMT and the MTRR). CONCLUSION: Our data provided evidence that acute exercise increases plasma Hcy concentration due to the augmented requirement for methylated compounds that increases liver SAM consumption. Also, Hcy remethylation and transsulfuration are coordinately regulated to maintain methyl balance.
Subject(s)
Homocysteine/blood , Liver/metabolism , Physical Conditioning, Animal/physiology , Animals , Body Weight/drug effects , Hyperhomocysteinemia/metabolism , Male , Methionine Adenosyltransferase/metabolism , Methylation , Rats , Rats, Wistar , S-Adenosylhomocysteine/metabolism , S-Adenosylmethionine/metabolism , Swimming/physiologyABSTRACT
Abstract Introduction: Salicylate at high doses induces tinnitus in humans and experimental animals. However, the mechanisms and loci of action of salicylate in inducing tinnitus are still not well known. The expression of Immediate Early Genes (IEG) is traditionally associated with long-term neuronal modifications but it is still not clear how and where IEGs are activated in animal models of tinnitus. Objectives: Here we investigated the expression of c-fos and Egr-1, two IEGs, in the Dorsal Cochlear Nucleus (DCN), the Inferior Colliculus (IC), and the Posterior Ventral Cochlear Nucleus (pVCN) of rats. Methods: Rats were treated with doses known to induce tinnitus in rats (300 mg/kg i.p. daily, for 3 days), and c-fos and Egr-1 protein expressions were analyzed using western blot and immunocytochemistry. Results: After administration of salicylate, c-fos protein expression increased significantly in the DCN, pVCN and IC when assayed by western blot. Immunohistochemistry staining showed a more intense labeling of c-fos in the DCN, pVCN and IC and a significant increase in c-fos positive nuclei in the pVCN and IC. We did not detect increased Egr-1 expression in any of these areas. Conclusion: Our data show that a high dose of salicylate activates neurons in the DCN, pVCN and IC. The expression of these genes by high doses of salicylate strongly suggests that plastic changes in these areas are involved in the genesis of tinnitus.
Resumo Introdução: Salicilato em doses elevadas induz zumbido nos seres humanos e em animais experimentais. No entanto, os mecanismos e loci de ação do salicilato na indução de zumbido ainda não são bem conhecidos. A expressão dos genes precoces imediatos (GPIs) está tradicionalmente associada a alterações neuronais em longo prazo, mas ainda não está claro como e onde os GPIs são ativados em modelos animais de zumbido. Objetivos: No presente estudo investigamos a expressão de c-fos e Egr-1, dois GPIs, no núcleo coclear dorsal (NCD), colículo inferior (CI) e núcleo coclear ventral posterior (NCVp) de ratos. Métodos: Os ratos foram tratados com doses que, conhecidamente, induzem zumbido em ratos (300 mg/kg IP/dia, por três dias) e as expressões das proteínas c-fos e Egr-1 foram analisadas por meio de Western blot e imunoistoquímica. Resultados: Após a administração de salicilato, a expressão da proteína c-fos aumentou significativamente no NCD, NCVp e CI, quando analisados por Western blot. A coloração imunoistoquímica mostrou uma marcação mais intensa de c-fos no NCD, NCVp e CI e um aumento significativo de núcleos positivos de c-fos no NCVp e CI. Não detectamos aumento da expressão de Egr-1 em qualquer dessas áreas. Conclusão: Nossos dados mostram que uma dose alta de salicilato ativa neurônios no NCD, NCVp e CI. A expressão desses genes por doses altas de salicilato sugere que as alterações plásticas nessas áreas estão envolvidas na gênese do zumbido.
Subject(s)
Animals , Male , Rats , Inferior Colliculi/drug effects , Salicylates/pharmacology , Gene Expression Regulation/drug effects , Genes, Immediate-Early/drug effects , Cochlear Nucleus/drug effects , Salicylates/administration & dosage , Blotting, Western , Genes, fos/drug effects , Rats, Wistar , Dose-Response Relationship, Drug , Early Growth Response Protein 1/drug effectsABSTRACT
INTRODUCTION: Salicylate at high doses induces tinnitus in humans and experimental animals. However, the mechanisms and loci of action of salicylate in inducing tinnitus are still not well known. The expression of Immediate Early Genes (IEG) is traditionally associated with long-term neuronal modifications but it is still not clear how and where IEGs are activated in animal models of tinnitus. OBJECTIVES: Here we investigated the expression of c-fos and Egr-1, two IEGs, in the Dorsal Cochlear Nucleus (DCN), the Inferior Colliculus (IC), and the Posterior Ventral Cochlear Nucleus (pVCN) of rats. METHODS: Rats were treated with doses known to induce tinnitus in rats (300mg/kg i.p. daily, for 3 days), and c-fos and Egr-1 protein expressions were analyzed using western blot and immunocytochemistry. RESULTS: After administration of salicylate, c-fos protein expression increased significantly in the DCN, pVCN and IC when assayed by western blot. Immunohistochemistry staining showed a more intense labeling of c-fos in the DCN, pVCN and IC and a significant increase in c-fos positive nuclei in the pVCN and IC. We did not detect increased Egr-1 expression in any of these areas. CONCLUSION: Our data show that a high dose of salicylate activates neurons in the DCN, pVCN and IC. The expression of these genes by high doses of salicylate strongly suggests that plastic changes in these areas are involved in the genesis of tinnitus.
Subject(s)
Cochlear Nucleus/drug effects , Gene Expression Regulation/drug effects , Genes, Immediate-Early/drug effects , Inferior Colliculi/drug effects , Salicylates/pharmacology , Animals , Blotting, Western , Dose-Response Relationship, Drug , Early Growth Response Protein 1/drug effects , Genes, fos/drug effects , Male , Rats , Rats, Wistar , Salicylates/administration & dosageABSTRACT
OBJECTIVE: The aim of this study was to investigate changes in homocysteine (Hcy) metabolism and redox balance in response to exercise treatment in a tumor-bearing rat model. METHODS: Male Wistar rats were exposed, or not, to a resistance exercise program 6 wk before inoculation with Walker-256 tumor cells or vehicle. After application, rats maintained their routine for 12 d and were then sacrificed for plasma and liver analyses. RESULTS: Impaired Hcy metabolism was evident after 12 d of tumor cell inoculation as demonstrated by significantly increased (P < 0.05) plasma total homocysteine (tHcy) concentration (53%) and decreased plasma cysteine, methionine, and vitamin B12 concentrations. Decreased hepatic cystathionine ß-synthase (CBS) and betaine-homocysteine S-methyltransferase mRNA levels were found in tumor-bearing rats but not in controls. Tumor inoculation also decreased levels of liver reduced glutathione (GSH) and increased hepatic oxidative stress compared with non-tumor controls. However, resistance exercise prevented the tumor-impaired transsulfuration pathway as demonstrated by the decreased plasma tHcy, hepatic CBS expression, and increased GSH in tumor-exercised versus tumor-sedentary rats. Remarkably, all measures of liver oxidative stress were suppressed by exercise training. Tumor weight was unchanged between groups. CONCLUSION: Resistance exercise prevented tHcy accumulation and liver oxidative damage caused by Walker-256 tumor cell inoculation; the modulatory effects of resistance exercise on Hcy metabolism appear to be at the level of transsulfuration pathway.
Subject(s)
Carcinoma 256, Walker/metabolism , Carcinoma 256, Walker/therapy , Homocysteine/metabolism , Liver/metabolism , Physical Conditioning, Animal/methods , Animals , Glutathione/metabolism , Male , Oxidation-Reduction , Oxidative Stress , Rats , Rats, WistarABSTRACT
The purpose of this study was to investigate (1) the impact of tumor growth on homocysteine (Hcy) metabolism, liver oxidative stress and cancer cachexia and, (2) the potential benefits of creatine supplementation in Walker-256 tumor-bearing rats. Three experiments were conducted. First, rats were killed on days 5 (D5), 10 (D10) and 14 (D14) after tumor implantation. In experiment 2, rats were randomly assigned to three groups designated as control (C), tumor-bearing (T) and tumor-bearing supplemented with creatine (TCr). A life span experiment was conducted as the third experiment. Creatine was supplied in drinking water for 21 days (8 g/L) in all cases. Tumor implantation consisted of a suspension of Walker-256 cells (8.0 × 10(7) cells in 0.5 mL of PBS). The progressive increase (P < 0.05) in tumor mass coincided with a progressively lower body weight and higher hepatic oxidative stress; plasma Hcy concentration was 80 % higher (P < 0.05) by 10 days of tumor implantation. Impaired Hcy metabolism was evidenced by decreased hepatic betaine-homocysteine methyltransferase (Bhmt), glycine N-methyltransferase (Gnmt) and cystathionine beta synthase (CBS) gene expression. In contrast, creatine supplementation promoted a 28 % reduction of tumor weight (P < 0.05). Plasma Hcy (C 6.1 ± 0.6, T 10.3 ± 1.5, TCr 6.3 ± 0.9, µmol/L) and hepatic oxidative stress were lower in the TCr group compared to T. Creatine supplementation was unable to decrease Hcy concentration and to increase SAM/SAH ratio in tumor tissue. These data suggest that creatine effects on hepatic impaired Hcy metabolism promoted by tumor cell inoculation are responsible to decrease plasma Hcy in tumor-bearing rats. In conclusion, Walker-256 tumor growth is associated with progressive hyperhomocysteinemia, body weight loss and liver oxidative stress in rats. Creatine supplementation, however, prevented these tumor-associated perturbations.
Subject(s)
Cachexia , Creatine/pharmacology , Hyperhomocysteinemia , Neoplasms, Experimental , Oxidative Stress/drug effects , Animals , Cachexia/drug therapy , Cachexia/metabolism , Cachexia/pathology , Creatine/pharmacokinetics , Hyperhomocysteinemia/metabolism , Hyperhomocysteinemia/pathology , Hyperhomocysteinemia/prevention & control , Male , Neoplasms, Experimental/drug therapy , Neoplasms, Experimental/metabolism , Neoplasms, Experimental/pathology , Rats , Rats, WistarABSTRACT
Purpose: Describe an animal model of dry induced by topical instillation of BAK and evaluate ocular surface biomarkers and histological findings. Methods: Male Wistar rats were used.Topical instillation of 0.2% BAK eyedrops twice a day during 7 days, in the right eye of each animal, while the other eye was taken as control. After 7 days treatment, we performed evaluation of tear film osmolarity, the red phenol thread and ocular surface staining with fluorescein and lissamine green. Afterwards, the animals were sacrificed for tissue extraction and histological evaluation under optical microscopy and H&E staining. Results: Compared with untreated controls, the BAK-group presented tear secretion significantly decreased, increased ocular surface staining by fluorescein and lissamine green and tear film hyperosmolarity (p <0,05). Histological evaluation revealed epithelial thinning and estromal oedema. Conclusions: A toxicity animal model of dry eye induced by topical instillation of benzalkonium chloride, which presents corneal and ocular surface alterations, decreased tear film volume and tear hyperosmolarity as seen in dry eye condition.
Objetivo: Descrever um modelo animal de olho seco induzido pela aplicação tópica de cloreto de benzalcônio (BAC) e avaliar marcadores de integridade da superfície ocular e os achados histológicos. Métodos: Foram utilizados ratos wistar machos adultos. Foi realizada a administração tópica de colírio de BAC 0,2% no olho direito de cada animal duas vezes por dia, durante 7 dias, sendo o olho contralateral tido como controle. Após o tratamento foi realizada a avaliação da osmolaridade do filme lacrimal, o teste de fenol vermelho e a coloração com fluoresceína e lisamina verde. Os animais foram sacrificados e os tecidos extraídos para o estudo histológico da córnea, por microscopia óptica, corada com hematoxilina eosina (H&E). Resultados: Comparados com os controles não tratados o grupo BAC apresentou diminuição significativa na secreção lacrimal, defeitos na integridade epitelial da superfície ocular marcada por corantes vitais, fluoresceína e lisamina verde além do aumento da osmolaridade do filme lacrimal (p < 0,05). À avaliação histológica observou-se diminuição da espessura do epitélio e edema estromal induzidos pela aplicação de BAC. Conclusão: O modelo animal de olho seco por toxicidade induzido pela aplicação tópica de cloreto de benzalcônio apresentou alterações estruturais da córnea e da superfície ocular, diminuição do volume lacrimal e hiperosmolaridade da lágrima características dessa condição.
Subject(s)
Animals , Rats , Benzalkonium Compounds/toxicity , Tears/metabolism , Models, Animal , Dry Eye Syndromes/chemically induced , Osmolar Concentration , Rats, WistarABSTRACT
AIM: To examine the effects of creatine (Cr) supplementation on liver fat accumulation in rats fed a choline-deficient diet. METHODS: Twenty-four rats were divided into 3 groups of 8 based on 4 weeks of feeding an AIN-93 control diet (C), a choline-deficient diet (CDD) or a CDD supplemented with 2% Cr. The CDD diet was AIN-93 without choline. RESULTS: The CDD significantly increased plasma homocysteine and TNFα concentration, as well as ALT activity. In liver, the CDD enhanced concentrations of total fat (55%), cholesterol (25%), triglycerides (87%), MDA (30%), TNFα (241%) and decreased SAM concentrations (25%) and the SAM/SAH ratio (33%). Cr supplementation prevented all these metabolic changes, except for hepatic SAM and the SAM/SAH ratio. However, no changes in PEMT gene expression or liver phosphatidylcholine levels were observed among the three experimental groups, and there were no changes in hepatic triglyceride transfer protein (MTP) mRNA level. On the contrary, Cr supplementation normalized expression of the transcription factors PPARα and PPARγ that were altered by the CDD. Further, the downstream targets and fatty acids metabolism genes, UCP2, LCAD and CPT1a, were also normalized in the Cr group as compared to CDD-fed rats. CONCLUSION: Cr supplementation prevented fat liver accumulation and hepatic injures in rats fed with a CDD for 4 weeks. Our results demonstrated that one-carbon metabolism may have a small role in mitigating hepatic fat accumulation by Cr supplementation. The modulation of key genes related to fatty acid oxidation pathway suggests a new mechanism by which Cr prevents liver fat accumulation.
Subject(s)
Carbon/metabolism , Creatine/administration & dosage , Dietary Supplements , Fatty Liver/prevention & control , Lipid Metabolism/drug effects , Animals , Carnitine O-Palmitoyltransferase/genetics , Carnitine O-Palmitoyltransferase/metabolism , Cholesterol/metabolism , Choline/administration & dosage , Choline Deficiency , Diet , Ion Channels/genetics , Ion Channels/metabolism , Liver/drug effects , Liver/metabolism , Male , Malondialdehyde/metabolism , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , PPAR alpha/genetics , PPAR alpha/metabolism , PPAR gamma/genetics , PPAR gamma/metabolism , Rats , Rats, Wistar , S-Adenosylmethionine/metabolism , Triglycerides/metabolism , Uncoupling Protein 2ABSTRACT
AIMS: The aim of this study is to examine the effects of taurine supplementation on homocysteine (Hcy) metabolism and liver injury in rats fed a choline-deficient diet. MAIN METHODS: Thirty rats were divided into three groups (n=10), to receive one of the following diets for 4 weeks: control diet (C), choline-deficient diet (CDD), or choline-deficient diet supplemented with taurine (CDDT). The CDD and the CDDT consisted of AIN-93 without the recommended choline content of 2.5%, and the CDDT was supplemented by the addition of 2.5% taurine. KEY FINDINGS: Four weeks of ingesting a CDD resulted in a significant increase in plasma Hcy (50%) as well as a decrease in liver S-adenosylmethionine (SAM) concentration and S-adenosylmethionine/S-adenosylhomocysteine ratio. No changes were found in plasma methionine and cysteine plasma levels compared to control group. Four weeks of ingesting a CDD also caused a significant (P<0.05) increase in hepatic total fat, hepatic malondialdehyde (MDA), and plasma alanine aminotransferase (ALT) levels. In addition, reduced hepatic glutathione (GSH) levels and reduced/oxidized glutathione ratios (GSH/GSSG) were found in rats fed a CDD compared to controls. Taurine supplementation of the CDD normalized genes involved in the remethylation pathway, BHMT and CHDH, which were impaired by CDD alone. However, taurine supplementation failed to prevent CDD-induced Hcy metabolism disturbances and hepatic injury. Also, taurine added to CDD caused decreased expression of PEMT, CHKa, and CHKb, key genes involved in phosphatidylcholine (PC) synthesis and liver fat accumulation. SIGNIFICANCE: Taurine supplementation failed to ameliorate impaired Hcy metabolism and liver injury caused by CDD intake.
Subject(s)
Choline Deficiency/drug therapy , Diet , Dietary Supplements , Homocysteine/blood , Liver/drug effects , Taurine/pharmacology , Alanine Transaminase/blood , Analysis of Variance , Animals , Choline Deficiency/pathology , Cysteine/blood , Gene Expression Regulation/drug effects , Liver/metabolism , Liver/pathology , Malondialdehyde/metabolism , Methionine/blood , RatsABSTRACT
Considerable effort has been invested in searching for less invasive methods of diagnosing endometriosis. Previous studies have indicated altered levels of the CALD1 gene (encoding the protein caldesmon) in endometriosis. The aims of our study were to investigate whether average CALD1 expression and caldesmon protein levels are differentially altered in the endometrium and endometriotic lesions and to evaluate the performance of the CALD1 gene and caldesmon protein as potential biomarkers for endometriosis. Paired biopsies of endometrial tissue (eutopic endometrium) and endometriotic lesions (ectopic endometrium) were obtained from patients with endometriosis to evaluate CALD1 gene expression and caldesmon protein levels by real-time PCR and Western blot analysis, respectively. In addition, immunostaining for caldesmon to determine cellular localization was also performed. Endometrium from women without endometriosis was used as a control. Increased CALD1 expression and caldesmon levels were detected in the endometriotic lesions. The electrophoretic profile of caldesmon by Western blot analysis was clearly different between the control group (endometrium of women without endometriosis) and the group of women with endometriosis (eutopic endometrium and endometriotic lesions). Caldesmon expression as determined by immunostaining showed no variation among the cell types in endometriotic lesions and eutopic endometrium. Stromal cells marked positively in eutopic endometrium from control patients and in the endometriotic lesions. The presence of caldesmon in the endometrium of patients with and without endometriosis permitted diagnoses with 95% sensitivity (specificity 100%) and 100% sensitivity (specificity 100%) for the disease and for minimal to mild endometriosis in the proliferative phase of the menstrual cycle, respectively. In the secretory phase, minimal to mild endometriosis was detected with 90% sensitivity and 93.3% specificity. Caldesmon is a possible predictor of endometrial dysregulation in patients with endometriosis. A potential limitation of our study is the fact that other endometrial diseases were not excluded, and therefore prospective studies are needed to confirm the potential of caldesmon as a biomarker exclusively for endometriosis.
Subject(s)
Calmodulin-Binding Proteins/metabolism , Endometriosis/diagnosis , Endometrium/metabolism , Ovarian Diseases/diagnosis , Peritoneal Diseases/diagnosis , Adult , Biomarkers/metabolism , Calmodulin-Binding Proteins/genetics , Case-Control Studies , Endometriosis/genetics , Endometriosis/metabolism , Endometrium/pathology , Female , Humans , Ovarian Diseases/genetics , Ovarian Diseases/metabolism , Peritoneal Diseases/genetics , Peritoneal Diseases/metabolism , Predictive Value of Tests , Real-Time Polymerase Chain Reaction , Sensitivity and Specificity , Stromal Cells/metabolism , Stromal Cells/pathologyABSTRACT
Adrenalectomy-induced hypophagia is associated with increased satiety-related responses, which involve neuronal activation of the nucleus of the solitary tract (NTS). Besides its effects on the pituitary-adrenal axis, corticotrophin-releasing factor (CRF) has been shown to play an important role in feeding behaviour, as it possesses anorexigenic effects. We evaluated feeding-induced CRF mRNA expression in the paraventricular nucleus (PVN) and the effects of pretreatment with CRF(2) receptor antagonist (Antisauvagine-30, AS30) on food intake and activation of NTS neurons in response to feeding in adrenalectomised (ADX) rats. Compared to the sham group, ADX increased CRF mRNA levels in the PVN of fasted animals, which was further augmented by refeeding. AS30 treatment did not affect food intake in the sham and ADX+corticosterone (B) groups; however, it reversed hypophagia in the ADX group. In vehicle-pretreated animals, refeeding increased the number of Fos and Fos/TH-immunoreactive neurons in the NTS in the sham, ADX and ADX+B groups, with the highest number of neurons in the ADX animals. Similarly to its effect on food intake, pretreatment with AS30 in the ADX group also reversed the increased activation of NTS neurons induced by refeeding while having no effect in the sham and ADX+B animals. The present results show that adrenalectomy induces an increase in CRF mRNA expression in the PVN potentiated by feeding and that CRF(2) receptor antagonist abolishes the anorexigenic effect and the increased activation of NTS induced by feeding in the ADX animals. These data indicate that increased activity of PVN CRF neurons modulates brainstem satiety-related responses, contributing to hypophagia after adrenalectomy.
