RESUMO
BACKGROUND: Skeletal muscle synthesizes, stores, and releases body L-glutamine (GLN). Muscle atrophy due to disabling diseases triggers the activation of proteolytic and pro-apoptotic cell signaling, thus impairing the body's capacity to manage GLN content. This situation has a poor therapeutic prognosis. OBJECTIVE: Evaluating if oral GLN supplementation can attenuate muscle wasting mediated by elevated plasma cortisol and activation of caspase-3, p38MAPK, and FOXO3a signaling pathways in soleus and gastrocnemius muscles of rats submitted to 14-day bilateral hindlimbs immobilization. METHODS: Animals were randomly distributed into six groups: non-immobilized rats (Control), control orally supplemented with GLN (1 g kg-1) in solution with L-alanine (ALA: 0.61 g kg-1; GLN+ALA), control orally supplemented with dipeptide L-alanyl-L-glutamine (DIP; 1.49 g kg-1), hindlimbs immobilized rats (IMOB), IMOB orally GLN+ALA supplemented (GLN+ALA-IMOB), and IMOB orally DIP supplemented (DIP-IMOB). Plasma and muscle GLN concentration, plasma cortisol level, muscle caspase-3 activity, muscle p38MAPK and FOXO3a protein content (total and phosphorylated forms), and muscle cross-sectional area (CSA) were measured. RESULTS: Compared to controls, IMOB rats presented: a) increased plasma cortisol levels; b) decreased plasma and muscle GLN concentration; c) increased muscle caspase-3 activity; d) increased total and phosphorylated p38MAPK protein content; e) increased FOXO3a and decreased phosphorylated FOXO3a protein content; f) reduced muscle weight and CSA befitting to atrophy. Oral supplementation with GLN+ALA and DIP was able to significantly attenuate these effects. CONCLUSIONS: These findings attest that oral GLN supplementation in GLN+ALA solution or DIP forms attenuates rats' skeletal muscle mass wasting caused by disuse-mediated muscle atrophy.
Assuntos
Glutamina , Hidrocortisona , Atrofia Muscular , Animais , Ratos , Caspase 3/metabolismo , Suplementos Nutricionais , Dipeptídeos/metabolismo , Dipeptídeos/farmacologia , Dipeptídeos/uso terapêutico , Glutamina/farmacologia , Músculo Esquelético , Atrofia Muscular/tratamento farmacológico , Atrofia Muscular/etiologia , Atrofia Muscular/metabolismo , Transdução de Sinais , Proteína Forkhead Box O3/efeitos dos fármacos , Proteína Forkhead Box O3/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Ischemic stroke is a major cause of death and disability, intensely demanding innovative and accessible therapeutic strategies. Approaches presenting a prolonged period for therapeutic intervention and new treatment administration routes are promising tools for stroke treatment. Here, we evaluated the potential neuroprotective properties of nasally administered human adipose tissue mesenchymal stem cell (hAT-MSC)-derived extracellular vesicles (EVs) obtained from healthy individuals who underwent liposuction. After a single intranasal EV (200 µg/kg) administered 24 h after a focal permanent ischemic stroke in rats, a higher number of EVs, improvement of the blood-brain barrier, and re-stabilization of vascularization were observed in the recoverable peri-infarct zone, as well as a significant decrease in infarct volume. In addition, EV treatment recovered long-term motor (front paws symmetry) and behavioral impairment (short- and long-term memory and anxiety-like behavior) induced by ischemic stroke. In line with these findings, our work highlights hAT-MSC-derived EVs as a promising therapeutic strategy for stroke.
Assuntos
Vesículas Extracelulares/transplante , Células-Tronco Mesenquimais , Transplante de Células-Tronco/métodos , Acidente Vascular Cerebral/terapia , Administração Intranasal , Adulto , Animais , Barreira Hematoencefálica , Encéfalo/irrigação sanguínea , Encéfalo/patologia , Teste de Labirinto em Cruz Elevado , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neovascularização Fisiológica , Ratos Wistar , Recuperação de Função Fisiológica , Acidente Vascular Cerebral/patologiaRESUMO
The phytoalexin Resveratrol (3,5,4'-trihydroxystilbene; RSV) has been related to numerous beneficial effects on health by its cytoprotection and chemoprevention activities. Liver fibrosis is characterized by the extracellular matrix accumulation after hepatic injury and can lead to cirrhosis. Hepatic stellate cells (HSC) play a crucial role during fibrogenesis and liver wound healing by changing their quiescent phenotype to an activated phenotype for protecting healthy areas from damaged areas. Strategies on promoting the activated HSC death, the quiescence return or the cellular activation stimuli decrease play an important role on reducing liver fibrosis. Here, we evaluated the RSV effects on some markers of activation in GRX, an HSC model. We further evaluated the RSV influence in the ability of GRX on releasing inflammatory mediators. RSV at 1 and 10 µM did not alter the protein content of α-SMA, collagen I and GFAP; but 50 µM increased the content of these activation-related proteins. Also, RSV did not change the myofibroblast-like morphology of GRX. Interestingly, RSV at 10 and 50 µM decreased the GRX migration and collagen-I gel contraction. Finally, we showed that RSV triggered the increase in the TNF-α and IL-10 content in culture media of GRX while the opposite occurred for the IL-6 content. Altogether, these results suggested that RSV did not decrease the activation state of GRX and oppositely, triggered a pro-activation effect at the 50 µM concentration. However, despite the increase of TNF- α in culture media, these results on IL-6 and IL-10 secretion were in accordance with the anti-inflammatory role of RSV in our model.
Assuntos
Antioxidantes/farmacologia , Citocinas/metabolismo , Células Estreladas do Fígado/efeitos dos fármacos , Inflamação/tratamento farmacológico , Cirrose Hepática/tratamento farmacológico , Resveratrol/farmacologia , Animais , Linhagem Celular , Proliferação de Células , Células Estreladas do Fígado/imunologia , Células Estreladas do Fígado/metabolismo , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/patologia , Cirrose Hepática/imunologia , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Camundongos , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/metabolismoRESUMO
Skeletal muscle disuse results in myofibrillar atrophy and protein degradation, via inflammatory and oxidative stress-mediated NF-kB signaling pathway activation. Nutritional interventions, such as l-glutamine (GLN) supplementation have shown antioxidant properties and cytoprotective effects through the modulation on the 70-kDa heat shock protein (HSP70) expression. However, these GLN-mediated effects on cell signaling pathways and biochemical mechanisms that control the myofibrillar protein content degradation in muscle disuse situations are poorly known yet. This study investigated the effects of oral GLN plus l-alanine (ALA; GLN â+ âALA-solution) supplementation, either in their free or dipeptide (L-alanyl-l-glutamine-DIP) form, on GLN-glutathione (GSH) axis and cytoprotection mediated by HSP70 protein expression in the slow-twitch soleus and fast-twitch gastrocnemius skeletal muscle of rats submitted to 14-days of hindlimb immobilization-induced disuse muscle atrophy. Forty-eight Wistar rats were distributed into 6 groups: hindlimb immobilized (IMOB group) and hindlimb immobilized orally supplemented with either GLN (1â¯gâ¯kg-1) plus ALA (0.61â¯gâ¯kg-1) â(GLN â+ âALA-IMOB group) or 1.49 âg âkg-1 of DIP (DIP-IMOB group) and; no-immobilized (CTRL) and no-immobilized supplemented GLN â+ âALA and DIP baselines groups. All animals, including CTRL and IMOB rats (water), were supplemented via intragastric gavage for 14 days, concomitantly to immobilization period. Plasma and muscle GLN levels, lipid (thiobarbituric acid reactive substances-TBARS) and protein (carbonyl) peroxidation, erythrocyte concentration of reduced GSH and GSH disulfide (GSSG), plasma and muscle pro-inflammatory TNF-α levels, muscle IKKα/ß-NF-kB signaling pathway and, the myofibrillar protein content (MPC) were measured. The MPC was significantly lower in IMOB rats, compared to CTRL, GLN â+ âALA, and DIP animals (p â< â0.05). This finding was associated with reduced plasma and muscle GLN concentration, equally in IMOB animals. Conversely, both GLN â+ âALA and DIP supplementation restored plasma and muscle GLN levels, which equilibrated GSH and intracellular redox status (GSSG/GSH ratio) in erythrocytes and skeletal muscle even as, increased muscle HSP70 protein expression; attenuating oxidative stress and TNF-α-mediated NF-kB pathway activation, fact that reverberated on reduction of MPC degradation in GLN â+ âALA-IMOB and DIP-IMOB animals (p â< â0.05). In conclusion, the findings shown herein support the oral GLN â+ âALA and DIP supplementations as a therapeutic and effective nutritional alternative to attenuate the deleterious effects of the skeletal muscle protein degradation induced by muscle disuse.
Assuntos
Glutamina/farmacologia , Inflamação/tratamento farmacológico , Músculo Esquelético/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Administração Oral , Animais , Antioxidantes/farmacologia , Creatina Quinase/genética , Suplementos Nutricionais , Modelos Animais de Doenças , Humanos , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Músculo Esquelético/patologia , NF-kappa B/genética , Proteólise/efeitos dos fármacos , Ratos , Ratos WistarRESUMO
Caveolin-1 (Cav-1) expression is increased in hepatic stellate cells (HSC) upon liver cirrhosis and it functions as an integral membrane protein of lipid rafts and caveolae that regulates and integrates multiple signals as a platform. This study aimed to evaluate the role of Cav-1 in HSC. Thus, the effects of exogenous expression of Cav-1 in GRX cells, a model of activated HSC, were determined. Here, we demonstrated through evaluating well-known HSC activation markers - such as α-smooth muscle actin, collagen I, and glial fibrillary acidic protein - that up regulation of Cav-1 induced GRX to a more activated phenotype. GRXEGFP-Cav1 presented an increased migration, an altered adhesion pattern, a reorganization f-actin cytoskeleton, an arrested cell cycle, a modified cellular ultrastructure, and a raised endocytic flux. Based on this, GRX EGFP-Cav1 represents a new cellular model that can be an important tool for understanding of events related to HSC activation. Furthermore, our results reinforce the role of Cav-1 as a molecular marker of HSC activation.
Assuntos
Caveolina 1/biossíntese , Pontos de Checagem do Ciclo Celular , Expressão Gênica , Células Estreladas do Fígado/metabolismo , Caveolina 1/genética , Linhagem Celular , Células Estreladas do Fígado/citologia , HumanosRESUMO
Hepatic stellate cells are liver-specific perivascular cells, identified as the major source of collagen in liver fibrosis, following their activation and conversion to myofibroblast-like cells. Lycopene is a carotenoid with biological activities and protective effects described in different pathologies, but little is known about its role in liver protection. We evaluated the influence of lycopene on the cell cycle and lipid metabolism and monitored the possible pathways involved in lycopene inhibition of stellate cell activation. Lycopene induced expression of the lipocyte phenotype, with an accumulation of fat droplets in cytoplasm, with high synthesis and turnover of phospholipids and triglycerides. Cell proliferation analysis showed that lycopene reduced the growth of GRX cells. Lycopene induced an arrest in the G0/G1 phase, followed by a decrease of cells in the G2/M phase, regardless of the concentration of lycopene used. Lycopene modulated relevant signaling pathways related to cholesterol metabolism, cellular proliferation, and lipid metabolism. Also, lycopene treatment increased the expression of RXR-α, RXR-ß, and PPARγ, important biomarkers of liver regeneration. These results show that lycopene was able to negatively modulate events related to the activation of hepatic stellate cells through mechanisms that involve changes in expression of cellular lipid metabolism factors, and suggest that this compound might provide a novel pharmacological approach for the prevention and treatment of fibrotic liver diseases.
Assuntos
Células Estreladas do Fígado/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Licopeno/farmacologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Humanos , Gotículas Lipídicas/metabolismo , Fígado/citologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , PPAR gama/genética , PPAR gama/metabolismo , Receptor X Retinoide alfa/genética , Receptor X Retinoide alfa/metabolismo , Receptor X Retinoide beta/genética , Receptor X Retinoide beta/metabolismo , Transdução de Sinais/efeitos dos fármacos , Triglicerídeos/metabolismoRESUMO
Obesity is a prevalent multifactorial chronic disorder characterized by metabolic dysregulation. Sustained pro-oxidative mediators trigger harmful consequences that reflect at systemic level and contribute for the establishment of a premature senescent phenotype associated with macromolecular damage (DNA, protein, and lipids). Telomeres are structures that protect chromosome ends and are associated with a six-protein complex called the shelterin complex and subject to regulation. Under pro-oxidant conditions, telomere attrition and the altered expression of the shelterin proteins are central for the establishment of many pathophysiological conditions such as obesity. Thus, considering that individuals with obesity display a systemic oxidative stress profile that may compromise the telomeres length or its regulation, the aim of this study was to investigate telomere homeostasis in patients with obesity and explore broad/systemic associations with the expression of shelterin genes and the plasma redox state. We performed a cross-sectional study in 39 patients with obesity and 27 eutrophic subjects. Telomere length (T/S ratio) and gene expression of shelterin components were performed in peripheral blood mononuclear cells by qPCR. The oxidative damage (lipid peroxidation and protein carbonylation) and non-enzymatic antioxidant system (total radical-trapping antioxidant potential/reactivity, sulfhydryl and GSH content) were evaluated in plasma. Our results demonstrate that independently of comorbidities, individuals with obesity had significantly shorter telomeres, augmented expression of negative regulators of the shelterin complex, increased lipid peroxidation and higher oxidized protein levels associated with increased non-enzymatic antioxidant defenses. Principal component analysis revealed TRF1 as a major contributor for firstly telomeres shortening. In conclusion, our study is first showing a comprehensive analysis of telomeres in the context of obesity, associated with dysregulation of the shelterin components that was partially explained by TRF1 upregulation that could not be reversed by the observed adaptive non-enzymatic antioxidant response.
Assuntos
Leucócitos Mononucleares/metabolismo , Obesidade/genética , Encurtamento do Telômero , Proteínas de Ligação a Telômeros/genética , Telômero/metabolismo , Proteína 1 de Ligação a Repetições Teloméricas/genética , Adulto , Estudos Transversais , Feminino , Regulação da Expressão Gênica , Glutationa/metabolismo , Humanos , Leucócitos Mononucleares/patologia , Peroxidação de Lipídeos , Masculino , Obesidade/metabolismo , Obesidade/patologia , Cultura Primária de Células , Análise de Componente Principal , Carbonilação Proteica , Complexo Shelterina , Transdução de Sinais , Telômero/ultraestrutura , Proteínas de Ligação a Telômeros/metabolismo , Proteína 1 de Ligação a Repetições Teloméricas/metabolismoRESUMO
OBJECTIVE: To evaluate the consequences of plasma from individuals with obesity on parameters associated with immunosenescence in unrelated healthy peripheral blood mononuclear cells (PBMC). METHODS: Freshly isolated PBMC were incubated in media supplemented with 10% of plasma from individuals with obesity or control subjects for the first 4 hours of 24 to 120 hours of culture. RESULTS: Plasma from individuals with obesity modulated the phenotype of healthy PBMC, leading to a higher rate of apoptosis, lower amounts of phospho-γH2AX and -p53, and mitochondrial dysfunction. After 120 hours, there was a higher secretion of inflammatory cytokines IL-1ß and IL-8. CD8+ T lymphocytes presented decreased expression of CD28, which is associated with the immunosenescent phenotype. CD14+ macrophages showed increased expression of CD80 and CD206, suggesting a modulation in the activation of macrophages. CONCLUSIONS: These results demonstrate that chronic systemic inflammation observed in obesity induces dysfunctional features in PBMC that are consistent with premature immunosenescence.
Assuntos
Imunossenescência , Inflamação/etiologia , Leucócitos Mononucleares/fisiologia , Obesidade/sangue , Transdução de Sinais/fisiologia , Adulto , Apoptose , Linfócitos T CD8-Positivos/fisiologia , Meios de Cultura , Feminino , Humanos , Interleucina-1beta/metabolismo , Interleucina-8/metabolismo , Macrófagos , Masculino , SoroRESUMO
Astrocytes are versatile cells involved in synaptic information processing, energy metabolism, redox homeostasis, inflammatory response, and structural support of the brain. Recently, we established a routine protocol of cultured astrocytes derived from adult and aged Wistar rats, which present several different responses compared to newborn astrocytes, commonly used to characterize the role of the astrocytes in the central nervous system. Previous studies hypothesized that astrocyte cultures prepared from adult animals derive from immature precursors present in the adult tissue throughout life. Since our group has already demonstrated that the glial functionality of adult astrocytes differs from newborn cultures, the aim of this study was to confirm that our in vitro astrocytes were derived from mature cells. Therefore, we evaluated cytoskeleton proteins, such as glial fibrillary acidic protein and vimentin, as well as Sox10, an essential marker of immature glial cells, in ex vivo tissue and in in vitro astrocytes from the same animals (1, 90, and 180 days old). In addition, we examined the mitochondrial functionality and the cellular redox homeostasis. Our results suggest that adult and aged astrocytes are derived from mature cells and that changes in mitochondrial parameters in ex vivo tissue were reproduced in in vitro astrocytes. J. Cell. Biochem. 118: 3111-3118, 2017. © 2017 Wiley Periodicals, Inc.
Assuntos
Astrócitos/metabolismo , Citoesqueleto/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Mitocôndrias/metabolismo , Fatores de Transcrição SOXE/metabolismo , Vimentina/metabolismo , Animais , Animais Recém-Nascidos , Astrócitos/citologia , Masculino , Oxirredução , Ratos , Ratos WistarRESUMO
Activated hepatic stellate cells (HSC) are the major source of collagen I in liver fibrosis. Eugenia uniflora L. is a tree species that is widely distributed in South America. E. uniflora L. fruit-popularly known as pitanga-has been shown to exert beneficial properties. Autophagy contributes to the maintenance of cellular homeostasis and survival under stress situation, but it has also been suggested to be an alternative cell death pathway. Mitochondria play a pivotal role on signaling cell death. Mitophagy of damaged mitochondria is an important cell defense mechanism against organelle-mediated cell death signaling. We previously found that purple pitanga extract induced mitochondrial dysfunction, cell cycle arrest, and death by apoptosis and necrosis in GRX cells, a well-established activated HSC line. We evaluated the effects of 72-h treatment with crescent concentrations of purple pitanga extract (5 to 100 µg/mL) on triggering autophagy in GRX cells, as this is an important mechanism to cells under cytotoxic conditions. We found that all treated cells presented an increase in the mRNA expression of autophagy-related protein 7 (ATG7). Concomitantly, flow cytometry and ultrastructural analysis of treated cells revealed an increase of autophagosomes/autolysosomes that consequentially led to an increased mitophagy. As purple pitanga extract was previously found to be broadly cytotoxic to GRX cells, we postulated that autophagy contributes to this scenario, where cell death seems to be an inevitable fate. Altogether, the effectiveness on inducing activated HSC death can make purple pitanga extract a good candidate on treating liver fibrosis.
Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Eugenia/química , Células Estreladas do Fígado/patologia , Extratos Vegetais/farmacologia , Animais , Autofagossomos/efeitos dos fármacos , Autofagossomos/metabolismo , Autofagossomos/ultraestrutura , Linhagem Celular , Células Estreladas do Fígado/efeitos dos fármacos , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/patologia , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Fitoterapia , Extratos Vegetais/uso terapêuticoRESUMO
Glioblastoma (GBM) is the most common and aggressive primary malignant brain tumor in adults. Hypoxia is a distinct feature in GBM and plays a significant role in tumor progression, resistance to treatment, and poor outcome. However, there is lack of studies relating type of cell death, status of Akt phosphorylation on Ser473, mitochondrial membrane potential, and morphological changes of tumor cells after hypoxia and reoxygenation. The rat glioma C6 cell line was exposed to oxygen deprivation (OD) in 5 % fetal bovine serum (FBS) or serum-free media followed by reoxygenation (RO). OD induced apoptosis on both 5 % FBS and serum-free groups. Overall, cells on serum-free media showed more profound morphological changes than cells on 5 % FBS. Moreover, our results suggest that OD combined with absence of serum provided a favorable environment for glioblastoma dedifferentiation to cancer stem cells, since nestin, and CD133 levels increased. Reoxygenation is present in hypoxic tumors through microvessel formation and cell migration to oxygenated areas. However, few studies approach these phenomena when analyzing hypoxia. We show that RO caused morphological alterations characteristic of cells undergoing a differentiation process due to increased GFAP. In the present study, we characterized an in vitro hypoxic microenvironment associated with GBM tumors, therefore contributing with new insights for the development of therapeutics for resistant glioblastoma.
Assuntos
Neoplasias Encefálicas/patologia , Glioblastoma/patologia , Hipóxia/patologia , Células-Tronco Neoplásicas/patologia , Neurônios/patologia , Microambiente Tumoral , Animais , Apoptose/fisiologia , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Glioblastoma/metabolismo , Hipóxia/metabolismo , Potencial da Membrana Mitocondrial/fisiologia , Células-Tronco Neoplásicas/metabolismo , Neurônios/metabolismo , Oxigênio/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , RatosRESUMO
Glioblastoma is the most frequent and malignant brain tumor. Treatment includes chemotherapy with temozolomide concomitant with surgical resection and/or irradiation. However, a number of cases are resistant to temozolomide, as well as the human glioblastoma cell line U138-MG. We investigated doxazosin's (an antihypertensive drug) activity against glioblastoma cells (C6 and U138-MG) and its neurotoxicity on primary astrocytes and organoptypic hippocampal cultures. For this study, the following methods were used: citotoxicity assays, flow cytometry, western-blotting and confocal microscopy. We showed that doxazosin induces cell death on C6 and U138-MG cells. We observed that doxazosin's effects on the PI3K/Akt pathway were similar as LY294002 (PI3K specific inhibitor). In glioblastoma cells treated with doxasozin, Akt levels were greatly reduced. Upon examination of activities of proteins downstream of Akt we observed upregulation of GSK-3ß and p53. This led to cell proliferation inhibition, cell death induction via caspase-3 activation and cell cycle arrest at G0/G1 phase in glioblastoma cells. We used in this study Lapatinib, a tyrosine kinase inhibitor, as a comparison with doxazosin because they present similar chemical structure. We also tested the neurocitotoxicity of doxazosin in primary astrocytes and organotypic cultures and observed that doxazosin induced cell death on a small percentage of non-tumor cells. Aggressiveness of glioblastoma tumors and dismal prognosis require development of new treatment agents. This includes less toxic drugs, more selective towards tumor cells, causing less damage to the patient. Therefore, our results confirm the potential of doxazosin as an attractive therapeutic antiglioma agent.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias Encefálicas/tratamento farmacológico , Doxazossina/farmacologia , Glioblastoma/tratamento farmacológico , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteína Supressora de Tumor p53/biossíntese , Animais , Astrócitos/efeitos dos fármacos , Caspase 3/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cromonas/farmacologia , Doxazossina/toxicidade , Ativação Enzimática/efeitos dos fármacos , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Glicogênio Sintase Quinase 3 beta/biossíntese , Hipocampo/efeitos dos fármacos , Humanos , Lapatinib , Morfolinas/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Quinazolinas/farmacologia , Ratos , Ratos WistarRESUMO
The activation of hepatic stellate cell (HSC), from a quiescent cell featuring cytoplasmic lipid droplets to a proliferative myofibroblast, plays an important role in liver fibrosis development. The GRX line is an activated HSC model that can be induced by all-trans-retinol to accumulate lipid droplets. Resveratrol is known for activating Sirtuin1 (SIRT1), a NAD(+)-dependent deacetylase that suppresses the activity of peroxisome proliferator-activated receptor gamma (PPARγ), an important adipogenic transcription factor involved in the quiescence maintenance of HSC. We evaluated the effects of 0.1 µM of resveratrol in retinol-induced GRX quiescence by investigating the interference of SIRT1 and PPARγ on cell lipogenesis. GRX lipid accumulation was evaluated through Oil-red O staining, triacylglycerides quantification, and [(14)C] acetate incorporation into lipids. mRNA expression and protein content of SIRT1 and PPARγ were measured by RT-PCR and immunoblotting, respectively. Resveratrol-mediated SIRT1 stimuli did not induce lipogenesis and reduced the retinol-mediated fat-storing capacity in GRX. In order to support our results, we established a cell culture model of transgenic super expression of PPARγ in GRX cells (GRXPγ). Resveratrol reduced lipid droplets accumulation in GRXPγ cells. These results suggest that the PPARγ/SIRT1 ratio plays an important role in the fate of HSC. Thus, whenever the PPARγ activity is greater than SIRT1 activity the lipogenesis is enabled.
Assuntos
Fibrose/genética , Gotículas Lipídicas/metabolismo , PPAR gama/biossíntese , Sirtuína 1/biossíntese , Animais , Proliferação de Células , Fibrose/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Humanos , Células de Kupffer/metabolismo , Lipogênese/efeitos dos fármacos , Fígado/metabolismo , Camundongos , Mioblastos/metabolismo , PPAR gama/metabolismo , RNA Mensageiro/biossíntese , Resveratrol , Sirtuína 1/metabolismo , Estilbenos/administração & dosagem , Vitamina A/metabolismoRESUMO
Resveratrol has been the focus of numerous studies reporting opposite effects that depend on its concentration. The GRX is an activated hepatic stellate cells model used to study liver fibrosis development and resolution. We recently showed that GRX treatment with RSV (0.1-50 µM) for 24 h triggered dose-dependent pro-oxidant effects, resulting in cytotoxicity and cell damage only at the highest concentration. Here, we evaluated whether the pro-oxidant effect of resveratrol treatment is accompanied by alterations on the GRX mitochondrial metabolism, and whether the concomitantly autophagy/mitophagy induction can influence on cell death or survival. We demonstrated that all concentrations of resveratrol promoted an increase of GRX cell death signals, altering the mitochondrial dynamics and function. Cells treated with all resveratrol concentrations presented higher autophagy/mitophagy features, but only treatments with 1 and 10 µM of resveratrol-induced mitochondrial biogenesis. Since cell damage was higher and there was no mitochondrial biogenesis in GRX treated with 50 µM of resveratrol, we suggest that these cells failed to remove and replace all damaged mitochondria. In conclusion, the cytotoxic effect of resveratrol that effectively promotes cell death could be related to the interrelation between the concomitant induction of apoptosis, autophagy/mitophagy and mitochondrial biogenesis in GRX.
Assuntos
Antioxidantes/farmacologia , Apoptose , Células Estreladas do Fígado/metabolismo , Mitofagia , Biogênese de Organelas , Estilbenos/farmacologia , Animais , Linhagem Celular , Células Estreladas do Fígado/efeitos dos fármacos , Camundongos , ResveratrolRESUMO
Resveratrol (RSV) is known for its antioxidant properties; however, this compound has been proposed to have cytotoxic and pro-oxidant effects depending on its concentration and time of exposure. We previously reported the cell cycle arrest effect of low doses of RSV in GRX cells, an activated hepatic stellate cell model. Here, we evaluated the effects of RSV treatment (0.1-50 µM) for 24 and 120 h on GRX viability and oxidative status. Only treatment with 50 µM of RSV reduced the amount of live cells. However, even low doses of RSV induced an increased reactive species production at both treatment times. While being diminished within 24 h, RSV induced an increase in the SOD activity in 120 h. The cellular damage was substantially increased at 24 h in the 50 µM RSV-treated group, as indicated by the high lipoperoxidation, which may be related to the significant cell death and low proliferation. Paradoxically, this cellular damage and lipoperoxidation were considerably reduced in this group after 120 h of treatment while the surviving cells proliferated. In conclusion, RSV induced a dose-dependent pro-oxidant effect in GRX cells. The highest RSV dose induced oxidative-related damage, drastically reducing cell viability; but this cytotoxicity seems to be attenuated during 120 h of treatment.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Células Estreladas do Fígado/efeitos dos fármacos , Estilbenos/farmacologia , Animais , Anti-Inflamatórios não Esteroides/química , Catalase/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Células Estreladas do Fígado/citologia , Células Estreladas do Fígado/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Resveratrol , Estilbenos/química , Superóxido Dismutase/metabolismo , Fatores de TempoRESUMO
Resveratrol (RSV) exerts anti-proliferative and pro-apoptotic actions in different cell lines. Hepatic stellate cells (HSCs) are major fibrogenic cell types that contribute to collagen accumulation during chronic liver disease. In the present study, the inhibitory effects of RSV on cell proliferation, cell cycle, and apoptosis were evaluated in the mouse hepatic stellate cell line GRX. Cells treated with 1 nM-1 muM of RSV demonstrated a decrease in cell growth of about 35% after 5 days. GRX cells, treated with RSV (100 nM or 1 muM), were analyzed by flow cytometry; RSV induced an increase in the number of GRX cells in the S- and sub-G1 phases. The increase in sub-G1 phase cells and the nuclear condensation and fragmentation shown by DAPI staining identified a possible pro-apoptotic effect of RSV on GRX cells. Furthermore, the RSV anti-proliferative effects could be explained by an S-phase accumulation caused by a decrease in the progression through the cell cycle or an inhibition of S or G2 phase transition. It is notable that these RSV actions are mediated at nanomolar levels, compatible with the concentrations of free RSV in biological fluids after ingestion of polyphenol-rich foods, suggesting a possible effect of these foods as an adjuvant treatment in chronic liver diseases.
Assuntos
Ciclo Celular/efeitos dos fármacos , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Estilbenos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Camundongos , Resveratrol , Fase S/efeitos dos fármacosRESUMO
Congenital hypothyroidism was induced in rats by adding 0.05% 6-propyl-2-thiouracil in the drinking water from day 9 of gestation, and continually up to postnatal day 15. Structural alterations observed by light microscopy of seminiferous tubules and by transmission electron microscopy of Sertoli cells of treated animals were consistent with hypothyroid condition. Hypothyroidism was also associated with high phospho-p38 mitogen-activated protein kinase and decreased phospho-extracellular signal-regulated kinase 1/2 levels. Furthermore, the phosphorylation and the immunoreactivity of cytoskeletal-associated vimentin were increased without altering vimentin expression, suggesting an accumulation of insoluble and phosphorylated vimentin. These alterations in intermediate filament dynamics could result in loss of Sertoli cell cytoskeletal integrity and be somewhat related to the deleterious effects of hypothyroidism in testis. In addition, the mitochondrial alterations observed could also be related to defective cytoskeletal dynamics implying in cell damage. Moreover, we observed decreased oxygen consumption and unaltered lipid peroxidation in hypothyroid testis. However, we demonstrated decreased enzymatic and non-enzymatic antioxidant defenses, supporting an increased mitochondrial reactive oxygen species (ROS) generation, contributing to biochemical changes in hypothyroid testis. In addition, the changes in the testis histoarchitecture could be ascribed to cytoskeletal alterations, decreased antioxidant defenses, and increased ROS generation, leading to oxidative stress in the organ.
Assuntos
Antioxidantes/metabolismo , Hipotireoidismo Congênito/metabolismo , Propiltiouracila , Testículo/metabolismo , Vimentina/metabolismo , Animais , Hipotireoidismo Congênito/induzido quimicamente , Hipotireoidismo Congênito/patologia , Citoesqueleto/metabolismo , Peroxidação de Lipídeos , Masculino , Mitocôndrias/metabolismo , Consumo de Oxigênio , Fosforilação , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Túbulos Seminíferos/metabolismo , Túbulos Seminíferos/patologia , Células de Sertoli/metabolismo , Células de Sertoli/patologia , Testículo/patologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Hyperthyroidism was induced in rats and somatic indices and metabolic parameters were analyzed in testis. In addition, the morphological analysis evidenced testes maturation and intense protein synthesis and processing, supporting the enhancement in vimentin synthesis in hyperthyroid testis. Furthermore, vimentin phosphorylation was increased, indicating an accumulation of phosphorylated vimentin associated to the cytoskeleton, which could be a consequence of the extracellular-regulated kinase (ERK) activation regulating the cytoskeleton. Biomarkers of oxidative stress demonstrated an increased basal metabolic rate measured by tissue oxygen consumption, as well as, increased TBARS levels. In addition, the enzymatic and non-enzymatic antioxidant defences appeared to respond according to the augmented oxygen consumption. We observed decreased total glutathione levels, with enhancement of reduced glutathione, whereas most of the antioxidant enzyme activities were induced. Otherwise, superoxide dismutase activity was inhibited. These results support the idea that an increase in mitochondrial ROS generation, underlying cellular oxidative damage, is a side effect of hyperthyroid-induced biochemical changes by which rat testis increase their metabolic capacity.