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1.
Nat Commun ; 11(1): 4071, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32792491

RESUMO

Arrest of oligodendrocyte (OL) differentiation and remyelination following myelin damage in multiple sclerosis (MS) is associated with neurodegeneration and clinical worsening. We show that Glutathione S-transferase 4α (Gsta4) is highly expressed during adult OL differentiation and that Gsta4 loss impairs differentiation into myelinating OLs in vitro. In addition, we identify Gsta4 as a target of both dimethyl fumarate, an existing MS therapy, and clemastine fumarate, a candidate remyelinating agent in MS. Overexpression of Gsta4 reduces expression of Fas and activity of the mitochondria-associated Casp8-Bid-axis in adult oligodendrocyte precursor cells, leading to improved OL survival during differentiation. The Gsta4 effect on apoptosis during adult OL differentiation was corroborated in vivo in both lysolecithin-induced demyelination and experimental autoimmune encephalomyelitis models, where Casp8 activity was reduced in Gsta4-overexpressing OLs. Our results identify Gsta4 as an intrinsic regulator of OL differentiation, survival and remyelination, as well as a potential target for future reparative MS therapies.


Assuntos
Glutationa Transferase/metabolismo , Oligodendroglia/citologia , Oligodendroglia/metabolismo , Animais , Apoptose/genética , Apoptose/fisiologia , Caspase 8/genética , Caspase 8/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Glutationa Transferase/genética , Homeostase/genética , Homeostase/fisiologia , Imuno-Histoquímica , Masculino , Microglia/citologia , Microglia/metabolismo , Microscopia Eletrônica de Transmissão , Mitocôndrias/metabolismo , Esclerose Múltipla/genética , Esclerose Múltipla/metabolismo , Fagocitose/genética , Fagocitose/fisiologia , Processamento de Proteína Pós-Traducional , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Remielinização/genética , Remielinização/fisiologia
3.
Nature ; 582(7813): 571-576, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32499656

RESUMO

Macrophages are the first cells of the nascent immune system to emerge during embryonic development. In mice, embryonic macrophages infiltrate developing organs, where they differentiate symbiotically into tissue-resident macrophages (TRMs)1. However, our understanding of the origins and specialization of macrophages in human embryos is limited. Here we isolated CD45+ haematopoietic cells from human embryos at Carnegie stages 11 to 23 and subjected them to transcriptomic profiling by single-cell RNA sequencing, followed by functional characterization of a population of CD45+CD34+CD44+ yolk sac-derived myeloid-biased progenitors (YSMPs) by single-cell culture. We also mapped macrophage heterogeneity across multiple anatomical sites and identified diverse subsets, including various types of embryonic TRM (in the head, liver, lung and skin). We further traced the specification trajectories of TRMs from either yolk sac-derived primitive macrophages or YSMP-derived embryonic liver monocytes using both transcriptomic and developmental staging information, with a focus on microglia. Finally, we evaluated the molecular similarities between embryonic TRMs and their adult counterparts. Our data represent a comprehensive characterization of the spatiotemporal dynamics of early macrophage development during human embryogenesis, providing a reference for future studies of the development and function of human TRMs.


Assuntos
Macrófagos/citologia , Análise de Célula Única , Linhagem da Célula , Embrião de Mamíferos/citologia , Cabeça , Hematopoese , Humanos , Antígenos Comuns de Leucócito/metabolismo , Fígado/citologia , Fígado/embriologia , Pulmão/citologia , Macrófagos/metabolismo , Microglia/citologia , Células Progenitoras Mieloides/citologia , RNA-Seq , Pele/citologia , Análise Espaço-Temporal , Transcriptoma , Saco Vitelino/citologia
4.
Chem Biol Interact ; 325: 109126, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32430275

RESUMO

Alzheimer's disease (AD) is a common neurodegenerative disease, and its pathogenesis is closely related to ß-amyloid (Aß) peptide. The deposition of Aß in the brain due to impaired Aß clearance is considered as an important cause of AD. The decrease in Aß clearance is closely related to the autophagy dysfunction in brains of AD patients. It is feasible to treat AD by increasing the autophagy level of cells such as microglia and neurons to accelerate Aß clearance. In this article we explored the ability of graphene oxide (GO) to clear Aß through activating autophagy. Our work demonstrated that GO could inhibit the mTOR signaling pathway by activating AMPK to induce the autophagy of microglial and neurons. As expected, with the improvement of autophagy ability of microglia, GO promoted microglia-mediated Aß phagocytosis. Under the conditions of co-culture of microglia and neurons, GO induced the autophagy of microglia and neurons, especially the autophagy of microglia, thereby promoting the clearance of Aß, and ultimately achieved the effect of protecting neurons. Moreover, GO was not only non-cytotoxic to microglia and neurons but also able to reduce the toxicity of Aß to neurons through its clearance. These results have shown the potential of GO in treating Alzheimer's disease.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Autofagia/efeitos dos fármacos , Grafite/farmacologia , Microglia/citologia , Microglia/efeitos dos fármacos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Linhagem Celular Tumoral , Ativação Enzimática/efeitos dos fármacos , Humanos , Camundongos , Microglia/metabolismo , Neurônios/metabolismo , Fagocitose/efeitos dos fármacos
5.
PLoS One ; 15(4): e0231597, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32287325

RESUMO

Resident microglia of the central nervous system are being increasingly recognized as key players in diseases such as neuropathic pain. Biochemical and behavioral studies in neuropathic pain rodent models have documented compelling evidence of the critical role of ATP mediated-P2X4R-brain-derived neurotrophic factor (BDNF) signaling pathway in the initiation and maintenance of pain hypersensitivity, a feature driving neuropathic pain-related behavior. The goal of this study was to develop and characterize an in vitro cell line model of activated microglia that can be subsequently utilized for screening neuropathic pain therapeutics. In the present study, we characterized the SIM-A9 microglia cell line for key molecules in the P2X4R-BDNF signaling axis using a combination of biochemical techniques and developed an ATP-activated SIM-A9 microglia model. We present three novel findings: first, SIM-A9 cells expressed P2X4R and BDNF proteins, second, ATP, but not LPS, was cytocompatible with SIM-A9 cells and third, exposure of cells to optimized ATP concentrations for defined periods increased intracellular expression of Iba1 and BDNF proteins. Increased Iba1 levels confirmed microglia activation and increased BDNF expression confirmed ATP-mediated stimulation of the P2X4R signaling pathway. We propose that this ATP-activated SIM-A9 cell line model system can be utilized for screening both small- as well as macro-molecular neuropathic pain therapeutics targeting BDNF and/or P2X4R knockdown.


Assuntos
Microglia/metabolismo , Neuralgia/metabolismo , Transdução de Sinais , Trifosfato de Adenosina/farmacologia , Animais , Fator Neurotrófico Derivado do Encéfalo/genética , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Técnicas de Cultura de Células/métodos , Lipopolissacarídeos/farmacologia , Camundongos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Microglia/citologia , Microglia/efeitos dos fármacos , Neuralgia/patologia , Receptores Purinérgicos P2X4/genética , Receptores Purinérgicos P2X4/metabolismo
6.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi ; 34(4): 509-517, 2020 Apr 15.
Artigo em Chinês | MEDLINE | ID: mdl-32291991

RESUMO

Objective: To explore the effect and mechanism of directive differentiation of microglia by SN50 on hypoxia-caused neurons injury in mice. Methods: The microglia were isolated and purified from brain tissue of new-born BALB/c mice through differential velocity adherent and vibration technique. The quantity of the microglia was identified by immunofluorescence staining of inducible nitric oxide synthetase (iNOS) and ionized calcium binding adapter molecule 1 (Iba1) and real-time fluorescence quantitative PCR (qRT-PCR) for special expression genes [iNOS, CD32, and interlenkin 10 (IL-10)]. Then the microglia were cultured with SN50, and the expressions of nuclear factor κB (NF-κB), differentiation-related genes (iNOS, CD11b, IL-10, and CD206), and apoptosis were detected by Western blot, qRT-PCR, and flow cytometry, respectively. The hypoxia model of neuron was established, and the cell apoptosis was evaluated by MTT after 0, 2, 6, 12, 24, and 48 hours of anoxic treatment. The apoptosis related markers (Bcl-2 and Caspase-3) were measured by Western blot and flow cytometry. In addition, the neurons after anoxic treatment were co-cultured with SN50 treated microglia (experimental group) and normal microglia (control group) for 24 hours. And the cell viability and apoptosis related markers (Bcl-2 and Caspase-3) were also measured. Results: Immunofluorescence staining and qRT-PCR analysis showed that the cells expressed the specific proteins and genes of microglia. Compared with the normal microglia, the relative expressions of NF-κB protein and iNOS and CD11b mRNAs in the microglia treated with SN50 significantly decreased ( P<0.05), the relative expressions of IL-10 and CD206 mRNAs significantly increased ( P<0.05), and the cell apoptosis rate had no significant change ( P>0.05). Compared with the normal neurons, the cell viability, the relative expressions of Bcl-2 and Caspase-3 proteins after anoxic treatment significantly decreased ( P<0.05), while the relative expressions of cleaved-Caspase-3 protein and cell apoptosis rate of neurons significantly increased ( P<0.05). In the co-culture system, the cell viability, the relative expressions of Bcl-2 and Caspase-3 proteins were significantly higher in experimental group than those in control group ( P<0.05), while the relative expressions of cleaved-Caspase-3 protein and cell apoptosis rate were significantly lower in experimental group than those in control group ( P<0.05). Conclusion: SN50 can induce the microglia differentiation into M2 type through NF-κB pathway. The SN50-induced microglia can protect neurons from hypoxic injury.


Assuntos
Diferenciação Celular , Hipóxia/patologia , Microglia/citologia , Neurônios/patologia , Peptídeos/farmacologia , Animais , Animais Recém-Nascidos , Apoptose , Células Cultivadas , Interleucina-10/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , NF-kappa B/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Receptores de IgG/metabolismo
7.
J Vis Exp ; (157)2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-32250348

RESUMO

Astrocytes and microglia are the most abundant glial cells. They are responsible for physiological support and homeostasis maintenance in the central nervous system (CNS). The increasing evidences of their involvement in the control of infectious diseases justify the emerging interest in the improvement of methodologies to isolate primary astrocytes and microglia in order to evaluate their responses to infections that affect the CNS. Considering the impact of Trypanosoma cruzi (T. cruzi) and Toxoplasma gondii (T. gondii) infection in the CNS, here we provide a method to extract, maintain, dissociate and infect murine astrocytes and microglia cells with protozoa parasites. Extracted cells from newborn cortices are maintained in vitro for 14 days with periodic differential media replacement. Astrocytes and microglia are obtained from the same extraction protocol by mechanical dissociation. After phenotyping by flow cytometry, cells are infected with protozoa parasites. The infection rate is determined by fluorescence microscopy at different time points, thus enabling the evaluation of differential ability of glial cells to control protozoan invasion and replication. These techniques represent simple, cheap and efficient methods to study the responses of astrocytes and microglia to infections, opening the field for further neuroimmunology analysis.


Assuntos
Astrócitos/citologia , Microglia/citologia , Doenças Parasitárias/patologia , Animais , Animais Recém-Nascidos , Astrócitos/parasitologia , Técnicas de Cultura de Células , Córtex Cerebral/citologia , Córtex Cerebral/parasitologia , Camundongos , Microglia/parasitologia , Doenças Parasitárias/parasitologia , Toxoplasma/fisiologia , Trypanosoma cruzi/fisiologia
8.
Neuron ; 106(5): 743-758.e5, 2020 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-32272058

RESUMO

The habenula complex is appreciated as a critical regulator of motivated and pathological behavioral states via its output to midbrain nuclei. Despite this, transcriptional definition of cell populations that comprise both the medial habenular (MHb) and lateral habenular (LHb) subregions in mammals remain undefined. To resolve this, we performed single-cell transcriptional profiling and highly multiplexed in situ hybridization experiments of the mouse habenula complex in naive mice and those exposed to an acute aversive stimulus. Transcriptionally distinct neuronal cell types identified within the MHb and LHb, were spatially defined, differentially engaged by aversive stimuli, and had distinct electrophysiological properties. Cell types identified in mice also displayed a high degree of transcriptional similarity to those previously described in zebrafish, highlighting the well-conserved nature of habenular cell types across the phylum. These data identify key molecular targets within habenular cell types and provide a critical resource for future studies.


Assuntos
Habenula/metabolismo , Neuroglia/metabolismo , Neurônios/metabolismo , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Células Ependimogliais/citologia , Células Ependimogliais/metabolismo , Perfilação da Expressão Gênica , Ontologia Genética , Habenula/citologia , Camundongos , Microglia/citologia , Microglia/metabolismo , Neuroglia/citologia , Neurônios/citologia , Oligodendroglia/citologia , Oligodendroglia/metabolismo , RNA-Seq , Análise de Célula Única , Peixe-Zebra
9.
Am J Chin Med ; 48(3): 559-577, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32345030

RESUMO

Depression is partially caused by inflammation in the central nervous system. Early study demonstrated that musk, glandular secretion from male musk deer, exerted an antidepressant-like effect. The aim of this study was to investigate if muscone, a bioactive ingredient in musk, could ameliorate neuroinflammation and depressive-like behaviors as well as explore the potential action mechanism. Mice were intraperitoneally (i.p.) injected with muscone for 2 weeks prior to administration of lipopolysaccharides (LPS, 1mg/kg, i.p.). Pre-treatment with muscone reversed the LPS-induced decrease in body weight within 24h and ameliorated depressive-like behaviors shown by sucrose preference, tail suspension test, and forced swimming test. LPS-induced activation of microglial cells and elevation in expression of inflammatory cytokines including IL-1ß, RANTES, and MCP-1 in the prefrontal cortex of mice were effectively abrogated by muscone, which significantly down-regulated expression of TLR4, MyD88, Caspase-1, NLRP3, renin, and Ang II. In addition, treatment of BV2 microglia cells with muscone markedly attenuated the LPS-induced rise in protein expression of TLR4, Ang II, and IL-1ß. This study revealed that muscone could ameliorate LPS-induced depressive-like behaviors by repressing neuroinflammation in the prefrontal cortex of mice caused by its suppression on microglia activation and production of inflammatory cytokines via acting on TLR4 pathway and RAS cascade.


Assuntos
Cicloparafinas/administração & dosagem , Cicloparafinas/farmacologia , Depressão/tratamento farmacológico , Lipopolissacarídeos/efeitos adversos , Animais , Peso Corporal/efeitos dos fármacos , Citocinas/metabolismo , Cervos , Depressão/induzido quimicamente , Mediadores da Inflamação/metabolismo , Injeções Intraperitoneais , Masculino , Camundongos , Microglia/citologia , Microglia/efeitos dos fármacos , Córtex Pré-Frontal/metabolismo , Córtex Pré-Frontal/patologia , Transdução de Sinais/efeitos dos fármacos , Receptor 4 Toll-Like/metabolismo
10.
Mol Immunol ; 121: 159-166, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32222586

RESUMO

Methamphetamine (METH) is a major public health and safety problem worldwide. METH is psychostimulant that activates microglia via the toll-like receptor (TLR) 4/MD2 complex, modulating the abundant production of pro-inflammatory cytokines in the central nervous system (CNS). The TLR4/MD2 complex on the surface of microglia recognizes pathogen-associated molecular patterns such as lipopolysaccharide (LPS) resulting in brain tissue inflammation and neuronal damage. Since METH has been associated with microglia-induced neurotoxicity, we hypothesized that METH impairs the expression of TLR4 and activation of NF-κB in NR-9460 microglia-like cells after LPS challenge. We demonstrated that METH decreases the distribution and expression of TLR4 receptors on the surface of microglia-like cells after incubation with endotoxin. Moreover, METH impairs the TLR4/MD2 complex signaling pathways, compromises the activation of NF-κB, and reduces the production of pro-inflammatory mediators in microglia-like cells upon LPS stimulation. Interestingly, microglia-like cells treated with METH and challenged with LPS showed considerable cellular morphological changes including enlarged nuclei and ruffled surface. Our results suggest that METH may have a significant impact on microglial-induced neuroinflammation, neurotoxicity, and the CNS defense against infection. It also highlights the importance of studying the effects of METH on the molecular and cellular components of users' CNS immunity. Finally, animal studies exploring the role of METH on the effectors functions of microglia after antigenic exposure are necessary to understand drug-related inflammation and neural damage in users.


Assuntos
Estimulantes do Sistema Nervoso Central/toxicidade , Encefalite/induzido quimicamente , Metanfetamina/toxicidade , Microglia/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Animais , Citocinas/imunologia , Citocinas/metabolismo , Encefalite/imunologia , Encefalite/patologia , Humanos , Lipopolissacarídeos/imunologia , Camundongos , Microglia/citologia , Microglia/imunologia , NF-kappa B/imunologia , NF-kappa B/metabolismo , Transdução de Sinais/imunologia , Receptor 4 Toll-Like/imunologia , Receptor 4 Toll-Like/metabolismo
11.
Nat Commun ; 11(1): 1577, 2020 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-32221280

RESUMO

Microglia, the brain-resident macrophages, exhibit highly dynamic functions in neurodevelopment and neurodegeneration. Human microglia possess unique features as compared to mouse microglia, but our understanding of human microglial functions is largely limited by an inability to obtain human microglia under homeostatic states. Here, we develop a human pluripotent stem cell (hPSC)-based microglial chimeric mouse brain model by transplanting hPSC-derived primitive macrophage progenitors into neonatal mouse brains. Single-cell RNA-sequencing of the microglial chimeric mouse brains reveals that xenografted hPSC-derived microglia largely retain human microglial identity, as they exhibit signature gene expression patterns consistent with physiological human microglia and recapitulate heterogeneity of adult human microglia. Importantly, the engrafted hPSC-derived microglia exhibit dynamic response to cuprizone-induced demyelination and species-specific transcriptomic differences in the expression of neurological disease-risk genes in microglia. This model will serve as a tool to study the role of human microglia in brain development and degeneration.


Assuntos
Encéfalo/citologia , Diferenciação Celular , Quimera/fisiologia , Células-Tronco Pluripotentes Induzidas/citologia , Microglia/citologia , Animais , Linhagem Celular , Cuprizona , Doenças Desmielinizantes/patologia , Feminino , Humanos , Imageamento Tridimensional , Camundongos , Microglia/transplante , RNA-Seq , Análise de Célula Única , Transcriptoma/genética
12.
Nat Commun ; 11(1): 1559, 2020 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-32214088

RESUMO

Microglia are highly motile cells that continuously monitor the brain environment and respond to damage-associated cues. While glucose is the main energy substrate used by neurons in the brain, the nutrients metabolized by microglia to support surveillance of the parenchyma remain unexplored. Here, we use fluorescence lifetime imaging of intracellular NAD(P)H and time-lapse two-photon imaging of microglial dynamics in vivo and in situ, to show unique aspects of the microglial metabolic signature in the brain. Microglia are metabolically flexible and can rapidly adapt to consume glutamine as an alternative metabolic fuel in the absence of glucose. During insulin-induced hypoglycemia in vivo or in aglycemia in acute brain slices, glutaminolysis supports the maintenance of microglial process motility and damage-sensing functions. This metabolic shift sustains mitochondrial metabolism and requires mTOR-dependent signaling. This remarkable plasticity allows microglia to maintain their critical surveillance and phagocytic roles, even after brain neuroenergetic homeostasis is compromised.


Assuntos
Encéfalo/imunologia , Metabolismo Energético/fisiologia , Microglia/metabolismo , Animais , Encéfalo/patologia , Receptor 1 de Quimiocina CX3C/genética , Movimento Celular , Ácidos Graxos/metabolismo , Glucose/deficiência , Glucose/metabolismo , Glutamina/metabolismo , Vigilância Imunológica , Camundongos , Camundongos Transgênicos , Microglia/citologia , Microglia/imunologia , NAD/metabolismo , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo
13.
Nat Commun ; 11(1): 986, 2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-32080187

RESUMO

Tissue microarchitecture and mechanics are important in development and pathologies of the Central Nervous System (CNS); however, their coordinating mechanisms are unclear. Here, we report that during colonization of the retina, microglia contacts the deep layer of high stiffness, which coincides with microglial bipolarization, reduction in TGFß1 signaling and termination of vascular growth. Likewise, stiff substrates induce microglial bipolarization and diminish TGFß1 expression in hydrogels. Both microglial bipolarization in vivo and the responses to stiff substrates in vitro require intracellular adaptor Kindlin3 but not microglial integrins. Lack of Kindlin3 causes high microglial contractility, dysregulation of ERK signaling, excessive TGFß1 expression and abnormally-patterned vasculature with severe malformations in the area of photoreceptors. Both excessive TGFß1 signaling and vascular defects caused by Kindlin3-deficient microglia are rescued by either microglial depletion or microglial knockout of TGFß1 in vivo. This mechanism underlies an interplay between microglia, vascular patterning and tissue mechanics within the CNS.


Assuntos
Microglia/fisiologia , Vasos Retinianos/inervação , Fator de Crescimento Transformador beta1/fisiologia , Actomiosina/fisiologia , Animais , Fenômenos Biomecânicos , Movimento Celular/fisiologia , Proteínas do Citoesqueleto/deficiência , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/fisiologia , Feminino , Hidrogéis , Integrinas/fisiologia , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/citologia , Comunicação Parácrina , Retina/crescimento & desenvolvimento , Vasos Retinianos/citologia , Vasos Retinianos/crescimento & desenvolvimento , Fator de Crescimento Transformador beta1/genética
14.
PLoS One ; 15(2): e0228750, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32032367

RESUMO

OBJECTIVE: Accumulating epidemiological studies have demonstrated that diabetes is an important risk factor for dementia. However, the underlying pathological and molecular mechanisms, and effective treatment, have not been fully elucidated. Herein, we investigated the effect of the dipeptidyl peptidase-4 (DPP-4) inhibitor, linagliptin, on diabetes-related cognitive impairment. METHOD: Streptozotocin (STZ)-induced diabetic mice were treated with linagliptin (3 mg/kg/24 h) for 17 weeks. The radial arm water maze test was performed, followed by evaluation of oxidative stress using DNP-MRI and the expression of NAD(P)H oxidase components and proinflammatory cytokines and of microglial activity. RESULTS: Administration of linagliptin did not affect the plasma glucose and body weight of diabetic mice; however, it improved cognitive impairment. Additionally, linagliptin reduced oxidative stress and the mRNA expression of NAD(P)H oxidase component and TNF-α, and the number and body area of microglia, all of which were significantly increased in diabetic mice. CONCLUSIONS: Linagliptin may have a beneficial effect on diabetes-related dementia by inhibiting oxidative stress and microglial activation, independently of glucose-lowering.


Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Inibidores da Dipeptidil Peptidase IV/uso terapêutico , Linagliptina/farmacologia , Microglia/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Animais , Glicemia/análise , Peso Corporal/efeitos dos fármacos , Encéfalo/metabolismo , Diabetes Mellitus Experimental/induzido quimicamente , Inibidores da Dipeptidil Peptidase IV/farmacologia , Linagliptina/uso terapêutico , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Microglia/citologia , Microglia/efeitos dos fármacos , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Estreptozocina/toxicidade , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
15.
PLoS One ; 15(2): e0228222, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32074109

RESUMO

Genetically modified swine disease models are becoming increasingly important for studying molecular, physiological and pathological characteristics of human disorders. Given the limited history of these model systems, there remains a great need for proven molecular reagents in swine tissue. Here, to provide a resource for neurological models of disease, we validated antibodies by immunohistochemistry for use in examining central nervous system (CNS) markers in a recently developed miniswine model of neurofibromatosis type 1 (NF1). NF1 is an autosomal dominant tumor predisposition disorder stemming from mutations in NF1, a gene that encodes the Ras-GTPase activating protein neurofibromin. Patients classically present with benign neurofibromas throughout their bodies and can also present with neurological associated symptoms such as chronic pain, cognitive impairment, and behavioral abnormalities. As validated antibodies for immunohistochemistry applications are particularly difficult to find for swine models of neurological disease, we present immunostaining validation of antibodies implicated in glial inflammation (CD68), oligodendrocyte development (NG2, O4 and Olig2), and neuron differentiation and neurotransmission (doublecortin, GAD67, and tyrosine hydroxylase) by examining cellular localization and brain region specificity. Additionally, we confirm the utility of anti-GFAP, anti-Iba1, and anti-MBP antibodies, previously validated in swine, by testing their immunoreactivity across multiple brain regions in mutant NF1 samples. These immunostaining protocols for CNS markers provide a useful resource to the scientific community, furthering the utility of genetically modified miniswine for translational and clinical applications.


Assuntos
Biomarcadores/metabolismo , Neurofibromatose 1/patologia , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Linhagem da Célula , Modelos Animais de Doenças , Proteína Glial Fibrilar Ácida/metabolismo , Proteínas dos Microfilamentos/metabolismo , Microglia/citologia , Microglia/metabolismo , Neurofibromatose 1/metabolismo , Neurofibromina 1/genética , Fator de Transcrição 2 de Oligodendrócitos/metabolismo , Oligodendroglia/citologia , Oligodendroglia/metabolismo , Suínos
16.
Phytomedicine ; 68: 153143, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32018209

RESUMO

BACKGROUND: Rhodiola rosea L. (Crassulaceae) has been used for years in the traditional medicine of several countries as an adaptogen drug, able to preserve homeostasis in response to stress stimuli. Currently R. rosea roots and rhizome are classified as a traditional herbal medicinal product for temporary relief of symptoms of stress, such as fatigue and sensation of weakness by the European Medicines Agency. HYPOTHESIS/PURPOSE: Increasing evidences suggest the involvement of neuroinflammation in response to stress. However, whether the modulation of neuroinflammatory parameters could be involved in the anti-stress effect of R. rosea has been barely studied. Thus, the aim of this work is to investigate the possible modulation of molecular inflammatory processes elicited by a R. rosea roots and rhizome ethanolic extract in an in vitro model of corticotropin releasing hormone (CRH)-stimulated BV2 microglial cells. METHODS: BV2 cells were stimulated with CRH 100 nM and changes in cell viability, cytokines production and heat shock protein 70 (HSP70) levels were evaluated. Intracellular pathways related to inflammation, such as nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB) nuclear translocation and mitogen-activated protein kinases (MAPK) activation were also analyzed. RESULTS: We found that R. rosea extract (2.7% m/m rosavin and 1% m/m salidroside) 20 µg/ml was able to counteract the neuroinflammatory effect of CRH by inhibiting NF-κB nuclear translocation with a mechanism of action involving the modulation of mitogen-activated protein kinase-activated protein kinase 2 (MKK2), extracellular signal-regulated kinase 1/2 (ERK 1/2) and c-Jun n-terminal kinase (JNK), resulting in a reduction of HSP70 expression. CONCLUSION: This work expands the knowledge of the intracellular mechanisms involved in R. rosea anti-stress activity and may be useful for the study of other adaptogen drugs.


Assuntos
Hormônio Liberador da Corticotropina/metabolismo , Inflamação/tratamento farmacológico , Extratos Vegetais/farmacologia , Rhodiola/química , Adaptação Biológica/efeitos dos fármacos , Animais , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Hormônio Liberador da Corticotropina/farmacologia , Glucosídeos/farmacologia , Proteínas de Choque Térmico HSP70/metabolismo , Inflamação/metabolismo , Inflamação/patologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Microglia/citologia , Microglia/efeitos dos fármacos , NF-kappa B/metabolismo , Fenóis/farmacologia , Extratos Vegetais/química , Raízes de Plantas/química , Plantas Medicinais/química , Rizoma/química , Estresse Fisiológico/efeitos dos fármacos
17.
J Vis Exp ; (156)2020 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-32091006

RESUMO

In the central nervous system, oligodendrocytes are well-known for their role in axon myelination, that accelerates the propagation of action potentials through saltatory conduction. Moreover, an increasing number of reports suggest that oligodendrocytes interact with neurons beyond myelination, notably through the secretion of soluble factors. Here, we present a detailed protocol allowing purification of oligodendroglial lineage cells from glial cell cultures also containing astrocytes and microglial cells. The method relies on overnight shaking at 37 °C, which allows selective detachment of the overlying oligodendroglial cells and microglial cells, and the elimination of microglia by differential adhesion. We then describe the culture of oligodendrocytes and production of oligodendrocyte-conditioned medium (OCM). We also provide the kinetics of OCM treatment or oligodendrocytes addition to purified hippocampal neurons in co-culture experiments, studying oligodendrocyte-neuron interactions.


Assuntos
Separação Celular/métodos , Técnicas de Cocultura , Meios de Cultivo Condicionados , Neuroglia/citologia , Oligodendroglia/citologia , Animais , Astrócitos/citologia , Células Cultivadas , Meios de Cultivo Condicionados/metabolismo , Feminino , Hipocampo/citologia , Humanos , Masculino , Microglia/citologia , Neurônios/fisiologia , Ratos , Ratos Wistar
18.
Carbohydr Res ; 488: 107908, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31927345

RESUMO

Three new triacetic acid lactone (TAL) glycosides, forsyphensides A-C (1-3) were isolated from the fruits of Forsythia suspensa. Their structures were elucidated by comprehensive spectroscopic techniques. The absolute configurations of their monosaccharides were determined by GC analysis. Notably, forsyphensides A-C were relatively rare TAL glycosides identified from plants. Compound 1 exhibited inhibitory activity against the lipopolysaccharide (LPS)-induced nitric oxide (NO) production in microglia BV2 cells with the inhibition rate of 69.40%.


Assuntos
Forsythia/química , Glicosídeos/química , Microglia/metabolismo , Pironas/química , Animais , Sequência de Carboidratos , Linhagem Celular , Glicosídeos/farmacologia , Lipopolissacarídeos/efeitos adversos , Camundongos , Microglia/citologia , Microglia/efeitos dos fármacos , Estrutura Molecular , Óxido Nítrico/metabolismo , Extratos Vegetais/química , Pironas/farmacologia
19.
Int J Mol Sci ; 21(2)2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31952234

RESUMO

Heat shock protein 27 (HSP27) is commonly involved in cellular stress. Increased levels of HSP27 as well as autoantibodies against this protein were previously detected in glaucoma patients. Moreover, systemic immunization with HSP27 induced glaucoma-like damage in rodents. Now, for the first time, the direct effects of an intravitreal HSP27 application were investigated. For this reason, HSP27 or phosphate buffered saline (PBS, controls) was applied intravitreally in rats (n = 12/group). The intraocular pressure (IOP) as well as the electroretinogram recordings were comparable in HSP27 and control eyes 21 days after the injection. However, significantly fewer retinal ganglion cells (RGCs) and amacrine cells were observed in the HSP27 group via immunohistochemistry and western blot analysis. The number of bipolar cells, on the other hand, was similar in both groups. Interestingly, a stronger neurofilament degeneration was observed in HSP27 optic nerves, while no differences were noted regarding the myelination state. In summary, intravitreal HSP27 injection led to an IOP-independent glaucoma-like damage. A degeneration of RGCs as well as their axons and amacrine cells was noted. This suggests that high levels of extracellular HSP27 could have a direct damaging effect on RGCs.


Assuntos
Proteínas de Choque Térmico HSP27/farmacologia , Filamentos Intermediários/efeitos dos fármacos , Nervo Óptico/efeitos dos fármacos , Retina/efeitos dos fármacos , Células Ganglionares da Retina/efeitos dos fármacos , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Eletrorretinografia , Proteínas de Choque Térmico HSP27/administração & dosagem , Filamentos Intermediários/metabolismo , Pressão Intraocular/efeitos dos fármacos , Masculino , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Microglia/citologia , Microglia/efeitos dos fármacos , Microglia/metabolismo , Bainha de Mielina/efeitos dos fármacos , Bainha de Mielina/metabolismo , Nervo Óptico/metabolismo , Nervo Óptico/fisiologia , Ratos Wistar , Retina/metabolismo , Retina/fisiologia , Células Ganglionares da Retina/metabolismo
20.
Phytomedicine ; 67: 153164, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31954258

RESUMO

BACKGROUND: Centipeda minima (L.) A.Br. (C. minima) has been used in traditional Chinese herbal medicine to treat nasal allergy, diarrhea, asthma and malaria for centuries. Recent pharmacological studies have demonstrated that the ethanol extract of C. minima (ECM) and several active components possess anti-bacterial, anti-arthritis and anti-inflammatory properties. However, the effects of ECM on neuroinflammation and the underlying mechanisms have never been reported. PURPOSE: The study aimed to examine the potential inhibitory effects of ECM on neuroinflammation and illustrate the underlying mechanisms. METHODS: High performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was performed to qualify the major components of ECM; BV2 and primary microglial cells were used to examine the anti-inflammatory activity of ECM in vitro. To evaluate the anti-inflammatory effects of ECM in vivo, the mice were orally administrated with ECM (100, 200 mg•kg-1•d-1) for 2 days before cotreatment with LPS (2 mg•kg-1•d-1, ip) for an additional 3 days. The mice were sacrificed the day after the last treatment and the hippocampus was dissected for further experiments. The expression of inflammatory proteins and the activation of microglia were respectively detected by real-time PCR, ELISA, Western blotting and immunofluorescence. RESULTS: HPLC-MS/MS analysis confirmed and quantified seven chemicals in ECM. In BV2 and primary microglial cells, ECM inhibited the LPS-induced production of tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß), thus protecting HT22 neuronal cells from inflammatory damage. Furthermore, ECM inhibited the LPS-induced activation of NF-κB signaling pathway and subsequently attenuated the induction of inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX2), NADPH oxidase 2 (NOX2) and NADPH oxidase 4 (NOX4), leading to the decreased production of nitrite oxide, prostaglandin E2 (PGE2) and reactive oxygen species (ROS). In an LPS-induced neuroinflammatory mouse model, ECM was found to exert anti-inflammatory activity by decreasing the production of proinflammatory mediators, inhibiting the phosphorylation of NF-κB, and reducing the expression of COX2, iNOS, NOX2 and NOX4 in the hippocampal tissue. Moreover, LPS-induced microglial activation was markedly attenuated in the hippocampus, while ECM at a high dose possesses a stronger anti-inflammatory activity than the positive drug dexamethansone (DEX). CONCLUSION: These findings demonstrate that ECM exerts antineuroinflammatory effects via attenuating the activation of NF-κB signaling pathway and inhibiting the production of proinflammatory mediators both in vitro and in vivo. C. minima might become a novel phytomedicine to treat neuroinflammatory diseases.


Assuntos
Anti-Inflamatórios/farmacologia , Asteraceae/química , NF-kappa B/metabolismo , Extratos Vegetais/farmacologia , Animais , Dinoprostona/metabolismo , Medicamentos de Ervas Chinesas/química , Medicamentos de Ervas Chinesas/farmacologia , Enzimas/metabolismo , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Inflamação/patologia , Lipopolissacarídeos/farmacologia , Masculino , Camundongos Endogâmicos C57BL , Microglia/citologia , Microglia/efeitos dos fármacos , Microglia/metabolismo , Extratos Vegetais/química , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Espectrometria de Massas em Tandem , Fator de Necrose Tumoral alfa/metabolismo
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