RESUMEN
<p><b>OBJECTIVE</b>To examine the role of Cd-induced reactive oxygen species (ROS) generation in the apoptosis of neuronal cells.</p><p><b>METHODS</b>Neuronal cells (primary rat cerebral cortical neurons and PC12 cells) were incubated with or without Cd post-pretreatment with rapamycin (Rap) or N-acetyl-L-cysteine (NAC). Cell viability was determined by MTT assay, apoptosis was examined using flow cytometry and fluorescence microscopy, and the activation of phosphoinositide 3'-kinase/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) and mitochondrial apoptotic pathways were measured by western blotting or immunofluorescence assays.</p><p><b>RESULTS</b>Cd-induced activation of Akt/mTOR signaling, including Akt, mTOR, p70 S6 kinase (p70 S6K), and eukaryotic initiation factor 4E binding protein 1 (4E-BP1). Rap, an mTOR inhibitor and NAC, a ROS scavenger, blocked Cd-induced activation of Akt/mTOR signaling and apoptosis of neuronal cells. Furthermore, NAC blocked the decrease of B-cell lymphoma 2/Bcl-2 associated X protein (Bcl-2/Bax) ratio, release of cytochrome c, cleavage of caspase-3 and poly(ADP-ribose) polymerase (PARP), and nuclear translocation of apoptosis-inducing factor (AIF) and endonuclease G (Endo G).</p><p><b>CONCLUSION</b>Cd-induced ROS generation activates Akt/mTOR and mitochondrial pathways, leading to apoptosis of neuronal cells. Our findings suggest that mTOR inhibitors or antioxidants have potential for preventing Cd-induced neurodegenerative diseases.</p>
Asunto(s)
Animales , Ratas , Apoptosis , Cadmio , Toxicidad , Caspasas , Metabolismo , Mitocondrias , Neuronas , Células PC12 , Proteínas Proto-Oncogénicas c-akt , Metabolismo , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno , Metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR , MetabolismoRESUMEN
To investigate 1alpha,25-(OH)2D3 regulation of matrix metalloproteinase-9 (MMP-9) protein expression during osteoclast formation and differentiation, receptor activator of nuclear factor kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) were administered to induce the differentiation of RAW264.7 cells into osteoclasts. The cells were incubated with different concentrations of 1alpha,25-(OH)2D3 during culturing, and cell proliferation was measured using the methylthiazol tetrazolium method. Osteoclast formation was confirmed using tartrate-resistant acid phosphatase (TRAP) staining and assessing bone lacunar resorption. MMP-9 protein expression levels were measured with Western blotting. We showed that 1alpha,25-(OH)2D3 inhibited RAW264.7 cell proliferation induced by RANKL and M-CSF, increased the numbers of TRAP-positive osteoclasts and their nuclei, enhanced osteoclast bone resorption, and promoted MMP-9 protein expression in a concentration-dependent manner. These findings indicate that 1alpha,25-(OH)2D3 administered at a physiological relevant concentration promoted osteoclast formation and could regulate osteoclast bone metabolism by increasing MMP-9 protein expression during osteoclast differentiation.
Asunto(s)
Animales , Ratones , Fosfatasa Ácida/metabolismo , Western Blotting , Calcitriol/farmacología , Agonistas de los Canales de Calcio/farmacología , Diferenciación Celular , Línea Celular , Proliferación Celular , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Isoenzimas/metabolismo , Metaloproteinasa 9 de la Matriz/genética , Osteoclastos/citología , Sales de Tetrazolio , TiazolesRESUMEN
The purpose of this study was to determine whether osteoprotegerin (OPG) could affect osteoclat differentiation and activation under serum-free conditions. Both duck embryo bone marrow cells and RAW264.7 cells were incubated with macrophage colony stimulatory factor (M-CSF) and receptor activator for nuclear factor kappaB ligand (RANKL) in serum-free medium to promote osteoclastogenesis. During cultivation, 0, 10, 20, 50, and 100 ng/mL OPG were added to various groups of cells. Osteoclast differentiation and activation were monitored via tartrate-resistant acid phosphatase (TRAP) staining, filamentous-actin rings analysis, and a bone resorption assay. Furthermore, the expression osteoclast-related genes, such as TRAP and receptor activator for nuclear factor kappaB (RANK), that was influenced by OPG in RAW264.7 cells was examined using real-time polymerase chain reaction. In summary, findings from the present study suggested that M-CSF with RANKL can promote osteoclast differentiation and activation, and enhance the expression of TRAP and RANK mRNA in osteoclasts. In contrast, OPG inhibited these activities under serum-free conditions.
Asunto(s)
Animales , Fosfatasa Ácida/genética , Proteínas Aviares/farmacología , Células de la Médula Ósea/efectos de los fármacos , Células Cultivadas , Patos , Embrión no Mamífero/efectos de los fármacos , Isoenzimas/genética , Factor Estimulante de Colonias de Macrófagos/metabolismo , Osteoclastos/citología , Osteoprotegerina/farmacología , Ligando RANK/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptor Activador del Factor Nuclear kappa-B/genéticaRESUMEN
<p><b>OBJECTIVE</b>To assess lipid peroxidation and ultrastructural modifications in rat brains following perinatal exposure to lead (Pb) and/or cadmium (Cd).</p><p><b>METHODS</b>Female rats were divided into four groups: control group, Pb (300 mg/L) group, Cd group (10 mg/L) and Pb+Cd (300 mg/L, 10 mg/L) group. The compounds were delivered in the drinking water throughout pregnancy and lactation.</p><p><b>RESULTS</b>The levels of compounds in blood and brain of the Pb+Cd group were similar to those of other groups, but the effects of Pb+Cd on pups' body and brain weights were higher than on other compounds. Electron microscopy revealed that Pb and Cd had effects on mitochondrial swelling, disruption and cristae loss, Nissl body dissolution, degenerated organelles and vacuoles, cytomembrane disappearance, and nuclear chromoplasm concentration. The activity of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), catalase (CAT), acetylcholinesterase (AChE) was decreased, whereas the activity of maleic dialdehyde (MDA) was increased.</p><p><b>CONCLUSION</b>Perinatal exposure to low doses of Pb and Cd can produce alterations in lipid peroxidation and ultrastructural modifications in rat brains, and exposure to both metals can result in greater damages.</p>