RESUMEN
Histamine is a biogenic amine implicated in various biological and pathological processes. Convenient cellular models are needed to screen and develop new antihistamine agents. This report aimed to characterize the response of neurons differentiated from mouse P19 embryonal carcinoma cells to histamine treatment, and to investigate the modulation of this response by antihistamine drugs, vegetal diamine oxidase, and catalase. The exposure of P19 neurons to histamine reduced cell viability to 65% maximally. This effect involves specific histamine receptors, since it was prevented by treatment with desloratadine and cimetidine, respectively, H1 and H2 antagonists, but not by the H3 antagonist ciproxifan. RT-PCR analysis showed that P19 neurons express H1 and H2 receptors, and the H3 receptor, although it seemed not involved in the histamine effect on these cells. The H4 receptor was not expressed. H1 and H2 antagonists as well as vegetal diamine oxidase diminished the intracellular Ca2+ mobilization triggered by histamine. The treatment with vegetal diamine oxidase or catalase protected against mortality and a significant reduction of H2O2 level, generated from the cells under the histamine action, was found upon treatments with desloratadine, cimetidine, vegetal diamine oxidase, or catalase. Overall, the results indicate the expression of functional histamine receptors and open the possibility of using P19 neurons as model system to study the roles of histamine and related drugs in neuronal pathogenesis. This model is less expensive to operate and can be easily implemented by current laboratories of analysis and by Contract Research Organizations.
Asunto(s)
Amina Oxidasa (conteniendo Cobre) , Productos Biológicos , Animales , Ratones , Histamina/farmacología , Histamina/metabolismo , Cimetidina/farmacología , Catalasa , Peróxido de Hidrógeno/farmacología , Antagonistas de los Receptores Histamínicos/farmacología , Receptores Histamínicos/genética , Antagonistas de los Receptores Histamínicos H1/farmacología , Neuronas/metabolismo , Productos Biológicos/farmacologíaRESUMEN
Copper coordinated with amino acid residues is essential for the function of many proteins. In addition, copper complexed to free l-Histidine, as [Cu(His)2], is used in the treatment of the neurodegenerative Menkes disease and of cardioencephalomyopathy. This study was aimed to coordinate copper(II) with four small ligands (l-Serine, l-Histidine, Urea and Biuret) and to evaluate structural features, stability, antioxidant activity and neuronal compatibility of the resulting complexes. All complexes were synthesized with CuCl2 and purified by precipitation in alcohol. Elemental composition, X-rays diffraction and FTIR indicated that the complexes were in form of [Cu(ligand)2] and exhibited tridentate (l-Histidine), bidentate (l-Serine and Biuret) or monodentate (Urea) coordination with copper. UV-Vis absorbance profiles in physiologically relevant solutions and cyclic voltammetry revealed that, contrarily to [Cu(Urea)2Cl2] and [Cu(Biuret)2Cl2], the [Cu(Ser)2] and [Cu(His)2Cl2] complexes were stable in different media including water, physiological saline and intestinal-like solutions. All complexes and their ligands had antioxidant capacity as evaluated by DPPH (1,1-diphenyl-2,2-picrylhydrazyl) and DPD (N,N-diethyl-p-phenylenediamine) methods, and the [Cu(His)2Cl2] complex was the most potent. Neuronal compatibility was assessed through cell viability measurements using cultured neurons derived from mouse P19 stem cells. Although only [Cu(His)2Cl2] showed a good neurocompatibility (about 90% at concentrations up to 200⯵M), the cytotoxicity of the other copper complexes was lower compared to equivalent concentrations of CuCl2. These findings open new perspectives for the use of these copper complexes as antioxidants and possibly as therapeutic agents for neurodegenerative diseases. Furthermore, study of these complexes may help to improve chelation therapy for copper dysfunctions.
Asunto(s)
Complejos de Coordinación , Cobre , Enfermedades Neurodegenerativas/tratamiento farmacológico , Neuronas/metabolismo , Animales , Línea Celular , Supervivencia Celular , Complejos de Coordinación/síntesis química , Complejos de Coordinación/química , Complejos de Coordinación/farmacocinética , Complejos de Coordinación/farmacología , Cobre/química , Cobre/farmacocinética , Cobre/farmacología , Ratones , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/patología , Neuronas/patologíaRESUMEN
The purpose of this study was to establish a drug screening method for small molecules extracted from traditional Chinese medicines (TCM) that have neuronal differentiation promoting effects, using P19 embryonic carcinoma cell as a cell-based model. First, the constructed plasmid (pTα1-Luc) was transfected into P19 cells to establish a screening model. Second, several TCMs were screened using the established model and all-trans-retinoic acid as a positive control. Finally, the underlying molecular mechanism was explored using immunofluorescence staining, qT-PCR, and Western blot analysis. Our results indicated that the drug screen model was established successfully and that both honokiol and hyperoside induced P19 differentiation into neurons, with the possible molecular mechanism being modulating the Wnt signaling pathway. In conclusion, the drug screening model developed in the present study provides a rapid, cell-based screening platform for identifying natural compounds with neuronal differentiation effects.
Asunto(s)
Compuestos de Bifenilo/farmacología , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Medicamentos Herbarios Chinos/farmacología , Células Madre de Carcinoma Embrionario/efectos de los fármacos , Lignanos/farmacología , Neuronas/efectos de los fármacos , Quercetina/análogos & derivados , Animales , Línea Celular Tumoral , Evaluación Preclínica de Medicamentos/métodos , Ratones , Quercetina/farmacología , Tretinoina/fisiología , Vía de Señalización WntRESUMEN
The purpose of this study was to establish a drug screening method for small molecules extracted from traditional Chinese medicines (TCM) that have neuronal differentiation promoting effects, using P19 embryonic carcinoma cell as a cell-based model. First, the constructed plasmid (pTα1-Luc) was transfected into P19 cells to establish a screening model. Second, several TCMs were screened using the established model and all-trans-retinoic acid as a positive control. Finally, the underlying molecular mechanism was explored using immunofluorescence staining, qT-PCR, and Western blot analysis. Our results indicated that the drug screen model was established successfully and that both honokiol and hyperoside induced P19 differentiation into neurons, with the possible molecular mechanism being modulating the Wnt signaling pathway. In conclusion, the drug screening model developed in the present study provides a rapid, cell-based screening platform for identifying natural compounds with neuronal differentiation effects.
Asunto(s)
Animales , Ratones , Compuestos de Bifenilo , Farmacología , Diferenciación Celular , Línea Celular Tumoral , Proliferación Celular , Evaluación Preclínica de Medicamentos , Métodos , Medicamentos Herbarios Chinos , Farmacología , Células Madre de Carcinoma Embrionario , Lignanos , Farmacología , Neuronas , Quercetina , Farmacología , Tretinoina , Fisiología , Vía de Señalización WntRESUMEN
Rnf10 is a member of the RING finger protein family. Recently, a number of RING finger proteins were reported to be involved in neuronal differentiation, development, and proliferation. In this study, we observed that the mRNA levels and protein expression of Rnf10 increase significantly upon the retinoic acid-induced neuronal differentiation of P19 cells. Knockdown of Rnf10 by RNA interference significantly impaired neuronal differentiation of P19 cells by attenuating the expression of neuronal markers. Cell cycle profiling revealed that Rnf10-depleted cells were unable to establish cell cycle arrest after RA treatment. In agreement with flow cytometry analysis, increased cell proliferation was observed after RA induction in Rnf10 knockdown cells as determined by a BrdU incorporation assay. Moreover, like Rnf10, the mRNA levels and protein expression of p21 and p27 also increased upon RA induction. Rnf10 knockdown only resulted in a reduction of p21 expression, while p27 and p57 expression remained unchanged, indicating that Rnf10 may regulate cell cycle exit through the p21 pathway. Ectopic p21 expression partially rescued the effect of Rnf10 depletion on the neuronal differentiation of P19 cells. Collectively, these results showed that increase in Rnf10 expression upon RA induction is necessary for the positive regulation of cyclin kinase inhibitor p21 expression, which leads to cell cycle arrest and is critical for neuronal differentiation.