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The aims of this study were to determine the effect of rutin on in vitro maturation (IVM) of oocytes from in vitro-grown sheep secondary follicles and to analyze the possible involvement of the mammalian target of rapamycin (mTOR) pathway in IVM under the influence of rutin. Secondary follicles were cultured for 18 days in α-Minimum Essential Medium (α-MEM) supplemented with bovine serum albumin (BSA), insulin, glutamine, hypoxanthine, transferrin, selenium, ascorbic acid, and leptin (control medium: α-MEM+). Next, the follicles were evaluated for morphology, antrum formation, and follicular diameter, and the rate of fully grown oocytes (≥110 µm). Fully grown oocytes were submitted to IVM in Tissue Culture Medium 199 (TCM199) supplemented with fetal bovine serum (FBS), luteinizing hormone (LH), and recombinant follicle-stimulating hormone (rFSH) (IVM control medium), or in this medium with 0.1, 1, or 10 µg mL-1rutin. At the end of IVM, the oocytes were evaluated for mitochondrial activity, the reactive oxygen species (ROS) and glutathione (GSH) levels, the percentage of meiotic resumption, DNA fragmentation, and mTOR pathway involvement. After 18 days of in vitro culture, 77.5% of the follicles were normal and 77.7% became antral follicles with a diameter of 380.41 µm. Furthermore, almost 70% of the oocytes grew in vitro, reaching a diameter ≥110 µm and were submitted to IVM. Supplementation with 10 µg mL-1rutin significantly increased the percentage of oocytes that resumed meiosis (47.27%) compared with the control medium (30.43%). There was a significant increase in the ROS and GSH levels in oocytes matured with 0.1 µg mL-1 rutin compared with the other rutin treatments (p < 0.05). Furthermore, oocytes matured with TCM199+ showed a higher (p < 0.05) percentage of DNA fragmentation (30%) than those that matured with 10 µg mL-1 rutin (0%). After IVM, oocytes matured in the presence or absence of rapamycin showed a similar percentage of meiotic resumption (61.76% for TCM199+ 10 µg mL-1 rutin and 70.73% for TCM + 10 µg mL-1 rutin + rapamycin; p > 0.05). In conclusion, supplementation with 10 µg mL-1 rutin increased meiosis resumption and reduced DNA damage.(AU)

Os objetivos deste estudo foram verificar o efeito da rutina sobre a maturação in vitro (MIV) de oócitos provenientes de folículos secundários de ovelhas cultivados in vitro e analisar o possível envolvimento da via mTOR na MIV, sob influência da rutina. Os folículos secundários foram cultivados por 18 dias em meio α-Mínimo Essencial (α-MEM) suplementado com albumina sérica bovina (BSA), insulina, glutamina, hipoxantina, transferrina, selênio, ácido ascórbico e leptina (meio controle: α-MEM+). Em seguida, os folículos foram avaliados quanto à morfologia, formação do antro e diâmetro folicular e taxa de oócitos totalmente crescidos (≥110 µm). Oócitos totalmente crescidos foram submetidos à MIV em meio de cultivo de tecidos 199 (TCM199) suplementado com soro fetal bovino (FBS), hormônio luteinizante (LH), hormônio folículo estimulante recombinante (rFSH) (meio controle MIV) ou neste meio com 0,1, 1 ou 10 µg.mL-1 de rutina. Ao final da MIV, os oócitos foram avaliados quanto à atividade mitocondrial, concentração de espécies reativas de oxigênio (ERO) e glutationa (GSH), porcentagem de retomada de meiose, fragmentação de DNA e envolvimento da via mTOR. Após 18 dias de cultivo in vitro, 77,5% dos folículos estavam normais e 77,7% tornaram-se folículos antrais, com 380,41 µm de diâmetro. Além disso, 70% dos oócitos que cresceram in vitro atingiram diâmetro ≥110 µm e foram submetidos à MIV. A concentração de 10 µg.mL-1 de rutina aumentou significativamente a porcentagem de oócitos que retomaram a meiose (47,27%) em comparação ao meio controle (30,43%). Houve um aumento significativo nas concentrações de ROS e GSH em oócitos maturados com 0.1 µg.mL-1 de rutina em comparação com os outros tratamentos com rutina (p < 0,05). Além disso, a maturação de oócitos em TCM199+ aumentou (p<0,05) o percentual de fragmentação de DNA (30%) comparado ao tratamento com 10 µg.mL-1 de rutina (0%). Após MIV, ambos os tratamentos maturados na presença ou ausência de rapamicina apresentaram porcentagem semelhante de retomada meiótica (61,76% para TCM199+10 µg.mL-1 de rutina e 70,73% para TCM199+ 10 µg.mL-1 de rutina + rapamicina) (p>0,05). Em conclusão, a concentração de 10 µg.mL-1 de rutina aumentou a retomada da meiose e reduziu os danos ao DNA.(AU)

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Oxidative stress is the result of an imbalance between the production of oxidant precursors and the capacity of antioxidant defense. Oxygen free radicals play an important role in causing diseases. In this study, the protective effect of ethanolic avocado on apoptosis caused by oxidative damage in the tissue of albino rats was investigated. 24 male albino rats of the Faculty of Veterinary Medicine in Mosul, Iraq, which were kept in standard conditions for at least 10 days before and through the experimental work, were examined. Four groups of rats include the control group (healthy group), the group of male rats with ethanolic avocado consumption; The third group of male rats that were treated with 0.5% of hydrogen peroxide H2O2; and the fourth group of male rats that were treated with both 0.5% H2O2 and avocado ethanolic extract (50 mg kg-1 BW) for four weeks. After fixing the tissues of the liver, kidney, lung, spleen and testis in 10% buffered formalin, they were stained with hematoxylin. TUNEL assay was performed using the TUNEL cell death assay kit to detect apoptotic cells. In this investigation, the histology results in four groups of rats showed that in the rats that were treated with avocado, there were minor tissue changes in their liver, kidney, and intestine, and the tissues of these organs were healthy. In TUNEL staining, it was also shown that there are no apoptotic cells in the liver, kidney and testis cells in avocado-treated rats. The results showed that ethanolic Avocado is useful against oxidative stress damage and it may be used to protect tissues against oxidative stress.(AU)

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Abstract Bulbine natalensis and Chorophytum comosum are potential medicinal source for the treatment of cancers. Chronic myeloid leukaemia is a hematopoietic stem cells disorder treated by tyrosine kinase inhibitors but often cause recurrence of the leukaemia after cessation of therapy, hence require alternative treatment. This study determines the anti-cancer effect of leaf, root and bulb methanolic and aqueous extracts of B. natalensis and C. comosum in chronic human myelogenous leukaemia (K562) cell line by MTT, Hoechst bis-benzimide nuclear and annexin V stain assays. The root methanolic extract of B. natalensis and C. comosum showed a high cytotoxicity of 8.6% and 16.7% respectively on the K562 cell line at 1,000 μg/ml concentration. Morphological loss of cell membrane integrity causing degradation of the cell and fragmentation were observed in the root methanolic extract of both plants. A high apoptosis (p < 0.0001) was induced in the K562 cells by both leaf and root extracts of the C. comosum compared to the B. natalensis. This study shows both plants possess apoptotic effect against in vitro myelogenous leukaemia which contributes to the overall anti-cancer properties of B. natalensis and C. comosum to justify future therapeutic applications against chronic myelogenous leukaemia blood cancer.

Resumo Bulbine natalensis Baker e Chorophytum comosum (Thunb.) Jacques são potenciais fontes medicinais para o tratamento de cânceres. A Leucemia Mieloide Crônica (LMC) é um distúrbio das células-tronco hematopoiéticas que é tratado com inibidores da tirosina quinase, mas frequentemente, causa recorrência da leucemia após a interrupção da terapia, portanto, requer um tratamento alternativo. Este estudo determinou o efeito anticancerígeno de extratos metanólicos e aquosos de folha, raiz e bulbo de B. natalensis e C. comosum na linhagem celular de leucemia mieloide humana crônica (K562) por ensaios de MTT, Hoechst bis-benzimida nuclear e anexina V. O extrato metanólico da raiz de B. natalensis e C. comosum apresentou alta citotoxidade de 8,6% e 16,7% respectivamente, na linhagem celular K562 com a concentração de 1,000 μg / ml. Perda morfológica da integridade da membrana celular causando degradação dos núcleos, citoplasma e encolhimento celular foi observada no extrato metanólico da raiz de ambas as plantas. Uma alta apoptose (p <0,0001) foi induzida nas células K562 por extratos de folhas e raízes de C. comosum em comparação com B. natalensis. Este estudo mostrou que ambas as plantas possuem efeito apoptótico contra leucemia mieloide in vitro que contribui para as propriedades anticâncer gerais de B. natalensis e C. comosum para justificar futuras aplicações terapêuticas contra câncer de sangue de LMC.

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This study aimed to investigate the impact of Vernonia amygdalina leaf extract on FLT3 regulation. V. amygdalina was extracted with 96% ethanol (VAE-96), and its cytotoxicity against FLT3- cell lines (MOLM-13 and MV-4-11) was evaluated. The results indicated that VAE-96 induced apoptosis in these cells and inhibited the phosphorylation of AKT, MAPK, and FLT3. Additionally, VAE-96 substantially diminished the activity of the FLT3 promoter and the expression of FLT3 mRNA. The extract was found to contain alkaloids, saponin, reduced sugar compounds, and polyphenols, including tannins and flavonoids, as per the phytoconstituents analysis. The potential of alkaloid fractions on MOLM-13 cells was indicated by the robust cytotoxic effect of the alkaloid fractions, which resulted in over 50% cell mortality at 30 µg/ml. Our results suggest that VAE-96 may be a beneficial agent for the prevention and treatment of AML with FLT3-ITD mutation.

Este estudo teve como objetivo investigar o impacto do extrato da folha de Vernonia amygdalina na regulação do FLT3. V. amygdalina foi extraída com etanol 96% (VAE-96) e sua citotoxicidade contra linhagens celulares FLT3 (MOLM-13 e MV-4-11) foi avaliada. Os resultados indicaram que o VAE-96 induziu apoptose nestas células e inibiu a fosforilação de AKT, MAPK e FLT3. Além disso, o VAE-96 diminuiu substancialmente a atividade do promotor FLT3 e a expressão do mRNA do FLT3. Descobriu-se que o extrato contém alcaloides, saponinas, compostos de açúcar reduzido e polifenóis, incluindo taninos e flavonoides, conforme análise de fitoconstituintes. O potencial das frações alcaloides nas células MOLM-13 foi indicado pelo intenso efeito citotóxico das frações alcaloides, que resultou em mais de 50% de mortalidade celular a 30 µg/ml. Nossos resultados sugerem que o VAE-96 pode ser um agente benéfico para a prevenção e tratamento da LMA com mutação FLT3-ITD.

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Purpose: This work aimed to investigate the effects of Tanshinone IIA (Tan IIA) on myocardial cell (MC) apoptosis in a rat model of heart failure (HF). Methods: Tan IIA was extracted from Salvia miltiorrhiza Bunge (SMB) using an ethanol reflux method. Fifty rats were randomly divided into five groups: sham (no treatment), mod (HF model establishment), low dose (LD: 0.1 mL/kg Tan IIA), medium dose (MD: 0.3 mL/kg Tan IIA), and high dose (HD: 0.5 mL/kg Tan IIA), with 10 rats in each group. The effects of different doses of Tan IIA on cardiac function, MC apoptosis, and the levels of proteins associated with the PI3K/Akt/mTOR signaling pathway were compared. Results: Mod group showed a significant decrease in systolic arterial pressure, mean arterial pressure, heart rate, left ventricular systolic pressure, left ventricular ejection fraction, left ventricular fractional shortening, and the levels of p-PI3K, p-Akt, and p-mTOR proteins versus sham group (p < 0.05). Additionally, the left ventricular end-diastolic diameter (LVIDd), end-systolic diameter, diastolic pressure, and MC apoptosis were significantly increased (p < 0.05). LD, MD, and HD groups exhibited significant improvements across various indicators of cardiac function and MC apoptosis versus mod group (p < 0.05). Conclusions: Tan IIA may improve cardiac function and inhibit MC apoptosis in rats with HF by modulating the PI3K/Akt/mTOR signaling pathway.

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Bisphenol A (BPA) may adversely affect human health by inducing oxidative stress and irreversible damage to cells. Bioactive compounds found in some functional foods, individually or in combination, can attenuate the negative effects of BPA exposure; an example is the multi-supplement containing guarana (Gua), selenium (Se), and L-carnitine (LC) —GSC— which has already demonstrated antioxidant, genoprotective, and immunomodulatory activities. This study aimed to determine the effect of GSC and its constituents on oxidative and genotoxic alterations triggered by BPA exposure in the retinal epithelial cell line. The cells exposed to BPA (0.001, 0.01, 0.1, 1, 3, and 10 µM) to determine the lowest concentration required to induce cyto-genotoxicity. ARPE-19 cells were then concomitantly exposed to the selected BPA concentration, GSC, and its components (Gua, 1.07 mg/mL; Se, 0.178 µg/mL; and LC, 1.43 mg/mL). Flow cytometry, biochemical assays, qRT-PCR, genotoxicity, apoptosis, and cellular proliferation. Based on our results, 10 µM of BPA could induce cyto-genotoxic and oxidative alterations. BPA did not alter the Bcl-2/BAX expression ratio but induced Casp3 and Casp8 overexpression, suggesting that apoptosis was induced mainly via the extrinsic pathway. GSC partially reversed the alterations triggered by BPA in ARPE-19 cells. However, Se had unexpected negative effects on ARPE-19 cells. The multi-supplement GSC may attenuate changes in oxidative and genotoxic markers related to exposure of ARPE-19 cells to BPA. our results revealed that the antioxidant, anti-apoptotic, and genoprotective properties of GSC were not universally shared by its individual, once Se did not exhibit any positive impact.

O Bisfenol A (BPA) pode afetar negativamente a saúde humana, induzindo estresse oxidativo e danos irreversíveis às células. Os compostos bioativos encontrados em alguns alimentos funcionais, individualmente ou em combinação, podem atenuar os efeitos negativos da exposição ao BPA; um exemplo é o multissuplemento contendo guaraná (Gua), selênio (Se) e L-carnitina (LC) —GSC— que já demonstrou atividades antioxidantes, genoprotetoras e imunomoduladoras. Este estudo teve como objetivo determinar o efeito do GSC e seus constituintes nas alterações oxidativas e genotóxicas desencadeadas pela exposição ao BPA na linhagem celular epitelial da retina (ARPE-19). Células foram tratadas em diferentes concentrações de BPA (0,001, 0,01, 0,1, 1, 3 e 10 µM) a fim de determinar a concentração mais baixa necessária para induzir citogenotoxicidade. As células ARPE-19 foram então expostas concomitantemente à concentração selecionada de BPA, o GSC e seus componentes (Gua, 1,07 mg/mL; Se, 0,178 µg/mL; e LC, 1,43 mg/mL). Ensaios de citometria de fluxo, bioquímicos, qRT-PCR, genotoxicidade, apoptose e proliferação celular foram executados. Com base em nossos resultados, 10 µM de BPA foi capaz de induzir alterações citogenotóxicas e oxidativas. O BPA não alterou a razão de expressão de Bcl-2/BAX, mas induziu a superexpressão de Casp3 e Casp8, sugerindo que a apoptose foi desencadeada principalmente pela via extrínseca. O GSC reverteu parcialmente as alterações desencadeadas pelo BPA nas células ARPE-19. No entanto, o Se teve efeitos negativos inesperados nas células ARPE-19. O GSC multissuplemento pode atenuar alterações nos marcadores oxidativos e genotóxicos relacionados à exposição das células ARPE-19 ao BPA. Nossos resultados revelaram que as propriedades antioxidantes, antiapoptóticas e genoprotetoras do GSC não foram universalmente compartilhadas pelo seu indivíduo, uma vez que o Se não apresentou nenhum impacto positivo.

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Purpose: The limitations in cancer treatment and the inadequacy of classical methods have made it necessary to discover therapeutics in cancer treatment. The cytotoxicity of thymoquinone, which has quite different properties in terms of biological activities, in ovarian cancer cells, and the changes in the expression levels of apoptotic genes (p53/caspase-3 (casp-3)) were investigated. Methods: In the study, thymoquinone (5, 50, 100, 250 and 500 µM and 24, 48, 72 hours) were applied to ovarian adenocarcinoma cancer cell line (OVCAR3), at different concentrations. Cytotoxic effect of thymoquinone on OVCAR-3 cells were analyzed by MTT method, and apoptotic and pro-apoptotic gene expression levels (p53, Casp-3) of thymoquinone in cancer cells were analyzed by quantitative real-time polymerase chain reaction. Results: Thymoquinone, whose effect has been revealed in many types of cancer, was shown to significantly reduce the viability of OVCAR3 cancer cells depending on the dose and time (p < 0.05). It was also determined that Casp-3 and p53 gene expressions increased in OVCAR3 cells. Conclusions: Considering the in-vitro cytotoxic activity and apoptotic gene expressions of thymoquinone, an important treatment agent, since it is a promising agent for the future of cancer treatment, more comprehensive studies may pave the way for its clinical use.

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Purpose: To evaluate the chemotherapeutic activity of temozolomide counter to mammary carcinoma. Methods: In-vitro anticancer activity has been conducted on MCF7 cells, and mammary carcinoma has been induced in Wistar rats by introduction of 7, 12-Dimethylbenz(a)anthracene (DMBA), which was sustained for 24 weeks. Histopathology, immunohistochemistry, cell proliferation study and apoptosis assay via TUNEL method was conducted to evaluate an antineoplastic activity of temozolomide in rat breast tissue. Results: IC50 value of temozolomide in MCF7 cell has been obtained as 103 µM, which demonstrated an initiation of apoptosis. The temozolomide treatment facilitated cell cycle arrest in G2/M and S phase dose dependently. The treatment with temozolomide suggested decrease of the hyperplastic abrasions and renovation of the typical histological features of mammary tissue. Moreover, temozolomide therapy caused the downregulation of epidermal growth factor receptor, extracellular signal-regulated kinase, and metalloproteinase-1 expression and upstream of p53 and caspase-3 proliferation to indicate an initiation of apoptotic events. Conclusions: The occurrence of mammary carcinoma has been significantly decreased by activation of apoptotic pathway and abrogation of cellular propagation that allowable for developing a suitable mechanistic pathway of temozolomide in order to facilitate chemotherapeutic approach.

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Puberty, governed by the endocrine system, marks the onset of reproductive functions in animals and humans through a series of physiological and biological transformations. Although the mycotoxin DON can disrupt hormonal balance and cause reproductive abnormalities, its impact on puberty-associated reproductive changes remains understudied. Considering the increased exposure of children and adolescents to DON, our study aimed to elucidate its influence on follicular integrity and the expression of pro-apoptotic proteins (BAX and Caspase-3) and anti-apoptotic protein (BCL-2) in juvenile rat ovarian tissue. We divided ten 28-day-old prepubertal Wistar rats into two dietary groups for 28 days: a control group with a mycotoxin-free diet and a DON group with a diet containing 10 mg DON/Kg. After the experiment, ovaries and uterus weights were recorded, and the ovaries underwent morphometric and immunohistochemical analysis. DON exposure led to significant reductions in both ovarian and uterine weights. Although DON intake did not change the number of ovarian follicles across developmental stages, we observed an increased expression of BAX and Caspase-3 and a decreased BCL-2 expression in most follicular stages and corpora lutea. In summary, DON exposure during puberty can interfere with apoptotic processes in diverse ovarian cell populations during early adulthood.(AU)

A puberdade é um processo regulado pelo sistema endócrino em que as diversas alterações fisiológicas e biológicas são responsáveis pelo início das funções reprodutivas em animais e humanos. DON pode interferir no sistema hormonal induzindo desordens endócrinas e danos reprodutivos. Entretanto, o potencial de DON em causar danos reprodutivos no período da puberdade tem sido negligenciado. Considerando que crianças e adolescentes têm um alto risco de exposição à DON, este estudo teve como objetivo avaliar o efeito desta micotoxina durante a puberdade sobre a integridade folicular e a expressão de proteínas pró-apoptóticas (BAX e Caspase-3) e anti-apoptóticas (BCL-2) em ovários de ratos jovens. Dez ratos Wistar pré-púberes (28 dias de idade) foram utilizados. Os animais foram expostos por 28 dias às seguintes dietas: 1) controle: dieta livre de micotoxinas e 2) DON: dieta contendo 10 mg DON/Kg de alimento. Ao final, os ovários e úteros foram pesados e os ovários submetidos à análise morfométrica e imuno-histoquímica. A ingestão da dieta contaminada induziu a uma redução significativa no peso dos ovários e úteros, no entanto não houve diferença no número de folículos ovarianos em todos os estágios de desenvolvimento folicular. Um aumento significativo da expressão de BAX e Caspase-3 e uma diminuição da expressão de BCL-2 foram observados na maioria dos estágios foliculares e do corpo lúteo nos animais alimentados com DON. Em conclusão, a exposição ao DON durante o período pré-púbere induz apoptose em diferentes populações de células ovarianas e pode interferir no desenvolvimento reprodutivo de animais adultos.(AU)

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In this study, it was aimed to determine the effect of sulforaphane (SFN) on rabbit semen cryopreservation. Semen collected from animals was divided into 5 equal volumes as Control, SFN 5 µM, SFN 10 µM, SFN 25 µM and SFN 50 µM groups. Afterwards, semen analyzes were performed. According to our results, there was no statistical difference between the groups at 4°C. However after freezing thawing, the highest total motility, progressive motility and rapid spermatozoa rate was seen in the 10 µM SFN group, while the lowest was observed in the 50 µM SFN group (P<0.05). Static sperm ratio was highest in the 50 µM group, while the lowest was observed in the 10 µM SFN group. When flow cytometry results examined the rate of acrosomal damaged and dead sperm was the lowest in the 10 µM SFN group, a statistical difference was observed between the control group (P<0.05). The highest rate of sperm with high mitochondrial membrane potential was seen in the 5 µM SFN and 10 µM SFN groups. Apoptosis and ROS rates were found to be lower in the experimental groups compared to the control groups (P<0.05). As a result, SFN supplementation at a dose of 10 µM increased the quality of sperm in the freezing and thawing processes of rabbit semen. In conclusion, 10 µM SFN improved the quality of cryopreservation of rabbit semen.(AU)

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Purpose: It was aimed to investigate the biochemical and immunohistochemical effects of ephedrine (EPH) in bilateral ovariectomized rats. Methods: Twenty-four Sprague Dawley female rats were divided into three groups: control group: The abdomen was opened and closed without any treatment; ischemia-reperfusion (IR) group: 2 h of ischemia followed by 2 h of reperfusion were allowed to cause IR injury; IR+EPH group: oral EPH solution (5 mg/kg) was administered for 28 days. Results: Biochemical parameters were statistically significant in group comparisons. Increased interleukin-6 (IL-6) expression, degenerative preantral and antral follicle cells and inflammatory cells around blood vessels were seen in IR group. Negative IL-6 expression was observed in seminal epithelial cells, preantral and antral follicle cells in IR+EPH group. While caspase-3 activity increased in granulosa cells and stromal cells in IR group, caspase-3 expression was negative in preantral and antral follicle cells in the germinal epithelium and cortex in IR+EPH group. Conclusion: The effect of apoptosis, which occurs with the signaling that starts in the cell nucleus, caused the cessation of the stimulating effect at the nuclear level after EPH administration, and a decrease in the antioxidative effect in IR damage and inflammation in the apoptotic process.

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Purpose: The effects of hesperidin application on the wound caused by esophageal burns were investigated in this study. Methods: Wistar albino rats were divided into three groups: Control group: only 1 mL of 0.09% NaCl was administered i.p. for 28 days; Burn group: An alkaline esophageal burn model was created with 0.2 mL of 25% NaOH orally by gavage­1 mL of 0.09% NaCl was administered i.p. for 28 days; Burn+Hesperidin group: 1 mL of 50 mL/kg of hesperidin was given i.p. for 28 days to rats after burn injury. Blood samples were collected for biochemical analysis. Esophagus samples were processed for histochemical staining and immunohistochemistry. Results: Malondialdehyde (MDA) and myeloperoxidase (MPO) levels were significantly increased in Burn group. Glutathione (GSH) content and histological scores of epithelialization, collagen formation, neovascularization was decreased. After hesperidin treatment, these values were significantly improved in the Burn+Hesperidin group. In the Burn group, epithelial cells and muscular layers were degenerated. Hesperidin treatment restored these pathologies in Burn+Hesperidin group. Ki-67 and caspase-3 expressions were mainly negative in control group; however, the expression was increased in the Burn group. In the Burn+Hesperidin group, Ki-67 and caspase-3 immune activities were reduced. Conclusion: Hesperidin dosage and application methods can be developed as an alternative treatment for burn healing and treatment.

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Thai Mucuna pruriens (L.) DC. var pruriens (T-MP) seed containing levodopa (L-DOPA) and antioxidant capacity has been shown to improve sexual behavior and male reproductive parameters in rats treated with ethanol (Eth). However, its protective effect on testicular apoptotic germ cells has never been reported. This study aimed to investigate the potential effects of T-MP seed extract on expressions of caspase, proliferating cell nuclear antigen (PCNA), and dopamine D2 receptor (D2R) proteins in Eth rats. Thirty-six male Wistar rats were divided into four groups (9 animals/group), including control, Eth, T-MP150+Eth, and T-MP300+Eth, respectively. Control rats received distilled water, and Eth rats received Eth (3g/kg BW; 40%v/v). The T-MP groups were treated with T-MP seed extract at a dose of 150 or 300 mg/kg before Eth administration for 56 consecutive days. The results showed that the seminiferous tubule diameter and epithelial height were significantly increased in both T-MP treated groups compared to the Eth group. Additionally, the caspase-9 and -3, and PCNA expressions were decreased, but D2R expression was markedly increased in T-MP groups. It was concluded that T-MP seed extract could protect testicular apoptosis induced by Eth via changes in caspase, PCNA, and D2R protein expressions.

Thai Mucuna pruriens (L.) DC. var pruriens (T-MP) contendo levodopa (L-DOPA) e capacidade antioxidante demonstrou melhorar o comportamento sexual e os parâmetros reprodutivos masculinos em ratos tratados com etanol (Eth). No entanto, seu efeito protetor sobre células germinativas apoptóticas testiculares nunca foi relatado. Este estudo teve como objetivo investigar os efeitos potenciais do extrato de semente de T-MP na expressão de proteínas de caspase, antígeno nuclear de proliferação celular (PCNA) e receptor de dopamina D2 (D2R) em ratos Eth. Trinta e seis ratos Wistar machos foram divididos em quatro grupos (9 animais/grupo), incluindo controle, Eth, T-MP150+Eth e T-MP300+Eth, respectivamente. Ratos controle receberam água destilada e ratos Eth receberam Eth (3g/kg PC; 40%v/v). Os grupos T-MP foram tratados com extrato de semente de T-MP na dose de 150 ou 300 mg/kg antes da administração de Eth por 56 dias consecutivos. Os resultados mostraram que o diâmetro dos túbulos seminíferos e a altura epitelial foram significativamente aumentados em ambos os grupos tratados com T-MP em comparação com o grupo Eth. Além disso, as expressões de caspase-9 e -3 e de PCNA diminuíram, mas a expressão de D2R aumentou acentuadamente nos grupos T-MP. Concluiu-se que o extrato de semente de T-MP pode proteger a apoptose testicular induzida por Eth através de alterações na expressão de proteínas caspase, PCNA e D2R.

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Purpose: Oxidative stress and apoptosis contribute to the pathological basis of doxorubicin (DOX)-induced cardiotoxicity. Columbianadin (CBN) is one of the main bioactive constituents isolated from the root of Angelica pubescens. Herein, we intended to explore the potential role and molecular basis of CBN in DOX-induced cardiotoxicity. Methods: C57BL/6 mice were subjected to DOX (15 mg/kg/day, i.p.) to generate DOX-induced cardiotoxicity. CBN (10 mg/kg/day, i.p.) was administered for four week following DOX injection. Results: DOX administered markedly dampened cardiac function, increased cardiac injury, excessive reactive oxygen species (ROS) production, and cardiomyocyte loss. These alterations induced by DOX significantly alleviated by CBN treatment. Mechanistically, our results demonstrated that the CBN exerts cardioprotection role against DOX by up-regulating silent information regulator 1 (Sirt1) and decreasing acetylation of forkhead box O1 (FOXO1). Moreover, Sirt1 inhibition with Ex-527 significantly blunt the beneficial effect of CBN on DOX-induced cardiotoxicity, including cardiac dysfunction, ROS, and apoptosis. Conclusion: Collectively, CBN attenuated oxidative stress and cardiomyocyte apoptosis in DOX-induced cardiotoxicity through maintaining Sirt1/FOXO1 signaling pathway. Our results demonstrated that CBN might be used to treat DOX-related cardiotoxicity.

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The objective of the present study was to evaluate the effect of lipopolysaccharide (LPS) administration on activation and apoptosis of primordial follicles. There was no difference in the total number of follicles as well as in the different types of follicles. Furthermore, the LPS challenge didn't modulate the expression of genes related with ovarian reserve (HAM), oocyte survival (Survivin), activation rate (Pten, KIT, KITL1, KITL2, AKT1, SIRT1), and follicular abnormalities. Therefore, the LPS exposure with 24h interval had no effect on activation rate and primordial follicles abnormalities, and also had no effect on expression of anti-apoptotic genes and genes related with ovarian reserve, oocyte survival, activation rate, and primordial follicles abnormalities.

O objetivo do presente estudo foi avaliar o efeito da administração de lipopolissacarídeo (LPS) na ativação e a apoptose de folículos primordiais. Dez novilhas saudáveis (Bos taurus taurus), com idade média de 14 meses, alojadas em sistema de confinamento e alimentadas com TMR, foram utilizadas neste experimento. Os animais foram distribuídos aleatoriamente em dois grupos: grupo LPS (LPS; n = 5), que recebeu duas injeções intravenosas de 0,5µg/kg de peso corporal de lipopolissacarídeo (Sigma Aldrich®) diluído em 2mL de solução salina (0,9% de NaCl), com intervalo de 24h; e grupo controle (CTR; n = 5), que recebeu duas injeções intravenosas de 2mL de solução salina (0,9% de NaCl), com intervalo de 24h. A primeira injeção de LPS foi realizada no d 1, e no d 5 os animais foram abatidos, os ovários foram pesados e as amostras dos ovários foram coletadas para avaliação histológica e molecular. Não houve diferença no número total de folículos, bem como nos diferentes tipos de folículos. Além disso, o desafio com LPS não modulou a expressão de genes relacionados à reserva ovariana (HAM), à sobrevivência oocitária (Survivin), à taxa de ativação (Pten, KIT, KITL1, KITL2, AKT1, SIRT1) e às anormalidades foliculares. Portanto, a exposição ao LPS com intervalo de 24h não teve efeito sobre a taxa de ativação e as anormalidades dos folículos primordiais, bem como não teve efeito sobre a expressão de genes antiapoptóticos e de genes relacionados com a reserva ovariana, a sobrevivência oocitária, a taxa de ativação e as anormalidades dos folículos primordiais.

Resumo

Purpose: The aim of this study was to determine the protective and antioxidative effects of intensive exercise on streptozotocin (STZ)-induced testicular damage, apoptotic spermatognial cells death, and oxidative stress. Methods: 36 male Sprague Dawley rats were divided into three groups: control, diabetes, and diabetes+intensive exercise (IE) groups. Testicular tissues were examined histopathologically and antioxidant enzymes, including catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), and malondialdehyde (MDA) activity, as well as serum testosterone level, were measured. Results: Seminiferous tubules and germ cells were found to be better in the testis tissue of the intense exercise group than in the diabetes group. Diabetes suppressed antioxidant enzymes CAT, SOD, GPx and testosterone levels were significantly decreased, and increased MDA level in the diabetic group compared to diabetes+IE group (p < 0.001). Following four weeks of treatment, intensive exercise improved the antioxidant defense, significantly decreased MDA activity, and increased testosterone levels in testicular tissue in the diabetic group compared to diabetes+IE group (p < 0.01). Conclusion: STZ-induced diabetes causes damage to the testis tissue. In order to prevent these damages, exercise practice has become very popular nowadays. In present study, our intensive exercise protocol, histological, and biochemical analysis of the effect of diabetes on the testicular tissues is shown.

Resumo

Colorectal cancer (CRC) is one of the most common cancers leading to comorbidities and mortalities globally. The rational of current study was to evaluate the combined epigallocatechin gallate and quercetin as a potent antitumor agent as commentary agent for therapeutic protocol. The present study investigated the effect of epigallocatechin Gallate (EGCG) (150mg) and quercetin (200mg) at different proportions on proliferation and induction of apoptosis in human colon cancer cells (HCT-116). Cell growth, colonogenic, Annexin V in addition cell cycle were detected in response to phytomolecules. Data obtained showed that, the colony formation was inhibited significantly in CRC starting from the lowest concentration tested of 10 µg/mL resulting in no colonies as visualized by a phase-contrast microscope. Data showed a significant elevation in the annexin V at 100 µg/mL EGCG(25.85%) and 150 µg/mL quercetin (48.35%). Moreover, cell cycle analysis showed that this combination caused cell cycle arrest at the G1 phase at concentration of 100 µg/mL (72.7%) and 150 µg/mL (75.25%). The combined effect of epigallocatechin Gallate and quercetin exert antiproliferative activity against CRC, it is promising in alternative conventional chemotherapeutic agent.

O câncer colorretal (CCR) é um dos cânceres mais comuns, levando a comorbidades e mortalidade em todo o mundo. O racional do presente estudo foi avaliar a combinação de galato de epigalocatequina e quercetina como um agente antitumoral potente como agente de comentário para protocolo terapêutico. O presente estudo investigou o efeito de galato de epigalocatequina (EGCG) (150 mg) e quercetina (200 mg) em diferentes proporções na proliferação e indução de apoptose em células de câncer de cólon humano (HCT-116). O crescimento celular, colonogênico, anexina V, além do ciclo celular foram detectados em resposta a fitomoléculas. Os dados obtidos mostraram que a formação de colônias foi inibida significativamente no CRC a partir da concentração mais baixa testada de 10 µg/mL, resultando em nenhuma colônia conforme visualizado por um microscópio de contraste de fase. Os dados mostraram uma elevação significativa na anexina V a 100 µg/mL de EGCG (25,85%) e 150 µg/mL de quercetina (48,35%). Além disso, a análise do ciclo celular mostrou que essa combinação causou parada do ciclo celular na fase G1 na concentração de 100 µg/mL (72,7%) e 150 µg/mL (75,25%). O efeito combinado da epigalocatequina galato e quercetina exerce atividade antiproliferativa contra o CCR, é promissor como agente quimioterápico alternativo convencional.

Resumo

Oral squamous cell carcinoma (OSCC) is a malignant tumour of Head and Neck Cancer (HNC). The recent therapeutic approaches used to treat cancer have adverse side effects. The natural agents exhibiting anticancer activities are generally considered to have a robust therapeutic potential. Curcuminoids, one of the major active compounds of the turmeric herb, are used as a therapeutic agent for several diseases including cancer. In this study, the cytotoxicity of curcuminoids was investigated against OSCC cell line HNO97. Our data showed that curcuminoids significantly inhibits the proliferation of HNO97 in a time and dose-dependent manner (IC50=35 μM). Cell cycle analysis demonstrated that curcuminoids increased the percentage of G2/M phase cell populations in the treated groups. Treating HNO97 cells with curcuminoids led to cell shrinking and increased detached cells, which are the typical appearance of apoptotic cells. Moreover, flow cytometry analysis revealed that curcuminoids significantly induced apoptosis in a time-dependent manner. Furthermore, as a response to curcuminoids treatment, comet tails were formed in cell nuclei due to the induction of DNA damage. Curcuminoids treatment reduced the colony formation capacity of HNO97 cells and induced morphological changes. Overall, these findings demonstrate that curcuminoids can in vitro inhibit HNC proliferation and metastasis and induce apoptosis.

O carcinoma de células escamosas oral (OSCC) é um tumor maligno do câncer de cabeça e pescoço (HNC). As recentes abordagens terapêuticas usadas para tratar o câncer têm efeitos colaterais adversos. Os agentes naturais que exibem atividades anticâncer são geralmente considerados como tendo um potencial terapêutico robusto. Curcuminoides, um dos principais compostos ativos da erva cúrcuma, são usados como agente terapêutico para várias doenças, incluindo câncer. Neste estudo, a citotoxicidade dos curcuminoides foi investigada contra a linha de células OSCC HNO97. Nossos dados mostraram que os curcuminoides inibem significativamente a proliferação de HNO97 de forma dependente do tempo e da dose (IC50 = 35 μM). A análise do ciclo celular demonstrou que os curcuminoides aumentaram a porcentagem de populações de células da fase G2 / M nos grupos tratados. O tratamento das células HNO97 com curcuminoides levou ao encolhimento celular e ao aumento das células destacadas, que são a aparência típica das células apoptóticas. Além disso, a análise de citometria de fluxo revelou que os curcuminoides induziram significativamente a apoptose de uma maneira dependente do tempo. Além disso, em resposta ao tratamento com curcuminoides, caudas de cometa foram formadas nos núcleos das células devido à indução de danos ao DNA. O tratamento com curcuminoides reduziu a capacidade de formação de colônias das células HNO97 e induziu alterações morfológicas. No geral, esses achados demonstram que os curcuminoides podem inibir in vitro a proliferação e metástase de HNC e induzir apoptose.

Resumo

This study investigated the use of melatonin to arrest the effects of apoptosis in vitrified zebrafish (D. rerio) embryos. Dechorionated embryos at 22-24 somite-stage were divided (n = 60/treatment) into a non-vitrified (Control Group, 0 M melatonin) and vitrified treatments with 0 M (T1), 1 µM (T2) and 1 mM of melatonin (T3). For vitrified treatments, a solution methanol/propylene glycol based was used and the embryos stored in -196 °C for a week. After thaw, survival rate, scanning electron microscopy, expression of anti (bcl-2) and pro-apoptotic (bax/caspase-3) genes, reactive oxygen species (ROS) formation and DNA fragmentation analyses were performed. No live embryos were obtained from vitrified treatments, observing a rapid degeneration immediately after thawing, with the vitelline layer rupture and leakage of its content, followed by breakdown of epithelial cells and melanisation of the tissue. Regarding the apoptotic process, T3 had the highest relative gene expression, for the three genes (P < 0.05) furthermore, T2 had similar expression of pro-apoptotic genes to CG (P < 0.05). ROS formation revealed that CG presented lower percentage of embryo surface area affected (3.80 ± 0.40%) (P < 0.05), in contrast, no differences were found among the other groups. T1 was most significantly (P < 0.05) damaged by DNA fragmentation. The vitrified groups with melatonin had similar damage levels of CG (P > 0.05). The inclusion of 1 µM of melatonin in the vitrifying solution, countered the effects of apoptotic process in post-thaw embryos, suggesting its utility in cryopreserving fish embryos.

Este estudo investigou o uso da melatonina para conter os efeitos da apoptose em embriões vitrificados de zebrafish (D. rerio). Embriões descorionados no estágio de 22-24 somitos foram divididos (n = 60 / tratamento) em tratamento não vitrificado (Grupo Controle, melatonina 0 M) e tratamentos vitrificados com 0 M (T1), 1 µM (T2) e 1 mM de melatonina (T3). Para os tratamentos vitrificados, utilizou-se uma solução à base de metanol/propilenoglicol e os embriões foram armazenados em -196 °C por uma semana. Após o descongelamento, foram realizadas análises de taxa de sobrevivência, microscopia eletrônica de varredura, expressão dos genes anti (bcl-2) e pró-apoptóticos (bax/caspase-3), formação de espécies reativas de oxigênio (EROS) e análises de fragmentação de DNA. Não foram obtidos embriões vivos a partir dos tratamentos vitrificados, observando uma rápida degeneração imediatamente após o descongelamento, com ruptura da camada vitelina e vazamento de seu conteúdo, seguida de quebra das células epiteliais e melanização do tecido. Em relação ao processo apoptótico. T3 apresentou expressão gênica relativa alta para os três genes (P <0,05), além disso, T2 apresentou expressão semelhante as dos genes pró-apoptóticos de GC (P <0,05). A formação de EROS revelou que GC apresentou menor percentual de área de superfície embrionária afetada (3,80 ± 0,40%) (P <0,05), ao contrário, não foram encontradas diferenças entre os outros grupos. T1 foi mais significativamente (P <0,05) danificado pela fragmentação do DNA. Os grupos vitrificados com melatonina apresentaram níveis de dano semelhantes ao do GC (P> 0,05). A inclusão de 1 µM de melatonina na solução de vitrificação, contrariou os efeitos do processo apoptótico em embriões pós-descongelamento, sugerindo sua utilidade na criopreservação de embriões de peixes.

Resumo

Os testículos, são mantidos no escroto a uma temperatura ~3-5°C abaixo da corporal. Quando a temperatura das gônadas se eleva, instala-se um quadro de estresse térmico (ET) testicular. O ET afeta a espermatogênese, e observam-se, já na primeira semana pós-ET, impactos na cinética, concentração e morfologia espermática. Classicamente, tais efeitos eram creditados à incapacidade da circulação local de atender ao aumento do metabolismo testicular devido ao aumento da temperatura local. Contudo, estudos recentes demonstraram que a hipóxia não era a causa da degeneração testicular. Atualmente, credita-se os efeitos deletérios do ET ao aumento local das espécies reativas de O2. Nesta situação, apesar da ativação de mecanismos antioxidantes (aumento das HSP e GPX1) e de proteção do DNA (aumento da P53), estes não são suficientes, sendo desencadeada a apoptose. Os efeitos deletérios do ET testicular podem ser mitigados pela melatonina, que pode ser tanto administrada aos animais ou adicionada ao sêmen para que desencadeie seus efeitos protetores.(AU)

The testes are kept in the scrotum at a ~3-5°C below body core temperature. When the temperature of the gonads increases, a process called heat stress (HS) takes place. The HS impairs spermatogenesis, and in the first week post-HS, impacts in sperm kinetics, concentration, and morphology are observed. Classically, such effects were credited to the incapacity of the local circulation to sustain the higher testicular metabolism due to the increased temperature. However, recent studies demonstrated that it was not the cause of testicular degeneration. The novel perspective credits the deleterious impacts of the HS to the local increase of the reactive oxygen species. Importantly, although there's an activation of antioxidant defenses (increase in HSP and GPX1) and DNA protection (increase in P53), such mechanisms are not sufficient, unfolding the apoptotic cascade. Lastly, some of the negative effects of HS can be mitigated by melatonin, which can either be given to the animals or added to the sperm to exert its protective effects.(AU)