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1.
J Toxicol Sci ; 45(9): 549-558, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32879254

RESUMO

Trimethyltin chloride (TMT) is a stabilizer by-product in the process of manufacturing plastic, which is a kind of very strong toxic substance, and has acute, cumulative and chronic toxicity. TMT may cause bradycardia in patients with occupational poisoning, the mechanism of which has not been reported. This study explored the mechanism of TMT resulting in bradycardia of C57BL/6 mice. TMT was administered to mice to measure heart rate, serum succinate dehydrogenase (SDH) level, and myocardial Na+/K+-ATPase activity and expression. The effects of TMT on myocardial apoptosis were observed by changing the expressions of caspase-3, Bax and Bcl-2 in myocardium. It was found that the heart rate and SDH activity in serum of mice gradually decreased with the increase of TMT dose compared with the control group. The activity and the expression of Na+/K+-ATPase in the heart tissue of mice exposed to TMT was measured and gradually decreased with the increase of dose and time. We measured the expression of Bcl-2, Bax, caspase-3 and cleaved caspase-3 in the heart tissues of TMT exposed mice and found that the expressions of Bax, caspase-3 and cleaved caspase-3 increased and the expressions of Bcl-2 decreased in the heart tissues of the TMT-exposed mice at different doses. With the extension of TMT exposure time, the expression of Bax and caspase-3 increased and the expression of Bcl-2 decreased in the heart tissues of TMT exposed mice. Our findings suggest the mechanisms of TMT resulting in bradycardia may be associated with the inhibited activity and decreased content of Na+/K+-ATPase, thus further leading to the changes of Bcl-2, Bax, caspase-3 and cleaved caspase-3 in the mice's ventricular tissues.


Assuntos
Apoptose/efeitos dos fármacos , Bradicardia/etiologia , Miocárdio/metabolismo , Miocárdio/patologia , ATPase Trocadora de Sódio-Potássio/genética , ATPase Trocadora de Sódio-Potássio/metabolismo , Compostos de Trimetilestanho/toxicidade , Animais , Apoptose/genética , Bradicardia/genética , Caspase 3/genética , Caspase 3/metabolismo , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo
2.
Int Heart J ; 61(4): 806-814, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32728001

RESUMO

This study aimed to explore the function of miR-24 in hypoxia/reoxygenation (H/R) -induced cardiomyocyte injury.We constructed a cardiomyocyte model of H/R using the primary cardiomyocytes isolated from Sprague-Dawley rats. To explore the role of miR-24, cells were transfected with a miR-24 mimic or miR-24 inhibitor. The RNA expression levels of miR-24 and Mapk14 were determined using qRT-PCR. The proliferation and apoptosis of cells were determined using a CCK8 assay and a flow cytometer. The TargetScan website was used to predict the targets of miR-24. A dual-luciferase reporter gene assay was conducted to verify whether Mapk14 is indeed a target of miR-24. A Western blot was applied for protein detection.H/R exposure decreased the expression of miR-24 in rat cardiomyocytes. Transfection of the miR-24 mimic into cardiomyocytes reduced H/R-induced injury as evidenced by an increase in proliferation and a decrease in the apoptotic rate. By contrast, transfection of the miR-24 inhibitor aggravated H/R-induced injury. The expression of Bcl-2 was increased while the levels of Bax and Active-caspase 3 were reduced in the H/R+miR-24 mimic group compared to those in the H/R group. H/R+miR-24 inhibitor group showed the opposite results. Mapk14 was identified as a target of miR-24. The mRNA level of Mapk14 and its protein (p38 MAPK) level were negatively affected by miR-24. Furthermore, we discovered that depletion of Mapk14 reduced the promoting effect of the miR-24 inhibitor on cell apoptosis.Overall, our results illustrated that miR-24 could attenuate H/R-induced injury partly by regulating Mapk14.


Assuntos
Hipóxia/metabolismo , MicroRNAs/genética , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Apoptose/fisiologia , Caspase 3/metabolismo , Genes Reporter/genética , Genes bcl-2/genética , Humanos , Ratos , Ratos Sprague-Dawley , Transfecção/métodos , Proteína X Associada a bcl-2/metabolismo
3.
J Stroke Cerebrovasc Dis ; 29(8): 104977, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32689608

RESUMO

BACKGROUND: Ischemic stroke is a severe neurological disorder that affected millions of people worldwide. Neuro-inflammation and apoptosis play an essential role in the pathogenesis of neuronal death during ischemic stroke. Alpha-pinene is a bicyclic terpenoid with anti-inflammatory and anti-apoptotic activities. Accordingly, the main purpose of this study was to assess the protective effect of α-pinene in ischemic stroke. MATERIALS AND METHODS: To induce ischemic stroke in male Wistar rats, the middle cerebral artery was occluded for 60 min followed by 24 h reperfusion. Alpha-pinene was injected intraperitoneally at the beginning of reperfusion. A day after reperfusion, the neurological deficits, volume of infarct area, and blood-brain barrier (BBB) permeability were evaluated. The mRNA expression of inflammatory cytokines as well as pro- and anti-apoptotic genes was assessed by using reverse transcription-polymerase chain reaction. The protein levels of inflammatory cytokines were also measured by ELISA method. RESULTS: The results showed that α-pinene (50 and 100 mg/kg) significantly improved sensorimotor function and decreased the volume of infarct area in the brain. The high permeability of BBB was also alleviated by α-pinene (50 and 100 mg/kg) in ischemic areas. Besides, α-pinene (100 mg/kg) attenuated neuro-inflammation through decreasing both the gene and protein expression of TNF-α and IL-1ß in the hippocampus, cortex, and striatum. Besides, α-pinene (100 mg/kg) suppressed apoptosis via downregulation of the pro-apoptotic Bax mRNA expression with a concomitant upregulation of anti-apoptotic Bcl-2 gene expression. CONCLUSIONS: Overall, it was concluded that α-pinene exerts neuroprotective effect during ischemic stroke through attenuating neuroinflammation and inhibition of apoptosis.


Assuntos
Anti-Inflamatórios/farmacologia , Apoptose/efeitos dos fármacos , Monoterpenos Bicíclicos/farmacologia , Encéfalo/efeitos dos fármacos , Citocinas/metabolismo , Infarto da Artéria Cerebral Média/tratamento farmacológico , Mediadores da Inflamação/metabolismo , Fármacos Neuroprotetores/farmacologia , Traumatismo por Reperfusão/prevenção & controle , Animais , Comportamento Animal/efeitos dos fármacos , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Encéfalo/fisiopatologia , Permeabilidade Capilar/efeitos dos fármacos , Citocinas/genética , Modelos Animais de Doenças , Infarto da Artéria Cerebral Média/metabolismo , Infarto da Artéria Cerebral Média/patologia , Infarto da Artéria Cerebral Média/fisiopatologia , Masculino , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos Wistar , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Traumatismo por Reperfusão/fisiopatologia , Transdução de Sinais , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo
4.
Mol Cell ; 79(1): 68-83.e7, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32533918

RESUMO

BAX is a pro-apoptotic protein that transforms from a cytosolic monomer into a toxic oligomer that permeabilizes the mitochondrial outer membrane. How BAX monomers assemble into a higher-order conformation, and the structural determinants essential to membrane permeabilization, remain a mechanistic mystery. A key hurdle has been the inability to generate a homogeneous BAX oligomer (BAXO) for analysis. Here, we report the production and characterization of a full-length BAXO that recapitulates physiologic BAX activation. Multidisciplinary studies revealed striking conformational consequences of oligomerization and insight into the macromolecular structure of oligomeric BAX. Importantly, BAXO enabled the assignment of specific roles to particular residues and α helices that mediate individual steps of the BAX activation pathway, including unexpected functionalities of BAX α6 and α9 in driving membrane disruption. Our results provide the first glimpse of a full-length and functional BAXO, revealing structural requirements for the elusive execution phase of mitochondrial apoptosis.


Assuntos
Apoptose , Mitocôndrias/patologia , Membranas Mitocondriais/metabolismo , Multimerização Proteica , Proteína X Associada a bcl-2/química , Proteína X Associada a bcl-2/metabolismo , Animais , Transporte Biológico , Permeabilidade da Membrana Celular , Citosol/metabolismo , Humanos , Camundongos , Mitocôndrias/metabolismo , Modelos Moleculares , Conformação Proteica , Proteínas Proto-Oncogênicas c-fos
5.
Life Sci ; 256: 117958, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32553929

RESUMO

PROPOSE: Understanding the protective effect of exercise against ethanol-induced toxicity through the oxidative stress signaling pathway, apoptosis, and cholesterol metabolism is important to prevent development of cardiovascular diseases. METHODS: Thirty-two male Wistar rats were randomly divided into four equal groups as follow: control, exercise training (ET), ethanol (4 g/kg of body weight/day) and ET + ethanol. The ET and ET + Ethanol groups ran on the treadmill at 65% maximum running speed for 60 min for five sessions per week for eight weeks. The ethanol and ET + Ethanol groups received ethanol for eight weeks. At the end of the study, animals were anesthetized and blood and tissues were sampled to examine the biochemical and molecular evaluation. RESULTS: The results showed that the antioxidant enzymes activity decreased and MDA levels increased in the heart and liver of animals in ethanol group compared to control group. The levels of these oxidative biomarkers improved by ET in ET + Ethanol group compared to ethanol group. It showed that ET could protect the heart and liver against oxidative damage induced by ethanol through up-regulating the expression of the Nrf2/Keap-1/HO-1 pathway. ET could exert a cardioprotective effect on ethanol-induced apoptosis through down-regulating the Bax and the caspase-3 and via up-regulating the Bcl-2 expression in the heart. ET could also improve the impairment of cholesterol metabolism induced by ethanol. CONCLUSION: Exercise can protect against ethanol-induced toxicity through moderating the expression of genes which are involved in oxidative status, apoptosis and cholesterol metabolism.


Assuntos
Apoptose , Etanol/toxicidade , Heme Oxigenase-1/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fígado/patologia , Miocárdio/patologia , Fator 2 Relacionado a NF-E2/metabolismo , Condicionamento Físico Animal , Animais , Antioxidantes/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/genética , Biomarcadores/sangue , Caspase 3/genética , Caspase 3/metabolismo , Colesterol/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Lipídeos/sangue , Fígado/efeitos dos fármacos , Masculino , Estresse Oxidativo/efeitos dos fármacos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo
6.
Chem Biol Interact ; 327: 109184, 2020 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-32590070

RESUMO

Scoulerine, an isoquinoline alkaloid isolated from Corydalis plants, has been reported to possess potent anti-proliferative and pro-apoptotic function in cancer cells. However, the effects and underlying mechanisms of scoulerine on colorectal cancer (CRC) progression remain elusive. CCK-8 and LDH assays were used to evaluate cell viability. Apoptosis was assessed by flow cytometry analysis, caspase-3/7 activity assay, and Western blot analysis of Bax, Bcl-2 and cytochrome c (Cyt C) expression. Oxidative stress level was examined by measuring reactive oxygen species (ROS) and glutathione (GSH) contents and superoxide dismutase (SOD) activity. Endoplasmic reticulum (ER) stress activation was detected by Western blot analysis of glucose-regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP) expression. Results showed that scoulerine dose-dependently suppressed CRC cell viability. Scoulerine induced apoptosis and increased caspase-3/7 activity in CRC cells. Bax and cytosolic Cyt C expression was enhanced while Bcl-2 and mitochondrial Cyt C expression was reduced in scoulerine-treated CRC cells. Additionally, scoulerine induced oxidative damage in CRC cells by increasing ROS generation and reducing GSH content and SOD activity. Scoulerine activated ER stress, as evidenced by the increased GRP78 and CHOP expression in CRC cells. Interestingly, blocking ROS production by ROS scavenger N-acetyl-cysteine (NAC) attenuated scoulerine-induced ER stress. Inhibition of ER stress by 4-phenyl butyric acid (4-PBA) abolished scoulerine-induced ROS generation in CRC cells. Blockage of ROS and ER stress attenuated scoulerine-induced cell viability reduction and apoptosis in CRC cells. In conclusion, scoulerine promoted cell viability reduction and apoptosis by activating ROS-dependent ER stress in CRC cells.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Alcaloides de Berberina/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/metabolismo , Citocromos c/metabolismo , Humanos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo
7.
Life Sci ; 256: 117924, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32522568

RESUMO

AIMS: Osteoarthritis (OA) is a common degenerative joint disease characterized by cartilage degeneration and joint inflammation. As its pathogenesis remains unclear, there are no effective treatments established. Circular RNA (circRNA), microRNA (miRNA), and other noncoding RNAs participate in OA development; however, the effects and mechanisms of circRNA and miRNA in OA remain unknown. MAIN METHODS: Cartilage miRNA was examined in patients with and without OA. KEY FINDINGS: CircRNA-9119 and phosphatase and tensin homolog (PTEN) expression decreased in OA-affected cartilage and interleukin (IL)-1ß-induced chondrocytes, and miR-26a expression significantly decreased in normal cells and tissues. CircRNA-9119 overexpression restored chondrocyte growth, whereas IL-1ß treatment impaired chondrocyte growth. Annexin V-FITC & PI flow cytometry and Bcl-2/Bax ratio measurement indicated that the apoptosis of IL-1ß-treated articular chondrocytes was decreased by circRNA-9119 upregulation. Bioinformatic prediction and the dual-luciferase reporter assay indicated that circRNA-9119 served as a miR-26a sponge and that miR-26a targeted the 3'-UTR of PTEN. Transfection of chondrocytes with a circRNA-9119-overexpressing vector revealed downregulation of miR-26a expression. Furthermore, circRNA-9119 overexpression induced PTEN expression. In addition, a miR-26a mimic induced IL-1ß-induced chondrocyte apoptosis, and circRNA-9119 overexpression inhibited IL-1ß-induced chondrocyte apoptosis. SIGNIFICANCE: CircRNA-9119 is an important regulator of IL-1ß-treated chondrocytes through the miR-26a/PTEN axis, possibly contributing to OA development.


Assuntos
Condrócitos/metabolismo , Interleucina-1beta/metabolismo , Osteoartrite/tratamento farmacológico , Substâncias Protetoras/metabolismo , RNA Circular/metabolismo , Regiões 3' não Traduzidas , Apoptose/efeitos dos fármacos , Cartilagem/metabolismo , Cartilagem Articular/metabolismo , Linhagem Celular , Condrócitos/citologia , Regulação para Baixo , Matriz Extracelular/metabolismo , Humanos , MicroRNAs/genética , PTEN Fosfo-Hidrolase/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Regulação para Cima , Proteína X Associada a bcl-2/metabolismo
8.
J Cancer Res Clin Oncol ; 146(7): 1751-1764, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32377840

RESUMO

PURPOSE: Although important for apoptosis, the signaling pathway involving MOAP-1(Modulator of Apoptosis 1), RASSF1A (RAS association domain family 1A), and Bax (Bcl-2 associated X protein) is likely to be dysfunctional in many types of human cancers due to mechanisms associated with gene mutation and DNA hyper-methylation. The purpose of the present study was to assess the potential impact of generating physiologically relevant signaling pathway mediated by MOAP-1, Bax, and RASSF1A (MBR) in cancer cells and chemo-drug resistant cancer cells. METHODS: The tricistronic expression construct that encodes MOAP-1, Bax, and RASSF1A (MBR) or its mutant, MOAP-1∆BH3L, Bax and RASSF1A (MBRX) was expressed from an IRES (Internal Ribosome Entry Site)-based tricistronic expression vector in human breast cancer cells, including MCF-7, MCF-7-CR (cisplatin resistant) and triple negative breast cancer cells, BMET05, for functional characterization through in vitro and in vivo models. RESULTS: Transient expression of MBR potently promoted dose-dependent apoptotic signaling and chemo-sensitization in the cancer cells, as evidenced by loss of cell viability, nuclei condensation and Annexin-V positive staining while stable expression of MBR in MCF-7 cells significantly reduced the number of MBR stable clone by 86% and the stable clone exhibited robust chemo-drug sensitivity. In contrast, MBRX stable clone exhibited chemo-drug resistance while transiently over-expressed MOAP-1ΔBH3L inhibited the apoptotic activity of MBR. Moreover, the spheroids derived from the MBR stable clone displayed enhanced chemo-sensitivity and apoptotic activity. In mouse xenograft model, the tumors derived from MBR stable clone showed relatively high level of tumor growth retardation associated with the increase in apoptotic activity, leading to the decreases in both tumor weight and volume. CONCLUSIONS: Expression of MBR in cancer cells induces apoptotic cell death with enhanced chemo-sensitization requiring the BH3L domain of MOAP-1. In animal model, the expression of MBR significantly reduces the growth of tumors, suggesting that MBR is a potent apoptotic sensitizer with potential therapeutic benefits for cancer treatment.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Reguladoras de Apoptose/genética , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Domínios e Motivos de Interação entre Proteínas , Proteínas Supressoras de Tumor/genética , Proteína X Associada a bcl-2/genética , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Modelos Animais de Doenças , Genes Reporter , Humanos , Camundongos , Modelos Biológicos , Ligação Proteica , Proteínas Supressoras de Tumor/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína X Associada a bcl-2/metabolismo
9.
Life Sci ; 254: 117760, 2020 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-32418889

RESUMO

AIM: The present study focused on the possible underlying protective mechanisms of UDCA against GNT-induced hepatic injury. METHODS: For achieving this goal, adult male rats were allocated into 4 groups: normal control (received vehicle), GNT (100 mg/kg, i.p. for 8 days), UDCA (60 mg/kg, P.O. for 15 days), and GNT + UDCA (received UDCA for 15 days and GNT started from the 7th day and lasted for 8 days). RESULTS: The results revealed that UDCA significantly improved GNT-induced hepatic injury, oxidative stress, apoptosis, and inflammatory response. Interestingly, UDCA inhibited apoptosis by marked down-regulation of the Bax gene, Caspase-3, and cleaved Caspase-3 protein expressions while the level of Bcl-xL gene significantly increased. Moreover, UDCA strongly inhibited the inflammatory response through the down-regulation of both NF-κB-p65 and TNF-α accompanied by IL-10 elevation. Furthermore, the obtained results ended with the restored of mitochondria function that confirmed by electron microscopy. Histological analysis showed that UDCA remarkably ameliorated the histopathological changes induced by GNT. SIGNIFICANCE: UDCA may be a promising agent that can be used to prevent hepatotoxicity observed in GNT treatment. This effect could be attributed to, at least in part, the ability of UDCA to modulate NF-κB-p65/TNF-α, Bax/Bcl-xl/Caspase-3, and eNOS/iNOS signaling pathways.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Gentamicinas/antagonistas & inibidores , Gentamicinas/toxicidade , Hepatócitos/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Ácido Ursodesoxicólico/farmacologia , Animais , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Interações Medicamentosas , Hepatócitos/metabolismo , Hepatócitos/patologia , Masculino , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Distribuição Aleatória , Ratos , Ratos Wistar , Fator de Transcrição RelA/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Proteína X Associada a bcl-2/metabolismo , Proteína bcl-X/metabolismo
10.
Mol Biol (Mosk) ; 54(2): 233-243, 2020.
Artigo em Russo | MEDLINE | ID: mdl-32392192

RESUMO

Obesity is a major disease that causes significant complications. Inhibition of preadipocyte proliferation has the potential to prevent obesity and metabolic diseases. Melatonin is a pineal gland hormone that has various effects on cells and tissues. In this research, we investigated whether melatonin induces apoptosis in 3T3-L1 preadipocytes. 3T3-L1 preadipocytes were cultured until confluence and then treated with 0, 10, 100, and 1000 µM melatonin for 1, 3, and 5 days. A cell viability assay kit was used for determining cell viability. Cell death marker proteins were assessed by Western blot analysis using GAPDH for control. Apoptotic morphological changes with nuclei fragmentation were observed using DAPI staining. Melatonin treatment decreased the phosphorylated extracellular signal-regulated kinases (p-ERK) activation while increasing the activation of caspase-3, 8, and 9. Furthermore, melatonin not only increased Bcl-2-associated X protein (Bax) but decreased B-cell lymphoma 2 (Bcl-2) expression as dose increases from 0 to 1000 µM. The melatonin treatment also suppressed the growth of preadipocytes with increasing concentration. These effects were attenuated by luzindole, a melatonin receptor antagonist and U0126, an inhibitor of p-ERK activation. In conclusion, melatonin can induce apoptosis of 3T3-L1 preadipocytes via p-ERK decrease.


Assuntos
Adipócitos/citologia , Apoptose , Melatonina/farmacologia , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Animais , Caspases/metabolismo , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Camundongos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo
11.
Nat Commun ; 11(1): 2598, 2020 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-32451402

RESUMO

DNA double-strand breaks (DSBs) are toxic to mammalian cells. However, during meiosis, more than 200 DSBs are generated deliberately, to ensure reciprocal recombination and orderly segregation of homologous chromosomes. If left unrepaired, meiotic DSBs can cause aneuploidy in gametes and compromise viability in offspring. Oocytes in which DSBs persist are therefore eliminated by the DNA-damage checkpoint. Here we show that the DNA-damage checkpoint eliminates oocytes via the pro-apoptotic BCL-2 pathway members Puma, Noxa and Bax. Deletion of these factors prevents oocyte elimination in recombination-repair mutants, even when the abundance of unresolved DSBs is high. Remarkably, surviving oocytes can extrude a polar body and be fertilised, despite chaotic chromosome segregation at the first meiotic division. Our findings raise the possibility that allelic variants of the BCL-2 pathway could influence the risk of embryonic aneuploidy.


Assuntos
Mutação , Oócitos/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Reparo de DNA por Recombinação/genética , Aneuploidia , Animais , Apoptose , Proteínas Reguladoras de Apoptose/deficiência , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Ciclo Celular/deficiência , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Segregação de Cromossomos , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/deficiência , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Feminino , Fertilização , Genes bcl-2 , Meiose/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oócitos/citologia , Proteínas de Ligação a Fosfato/deficiência , Proteínas de Ligação a Fosfato/genética , Proteínas de Ligação a Fosfato/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/deficiência , Proteínas Proto-Oncogênicas c-bcl-2/genética , Transdução de Sinais , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Proteína X Associada a bcl-2/deficiência , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo
12.
Clin Sci (Lond) ; 134(11): 1191-1218, 2020 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-32432676

RESUMO

Myocardial infarction (MI) is the leading cause of mortality worldwide. Interleukin (IL)-33 (IL-33) is a cytokine present in most cardiac cells and is secreted on necrosis where it acts as a functional ligand for the ST2 receptor. Although IL-33/ST2 axis is protective against various forms of cardiovascular diseases, some studies suggest potential detrimental roles for IL-33 signaling. The aim of the present study was to examine the effect of IL-33 administration on cardiac function post-MI in mice. MI was induced by coronary artery ligation. Mice were treated with IL-33 (1 µg/day) or vehicle for 4 and 7 days. Functional and molecular changes of the left ventricle (LV) were assessed. Single cell suspensions were obtained from bone marrow, heart, spleen, and peripheral blood to assess the immune cells using flow cytometry at 1, 3, and 7 days post-MI in IL-33 or vehicle-treated animals. The results of the present study suggest that IL-33 is effective in activating a type 2 cytokine milieu in the damaged heart, consistent with reduced early inflammatory and pro-fibrotic response. However, IL-33 administration was associated with worsened cardiac function and adverse cardiac remodeling in the MI mouse model. IL-33 administration increased infarct size, LV hypertrophy, cardiomyocyte death, and overall mortality rate due to cardiac rupture. Moreover, IL-33-treated MI mice displayed a significant myocardial eosinophil infiltration at 7 days post-MI when compared with vehicle-treated MI mice. The present study reveals that although IL-33 administration is associated with a reparative phenotype following MI, it worsens cardiac remodeling and promotes heart failure.


Assuntos
Eosinófilos/metabolismo , Ventrículos do Coração/patologia , Ventrículos do Coração/fisiopatologia , Interleucina-33/farmacologia , Infarto do Miocárdio/fisiopatologia , Sístole/efeitos dos fármacos , Remodelação Ventricular/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Citocinas/sangue , Fragmentação do DNA/efeitos dos fármacos , Diástole/efeitos dos fármacos , Eosinofilia/patologia , Eosinófilos/efeitos dos fármacos , Fibrose , Ventrículos do Coração/efeitos dos fármacos , Hipertrofia Ventricular Esquerda/patologia , Mediadores da Inflamação/sangue , Interleucina-33/administração & dosagem , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/enzimologia , Infarto do Miocárdio/genética , Infarto do Miocárdio/patologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Esplenomegalia/patologia , Regulação para Cima/efeitos dos fármacos , Remodelação Ventricular/genética , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo
13.
Arch Biochem Biophys ; 688: 108402, 2020 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-32418909

RESUMO

A/J mouse is a model of age-related hearing loss (AHL). Mutation in the citrate synthase (Cs) gene of the mouse plays an important role in the hearing loss and degeneration of cochlear cells. To investigate the pathogenesis of cochlear cell damage in A/J mice resulted from Cs mutation, we downregulated the expression level of CS in HEI-OC1, a cell line of mouse cochlea, by shRNA. The results showed that low CS expression led to low ability of cell proliferation. Further study revealed an increase level of reactive oxygen species (ROS), activation of ATF6 mediated endoplasmic reticulum stress (ERS) and high expression levels of caspase12 and Bax in the cells. Moreover, the AEBSF, an ATF6 inhibitor, could reduce the expression levels of caspase-12 and Bax by inhibiting the hydrolysis of ATF6 in the cells. Finally, antioxidant alpha-lipoic acid (ALA) reduced the ROS levels and the apoptotic signals in the cell model with low CS expression. We therefore conclude that the ERS mediated apoptosis, which is triggered by ROS, may be involved in the cell degeneration in the cochleae of A/J mice.


Assuntos
Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Citrato (si)-Sintase/metabolismo , Estresse do Retículo Endoplasmático/fisiologia , Estresse Oxidativo/efeitos dos fármacos , Ácido Tióctico/farmacologia , Fator 6 Ativador da Transcrição/antagonistas & inibidores , Animais , Apoptose/fisiologia , Caspase 12/metabolismo , Linhagem Celular , Proliferação de Células/fisiologia , Regulação para Baixo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Camundongos , Mitocôndrias/metabolismo , Modelos Biológicos , Estresse Oxidativo/fisiologia , Presbiacusia/fisiopatologia , Espécies Reativas de Oxigênio/metabolismo , Sulfonas/farmacologia , Proteína X Associada a bcl-2/metabolismo
14.
Nucleic Acids Res ; 48(11): 6326-6339, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32374860

RESUMO

Nucleotide excision repair (NER) is a major DNA repair pathway for a variety of DNA lesions. XPB plays a key role in DNA opening at damage sites and coordinating damage incision by nucleases. XPB is conserved from archaea to human. In archaea, XPB is associated with a nuclease Bax1. Here we report crystal structures of XPB in complex with Bax1 from Archaeoglobus fulgidus (Af) and Sulfolobus tokodaii (St). These structures reveal for the first time four domains in Bax1, which interacts with XPB mainly through its N-terminal domain. A Cas2-like domain likely helps to position Bax1 at the forked DNA allowing the nuclease domain to incise one arm of the fork. Bax1 exists in monomer or homodimer but forms a heterodimer exclusively with XPB. StBax1 keeps StXPB in a closed conformation and stimulates ATP hydrolysis by XPB while AfBax1 maintains AfXPB in the open conformation and reduces its ATPase activity. Bax1 contains two distinguished nuclease active sites to presumably incise DNA damage. Our results demonstrate that protein-protein interactions regulate the activities of XPB ATPase and Bax1 nuclease. These structures provide a platform to understand the XPB-nuclease interactions important for the coordination of DNA unwinding and damage incision in eukaryotic NER.


Assuntos
Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , DNA Helicases/química , DNA Helicases/metabolismo , Reparo do DNA , Proteína X Associada a bcl-2/química , Proteína X Associada a bcl-2/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Archaeoglobus fulgidus/química , Domínio Catalítico , Cristalografia por Raios X , DNA/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Soluções , Eletricidade Estática , Sulfolobus/química
15.
Chem Biol Interact ; 323: 109075, 2020 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-32229109

RESUMO

The use of orchids in herbal medicine has a very long history. Dendrobium species are known to produce a variety of secondary metabolites such as phenanthrens, bibenzyls, fluorenones and sesquiterpenes, and alkaloids and are responsible for their wide variety of medicinal properties. For decades, bibenzyls, which are the main bioactive components derived from Dendrobium species, have been subjected to extensive investigation as likely candidates for cancer treatment. The present study was undertaken to investigate the effect of moscatilin, a bibenzyl derivative from the orchid Dendrobium loddigesii on human melanoma cells. In A375 cells compound moscatilin showed a clear dose-response relationship in the range of 6.25-50 µM concentrations. In addition, we demonstrated an apoptotic response after treatment of cancer cells with this bibenzyl compound at 6.25 and 12.5 µM concentrations that probably involves PTEN activity, inhibition of Hsp70 expression and reactive oxygen species production. Alternatively, the inhibition of the caspase cascade at higher concentrations, 25 and 50 µM, correlated with additional reactive oxygen species increase, probably switched the mode of moscatilin-induced cell death from apoptosis to necrosis.


Assuntos
Apoptose/efeitos dos fármacos , Compostos de Benzil/uso terapêutico , Dendrobium/química , Melanoma/tratamento farmacológico , Melanoma/patologia , Compostos de Benzil/química , Compostos de Benzil/farmacologia , Caspase 3/metabolismo , Caspase 9/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , DNA de Neoplasias/metabolismo , Glutationa/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , L-Lactato Desidrogenase/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteína X Associada a bcl-2/metabolismo
16.
Zhongguo Zhong Yao Za Zhi ; 45(6): 1418-1422, 2020 Mar.
Artigo em Chinês | MEDLINE | ID: mdl-32281356

RESUMO

Polyphyllin D is a steroid saponin monomer in Polyphyllin, with antibacterial, analgesic, sedative, anti-tumor and other pharmacological effects, but is rarely reported in pancreatic cancer. This study detected apoptosis-relevant indicators, in order to explore the effect of polyphyllin D on the proliferation and apoptosis of human pancreatic cancer Panc-1 cells and relevant mechanisms of action. After pancreatic cancer Panc-1 cells were treated with polyphyllin D(0, 1, 2, 3, 4, 5 µg·µL~(-1)) for 24, 48 and 72 hours, CCK-8 method was used to detect the effect of polyphyllin D on the proliferation of pancreatic cancer Panc-1 cells. Flow cytometry was used to detect cell cycle and changes in mitochondrial membrane potential(MMP). The apoptosis was detected by Annexin V-FITC/PI staining, and Western blot was used to detect the protein expressions of cytochrome C(Cyto C), Bax, Bcl-2, cleaved caspase-3 and cleaved caspase-9. The results indicated that compared with the control group, polyphyllin D could inhibit the proliferative activity of Panc-1 cells in a time and concentration-dependent manner. Flow cytometry results showed that polyphyllin D could block the cells in S and G_2/M phase in a concentration manner, the MMP of the cells was significantly reduced, and the apoptosis rate increased with the concentration of polyphyllin D. Western blot results showed that polyphyllin D could concentration-dependently up-regulate the protein expression levels of Bax, Cyto C, cleaved caspase-3 and cleaved caspase-9, and down-regulate the protein expression level of Bcl-2. The above findings suggested that polyphyllin D could effectively inhibit the proliferation of Panc-1 cells, and its mechanism may be related to the blocking of cell growth cycle and the apoptosis induced by mitochondrial pathway.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose , Diosgenina/análogos & derivados , Neoplasias Pancreáticas/patologia , Saponinas/farmacologia , Caspase 3/metabolismo , Caspase 9/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Diosgenina/farmacologia , Humanos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo
17.
PLoS One ; 15(4): e0223208, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32302311

RESUMO

The aim of this study was to investigate whether exogenous erythropoietin (EPO) administration attenuates N-methyl-D-aspartate (NMDA)-mediated excitotoxic retinal damage in Wistar rats. The survival rate of retinal ganglion cells (RGCs) were investigated by flat mount analysis and flow cytometry. A total of 125 male Wistar rats were randomly assigned to five groups: negative control, NMDA80 (i.e., 80 nmoles NMDA intravitreally injected), NMDA80 + 10ng EPO, NMDA80 + 50ng EPO, and NMDA80 + 250ng EPO. The NMDA80 + 50ng EPO treatment group was used to evaluate various administrated points (pre-/co-/post- administration of NMDA80). Meanwhile, the transferase dUTP Nick-End Labeling (TUNEL) assay of RGCs, the inner plexiform layer (IPL) thickness and the apoptotic signal transduction pathways of µ-calpain, Bax, and caspase 9 were assessed simultaneously using an immunohistochemical method (IHC). When EPO was co-administered with NMDA80, attenuated cell death occurred through the downregulation of the apoptotic indicators: µ-calpain was activated first (peak at ~18hrs), followed by Bax and caspase 9 (peak at ~40hrs). Furthermore, the images of retinal cross sections have clearly demonstrated that thickness of the inner plexiform layer (IPL) was significantly recovered at 40 hours after receiving intravitreal injection with NMDA80 and 50ng EPO. Exogenous EPO may protect RGCs and bipolar cell axon terminals in IPL by downregulating apoptotic factors to attenuate NMDA-mediated excitotoxic retinal damage.


Assuntos
Apoptose , Eritropoetina/farmacologia , N-Metilaspartato/farmacologia , Fármacos Neuroprotetores/farmacologia , Células Ganglionares da Retina/efeitos dos fármacos , Animais , Caspase 9/genética , Caspase 9/metabolismo , Regulação para Baixo , Masculino , Ratos , Ratos Wistar , Receptores de N-Metil-D-Aspartato/agonistas , Receptores de N-Metil-D-Aspartato/metabolismo , Células Ganglionares da Retina/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo
18.
Int. j. morphol ; 38(2): 427-434, abr. 2020. tab, graf
Artigo em Inglês | LILACS | ID: biblio-1056458

RESUMO

Granulosa cells (GCs) are essential components of follicles and play a role in regulating follicle development. The aim of this study was to investigate certain cellular components involved in the proliferation, differentiation and functional characteristics of granulosa cells in the success of fertilization of human oocytes during invitro fertilization (IVF) via immunohistochemical techniques. In this study, 30 patients who were diagnosed as primary or secondary infertility, polycystic ovary syndrome in the IVF center of Memorial Hospital, Department of Obstetrics and Gynecology were included. The amount of Anti Müllerian Hormone (AMH) in blood and granulosa cell diameter and cell core diameter were measured in 20 cells collected from each patient. In addition, degeneration scoring and BAX, ADAMTS-1, IL-10 expressions in granulosa cells were evaluated (p <0.01). It was thought that apoptosis induced by human GCs might be an indicator of egg quality. Moderate expression of ADAMTS-1 was thought to be related to failure of ovulation, deterioration of oocyte quality and decreased fertilization rate. This decrease in AMH levels may be associated with defects in granulosa cells. Therefore, significantly lower AMH secretion and increase in IL10 expression levels in healthy people can be explained by the increase of granulocyte cells.


Las células de la granulosa (GC) son componentes esenciales de los folículos y tienen un papel en la regulación del desarrollo de éste. El objetivo del estudio fue investigar ciertos componentes celulares involucrados en la proliferación, diferenciación y características funcionales de las células de la granulosa en el éxito de la fertilización de los ovocitos humanos durante la fertilización in vitro (FIV) a través de técnicas inmunohistoquímicas. En este estudio, se incluyeron 30 pacientes diagnosticados con infertilidad primaria o secundaria, síndrome de ovario poliquístico en el centro de FIV del Departamento de Obstetricia y Ginecología del Hospital Memorial. La cantidad de Hormona Anti Mülleriana (AMH) en la sangre, el diámetro de las células de la granulosa y el diámetro del núcleo celular se midieron en 20 células obtenidas de cada paciente. Además, se evaluó la puntuación de degeneración y las expresiones BAX, ADAMTS-1, IL-10 en células de granulosa (p <0,01). Se estimó que la apoptosis inducida por los GC humanos podría ser un indicador de la calidad del huevo. Se estimó que la expresión moderada de ADAMTS-1 estaba relacionada con el fracaso de la ovulación, el deterioro de la calidad de los ovocitos y la disminución de la tasa de fertilización. La disminución en los niveles de AMH puede estar asociada con defectos en las células de la granulosa. Por lo tanto, el aumento de las células de granulocitos puede explicar la disminución significativa de la secreción de AMH y el aumento de los niveles de expresión de IL10 en personas sanas.


Assuntos
Humanos , Feminino , Fertilização In Vitro/métodos , Interleucina-10/metabolismo , Proteína X Associada a bcl-2/metabolismo , Proteína ADAMTS1/metabolismo , Células da Granulosa/metabolismo , Imuno-Histoquímica
19.
Toxicol Appl Pharmacol ; 396: 114996, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32278510

RESUMO

Antineoplastic drugs cause severe cytotoxicity for normal cells, especially hematopoietic stem cells (HSCs). However, bleomycin (BLM) is glycopeptide antibiotic that is effective on various cancers and has either low or no myelosuppression effects. The aim of the present study was to investigate the effect of BLM on 5-Azacitidine (5-AZA) induced cytotoxicity in bone marrow HSCs. 5-AZA reduced HSC cell viability in a time and dose-dependent manner with an IC50 value of 16 µM. However, pretreatment of the cells with BLM for 4 h induced an antagonistic cytotoxicity with an increased IC50 of 64 µM. 5-AZA decreased the colony formation ability of HSC cells in semi-solid agar culture and this effect was attenuated by BLM. 5-AZA significantly downregulated high mobility group Box1 (HMGB1) and Bcl-2 gene expression but upregulated Bax gene expression, while BLM impeded the action of 5-AZA. Pretreatment with BLM remarkably decreased HMGB1 release into culture media that was induced by 5-AZA. The cells were distribution at the sub/G1 phase. Annexin/PI staining of the cells, poly (ADP-ribose) polymerase (PARP) cleavage, and anion superoxide production indicated that BLM limited 5-AZA induced apoptotic cell death. In conclusion, BLM in combination with 5-AZA effectively reduces the adverse cytotoxic effects of 5-AZA on bone marrow hematopoietic stem cells, providing a new chemotherapeutic strategy.


Assuntos
Antineoplásicos/toxicidade , Apoptose/efeitos dos fármacos , Azacitidina/toxicidade , Bleomicina/farmacologia , Proteína HMGB1/metabolismo , Células-Tronco Hematopoéticas/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína X Associada a bcl-2/metabolismo , Animais , Azacitidina/antagonistas & inibidores , Relação Dose-Resposta a Droga , Expressão Gênica/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Espécies Reativas de Oxigênio/metabolismo
20.
Psychopharmacology (Berl) ; 237(7): 2125-2137, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32333135

RESUMO

RATIONALE: Since the precise mechanisms of posttraumatic stress disorder (PTSD) remain unknown, effective treatment interventions have not yet been established. Numerous clinical studies have led to the hypothesis that elevated glucocorticoid levels in response to extreme stress might trigger a pathophysiological cascade which consequently leads to functional and morphological changes in the hippocampus. OBJECTIVES: To elucidate the pathophysiology of PTSD, we examined the alteration of hippocampal gene expression through the glucocorticoid receptor (GR) in the single prolonged stress (SPS) paradigm, a rat model of PTSD. METHODS: We measured nuclear GRs by western blot, and the binding of GR to the promoter of Bcl-2 and Bax genes by chromatin immunoprecipitation-qPCR as well as the expression of these 2 genes by RT-PCR in the hippocampus of SPS rats. In addition, we examined the preventive effects of a GR antagonist on SPS-induced molecular, morphological, and behavioral alterations (hippocampal gene expression of Bcl-2 and Bax, hippocampal apoptosis using TUNEL staining, impaired fear memory extinction (FME) using the contextual fear conditioning paradigm). RESULTS: Exposure to SPS increased nuclear GR expression and GR binding to Bcl-2 gene, and decreased Bcl-2 mRNA expression. Administration of GR antagonist immediately after SPS prevented activation of the glucocorticoid cascade, hippocampal apoptosis, and impairment FME in SPS rats. CONCLUSION: The activation of GRs in response to severe stress may trigger the pathophysiological cascade leading to impaired FME and hippocampal apoptosis. In contrast, administration of GR antagonist could be useful for preventing the development of PTSD.


Assuntos
Modelos Animais de Doenças , Hipocampo/metabolismo , Receptores de Glucocorticoides/metabolismo , Transtornos de Estresse Pós-Traumáticos/metabolismo , Transtornos de Estresse Pós-Traumáticos/psicologia , Animais , Medo/efeitos dos fármacos , Medo/fisiologia , Medo/psicologia , Hipocampo/patologia , Masculino , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Transtornos de Estresse Pós-Traumáticos/patologia , Proteína X Associada a bcl-2/metabolismo
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