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Int. j. morphol ; 41(6): 1870-1880, dic. 2023. ilus, tab
Article in English | LILACS | ID: biblio-1528799


SUMMARY: The aim of this study is to reveal the gonadoprotective effects of myricetin (MYC), which has many biological properties, on cisplatin (CP)-induced testicular damage in rats. For this purpose, 40 male Wistar albino rats were divided into 4 groups as Control (group given no treatment), MYC (group given 5 mg/kg/i.p myricetin for 7 days), CP (group given 7 mg/kg/i.p cisplatin at 7th day) and MYC + CP (group given 5 mg/kg/i.p myricetin for 7 days before 7 mg/kg/i.p cisplatin injection). After administrations, testicular tissues of animals were extracted and processed according to tissue processing protocol. Hematoxylin & Eosin staining were performed to evaluate the histopathological changes and Johnsen'sTesticular Biopsy Score (JTBS) was applied and mean seminiferous tubule diameters (MSTD) were measured to compare experimental groups in terms of histopathological changes. Moreover, TLR4, NF-kB, HSP70 and HSP90 expression levels were detected by immunohistochemical staining and the density of immunoreactivity were measured to determine the difference in the expression levels of these factors among groups. Additionally, testicular apoptosis was detected via TUNEL assay. JTBS and MSTD data were significantly lower in CP group compared to other groups and MYC administrations significantly protects testicular tissue against CP-induced damage. Moreover, TLR4, NF-kB, HSP70 and HSP90 expressions and apoptotic cells significantly increased in the CP group (p<0.05). However, MYC administrations exerted a strong gonadoprotective effect on testicular tissue in terms of these parameters in MYC+CP group (p<0.05). According to our results, we suggested that MYC can be considered as a protective agent against cisplatin-induced testicular damage.

El objetivo de este estudio es revelar los efectos gonadoprotectores de la miricetina (MYC), que tiene muchas propiedades biológicas, sobre el daño testicular inducido por cisplatino (CP) en ratas. Para este propósito, se dividieron 40 ratas albinas Wistar macho en 4 grupos: Control (grupo que no recibió tratamiento), MYC (grupo que recibió 5 mg/kg/i.p de miricetina durante 7 días), CP (grupo que recibió 7 mg/kg/i.p de cisplatino al séptimo día) y MYC + CP (grupo que recibió 5 mg/ kg/i.p de miricetina durante 7 días antes de la inyección de 7 mg/ kg/i.p de cisplatino). Después de las administraciones, se extrajeron y procesaron tejidos testiculares de animales según el protocolo de procesamiento de tejidos. Se realizó tinción con hematoxilina y eosina para evaluar los cambios histopatológicos y se aplicó la puntuación de biopsia testicular de Johnsen (JTBS) y se midieron los diámetros medios de los túbulos seminíferos (MSTD) para comparar los grupos experimentales en términos de cambios histopatológicos. Además, los niveles de expresión de TLR4, NF-kB, HSP70 y HSP90 se detectaron mediante tinción inmunohistoquímica y se midió la densidad de inmunorreactividad para determinar la diferencia en los niveles de expresión de estos factores entre los grupos. Además, se detectó apoptosis testicular mediante el ensayo TUNEL. Los datos de JTBS y MSTD fueron significativamente más bajos en el grupo CP en comparación con otros grupos y las administraciones de MYC protegen significativamente el tejido testicular contra el daño inducido por CP. Además, las expresiones de TLR4, NF-kB, HSP70 y HSP90 y las células apoptóticas aumentaron significativamente en el grupo CP (p<0,05). Sin embargo, las administraciones de MYC ejercieron un fuerte efecto gonadoprotector sobre el tejido testicular en términos de estos parámetros en el grupo MYC+CP (p<0,05). Según nuestros resultados, sugerimos que MYC puede considerarse como un agente protector contra el daño testicular inducido por cisplatino.

Animals , Male , Rats , Testis/drug effects , Testis/injuries , Flavonoids/administration & dosage , Cisplatin/toxicity , Flavonoids/pharmacology , Immunohistochemistry , NF-kappa B , Rats, Wistar , Heat-Shock Response , In Situ Nick-End Labeling , Toll-Like Receptor 4 , Inflammation , Antineoplastic Agents/toxicity
Int. j. morphol ; 39(4): 1123-1131, ago. 2021. ilus, tab
Article in English | LILACS | ID: biblio-1385439


SUMMARY: Adriamycin (ADR) is an anthracycline antibiotic used for treatment of many types of cancer. However, its applications may damage to healthy tissues. Chloroquine (CLQ) is an anti-inflammatory agent used in treatment of many inflammation associated diseases such as malaria and rheumatoid arthritis. Moreover, it is used in the treatment of pneumonia caused by COVID-19. The aim of this study is to determine possible therapeutic effects of Chloroquine on Adriamycin-induced testicular toxicity in rats. We investigated the effect of CLQ on testicular injury caused by ADR. Rats were divided into four groups: Control, ADR, CLQ, ADR+CLQ. After administrations, animals were sacrificed, and testis tissues were extracted from the animals for the further examinations. Histopathological changes in testis tissues were evaluated and TNF-α and IL-6 immunostaining were performed to determine the expression levels of these cytokines. TUNEL method were used for evaluation of apoptotic index. Moreover, serum testosterone levels were measured by ELISA assay. We observed that ADR group showed histopathological deterioration when compared to the Control group and CLQ treatment ameliorated this damage induced by Adriamycin.An increase in TNF-α and IL-6 immunoreactivities and in the number of apoptotic cells and a decrease in serum testosterone levels were determined in the ADR group compared to the Control and CLQ group. Furthermore, our examinations showed an improvement in testicular tissue in ADR+CLQ group in terms of these parameters when compared to the ADR group. We suggest that CLQ can be used as a protective agent to reduce the toxic effects of Adriamycin as a result of its anti-inflammatory and anti-apoptotic properties.

RESUMEN: La adriamicina (ADR) es un antibiótico de antraciclina que se usa para el tratamiento de muchos tipos de cáncer. Sin embargo, sus aplicaciones pueden dañar los tejidos sanos. La cloroquina (CLQ) es un agente antiinflamatorio que se utiliza en el tratamiento de enfermedades asociadas a la inflamación, tal como la malaria y la artritis reumatoide. También se utiliza en el tratamiento de la neumonía causada por COVID-19. El objetivo de este estudio fue determinar los posibles efectos terapéuticos de la cloroquina sobre la toxicidad testicular inducida por adriamicina en ratas. Investigamos el efecto de CLQ sobre la lesión testicular causada por ADR. Las ratas se dividieron en cuatro grupos: Control, ADR, CLQ, ADR + CLQ. Después de las administraciones, se sacrificaron los animales y se extrajeron los testículos de los animales para los exámenes adicionales. Se evaluaron los cambios histopatológicos en los tejidos testiculares y se realizó la inmunotinción de TNF-α e IL-6 para determinar los niveles de expresión de estas citocinas. Se utilizó el método TUNEL para la evaluación del índice apoptótico. Además, los niveles de testosterona en suero se midieron mediante un ensayo ELISA. El grupo ADR mostró un deterioro histopatológico en comparación con el grupo Control y observamos que el tratamiento con CLQ mejoró el daño inducido por Adriamicina. Un aumento en las inmunorreactividades de TNF-α e IL-6 y en el número de células apoptóticas además de una disminución en los niveles séricos de testosterona se determinaron en el grupo de ADR en comparación con el grupo de control y CLQ. Además, nuestros exámenes mostraron una mejora en el tejido testicular en el grupo ADR + CLQ en términos de estos parámetros en comparación con el grupo ADR. Sugerimos que CLQ se puede utilizar como agente protector para reducir los efectos tóxicos de la Adriamicina, gracias a sus propiedades antiinflamatorias y antiapoptóticas.

Animals , Male , Rats , Testicular Diseases/chemically induced , Testicular Diseases/drug therapy , Doxorubicin/adverse effects , Chloroquine/administration & dosage , Enzyme-Linked Immunosorbent Assay , Immunohistochemistry , Interleukin-6 , Tumor Necrosis Factor-alpha , Rats, Wistar , Apoptosis/drug effects , In Situ Nick-End Labeling , Inflammation , Antibiotics, Antineoplastic/adverse effects
Rev. bras. anestesiol ; 70(6): 627-634, Nov.-Dec. 2020. graf
Article in English | LILACS | ID: biblio-1155766


Abstract Background and objectives The mechanisms by which local anesthetics cause neurotoxicity are very complicated. Apoptosis and autophagy are highly coordinated mechanisms that maintain cellular homeostasis against stress. Studies have shown that autophagy activation serves as a protective mechanism in vitro. However, whether it also plays the same role in vivo is unclear. The aim of this study was to explore the role of autophagy in local anesthetic-induced neurotoxicity and to elucidate the mechanism of neurotoxicity in an intrathecally injected rat model. Methods Eighteen healthy adult male Sprague-Dawley rats were randomly divided into three groups. Before receiving an intrathecal injection of 1% bupivacaine, each rat received an intraperitoneal injection of vehicle or rapamycin (1 once a day for 3 days. The pathological changes were examined by Haematoxylin and Eosin (HE) staining. Apoptosis was analysed by TdT-mediated dUTP Nick-End Labelling (TUNEL) staining. Caspase-3, Beclin1 and LC3 expression was examined by Immunohistochemical (IHC) staining. Beclin1 and LC3 expression and the LC3-II/LC3-I ratio were detected by western blot analysis. Results After bupivacaine was injected intrathecally, pathological damage occurred in spinal cord neurons, and the levels of apoptosis and caspase-3 increased. Enhancement of autophagy with rapamycin markedly alleviated the pathological changes and decreased the levels of apoptosis and caspase-3 while increasing the expression of LC3 and Beclin1 and the ratio of LC3-II to LC3-I. Conclusions Enhancement of autophagy decreases caspase-3-dependent apoptosis and improves neuronal survivalin vivo. Activation of autophagy may be a potential therapeutic strategy for local anaesthetic-induced neurotoxicity.

Resumo Introdução e objetivos Os mecanismos de neurotoxicidade dos anestésicos locais são complexos. A apoptose e a autofagia são mecanismos altamente organizados que mantêm a homeostase celular durante o estresse. Estudos revelam que a ativação da autofagia atua como mecanismo de proteção in vitro. Não está claro se a autofagia também desempenha essa função in vivo. O objetivo deste estudo foi analisar o papel da autofagia na neurotoxicidade induzida por anestésico local e esclarecer o mecanismo dessa neurotoxicidade utilizando um modelo de injeção intratecal em ratos. Métodos Dezoito ratos Sprague‐Dawley machos adultos saudáveis foram divididos aleatoriamente em três grupos. Antes de receber a injeção intratecal de bupivacaína a 1%, cada rato recebeu injeção intraperitoneal de veículo ou rapamicina (1‐1) uma vez ao dia durante 3 dias. As alterações patológicas foram examinadas por coloração com Hematoxilina e Eosina (HE). A apoptose foi analisada por coloração com o método dUTP Nick‐End Labeling (TUNEL) mediado por TdT. A expressão de caspase‐3, Beclin1 e LC3 foram examinadas por coloração Imunohistoquímica (IHQ). A expressão de Beclin1 e LC3 e a razão LC3‐II/LC3‐I foram detectadas por análise de western blot. Resultados Após a injeção intratecal de bupivacaína, ocorreu lesão patológica nos neurônios da medula espinhal e os níveis de apoptose e caspase‐3 aumentaram. A ativação da autofagia causada pela rapamicina mitigou de forma expressiva as alterações patológicas e diminuiu os níveis de apoptose e caspase‐3, aumentando a expressão de LC3 e Beclin1 e a razão LC3‐II/LC3‐I. Conclusões O aumento da autofagia diminui a apoptose dependente da caspase‐3 e melhora a sobrevivência neuronal in vivo. A ativação da autofagia pode ser uma estratégia terapêutica potencial para a neurotoxicidade induzida por anestésicos locais.

Animals , Male , Rats , Autophagy/drug effects , Bupivacaine/toxicity , Neurotoxicity Syndromes/prevention & control , Caspase 3/metabolism , Anesthetics, Local/toxicity , Neurons/drug effects , Spinal Cord/drug effects , Autophagy/physiology , Bupivacaine/administration & dosage , Random Allocation , Rats, Sprague-Dawley , Apoptosis/drug effects , Sirolimus/administration & dosage , In Situ Nick-End Labeling , Beclin-1/metabolism , Microtubule-Associated Proteins/metabolism , Neurons/pathology
Rev. bras. ortop ; 55(6): 764-770, Nov.-Dec. 2020. tab, graf
Article in English | LILACS | ID: biblio-1156206


Abstract Objective To analyze the anatomical variations of the motor branches of the radial nerve in the elbow region. The origin, course, length, branches, motor points and relationships with neighboring structures were evaluated. Materials and Methods Thirty limbs from15 adult cadavers were dissected and prepared by intra-arterial injection of a 10% glycerin and formaldehyde solution. Results The first branch of the radial nerve in the forearm went to the brachioradialis muscle (BR), originating proximally to the division of the radial nerve into superficial branch of the radial nerve (SBRN) and posterior interosseous nerve (PIN) in all limbs. The branches to the extensor carpi radialis longus muscle (ECRL) detached from the proximal radial nerve to its division into 26 limbs, in 2, at the dividing points, in other 2, from the PIN. In six limbs, the branches to the BR and ECRL muscles originated from a common trunk. We identified the origin of the branch to the extensor carpi radialis brevis muscle (ECRB) in the PIN in 14 limbs, in the SBRN in 12, and in the radial nerve in only 4. The branch to the supinator muscle originated from the PIN in all limbs. Conclusion Knowledge of the anatomy of the motor branches of the radial nerve is important when performing surgical procedures in the region (such as the approach of the proximal third and the head of the radius, release of compressive syndromes of the posterior interosseous nerve and radial tunnel, and distal nerve transfers) in order to understand the order of recovery of muscle function after a nerve injury.

Resumo Objetivo Analisar as variações anatômicas dos ramos motores do nervo radial na região do cotovelo. Foram avaliadas a origem, curso, comprimento, ramificações, pontos motores e relações com estruturas vizinhas. Materiais e Métodos Foram dissecados 30 membros de 15 cadáveres adultos, preparados por injeção intra-arterial de uma solução de glicerina e formol a 10%. Resultados O primeiro ramo do nervo radial no antebraço foi para o músculo braquiorradial (BR), que se origina proximalmente à divisão do nervo radial em ramo superficial do nervo radial (RSNR) e nervo interósseo posterior (NIP) em todos os membros. Os ramos para o músculo extensor radial longo do carpo (ERLC) se desprenderam do nervo radial proximalmente à sua divisão em 26 membros, em 2, nos pontos de divisão, em outros 2, do NIP. Em seis, os ramos para os músculos BR e ERLC originavam-se de um tronco comum. Identificamos a origem do ramo para o músculo extensor radial curto do carpo (ERCC) no NIP em 14 membros, no RSNR em 12, e no nervo radial em apenas 4. O ramo para o músculo supinador originou-se do NIP em todos os membros. Conclusão O conhecimento da anatomia dos ramos motores do nervo radial é importante quando se realizam procedimentos cirúrgicos na região, como a abordagem do terço proximal e da cabeça do rádio, a liberação das síndromes compressivas do nervo interósseo posterior e do túnel radial, as transferências nervosas distais, e para entender a ordem de recuperação da função muscular após uma lesão nervosa.

Radial Nerve , Radius , Surgical Procedures, Operative , Wrist , Cadaver , Nerve Transfer , In Situ Nick-End Labeling , Elbow , Extremities , Forearm , Forearm Injuries , Glycerol , Head , Anatomy , Injections, Intra-Arterial
Acta cir. bras ; 34(11): e201901102, Nov. 2019. tab, graf
Article in English | LILACS | ID: biblio-1054682


Abstract Purpose: To investigate the effect of Picroside II on testicular ischemia and reperfusion (l/R) injury and the underlying mechanism. Methods: Sprague-Dawley rats were randomly divided into 4 groups: sham operated group (Sham), Sham with Picroside II treatment group (Sham+ Pic II), l/R group (l/R) and l/R with Picroside II treatment group (I/R+ Pic II). l/R model was established by rotating the left testis 720° in a clock-wise direction for 4 hours. The histopathologic and spermatogenetic evaluation was performed. The apoptosis changes and the levels of HO-1 (heme oxygenase-1), MPO (myeloperoxidase), NOX (NADPH oxidase), SOD (superoxide dismutase), XO (xanthine oxidase) and NOS (nitric oxide synthase) were measured. Results: The seminiferous tubules were damaged in l/R rats, but Picroside II alleviated the changes induced by l/R. The increased level of apoptosis was decreased by Picroside II (P=0.01, 9.05±0.35 vs. 4.85±0.25). The activities of HO-1, MPO, NOX, XO and MDA content were increased and the SOD activity was decreased in l/R (P<0.05) and could be reversed by Picroside II (P=0.03, 405.5±7.5 vs. 304±17U/mgprot; P=0.02, 0.99±0.05 vs. 0.52±0.04 mgprot; P=0.01, 260+7 vs. 189±2 mgprot; P=0.04, 10.95+0.55 vs. 8.75+0.35 U/mgprot; P=0.045, 6.8+0.7 vs. 3.75+0.35 mgprot; P=0.04, 44.5+3.5 vs. 57.5+3.5 mgprot). Western blot showed that the expression of iNOS, nNOS and eNOS were increased in l/R (P<0.05); however, they were decreased after Picroside II treatment (P<0.05). Conclusion: Picroside II attenuated testicular I/R injury in rats mainly through suppressing apoptosis and oxidative stress through reduction of nitric oxide synthesis.

Animals , Male , Testis/blood supply , Reperfusion Injury/prevention & control , Cinnamates/pharmacology , Apoptosis/drug effects , Oxidative Stress/drug effects , Iridoid Glucosides/pharmacology , Nitric Oxide/biosynthesis , Reperfusion Injury/metabolism , Reperfusion Injury/pathology , Random Allocation , Blotting, Western , Rats, Sprague-Dawley , Peroxidase/analysis , In Situ Nick-End Labeling , Heme Oxygenase-1/analysis , Malondialdehyde/analysis , NADP/analysis
Arch. argent. pediatr ; 117(4): 237-244, ago. 2019. ilus, tab
Article in English, Spanish | BINACIS, LILACS | ID: biblio-1054927


Objetivos: Al ser un antioxidante, el licopeno protege a las células contra el daño causado por los radicales libres, fortalece los enlaces intercelulares y mejora el metabolismo celular. Este estudio analiza los efectos del licopeno sobre los trastornos neurodegenerativos por hiperoxia en ratas recién nacidas a término. Métodos: Estas ratas se dividieron en cuatro grupos: grupo 1 de referencia con normoxia, grupo 2 con normoxia + licopeno, grupo 3 de referencia con hiperoxia y grupo 4 con hiperoxia + licopeno. Los grupos 1 y 2 se supervisaron en condiciones de aire ambiental, y los grupos 3 y 4 se supervisaron con un nivel de oxígeno > 85 % O2. Los grupos 2 y 4 recibieron inyecciones intraperitoneales de licopeno de 50 mg/kg/día; los otros grupos recibieron inyecciones intraperitoneales de aceite de maíz con el mismo volumen. Las ratas se sacrificaron en el día 11, después de 10 días con hiperoxia. Se extrajeron los cerebros, y se evaluaron los parámetros del sistema oxidativo. Resultados: Se detectaron lesiones cerebrales por hiperoxia en sustancia blanca, regiones corticales y tálamo. Aumentó la cantidad de células apoptóticas y disminuyó la cantidad de células PCNA positivas en los grupos 3 y 4, comparados con el grupo 1. No se observó una mejora significativa en la cantidad de células apoptóticas y células PCNA positivas en los grupos 3 y 4; además, aumentó la apoptosis. Conclusión: Se halló que el licopeno no mostró efectos terapéuticos para el daño cerebral en ratas recién nacidas. Además, se demostró que el licopeno podría causar efectos tóxicos.

Objectives. In addition to protecting cells against free radical harm thanks to its anti-oxidant nature, lycopene strengthens the bonds among cells and improves cell metabolism. This study focuses on analyzing therapeutic effects of lycopene in hyperoxia-induced neurodegenerative disorders in newborn rats. Methods. Term newborn rats were divided into four groups as the normoxia control group (group-1), normoxia+lycopene group (group-2), hyperoxia control group (group-3) and hyperoxia+lycopene group (group-4). Group-1 and group-2 were monitored in room air while the group-3 and group-4 were monitored at > 85% O2. The group-2 and group-4 were injected with lycopene intrapertioneally (i.p. ) at 50mg/kg/day while the other groups were injected with corn oil i.p. at the same volume. The rats we sacrificed on the 11th day following the 10-day hyperoxia. The brains were removed and oxidant system parameters were assessed. Results. Injury resulting from hyperoxia was detected in the white matter, cortical regions, and thalamus of the brains. It was observed that the number of apoptotic cells increased and the number of proliferating cell nuclear antigen (PCNA) positive cells decreased in the groups-3 and 4 compared to the group-1. No significant improvement in the number of apoptotic cells and PCNA positive cells was observed in the groups-3 and 4, and apoptosis increased as well. Conclusion. This study found that lycopene, did not show any therapeutic effects for brain damage treatment in newborn rats. In addition, this study demonstrated that lycopene might lead to toxic effects.

Animals , Rats , Hyperoxia , Lycopene , Rats , Enzyme-Linked Immunosorbent Assay , In Situ Nick-End Labeling , Free Radicals
Arq. bras. cardiol ; 112(2): 180-188, Feb. 2019. graf
Article in English | LILACS | ID: biblio-983821


Abstract Background: In menopause, there is greater cellular exposure to oxidative stress, related to the decreased antioxidative effects of estrogen. These metabolic changes favor the progression of cardiovascular diseases, such as atherosclerosis. Abnormal function of the aorta - the most important artery - is associated with many cardiovascular diseases. Collagen, especially types I and III, is one of the most important aortic wall components and it can be affected by many factors, including menopause. The 8-OHdG is one of the main markers of DNA oxidative damage induced by reactive oxygen species (ROS). Objective: We aimed to investigate effects of moderate aerobic training on the ascending aorta of LDL-knockout (LDL-KO) and ovariectomized female mice. Methods: A total of 15 C57BL/6 mice and 15 LDL-KO mice were divided into experimental groups. The thickness and volume density of types I and III collagen fibers were performed by morphoquantitative analysis, whereas the MMP-2 and MMP-9 and 8-OHdG were detected by immunohistochemistry and apoptosis was detected by the TUNEL assay. The significance level for all tests was p < 0.05. Results: Exercise causes an increase in the thickness of the aorta in LDL-KO groups, particularly accentuated in the ovariectomized groups. The type I collagen fibers showed an increase in volume density influenced by training in both Control groups and in the LDL-KO group. Type III collagen density decreased in both groups. The MMP-2 showed moderade immunostaining in the tunica media in LDL-KO groups, which did not occur in the control groups and the MMP-9 stained irregularly in all tissues. The marker 8-OhdG was stronger in the exercise training groups. Additionally, the ovariectomy, the exercise training and the LDL-KO treatments increased apoptosis. Conclusion: These results suggest that moderate-intensity aerobic exercise in ovariectomized mice associated to an increase in LDL rate possibly increases oxidative stress and apoptosis induction.

Resumo Fundamento: Na menopausa, há maior exposição celular ao estresse oxidativo, relacionada à diminuição dos efeitos antioxidantes do estrogênio. Essas alterações metabólicas favorecem a progressão das doenças cardiovasculares, como a aterosclerose. A função anormal da aorta - a artéria mais importante - está associada a muitas doenças cardiovasculares. O colágeno, especialmente os tipos I e III, é um dos mais importantes componentes da parede da aorta e pode ser afetado por muitos fatores, incluindo a menopausa. Por sua vez, 8-OHdG é um dos principais marcadores de danos oxidativos do DNA induzidos por espécies reativas de oxigênio (EROS). Objetivo: Investigar os efeitos do treinamento aeróbico moderado na aorta ascendente de camundongos fêmeas, nocaute para LDL (LDL-KO) e ovariectomizadas. Métodos: Um total de 15 animais C57BL/6 e 15 animais LDL-KO foram divididos em grupos experimentais. A espessura e a densidade de volume das fibras de colágeno tipos I e III foram realizadas por análise morfoquantitativa; MMP-2 e MMP-9 e 8-OHdG foram detectadas por imunohistoquímica; e a apoptose foi detectada pelo ensaio TUNEL. O nível de significância adotado para todos os testes realizados foi p < 0,05. Resultados: o exercício causa aumento da espessura da aorta em grupos LDL-KO, particularmente acentuada em grupos ovariectomizados. As fibras de colágeno de tipo I mostraram aumento da densidade de volume influenciado pelo treinamento em animais controle e LDL-KO. A densidade do colágeno tipo III diminuiu em ambos os grupos. A MMP-2 mostrou imunomarcação moderada na túnica média em animais LDL-KO; em grupos controle, a MMP-9 marcou irregularmente em todos os tecidos. O marcador 8-OHdG foi mais forte nos grupos de treinamento de exercícios. Além disso, a ovariectomia, o treinamento físico e os tratamentos de LDL-KO aumentaram a apoptose. Conclusão: Esses resultados sugerem que exercícios aeróbicos de intensidade moderada em camundongos ovariectomizados associados ao aumento da taxa de LDL, possivelmente, aumentam o estresse oxidativo e a indução da apoptose.

Animals , Female , Rats , Aorta/metabolism , Physical Conditioning, Animal/physiology , Ovariectomy , Collagen/analysis , Matrix Metalloproteinase 2/analysis , Matrix Metalloproteinase 9/analysis , Aorta/pathology , Menopause/metabolism , Receptors, LDL/blood , Immunohistochemistry , Tunica Media/pathology , Apoptosis/physiology , Mice, Knockout , Oxidative Stress/physiology , In Situ Nick-End Labeling , Sedentary Behavior
Acta cir. bras ; 34(4): e201900402, 2019. tab, graf
Article in English | LILACS | ID: biblio-1001091


Abstract Purpose: To evaluate the effect of amniotic fluid in liver preservation in organ transplantation, and compare it with standard preservation solutions. Methods: The groups consisted of Group 1: Ringer Lactate (RL) group, Group 2: HTK group, Group 3: UW group, Group 4: AF group. The livers of rats from Group 1, 2, 3, and 4 were perfused and placed into falcon tubes containing RL, HTK, UW, and AF solutions at +4‎°C, respectively. The tubes were stored for 12 hours in the refrigerator at +4°C. Tissue samples were taken at the 6th and 12th hours for histopathological examinations of the perfused livers, and storage solutions for biochemical analyzes at 6th and 12th hours. Results: AF was shown to maintain organ viability by reducing the number of cells undergoing apoptosis. Histopathological changes such as sinusoidal dilatation, hydropic degeneration, and focal necrosis were found to be similar to the groups in which the standard organ preservation solutions were used. Additionally, the results of INOS, IL-10, and TNF-α,which were evaluated immunohistochemically, have been shown to be similar to the UW and HTK groups. Conclusions: AF provided conservation similar to UW and HTK in the 12-hour liver SCS process. The fact that apoptosis values are comparable to standard preservation solutions supports the success of AF in the cold storage of the liver.

Animals , Male , Cryopreservation/methods , Organ Preservation Solutions/pharmacology , Amniotic Fluid , Liver/blood supply , Liver/pathology , Organ Preservation/methods , Potassium Chloride/pharmacology , Procaine/pharmacology , Reference Values , Time Factors , Tissue Survival , Immunohistochemistry , Reperfusion Injury/prevention & control , Random Allocation , Reproducibility of Results , Tumor Necrosis Factor-alpha/analysis , Interleukin-10/analysis , Rats, Wistar , In Situ Nick-End Labeling , Nitric Oxide Synthase Type II/analysis , Ringer's Solution/pharmacology , Glucose/pharmacology , Mannitol/pharmacology
Clinics ; 74: e787, 2019. graf
Article in English | LILACS | ID: biblio-1011911


OBJECTIVES: Intestinal obstruction has a high mortality rate when therapeutic treatment is delayed. Resuscitation in intestinal obstruction requires a large volume of fluid, and fluid combinations have been studied. Therefore, we evaluated the effects of hypertonic saline solution (HS) with pentoxifylline (PTX) on apoptosis, oxidative stress and survival rate. METHODS: Wistar rats were subjected to intestinal obstruction and ischemia through a closed loop ligation of the terminal ileum and its vessels. After 24 hours, the necrotic bowel segment was resected, and the animals were randomized into four groups according to the following resuscitation strategies: Ringer's lactate solution (RL) (RL-32 ml/kg); RL+PTX (25 mg/kg); HS+PTX (HS, 7.5%, 4 ml/kg), and no resuscitation (IO-intestinal obstruction and ischemia). Euthanasia was performed 3 hours after resuscitation to obtain kidney and intestine samples. A malondialdehyde (MDA) assay was performed to evaluate oxidative stress, and histochemical analyses (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling [TUNEL], Bcl-2 and Bax) were conducted to evaluate kidney apoptosis. Survival was analyzed with another series of animals that were observed for 15 days. RESULTS: PTX in combination with RL or HS reduced the MDA levels (nmol/mg of protein), as follows: kidney IO=0.42; RL=0.49; RL+PTX=0.31; HS+PTX=0.34 (p<0.05); intestine: IO=0.42; RL=0.48; RL+PTX=0.29; HS+PTX=0.26 (p<0.05). The number of labeled cells for TUNEL and Bax was lower in the HS+PTX group than in the other groups (p<0.05). The Bax/Bcl-2 ratio was lower in the HS+PTX group than in the other groups (p<0.05). The survival rate on the 15th day was higher in the HS+PTX group (77%) than in the RL+PTX group (11%). CONCLUSION: PTX in combination with HS enhanced survival and attenuated oxidative stress and apoptosis. However, when combined with RL, PTX did not reduce apoptosis or mortality.

Animals , Male , Pentoxifylline/pharmacology , Resuscitation/methods , Saline Solution, Hypertonic/pharmacology , Apoptosis/drug effects , Oxidative Stress/drug effects , Intestinal Obstruction/metabolism , Immunohistochemistry , Lipid Peroxidation/drug effects , Random Allocation , Reproducibility of Results , Rats, Wistar , In Situ Nick-End Labeling , Disease Models, Animal , Kaplan-Meier Estimate , Intestinal Obstruction/mortality , Intestinal Obstruction/prevention & control , Intestine, Small/drug effects , Intestine, Small/metabolism , Kidney/drug effects , Kidney/metabolism , Malondialdehyde/analysis
Experimental Neurobiology ; : 216-228, 2019.
Article in English | WPRIM | ID: wpr-739543


The complement cascade is a central component of innate immunity which plays a critical role in brain inflammation. Complement C3a receptor (C3aR) is a key mediator of post-ischemic cerebral injury, and pharmacological antagonism of the C3a receptor is neuroprotective in stroke. Cerebral ischemia injures brain endothelial cells, causing blood brain barrier (BBB) disruption which further exacerbates ischemic neuronal injury. In this study, we used an in vitro model of ischemia (oxygen glucose deprivation; OGD) to investigate the protective effect of a C3aR antagonist (C3aRA, SB290157) on brain endothelial cells (bEnd.3). Following 24 hours of reperfusion, OGD-induced cell death was assessed by TUNEL and Caspase-3 staining. Western blot and immunocytochemistry were utilized to demonstrate that OGD upregulates inflammatory, oxidative stress and antioxidant markers (ICAM-1, Cox-2, Nox-2 and MnSOD) in endothelial cells and that C3aRA treatment significantly attenuate these markers. We also found that C3aRA administration restored the expression level of the tight junction protein occludin in endothelial cells following OGD. Interestingly, OGD/reperfusion injury increased the phosphorylation of ERK1/2 and C3aR inhibition significantly reduced the activation of ERK suggesting that endothelial C3aR may act via ERK signaling. Furthermore, exogenous C3a administration stimulates these same inflammatory mechanisms both with and without OGD, and C3aRA suppresses these C3a-mediated responses, supporting an antagonist role for C3aRA. Based on these results, we conclude that C3aRA administration attenuates inflammation, oxidative stress, ERK activation, and protects brain endothelial cells following experimental brain ischemia.

Blood-Brain Barrier , Blotting, Western , Brain Ischemia , Brain , Caspase 3 , Cell Death , Complement C3a , Complement System Proteins , Encephalitis , Endothelial Cells , Glucose , Immunity, Innate , Immunohistochemistry , In Situ Nick-End Labeling , In Vitro Techniques , Inflammation , Ischemia , Neurons , Occludin , Oxidative Stress , Phosphorylation , Reperfusion , Stroke , Tight Junctions
International Journal of Stem Cells ; : 151-161, 2019.
Article in English | WPRIM | ID: wpr-764052


BACKGROUND AND OBJECTIVES: The feature of chronic kidney failure (CKF) is loss of kidney functions due to erosion of healthy tissue and fibrosis. Recent studies showed that Mesenchymal stem cells (MSCs) differentiated into tubular epithelial cells thus renal function and structures renewed. Furthermore, MSCs protect renal function in CKF. Therefore, we aimed to investigate whether human amnion-derived mesenchymal stem cells (hAMSCs) can repair fibrosis and determine the effects on proliferation and apoptosis mechanisms in chronic kidney failure. METHODS AND RESULTS: In this study, rat model of CKF was constituted by applying Aristolochic acid (AA). hAMSCs were isolated from term placenta amnion membrane and transplanted into tail vein of rats. At the end of 30 days and 60 days of recovery period, we examined expressions of PCNA, p57 and Parp-1 by western blotting. Immunoreactivity of PCNA, Ki67, IL-6 and Collagen type I were detected by immunohistochemistry. Besides, apoptosis was detected by TUNEL. Serum creatinine and urea were measured. Expressions of PCNA and Ki67 increased in hAMSC groups compared with AA group. Furthermore, expressions of PARP-1 apoptosis marker and p57 cell cycle inhibitory protein increased in AA group significantly according to control, hAMSC groups and sham groups. IL-6 proinflammatory cytokine increased in AA group significantly according to control, hAMSCs groups and sham groups. Expressions of Collagen type I protein reduced in hAMSCs groups compared to AA group. After hAMSC treatment, serum creatinine and urea levels significantly decreased compared to AA group. After injection of hAMSC to rats, Masson’s Trichrome and Sirius Red staining showed fibrosis reduction in kidney. CONCLUSIONS: According to our results hAMSCs can be ameliorate renal failure.

Animals , Humans , Rats , Amnion , Apoptosis , Blotting, Western , Cell Cycle , Collagen Type I , Creatinine , Epithelial Cells , Fibrosis , Immunohistochemistry , In Situ Nick-End Labeling , Interleukin-6 , Kidney , Kidney Failure, Chronic , Membranes , Mesenchymal Stem Cells , Models, Animal , Placenta , Proliferating Cell Nuclear Antigen , Renal Insufficiency , Renal Insufficiency, Chronic , Tail , Urea , Veins
Clinical and Experimental Reproductive Medicine ; : 14-21, 2019.
Article in English | WPRIM | ID: wpr-763349


OBJECTIVE: The usual seminal profile has been customarily used for diagnosing male infertility based on an examination of semen samples. However, sperm DNA fragmentation has also been causally linked to reproductive failure, suggesting that it should be evaluated as part of male infertility assessments. To compare the ability of the five most widely utilized methodologies of measuring DNA fragmentation to predict male infertility and reactive oxygen species by Oxisperm kit assay. METHODS: In this case-control study, which received ethical committee approval, the participants were divided into fertile and infertile groups (50 patients in each group). RESULTS: The alkaline comet test showed the best ability to predict male infertility, followed by the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay, the sperm chromatin dispersion (SCD) test, and the sperm chromatin structure assay (SCSA), while the neutral comet test had no predictive power. For our patient population, the projected cut-off point for the DNA fragmentation index was 22.08% using the TUNEL assay, 19.90% using SCSA, 24.74% using the SCD test, 48.47% using the alkaline comet test, and 36.37% using the neutral comet test. Significant correlations were found between the results of the SCD test and those obtained using SCSA and TUNEL (r =0.70 and r =0.68, respectively; p<0.001), and a statistically significant correlation was also found between the results of SCSA and the TUNEL assay (r =0.77, p<0.001). Likewise, the results of the alkaline comet test showed significant correlations with those of the SCD, SCSA, and TUNEL tests (r =0.59, r =0.57, and r =0.72, respectively; p<0.001). CONCLUSION: The TUNEL assay, SCSA, SCD, and the alkaline comet test were effective for distinguishing between fertile and infertile patients, and the alkaline comet test was the best predictor of male infertility.

Humans , Male , Male , Case-Control Studies , Chromatin , DNA Fragmentation , DNA Nucleotidylexotransferase , DNA , In Situ Nick-End Labeling , Infertility , Infertility, Male , Methods , Reactive Oxygen Species , Semen , Sensitivity and Specificity , Spermatozoa
Chinese Medical Journal ; (24): 1071-1078, 2019.
Article in English | WPRIM | ID: wpr-772221


BACKGROUND@#Colorectal cancer is the third most common cancer worldwide and still lack of effective therapy so far. Petasin, a natural product found in plants of the genus Petasites, has been reported to possess anticancer activity. The present study aimed to investigate the anticolon cancer activity of petasin both in vitro and in vivo. The molecular mechanism of petasin was also further explored.@*METHODS@#Caco-2, LoVo, SW-620, and HT-29 cell lines were used to detect the inhibitory effect of petasin on colon cancer proliferation. Cell viability was determined using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. Cell apoptosis was analyzed by flow cytometry. Hoechst 33258 staining was used to visualize morphological changes. Cell migration was assessed using a wound-healing migration assay, and cell invasion was investigated using Transwell chambers. Western blotting assays were employed to evaluate the expression levels of proteins in the protein kinase B/mammalian target of rapamycin (Akt/mTOR) signaling pathway. Finally, in vivo activity of petasin was evaluated using the SW-620 subcutaneous tumor model established in Balb/c nude mice. Twelve rats were randomly divided into control group and 10 mg/kg petasin group. The tumor volume was calculated every 7 days for 28 days. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was performed to assess the apoptotic effect of petasin. Differences between two groups were assessed by analysis of independent-sample t tests.@*RESULTS@#Petasin significantly inhibited the proliferation of human colon carcinoma cell lines, induced apoptosis, and suppressed migration and invasion in SW-620 cells. Western blotting results showed that petasin decreased the phosphorylation of Akt (1.01 ± 0.16 vs. 0.74 ± 0.06, P = 0.042), mTOR (0.71 ± 0.12 vs. 0.32 ± 0.11, P = 0.013), and P70S6K (1.23 ± 0.21 vs. 0.85 ± 0.14, P = 0.008), elevated the expression of caspase-3 (0.41 ± 0.09 vs. 0.74 ± 0.12, P = 0.018) and caspase-9 (1.10 ± 0.27 vs. 1.98 ± 0.22, P = 0.009), decreased the Bcl-2 protein (2.75 ± 0.47 vs. 1.51 ± 0.36, P = 0.008), downregulated the expression of matrix metalloproteinase (MMP)-3 (1.51 ± 0.31 vs. 0.82 ± 0.11, P = 0.021) and MMP-9 (1.56 ± 0.32 vs. 0.94 ± 0.15, P = 0.039) in SW-620 cell. In vivo, 10 mg/kg petasin inhibited tumor growth in Balb/c nude mice (924.18 ± 101.23 vs. 577.67 ± 75.12 mm at day 28, P = 0.001) and induced apoptosis (3.6 ± 0.7% vs. 36.0 ± 4.9%, P = 0.001) in tumor tissues.@*CONCLUSIONS@#Petasin inhibits the proliferation of colon cancer SW-620 cells via inactivating the Akt/mTOR pathway. Our findings suggest petasin as a potential candidate for colon cancer therapy.

Animals , Humans , Mice , Antineoplastic Agents , Therapeutic Uses , Apoptosis , Caco-2 Cells , Cell Line, Tumor , Cell Proliferation , HT29 Cells , In Situ Nick-End Labeling , Matrix Metalloproteinase 3 , Metabolism , Matrix Metalloproteinase 9 , Metabolism , Mice, Inbred BALB C , Mice, Nude , Phosphorylation , Proto-Oncogene Proteins c-akt , Genetics , Metabolism , Sesquiterpenes , Therapeutic Uses , Signal Transduction , TOR Serine-Threonine Kinases , Genetics , Metabolism
Asian Journal of Andrology ; (6): 438-444, 2019.
Article in English | WPRIM | ID: wpr-1009692


Lack of standardized, reproducible protocols and reference values is among the challenges faced when using new or upgraded versions of instruments in reproductive laboratories and flow cytometry. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay combined with flow cytometry routinely used for diagnostic measurement of sperm DNA fragmentation (SDF) is a unique example. Any change in the setting of the standard instrument, including upgrades of hardware or software, can lead to different results and may affect clinicians' decision for treatment. Therefore, we compared TUNEL results of SDF obtained from a standard (C6) flow cytometer with a newer version of the same instrument (C6 Plus) and examined the cutoff, sensitivity, and specificity without calibration (adjustment) and after adjustment. Identical sperm preparation and matched acquisition settings were used to examine the performance of two flow cytometers. The strength of agreement of the results between the two observers was also assessed. After adjustment of the settings, overall concordance became high and the two cytometers showed 100% positive and negative predictive value with 100% area under the curve. The overall correlation coefficient observed between C6 and C6 Plus was highly significant (P < 0.0001; r = 0.992; 95% confidence interval [CI]: 0.982-0.997). After adjustment, the two cytometers showed very high precision of 98% and accuracy of >99%. The interobserver agreement on C6 flow cytometer for the two observers was 0.801 ± 0.062 and 0.746 ± 0.044 for C6 Plus. We demonstrated a strong agreement between the samples tested on the two flow cytometers after calibration and established the robustness of both instruments.

Adult , Humans , Male , Calibration , DNA Fragmentation , Flow Cytometry/instrumentation , In Situ Nick-End Labeling , Observer Variation , Reference Values , Reproducibility of Results , Semen Analysis/methods , Sensitivity and Specificity , Spermatozoa/chemistry
The Korean Journal of Physiology and Pharmacology ; : 21-28, 2019.
Article in English | WPRIM | ID: wpr-728029


Swertiamarin (STM) is an iridoid compound that is present in the Gentianaceae swertia genus. Here we investigated antiapoptotic effects of STM on carbon tetrachloride (CCl₄)-induced liver injury and its possible mechanisms. Adult male Sprague Dawley rats were randomly divided into a control group, an STM 200 mg/kg group, a CCl₄ group, a CCl₄+STM 100 mg/kg group, and a CCl₄+STM 200 mg/kg group. Rats in experimental groups were subcutaneously injected with 40% CCl₄ twice weekly for 8 weeks. STM (100 and 200 mg/kg per day) was orally given to experimental rats by gavage for 8 consecutive weeks. Hepatocyte apoptosis was determined by TUNEL assay and the expression levels of Bcl-2, Bax, and cleaved caspase-3 proteins were evaluated by western blot analysis. The expression of TGF-β1, collagen I, collagen III, CTGF and fibronectin mRNA were estimated by qRT-PCR. The results showed that STM significantly reduced the number of TUNEL-positive cells compared with the CCl₄ group. The levels of Bax and cleaved caspase-3 proteins, and TGF-β1, collagen I, collagen III, CTGF, and fibronectin mRNA were significantly reduced by STM compared with the CCl₄ group. In addition, STM markedly abrogated the repression of Bcl-2 by CCl₄. STM also attenuated the activation of the PI3K/Akt pathway in the liver. These results suggested that STM ameliorated CCl₄-induced hepatocyte apoptosis in rats.

Adult , Animals , Humans , Male , Rats , Apoptosis , Blotting, Western , Carbon Tetrachloride , Carbon , Caspase 3 , Collagen , Fibronectins , Gentianaceae , Hepatocytes , In Situ Nick-End Labeling , Liver , Rats, Sprague-Dawley , Repression, Psychology , RNA, Messenger , Swertia
Int. j. morphol ; 36(1): 175-179, Mar. 2018. tab, graf
Article in English | LILACS | ID: biblio-893207


SUMMARY: Traumatic injury to the spinal cord results in the delayed dysfunction and neuronal death. Impaired mitochondrial function, generation of reactive oxygen species (ROS), and lipid peroxidation occur soon after traumatic spinal cord injury (SCI), while the activation of compensatory molecules that neutralize ROS occurs at later time points. The aim of the current study was to investigate the putative neuroprotective effect of Ganoderma lucidum in a rat model of SCI. In order to induce SCI, a standard weight-drop method that induced a moderately severe injury (100 g/cm force) at T10, was used. Injured animals were given either 20 mL/kg Ganoderma lucidum or saline 30 min post injury per day by gastric gavage. At seven days postinjury, rats were decapitated. Spinal cord samples were taken for histological examination or determination of malondialdehyde (MDA) and glutathione (GSH) levels, myeloperoxidase (MPO) activity. SCI caused a significant decrease in spinal cord GSH content, which was accompanied with significant increases in MDA levels, MPO activity. On the other hand, Ganoderma lucidum treatment reversed all these biochemical parameters as well as SCI-induced histopathological alterations. Furthermore, impairment of the neurological functions due to SCI was improved by meloxicam treatment. The present study suggests that Ganoderma Lucidum, reduces SCI-induced oxidative stress and exerts neuroprotection by inhibiting lipid peroxidation, GSH depletion.

RESUMEN: La lesión traumática de la médula espinal provoca disfunción retrasada y muerte neuronal. La función mitocondrial deteriorada, la generación de especies reactivas de oxígeno (ERO) y la peroxidación lipídica ocurren poco después de una lesión traumática de la médula espinal (LTE), mientras que la activación de moléculas compensatorias que neutralizan ERO ocurre posteriormente. El objetivo del presente estudio fue investigar el efecto neuroprotector de Ganoderma lucidum en un modelo de LTE en ratas. Con el fin de inducir LTE, se utilizó un método estándar de pérdida de peso que indujo una lesión moderadamente grave (100 g / cm de fuerza) a T10. A los animales lesionados se les administró 20 ml / kg de Ganoderma lucidum o solución salina, por sonda gástrica, 30 minutos después de la lesión. A los siete días después de la lesión, las ratas fueron eutanasiadas por decapitación. Se tomaron muestras de médula espinal para el examen histológico y para la determinación de los niveles de malondialdehído (MDA) y glutatión (GSH), y la actividad de mieloperoxidasa (MPO). LTE causó una disminución significativa en el contenido de GSH de la médula espinal, además de aumentos significativos en los niveles de MDA y la actividad de MPO. Por otro lado, el tratamiento con Ganoderma lucidum invirtió todos estos parámetros bioquímicos así como las alteraciones histopatológicas inducidas por LTE. El deterioro de las funciones neurológicas debidas a LTE mejoró con el tratamiento con meloxicam. El presente estudio sugiere que Ganoderma lucidum, reduce el estrés oxidativo inducido por LTE y ejerce la neuroprotección mediante la inhibición de la peroxidación de los lípidos y agotamiento del GSH.

Animals , Rats , Neuroprotective Agents/administration & dosage , Reishi/chemistry , Spinal Cord Injuries/drug therapy , Glutathione/analysis , Immunohistochemistry , In Situ Nick-End Labeling , Malondialdehyde/analysis , Oxidative Stress/drug effects , Peroxidase/drug effects , Rats, Sprague-Dawley , Spinal Cord Injuries/pathology
Biol. Res ; 51: 56, 2018. graf
Article in English | LILACS | ID: biblio-1011400


BACKGROUND: Glioma is the most prevalent malignant tumor in human central nervous systems. Recently, the development of resistance to radiotherapy in glioma patients markedly vitiates the therapy outcome. MiR-153-3p has been reported to be closely correlated with tumor progression, but its effect and molecular mechanism underlying radioresistance remains unclear in glioma. METHODS: The expression of miR-153-3p was determined in radioresistant glioma clinical specimens as well as glioma cell lines exposed to irradiation (IR) using quantitative real-time PCR. Cell viability, proliferation and apoptosis were then evaluated by MTT assay, colony formation assay, Flow cytometry analysis and caspase-3 activity assay in glioma cells (U87 and U251). Tumor forming was evaluated by nude mice model in vivo. TUNEL staining was used to detect cell apoptosis in nude mice model. The target genes of miR-153-3p were predicted and validated using integrated bioinformatics analysis and a luciferase reporter assay. RESULTS: Here, we found that miR-153-3p was down-regulated in radioresistant glioma clinical specimens as well as glioma cell lines (U87 and U251) exposed to IR. Enhanced expression of miR-153-3p promoted the radiosensitivity, promoted apoptosis and elevated caspase-3 activity in glioma cells in vitro, as well as the radiosensitivity in U251 cell mouse xenografs in vivo. Mechanically, B cell lymphoma-2 gene (BCL2) was identified as the direct and functional target of miR-153-3p. Moreover, restoration of BCL2 expression reversed miR-153-3p-induced increase of radiosensitivity, apoptosis and caspase-3 activity in U251 cells in vitro. In addition, clinical data indicated that the expression of miR-153-3p was significantly negatively associated with BCL2 in radioresistance of glioma samples. CONCLUSIONS: Our findings suggest that miR-153-3p is a potential target to enhance the effect of radiosensitivity on glioma cells, thus representing a new potential therapeutic target for glioma.

Humans , Male , Female , Adult , Middle Aged , Radiation Tolerance/genetics , Genes, bcl-2/physiology , MicroRNAs/radiation effects , MicroRNAs/physiology , Glioma/genetics , Time Factors , Down-Regulation , Gene Expression Regulation, Neoplastic , Cell Survival/radiation effects , Blotting, Western , Analysis of Variance , Gene Targeting/methods , Genes, bcl-2/radiation effects , In Situ Nick-End Labeling , MicroRNAs/analysis , Cell Line, Tumor , Cell Proliferation/radiation effects , Caspase 3/analysis , Real-Time Polymerase Chain Reaction , Flow Cytometry , Glioma/radiotherapy
Acta odontol. latinoam ; 31(2): 110-116, 2018. ilus, graf
Article in English | LILACS | ID: biblio-970843


The in vivo response of osteocytes to different force magnitudes soon after they are applied remains to be elucidated. The aim of this study was to examine the early effects of applying a very light (LF: 0,16 N) and a very strong (SF: 2,26 N) orthodontic force during one hour on apoptosis and osteopontin (OPN) expression on alveolar bone osteocytes, in rats. Results: LF: compared to the control group, they showed a significant increase in OPN expression, and a significant decrease in the number of TUNELpositive osteocytes. SF: compared to the control group, they showed a significant increase in OPN expression and a significant decrease in the number of TUNELpositive osteocytes. Our results show that osteocytes respond very early to the application of tension and pressure forces of different magnitudes, and application of forces decreases the number of apoptotic osteocytes and increases OPN expression. These results allow concluding that osteocytes activate rapidly when subjected to locally applied forces, whether these forces be pressure or tension, light or strong forces. Grants: UBACyT 200201301002270 BA and School of Dentistry, University of Buenos Aires (AU)

Hasta el momento no se ha dilucidado la respuesta temprana in vivo de los osteocitos a la aplicación de fuerzas de diferentes magnitudes sobre el hueso. El objetivo de este estudio fue examinar la respuesta temprana de la aplicación de una fuerza ortodóncica muy liviana (FL: 0,16 N) y muy fuerte (FF: 2,26 N) durante una hora sobre la expresión de apoptosis y osteopontina (OPN) en los osteocitos del hueso alveolar, en ratas. Resultados: FL: en comparación con el grupo control, mostraron un aumento significativo en la expresión de OPN y una disminución significativa en el número de osteocitos TUNELpositivos. FF: en comparación con el grupo control, mostraron un aumento significativo en la expresión de OPN y una disminución signi ficativa en el número de osteocitos TUNELpositivos. Nuestros resultados muestran que los osteocitos responden muy temprano a la aplicación de fuerzas de tensión y presión de diferentes magnitudes, y la aplicación de fuerzas disminuye el número de osteocitos apoptóticos y aumenta la expresión de OPN. Estos resultados permiten concluir que los osteocitos se activan rápidamente cuando se los somete a fuerzas aplicadas localmente, ya sean estas fuerzas de presión o tensión, livianas o fuertes (AU)

Animals , Rats , Osteocytes , Stress, Mechanical , Tooth Movement Techniques , Apoptosis , Osteopontin , Immunohistochemistry , Data Interpretation, Statistical , In Situ Nick-End Labeling , Mechanotransduction, Cellular , Alveolar Process
Bauru; s.n; 2018. 95 p. ilus, graf, tab.
Thesis in English | LILACS, BBO | ID: biblio-884465


Current knowledge supports the application of TiF4 varnish to protect against tooth caries and erosion; however, it is indispensable to know its cytotoxic potential and the mechanism involved on it before applying in patients. Therefore, this study aimed to evaluate 1) The cytotoxic effect of titanium tetrafluoride (TiF4) varnish compared with sodium fluoride (NaF) varnish on murine fibroblast (NIH/3T3), varying the fluoride concentration and time of treatment and 2) The percentage of apoptosis and its mechanism (both mitochondrial mediated by the Bcl-2 family- and death receptorpathways) in human gingival fibroblasts (HGF) and murine fibroblasts (NIH/3T3) treated with TiF4 varnish compared to NaF varnish for 6 h. Step 1) NIH/3T3 were exposed to NaF or TiF4 varnishes containing 0.95, 1.95 or 2.45% F, for 6, 12 or 24 h. MTT viability (n=6) and Hoescht/PI stain assays (n=3) as well as the cells morphology (HE, only for 24 h, n=3) and stiffness (AFM, only for 2.45% F, 6 or 12 h) were analyzed. Both varnishes, at 1.90 and 2.45% F, reduced cells viability by similar extent (33-86% at 6 h, 35-93% at 12 h, and 87-98% at 24 h) compared to control, regardless of the type of fluoride. TiF4 and NaF (2.45% F) reduced cell stiffness to a similar extent, but only TiF4 differed from control. Step 2) HGF and NIH/3T3 were exposed to NaF or TiF4 (2.45% F) varnishes for 6 h. Cells were examined by the TUNEL method using fluorescence microscope. The caspases-3, -8 and -9 activities were assessed. The cDNA for cytocrome c, Bax, Bad, Bcl-2, VDAC-1 and Fas-L was amplified by quantitative PCR (qPCR). Bax, Bcl-2 and Fas-L were further detected by western blot. Both fluorides similarly increased the percentage of apoptosis, while they failed in activating caspases-3, -8 and -9 for both types of cells. Bax/Bcl-2 ratio, cytochrome C and VDAC-1 gene expressions were not altered by both fluoride treatments. However, NaF varnish increased the amplification of Fas-L gene for NIH/3T3 and HGF, while TiF4 varnish induced lower Bad/Bcl-2 ratio expression compared to control for NIH/3T3, but not for HGF. No effect of the fluorides was detected in the proteins analysis. TiF4 and NaF have similar cytotoxicity on NIH/3T3, which is dependent on the F concentration and the exposure time. Both fluorides, at the studied conditions, similarly induce a low percentage of apoptosis, with consequent modest activation of Bcl-2 and Fas-L-dependent signaling pathways.(AU)

Conhecimento atual suporta a aplicação de verniz de TiF4 para proteção contra cárie e erosão dentárias; entretanto, é indispensável conhecer o seu potencial citotóxico e o mecanismo envolvido antes de aplicá-lo em pacientes. Portanto, o objetivo deste estudo foi avaliar 1) o efeito citotóxico do verniz de tetrafluoreto de Titânio (TiF4) comparado ao fluoreto de sódio (NaF), em fibroblastos NIH/3T3, variando a concentração de fluoreto e o tempo de tratamento 2) a porcentagem de apoptose e seus mecanismos (ambos mitocondrial mediado pela família Bcl-2 e pelo receptor de morte celular) em fibroblastos gengivais humanos (FGH) e fibroblastos murinos (NIH/3T3) tratados com verniz de TiF4 comparado com verniz de NaF por 6 h. Etapa 1) NIH/3T3 foram expostos a vernizes de NaF e TiF4 contendo 0,95, 1,95 ou 2,45% F, por 6, 12 ou 24 h. Ensaios de viabilidade por MTT (n=6) e Hoechst 33342/iodeto de propídeo (n=3) bem como a morfologia (HE, apenas para 24 h, n = 3) e a rigidez celular (MFA, apenas para 2,45% F, 6 ou 12 h) foram realizados. Ambos os vernizes com 1,90 e 2,45% F reduziram a viabilidade das células de forma semelhante (33-86% em 6 h, 35-93% em 12 h e 87-98% em 24 h) em comparação com o controle, independentemente do tipo de fluoreto. TiF4 e NaF (2,45%) reduziram de forma similar a rigidez celular, mas somente TiF4 diferiu do controle no período de 6 h. Etapa 2) FGH e NIH/3T3 foram tratadas com verniz de NaF ou TiF4 por 6h. As células foram examinadas pelo método de TUNEL, usando microscopia de fluorescência. A atividade das caspases -3, -8 e -9 foram avaliadas. O cDNA para citocromo C, Bax, Bad, Bcl-2, VDAC-1 e Fas-L foi amplificado e quantificado por PCR em tempo real (qPCR). A expressão das proteínas Bax, Bcl-2 e Fas-L foi quantificada por western blot. Ambos os fluoretos aumentaram de forma semelhante a porcentagem de apoptose, enquanto falharam na ativação de caspases-3, -8 e -9 para ambos tipos celulares. A expressão gênica da relação Bax/Bcl-2, do citocromo C e do VDAC-1 não foram alteradas por ambos fluoretos. No entanto, o verniz NaF aumentou a amplificação do gene Fas-L para ambas as células, enquanto que o verniz TiF4 induziu menor expressão da razão Bad/Bcl-2 em comparação com o controle para NIH/3T3, mas não para FGH. Nenhum efeito foi detectado na análise de proteínas. TiF4 e NaF apresentam citotoxicidade similar em NIH/3T3, a qual é dependente da concentração de F e do tempo de exposição. Ambos os fluoretos, nas condições estudadas, induzem uma baixa porcentagem de apoptose, com consequente modesta ativação das vias de sinalização dependentes de Bcl-2 e Fas-L.(AU)

Humans , Animals , Mice , Cariostatic Agents/pharmacology , Fibroblasts/drug effects , Fluorides, Topical/pharmacology , Titanium/pharmacology , Apoptosis/drug effects , Blotting, Western , Cell Survival/drug effects , Gingiva/cytology , In Situ Nick-End Labeling , Microscopy, Fluorescence , NIH 3T3 Cells , Polymerase Chain Reaction , Reproducibility of Results , Time Factors
Journal of Dental Hygiene Science ; (6): 76-84, 2018.
Article in Korean | WPRIM | ID: wpr-714096


Wet wipes are being increasingly used because of their convenience. Particularly, oral wet wipes are useful for regular cleaning of a baby's mouth after birth. Therefore, the consumption of oral wet wipes has increased over the past few years and a variety of products are commercially available. However, product information on safety is not sufficiently provided and still raises doubts regarding adverse effects. To confirm the safety of wet wipes as an oral hygiene item and provide information for their use, we investigated the cytotoxicity of oral wet wipes and verified the underlying mechanism. The anti-bacterial effect of oral wet wipes was analyzed using the disk diffusion method. The cytotoxic effects of oral wet wipes were observed based on morphological changes using microscopy and determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in gingival epithelial cells and gingival fibroblasts. Evaluation of apoptosis by oral wet wipes was explored using propidium iodide flow cytometric analysis and a terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL) assay. Apoptosis-related molecules were also analyzed using western blotting. Five types of oral wet wipes were tested, and two products from Fisher-Price and Dr. Kennedy revealed strong cytotoxic effects on gingiva epithelial cells and gingiva fibroblasts, although they also showed intense anti-bacterial effects on oral bacteria. Cell cycle arrest in the G2/M phase and apoptosis were observed based on treatment of extracts from Fisher-Price and Dr. KENNEDY. Relatively high TUNEL levels, reduction of proliferating cell nuclear antigen and cyclin-dependent kinase 4 expression, and fragmentation of poly (ADP-ribose) polymerase were also elucidated. These results suggest that commercial oral wet wipes could exert cytotoxic influences on oral tissue, although there are anti-bacterial effects, and careful attention is required, especially for infants and toddlers.

Humans , Infant , Apoptosis , Bacteria , Blotting, Western , Cell Cycle , Cell Cycle Checkpoints , Cell Survival , Cyclin-Dependent Kinase 4 , Deoxyuridine , Diffusion , DNA Nucleotidylexotransferase , Epithelial Cells , Fibroblasts , Gingiva , In Situ Nick-End Labeling , Methods , Microscopy , Mouth , Oral Hygiene , Parturition , Proliferating Cell Nuclear Antigen , Propidium