RESUMEN
Although stem cells are a promising avenue for harnessing the potential of adipose tissue, conventional two-dimensional (2D) culture methods have limitations. This study explored the use of three-dimensional (3D) cultures to preserve the regenerative potential of adipose-derived stem cells (ADSCs) and investigated their cellular properties. Flow cytometric analysis revealed significant variations in surface marker expressions between the two culture conditions. While 2D cultures showed robust surface marker expressions, 3D cultures exhibited reduced levels of CD44, CD90.2, and CD105. Adipogenic differentiation in 3D organotypic ADSCs faced challenges, with decreased organoid size and limited activation of adipogenesis-related genes. Key adipocyte markers, such as lipoprotein lipase (LPL) and adipoQ, were undetectable in 3D-cultured ADSCs, unlike positive controls in 2D-cultured mesenchymal stem cells (MSCs). Surprisingly, 3D-cultured ADSCs underwent mesenchymal-epithelial transition (MET), evidenced by increased E-cadherin and EpCAM expression and decreased mesenchymal markers. This study highlights successful ADSC organoid formation, notable MSC phenotype changes in 3D culture, adipogenic differentiation challenges, and a distinctive shift toward an epithelial-like state. These findings offer insights into the potential applications of 3D-cultured ADSCs in regenerative medicine, emphasizing the need for further exploration of underlying molecular mechanisms.
Asunto(s)
Adiposidad , Sistemas Microfisiológicos , Animales , Ratones , Obesidad , Organoides , AdipocitosRESUMEN
Endoplasmic reticulum (ER) stress is involved in non-alcoholic fatty liver disease (NAFLD), but the relationship between oxidative stress, another well-known risk factor of NAFLD, and ER stress has yet to be elucidated. In this study, we treated mice with tunicamycin (TM) (2 mg/kg body weight) for 48 h to induce ER stress in the liver and examined the metabolic pathway that synthesizes the endogenous antioxidant, glutathione (GSH). Tunicamycin (TM) treatment significantly increased mRNA levels of CHOP and GRP78, and induced lipid accumulation in the liver. Lipid peroxidation in the liver tissue also increased from TM treatment (CON vs. TM; 3.0 ± 1.8 vs. 11.1 ± 0.8 nmol MDA/g liver, p < 0.001), which reflects an imbalance between the generation of reactive substances and antioxidant capacity. To examine the involvement of GSH synthetic pathway, we determined the metabolomic changes of sulfur amino acids in the liver. TM significantly decreased hepatic S-adenosylmethionine concentration in the methionine cycle. The levels of cysteine in the liver were increased, while taurine concentration was maintained and GSH levels profoundly decreased (CON vs. TM; 8.7 ± 1.5 vs. 5.4 ± 0.9 µmol GSH/g liver, p < 0.001). These results suggest that abnormal cysteine metabolism by TM treatment resulted in a decrease in GSH, followed by an increase in oxidative stress in the liver. In HepG2 cells, decreased GSH levels were examined by TM treatment in a dose dependent manner. Furthermore, pretreatment with TM in HepG2 cells potentiated oxidative cell death, by exacerbating the effects of tert-butyl hydroperoxide. In conclusion, TM-induced ER stress was accompanied by oxidative stress by reducing the GSH synthesis, which made the liver more susceptible to oxidative stress.
Asunto(s)
Proteínas de Choque Térmico/genética , Hígado/metabolismo , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Estrés Oxidativo/efectos de los fármacos , Factor de Transcripción CHOP/genética , Aminoácidos Sulfúricos/metabolismo , Animales , Antioxidantes/administración & dosificación , Vías Biosintéticas/efectos de los fármacos , Cisteína/metabolismo , Chaperón BiP del Retículo Endoplásmico , Estrés del Retículo Endoplásmico/efectos de los fármacos , Glutatión/biosíntesis , Glutatión/genética , Células Hep G2 , Humanos , Peroxidación de Lípido/efectos de los fármacos , Hígado/efectos de los fármacos , Ratones , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/patología , S-Adenosilmetionina/metabolismo , Taurina/metabolismo , Tunicamicina/administración & dosificación , terc-Butilhidroperóxido/farmacologíaRESUMEN
Silymarin is a flavonoid extracted from the milk thistle Silybum marianum. It has been reported to prevent liver injuries induced by various chemicals or toxins. Our recent study suggested that silymarin induces hepatic synthesis of glutathione by increasing cysteine availability, which may consequently contribute to increased antioxidant capacity of the liver. In the present study, we investigated the effects of silymarin on acute liver injury induced by restraint stress. Silymarin (100 mg/kg) was orally administered to BALB/c mice every 12 h (3 times in total). After the last dose, mice were subjected to restraint stress for 6 h, and serum levels of aspartate and alanine aminotransferases, and hepatic levels of lipid peroxidation were determined. Hepatic levels of sulfur-containing metabolites such as methionine, S-adenosylmethionine, cysteine, and glutathione were also measured. The level of pro-inflammatory mediators in both liver and serum was determined. To study the mechanism of the effects of silymarin, we assessed Jun N-terminal kinase (JNK) activation and apoptotic signaling. Restraint stress induced severe oxidative stress and increased mRNA levels of pro-inflammatory mediators; both effects of restraint stress were significantly inhibited by silymarin. Moreover, administration of silymarin significantly prevented acute liver injury induced by restraint stress by blocking JNK activation and subsequently apoptotic signaling. In conclusion, these results suggest that the inhibition of restraint stress-induced liver injury by silymarin is due at least in part to its anti-oxidant activity and its ability to suppress the inflammatory response.
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Lesión Pulmonar Aguda/tratamiento farmacológico , Antioxidantes/administración & dosificación , Inflamación/tratamiento farmacológico , Silimarina/administración & dosificación , Lesión Pulmonar Aguda/patología , Animales , Antioxidantes/química , Apoptosis/efectos de los fármacos , Humanos , Inflamación/patología , Peroxidación de Lípido/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/metabolismo , Ratones , Silybum marianum/química , Estrés Oxidativo/efectos de los fármacos , Silimarina/químicaRESUMEN
The comprehensive genomic impact of ionizing radiation (IR), a carcinogen, on healthy somatic cells remains unclear. Using large-scale whole-genome sequencing (WGS) of clones expanded from irradiated murine and human single cells, we revealed that IR induces a characteristic spectrum of short insertions or deletions (indels) and structural variations (SVs), including balanced inversions, translocations, composite SVs (deletion-insertion, deletion-inversion, and deletion-translocation composites), and complex genomic rearrangements (CGRs), including chromoplexy, chromothripsis, and SV by breakage-fusion-bridge cycles. Our findings suggest that 1 Gy IR exposure causes an average of 2.33 mutational events per Gb genome, comprising 2.15 indels, 0.17 SVs, and 0.01 CGRs, despite a high level of inter-cellular stochasticity. The mutational burden was dependent on total irradiation dose, regardless of dose rate or cell type. The findings were further validated in IR-induced secondary cancers and single cells without clonalization. Overall, our study highlights a comprehensive and clear picture of IR effects on normal mammalian genomes.
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Reordenamiento Génico , Translocación Genética , Humanos , Animales , Ratones , Mutación , Genómica , Inversión Cromosómica , MamíferosRESUMEN
Histone modifying factors are functional components of chromatin and play a role in gene regulation. The expression level of JMJD2B, a histone demethylase, is notably up-regulated in cancer tissues. Upregulation of JMJD2B promotes cancer cell proliferation under hypoxic conditions through target gene expression. Here, we describe the patterns of histone methylation and JMJD2B expression under various stressed conditions, such as hypoxia and radiation, in a gastric cancer cell line. JMJD2B expression in AGS cells was actively regulated by hypoxia and radiation. Chromatin immunoprecipitation experiments demonstrated that binding of JMJD2B on the cyclin A1 (CCNA1) promoter resulted in CCNA1 upregulation under hypoxic conditions. Furthermore, we confirmed that AGS cell proliferation was directly affected by JMJD2B and CCNA1 expression by performing experiments with JMJD2B depleted cells. Interestingly, the effects of JMJD2B on cell growth under hypoxia were remarkably repressed after gamma-ray irradiation. These results suggest that JMJD2B may play a central role in gastric cancer cell growth and might constitute a novel therapeutic target to overcome hypoxia-induced radio-resistance, thereby improving the efficiency of radiation therapy.
Asunto(s)
Proliferación Celular/efectos de la radiación , Rayos gamma , Regulación Enzimológica de la Expresión Génica/efectos de la radiación , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Histona Demetilasas con Dominio de Jumonji/biosíntesis , Proteínas de Neoplasias/metabolismo , Tolerancia a Radiación/efectos de la radiación , Neoplasias Gástricas/enzimología , Hipoxia de la Célula/efectos de la radiación , Línea Celular Tumoral , Ciclina A1/genética , Ciclina A1/metabolismo , Regulación Enzimológica de la Expresión Génica/genética , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Histona Demetilasas con Dominio de Jumonji/genética , Proteínas de Neoplasias/genética , Tolerancia a Radiación/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , Neoplasias Gástricas/terapiaRESUMEN
Huntington's disease (HD) is an inherited neurodegenerative disorder caused by expanded polyglutamine repeats in the huntingtin (Htt) protein. Mutant Htt may damage and kill striatal neurons by a mechanism involving reduced production of brain-derived neurotrophic factor (BDNF) and increased oxidative and metabolic stress. Because electroconvulsive shock (ECS) can stimulate the production of BDNF and protect neurons against stress, we determined whether ECS treatment would modify the disease process and provide a therapeutic benefit in a mouse model of HD. ECS (50 mA for 0.2 s) or sham treatment was administered once weekly to male N171-82Q Htt mutant mice beginning at 2 months of age. Endpoints measured included motor function, striatal and cortical pathology, and levels of protein chaperones and BDNF. ECS treatment delayed the onset of motor symptoms and body weight loss and extended the survival of HD mice. Striatal neurodegeneration was attenuated and levels of protein chaperones (Hsp70 and Hsp40) and BDNF were elevated in striatal neurons of ECS-treated compared with sham-treated HD mice. Our findings demonstrate that ECS can increase the resistance of neurons to mutant Htt resulting in improved functional outcome and extended survival. The potential of ECS as an intervention in subjects that inherit the mutant Htt gene merits further consideration.
Asunto(s)
Progresión de la Enfermedad , Electrochoque , Enfermedad de Huntington/patología , Enfermedad de Huntington/terapia , Mutación/genética , Proteínas de Transporte de Serotonina en la Membrana Plasmática/genética , Animales , Factor Neurotrófico Derivado del Encéfalo/genética , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Enfermedad de Huntington/genética , Masculino , Ratones , Ratones Transgénicos , Degeneración Nerviosa/genética , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/patología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismo , Transducción de Señal , Análisis de SupervivenciaRESUMEN
Nuclear factor E2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway is an important cellular stress response pathway involved in neuroprotection. We previously screened several natural phytochemicals and identified plumbagin as a novel activator of the Nrf2/ARE pathway that can protect neurons against ischemic injury. Here we extended our studies to natural and synthetic derivatives of plumbagin. We found that 5,8-dimethoxy-1,4-naphthoquinone (naphthazarin) is a potent activator of the Nrf2/ARE pathway, up-regulates the expression of Nrf2-driven genes in primary neuronal and glial cultures, and protects neurons against glutamate-induced excitotoxicity.
Asunto(s)
Muerte Celular , Ácido Glutámico/efectos adversos , Factor 2 Relacionado con NF-E2/metabolismo , Naftoquinonas/farmacología , Neuronas/efectos de los fármacos , Animales , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Astrocitos/patología , Proteínas Portadoras/metabolismo , Supervivencia Celular , Relación Dosis-Respuesta a Droga , Genes Reporteros , Células Hep G2 , Humanos , Proteínas de Microfilamentos/metabolismo , Factor 2 Relacionado con NF-E2/genética , Neuronas/metabolismo , Neuronas/patología , Fármacos Neuroprotectores/farmacología , Cultivo Primario de Células , Proteolisis , Ratas , Ratas Sprague-DawleyRESUMEN
We studied the roles of JMJD1A and its target gene ADM in the growth of hepatocellular carcinomas (HCCs) and breast cancer cells under hypoxic conditions. Hypoxia stimulated HepG2 and Hep3B cell proliferation but had no effect on MDA-MB-231 cell proliferation. Interestingly, the JMJD1A and ADM expressions were enhanced by hypoxia only in HepG2 and Hep3B cells. Our ChIP results showed that hypoxia-induced HepG2 and Hep3B cell proliferation is mediated by JMJD1A upregulation and subsequent decrease in methylation in the ADM promoter region. Furthermore, JMJD1A gene silencing abrogated the hypoxia-induced ADM expression and inhibited HepG2 and Hep3B cell growth. These data suggest that JMJD1A might function as a proliferation regulator in some cancer cell types.
Asunto(s)
Adrenomedulina/genética , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Histona Demetilasas con Dominio de Jumonji/genética , Adrenomedulina/metabolismo , Animales , Western Blotting , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Hipoxia de la Célula , Línea Celular Tumoral , Metilación de ADN , Femenino , Células Hep G2 , Humanos , Histona Demetilasas con Dominio de Jumonji/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Regiones Promotoras Genéticas/genética , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Radiation therapy is the most widely used treatment for cancer, but it causes the side effect of mucositis due to intestinal damage. We examined the protective effect of genistein in tumor-bearing mice after abdominal irradiation by evaluation of apoptosis and intestinal morphological changes. METHODS: Mouse colon cancer CT26 cells were subcutaneously injected at the flank of BALB/c mice to generate tumors. The tumor-bearing mice were treated with abdominal radiation at 5 and 10 Gy, and with genistein at 200 mg/kg body weight per day for 1 d before radiation. The changes in intestinal histology were evaluated 12 h and 3.5 d after irradiation. To assess the effect of the combination treatment on the cancer growth, the tumor volume was determined at sacrifice before tumor overgrowth occurred. RESULTS: Genistein significantly decreased the number of apoptotic nuclei compared with that in the irradiation group 12 h after 5 Gy irradiation. Evaluation of histological changes showed that genistein ameliorated intestinal morphological changes such as decreased crypt survival, villus shortening, and increased length of the basal lamina 3.5 d after 10 Gy irradiation. Moreover, the genistein-treated group exhibited more Ki-67-positive proliferating cells in the jejunum than the irradiated control group, and crypt depths were greater in the genistein-treated group than in the irradiated control group. The mean weight of the CT26 tumors was reduced in the group treated with genistein and radiation compared with the control group. CONCLUSION: Genistein had a protective effect on intestinal damage induced by irradiation and delayed tumor growth. These results suggest that genistein is a useful candidate for preventing radiotherapy-induced intestinal damage in cancer patients.
Asunto(s)
Apoptosis/efectos de los fármacos , Genisteína/uso terapéutico , Glycine max/química , Mucosa Intestinal/efectos de los fármacos , Intestino Delgado/efectos de los fármacos , Neoplasias/radioterapia , Traumatismos por Radiación/prevención & control , Animales , Línea Celular Tumoral , Femenino , Genisteína/farmacología , Mucosa Intestinal/lesiones , Mucosa Intestinal/patología , Mucosa Intestinal/efectos de la radiación , Intestino Delgado/lesiones , Intestino Delgado/patología , Intestino Delgado/efectos de la radiación , Yeyuno/efectos de los fármacos , Yeyuno/lesiones , Yeyuno/patología , Yeyuno/efectos de la radiación , Masculino , Ratones , Ratones Endogámicos BALB C , Mucositis/etiología , Mucositis/prevención & control , Neoplasias/patología , Fitoterapia , Extractos Vegetales/farmacología , Extractos Vegetales/uso terapéuticoRESUMEN
BACKGROUND: To assess the radiosensitivity of liver tumors harboring different genetic mutations, mouse liver tumors were generated in vivo through the hydrodynamic injection of clustered regularly interspaced short palindromic repeat/caspase 9 (CRISPR/Cas9) constructs encoding single-guide RNAs (sgRNAs) targeting Tp53, Pten, Nf1, Nf2, Tsc2, Cdkn2a, or Rb1. METHODS: The plasmid vectors were delivered to the liver of adult C57BL/6 mice via hydrodynamic tail vein injection. The vectors were injected into 10 mice in each group. Organoids were generated from mouse liver tumors. The radiation response of the organoids was assessed using an ATP cell viability assay. RESULTS: The mean survival period of mice injected with vectors targeting Nf2 (4.8 months) was lower than that of other mice. Hematoxylin and eosin staining, immunohistochemical (IHC) staining, and target sequencing analyses revealed that mouse liver tumors harbored the expected mutations. Tumor organoids were established from mouse liver tumors. Histological evaluation revealed marked morphological similarities between the mouse liver tumors and the generated tumor organoids. Moreover, IHC staining indicated that the parental tumor protein expression pattern was maintained in the organoids. The results of the ATP cell viability assay revealed that the tumor organoids with mutated Nf2 were more resistant to high-dose radiation than those with other gene mutations. CONCLUSIONS: This study developed a radiation response assessment system for mouse tumors with mutant target genes using CRISPR/Cas9 and organoids. The Tp53 and Pten double mutation in combination with the Nf2 mutation increased the radiation resistance of tumors. The system used in this study can aid in elucidating the mechanism underlying differential intrinsic radiation sensitivity of individual tumors.
Asunto(s)
Sistemas CRISPR-Cas , Neoplasias Hepáticas , Ratones , Animales , Sistemas CRISPR-Cas/genética , Ratones Endogámicos C57BL , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/radioterapia , Neoplasias Hepáticas/metabolismo , Mutación , Organoides/metabolismo , Organoides/patología , Adenosina TrifosfatoRESUMEN
Although ionizing radiation (IR) is widely used for therapeutic and research purposes, studies on low-dose ionizing radiation (LDIR) are limited compared with those on other IR approaches, such as high-dose gamma irradiation and ultraviolet irradiation. High-dose IR affects DNA damage response and nucleotide-protein crosslinking, among other processes; however, the molecular consequences of LDIR have been poorly investigated. Here, we developed a method to profile RNA species crosslinked to an RNA-binding protein, namely, human antigen R (HuR), using LDIR and high-throughput RNA sequencing. The RNA fragments isolated via LDIR-crosslinking and immunoprecipitation sequencing were crosslinked to HuR and protected from RNase-mediated digestion. Upon crosslinking HuR to target mRNAs such as PAX6, ZFP91, NR2F6, and CAND2, the transcripts degraded rapidly in human cell lines. Additionally, PAX6 and NR2F6 downregulation mediated the beneficial effects of LDIR on cell viability. Thus, our approach provides a method for investigating post-transcriptional gene regulation using LDIR.
RESUMEN
"Neurohormesis" refers to a response to a moderate level of stress that enhances the ability of the nervous systems to resist more severe stress that might be lethal or cause dysfunction or disease. Neurohormetic phytochemicals, such as, resveratrol, sulforaphane, curcumin, and catechins, protect neurons against injury and disease. Naphthoquinones, such as, juglone and plumbagin, induce robust hormetic stress responses. However, the possibility that subtoxic dose of 5,8-dihydroxy-1,4-naphthoquinone (naphthazarin) may protect against brain diseases via the activation of an adaptive stress response pathway in the brain has not been investigated. In this study, we examined the neurohormetic effect of a subtoxic dose of naphthazarin in a Parkinson's disease model. It was found that, under these conditions, naphthazarin enhanced movement ability, prevented loss of dopaminergic neurons, and attenuated neuroinflammation in a 1-methyl-4-phenyl-1,2,3,4-tetrahydropyridine-induced Parkinson's disease model. Furthermore, it was found that the neuroprotective effect of naphthazarin was mediated by the suppression of astroglial activation in response to 1-methyl-4-phenylpyridine treatment. In conclusion, we suggest that naphthazarin, in view of its hormetic effect on neuroprotection, be viewed as a potential treatment for Parkinson's disease and other neurodegenerative diseases associated with neuroinflammation.
Asunto(s)
Astrocitos/efectos de los fármacos , Naftoquinonas/farmacología , Fármacos Neuroprotectores/farmacología , Trastornos Parkinsonianos/tratamiento farmacológico , Animales , Astrocitos/metabolismo , Western Blotting , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas/efectos de los fármacos , Trastornos Parkinsonianos/patologíaRESUMEN
Pregabalin, a Ca(2+) channel α(2)δ-subunit antagonist with analgesic and antiepileptic activity, reduced neuronal loss and improved functional outcome in a mouse model of focal ischemic stroke. Pregabalin administration (5-10mg/kg, i.p.) 30-90 min after transient middle cerebral artery occlusion/reperfusion reduced infarct volume, neuronal death in the ischemic penumbra and neurological deficits at 24h post-stroke. Pregabalin significantly decreased the amount of Ca(2+)/calpain-mediated α-spectrin proteolysis in the cerebral cortex measured at 6h post-stroke. Together with the extensive clinical experience with pregabalin for other neurological indications, our findings suggest the potential for a therapeutic benefit of pregabalin in stroke patients.
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Calcio/antagonistas & inhibidores , Calcio/fisiología , Proteolisis/efectos de los fármacos , Accidente Cerebrovascular/tratamiento farmacológico , Ácido gamma-Aminobutírico/análogos & derivados , Animales , Relación Dosis-Respuesta a Droga , Masculino , Ratones , Ratones Endogámicos C57BL , Pregabalina , Accidente Cerebrovascular/enzimología , Accidente Cerebrovascular/patología , Resultado del Tratamiento , Ácido gamma-Aminobutírico/farmacología , Ácido gamma-Aminobutírico/uso terapéuticoRESUMEN
Toll-like receptors (TLRs) play essential roles in innate immunity, and increasing evidence indicates that these receptors are expressed in neurons, astrocytes, and microglia in the brain, where they mediate responses to infection, stress, and injury. To address the possibility that TLR2 heterodimer activation could affect progenitor cells in the developing brain, we analyzed the expression of TLR2 throughout mouse cortical development, and assessed the role of TLR2 heterodimer activation in neuronal progenitor cell (NPC) proliferation. TLR2 mRNA and protein was expressed in the cortex in embryonic and early postnatal stages of development, and in cultured cortical NPC. While NPC from TLR2-deficient and wild type embryos had the same proliferative capacity, TLR2 activation by the synthetic bacterial lipopeptides Pam(3)CSK(4) and FSL1, or low molecular weight hyaluronan, an endogenous ligand for TLR2, inhibited neurosphere formation in vitro. Intracerebral in utero administration of TLR2 ligands resulted in ventricular dysgenesis characterized by increased ventricle size, reduced proliferative area around the ventricles, increased cell density, an increase in phospho-histone 3 cells, and a decrease in BrdU(+) cells in the sub-ventricular zone. Our findings indicate that loss of TLR2 does not result in defects in cerebral development. However, TLR2 is expressed and functional in the developing telencephalon from early embryonic stages and infectious agent-related activation of TLR2 inhibits NPC proliferation. TLR2-mediated inhibition of NPC proliferation may therefore be a mechanism by which infection, ischemia, and inflammation adversely affect brain development.
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Células Madre Embrionarias/efectos de los fármacos , Neuronas/efectos de los fármacos , Receptor Toll-Like 2/agonistas , Animales , Animales Recién Nacidos , Recuento de Células , Proliferación Celular , Células Cultivadas , Ventrículos Cerebrales/anomalías , Ventrículos Cerebrales/citología , Diglicéridos/farmacología , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Histonas/metabolismo , Ácido Hialurónico/farmacología , Lipopéptidos/farmacología , Ratones , Ratones Noqueados , Neuronas/citología , Neuronas/metabolismo , Oligopéptidos/farmacología , Fosforilación , ARN Mensajero/biosíntesis , Telencéfalo/citología , Telencéfalo/embriología , Telencéfalo/crecimiento & desarrollo , Telencéfalo/metabolismo , Receptor Toll-Like 2/biosíntesis , Receptor Toll-Like 2/genéticaRESUMEN
Many phytochemicals function as noxious agents that protect plants against insects and other damaging organisms. However, at subtoxic doses, the same phytochemicals may activate adaptive cellular stress response pathways that can protect cells against a variety of adverse conditions. We screened a panel of botanical pesticides using cultured human and rodent neuronal cell models, and identified plumbagin as a novel potent activator of the nuclear factor E2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway. In vitro, plumbagin increases nuclear localization and transcriptional activity of Nrf2, and induces the expression of the Nrf2/ARE-dependent genes, such as heme oxygenase 1 in human neuroblastoma cells. Plumbagin specifically activates the Nrf2/ARE pathway in primary mixed cultures from ARE-human placental alkaline phosphatase reporter mice. Exposure of neuroblastoma cells and primary cortical neurons to plumbagin provides protection against subsequent oxidative and metabolic insults. The neuroprotective effects of plumbagin are abolished by RNA interference-mediated knockdown of Nrf2 expression. In vivo, administration of plumbagin significantly reduces the amount of brain damage and ameliorates-associated neurological deficits in a mouse model of focal ischemic stroke. Our findings establish precedence for the identification and characterization of neuroprotective phytochemicals based upon their ability to activate adaptive cellular stress response pathways.
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Regulación de la Expresión Génica/efectos de los fármacos , Hipoxia/tratamiento farmacológico , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Naftoquinonas/uso terapéutico , Fármacos Neuroprotectores/uso terapéutico , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Células Cultivadas , Corteza Cerebral/citología , Infarto Cerebral/etiología , Infarto Cerebral/prevención & control , Modelos Animales de Enfermedad , Embrión de Mamíferos , Glucosa/deficiencia , Hemo-Oxigenasa 1/genética , Hemo-Oxigenasa 1/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Factor 2 Relacionado con NF-E2/genética , Naftoquinonas/metabolismo , Naftoquinonas/farmacología , Neuroblastoma , Examen Neurológico , Neuronas , Fármacos Neuroprotectores/metabolismo , Fármacos Neuroprotectores/farmacología , Estrés Oxidativo/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Factor de Transcripción AP-1/genética , Factor de Transcripción AP-1/metabolismo , Transfección/métodosRESUMEN
Senescence maker protein 30 (SMP30) is decreased in an androgen-independent manner in kidney and liver with age. However, regulation of SMP30 expression in the brain has not been examined in aging and neurodegenerative diseases. To investigate SMP30 expression in the brain, we utilized aging and kainate (KA)-induced neurodegenerative disease models. Interestingly, expression of SMP30 was unlikely to decrease in the aged brain, but total levels of SMP30 protein were increased at 4 weeks after KA injury. Increased glial fibrillary acidic protein (GFAP) with elevated SMP30 expression was observed at the same time post-KA, indicating that regulation of SMP30 expression in the brain may be associated with astrocytosis. We confirmed that KA induced GFAP expression with increased SMP30 in rat astrocyte cells. Moreover, we found that ERK1/2 activation was involved in the up-regulation of SMP30 in astrocytes. Our results suggest that elevated SMP30 in activated astrocytes plays an important supportive role after brain damage.
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Envejecimiento/metabolismo , Proteínas de Unión al Calcio/biosíntesis , Gliosis/metabolismo , Hipocampo/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/fisiología , Proteína Quinasa 3 Activada por Mitógenos/fisiología , Convulsiones/metabolismo , Animales , Proteínas de Unión al Calcio/deficiencia , Proteínas de Unión al Calcio/genética , Hidrolasas de Éster Carboxílico , Línea Celular/efectos de los fármacos , Línea Celular/metabolismo , Cerebelo/crecimiento & desarrollo , Cerebelo/metabolismo , Corteza Cerebral/crecimiento & desarrollo , Corteza Cerebral/metabolismo , Regulación de la Expresión Génica , Proteína Ácida Fibrilar de la Glía , Gliosis/inducido químicamente , Gliosis/genética , Hipocampo/efectos de los fármacos , Hipocampo/patología , Péptidos y Proteínas de Señalización Intracelular/deficiencia , Péptidos y Proteínas de Señalización Intracelular/genética , Ácido Kaínico/toxicidad , Masculino , Ratones , Ratones Endogámicos ICR , Ratones Noqueados , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos/antagonistas & inhibidores , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Estrés Oxidativo , Ratas , Ratas Endogámicas F344 , Convulsiones/inducido químicamente , Convulsiones/genética , Convulsiones/patología , Transducción de Señal/efectos de los fármacos , Organismos Libres de Patógenos EspecíficosRESUMEN
Endocrine-disrupting chemicals (EDC) produce adverse effects on reproductive and immune function or neurological behavior, and may also induce cancer. The environmental EDC bisphenol A (BPA) is widely used in the manufacture of plastics and epoxy resins. BPA affects reproductive organ growth and development, but the potential adverse effects of BPA on neuronal development are not fully understood. Here, BPA concentration-dependently decreased proliferation of murine-derived multipotent neural progenitor cells (NPC), and high concentrations produced cytotoxicity. In contrast, low concentrations of BPA, which possess estrogenic activity, stimulated NPC differentiation into a neuronal phenotype. BPA treatment did not affect neonatal brain development in F1 mice. However, BPA treatment (20 mg/kg) accelerated formation of the dentate gyrus in postnatal day 1 mice. Prenatal and postnatal BPA treatment did not affect adult hippocampal neurogenesis in the dentate gyrus in 8-wk-old mice. Data indicate that BPA stimulates neuronal differentiation and might disrupt neonatal brain development.
Asunto(s)
Estrógenos no Esteroides/toxicidad , Hipocampo/citología , Hipocampo/crecimiento & desarrollo , Neuronas/citología , Fenoles/toxicidad , Animales , Animales Recién Nacidos , Compuestos de Bencidrilo , Diferenciación Celular , Relación Dosis-Respuesta a Droga , Estrógenos no Esteroides/administración & dosificación , Femenino , Masculino , Ratones , Ratones Endogámicos ICR , Células Madre Multipotentes , Fenoles/administración & dosificaciónRESUMEN
Senescence marker protein 30 (SMP30) is identified as an important aging marker molecule and known to play multifunctional roles as an intracellular calcium regulatory protein in the signaling process. To elucidate the functional significance of SMP30, we established the stably transfected P19 cell line with SMP30 expression vector. Overexpression of SMP30 slightly suppressed the proliferation of P19 cells. However, SMP30 overexpression was cytoprotective against calcium-mediated stress such as calcium ionophore (A23187), and thapsigargin. We found that SMP30 overexpression reduced the elevated intracellular calcium levels induced by A23187, but not by thapsigargin. In addition, SMP30 transfected P19 cells were more protective to tert-butylhydroperoxide induced cytotoxicity, indicating the antioxidative properties of SMP30. Taken together, our results suggest that external calcium regulation and antioxidant properties are involved in the cytoprotective mechanism of SMP30.
Asunto(s)
Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/fisiología , Calcio/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/fisiología , Estrés Oxidativo/fisiología , Animales , Antioxidantes/metabolismo , Western Blotting , Calcimicina/antagonistas & inhibidores , Calcimicina/toxicidad , Señalización del Calcio/fisiología , Carcinógenos/antagonistas & inhibidores , Carcinógenos/toxicidad , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Ratones , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tapsigargina/antagonistas & inhibidores , Tapsigargina/toxicidad , TransfecciónRESUMEN
Endocrine disruptors (EDs) exert adverse effects on reproductive and immune function or neurological behavior. Bisphenol A (BPA), one of the environmental EDs, is widely used in the manufacture of plastics and epoxy resins. Studies reported that BPA affects reproductive organ growth and development. However, the potential adverse effects of BPA on neuronal development have not been fully explored. In this study, the potent harmful effects of BPA were investigated on the murine-derived multipotent neural progenitor cells (NPCs). Pretreatment of BPA significantly decreased proliferation of NPCs in a concentration-dependent manner. Moreover, at a high concentration (> 400 microM), BPA was cytotoxic to NPCs. However, the low concentrations of BPA, previously shown to exert estrogenic actions, did not affect the proliferation of NPCs. BPA altered the activation of extracellular signal-regulated kinases and c-Jun-N-Kinases in a different manner without affecting activities of p38 kinases. It was also found that reactive oxygen species (ROS) were elevated in NPCs exposed to high concentrations of BPA, indicating oxidative stress-related cytotoxicity. These data show adverse effects of BPA on the nervous system and potentially on neonatal brain development.
Asunto(s)
Muerte Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Disruptores Endocrinos/toxicidad , Neuronas/efectos de los fármacos , Fenoles/toxicidad , Especies Reactivas de Oxígeno/metabolismo , Células Madre/efectos de los fármacos , Animales , Compuestos de Bencidrilo , Encéfalo/efectos de los fármacos , Encéfalo/crecimiento & desarrollo , Línea Celular , Relación Dosis-Respuesta a Droga , Ratones , Neuronas/enzimología , Estrés Oxidativo , Células Madre/enzimologíaRESUMEN
Nrf2/skn-1, a transcription factor known to mediate adaptive responses of cells to stress, also regulates energy metabolism in response to changes in nutrient availability. The ability to locate food sources depends upon chemosensation. Here we show that Nrf2/skn-1 is expressed in olfactory interneurons, and is required for proper integration of multiple food-related sensory cues in Caenorhabditis elegans. Compared to wild type worms, skn-1 mutants fail to perceive that food density is limiting, and display altered chemo- and thermotactic responses. These behavioral deficits are associated with aberrant AIY interneuron morphology and migration in skn-1 mutants. Both skn-1-dependent AIY autonomous and non-autonomous mechanisms regulate the neural circuitry underlying multisensory integration of environmental cues related to energy acquisition.