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1.
Chem Biol Interact ; 348: 109653, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34516974

RESUMO

Angiotensin II, the effector peptide of the renin-angiotensin system, is not only a pivotal peptide implicated in the regulation of blood pressure but also a key mediator of the inflammatory processes that play an important role in the pathology of hypertension-related cSVD. Harpagide is the major bioactive constituent of Scrophulariae Radix widely used in traditional Chinese medicine for numerous diseases including hypertension. The present study aimed to investigate the effect of harpagide on Ang II-induced neuroinflammation and the potential mechanism. Pretreated with harpagide or resatorvid (the TLR4 pathway inhibitor), BV2 cells were treated with Ang II or LPS (the TLR4 activator). NO, pro-inflammatory cytokines, the proteins on TLR4/MyD88/NF-κB signaling pathway and the expression of CD86, CD206, TREM2 in BV2 cells were detected respectively. Subsequently, the effects of harpagide on neurotoxicity and BBB destruction triggered by Ang II-induced neuroinflammation were investigated in the co-cultures of BV2 microglia/HT22 hippocampal neurons, BV2 microglia/bEnd.3 endotheliocyte and BV2 microglia/BBB monolayer model. We found that Ang II converted microglia into M1 state and resulted in neuroinflammation through activating TLR4/MyD88/NF-κB signaling pathway. It also triggered the imbalance of TLR4/TREM2 in microglia. Ang II-mediated inflammation microglia further led to neuronal apoptosis and BBB damage. Harpagide showed the effect of alleviating Ang II-mediated neuroinflammation as well as the resulting neurotoxicity and BBB destruction through inhibiting the TLR4/MyD88/NF-κB pathway. The anti-inflammatory and neuroprotective effect of harpagide suggested that it might be a potential therapeutic strategy in hypertensive cSVD.


Assuntos
Angiotensina II/farmacologia , Apoptose/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Glicosídeos Iridoides/farmacologia , Microglia/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Piranos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Barreira Hematoencefálica/efeitos dos fármacos , Linhagem Celular , Humanos , Microglia/citologia , Microglia/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Receptor 4 Toll-Like/metabolismo
2.
Nat Commun ; 12(1): 5289, 2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34489419

RESUMO

Microglia are brain-resident immune cells with a repertoire of functions in the brain. However, the extent of their interactions with the vasculature and potential regulation of vascular physiology has been insufficiently explored. Here, we document interactions between ramified CX3CR1 + myeloid cell somata and brain capillaries. We confirm that these cells are bona fide microglia by molecular, morphological and ultrastructural approaches. Then, we give a detailed spatio-temporal characterization of these capillary-associated microglia (CAMs) comparing them with parenchymal microglia (PCMs) in their morphological activities including during microglial depletion and repopulation. Molecularly, we identify P2RY12 receptors as a regulator of CAM interactions under the control of released purines from pannexin 1 (PANX1) channels. Furthermore, microglial elimination triggered capillary dilation, blood flow increase, and impaired vasodilation that were recapitulated in P2RY12-/- and PANX1-/- mice suggesting purines released through PANX1 channels play important roles in activating microglial P2RY12 receptors to regulate neurovascular structure and function.


Assuntos
Encéfalo/irrigação sanguínea , Conexinas/genética , Microglia/metabolismo , Células Mieloides/metabolismo , Proteínas do Tecido Nervoso/genética , Receptores Purinérgicos P2Y12/genética , Animais , Encéfalo/citologia , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Receptor 1 de Quimiocina CX3C/genética , Receptor 1 de Quimiocina CX3C/metabolismo , Contagem de Células , Circulação Cerebrovascular/fisiologia , Conexinas/deficiência , Eletrodos Implantados , Feminino , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Masculino , Camundongos , Camundongos Knockout , Microglia/citologia , Células Mieloides/citologia , Proteínas do Tecido Nervoso/deficiência , Neuroimagem/instrumentação , Neuroimagem/métodos , Receptores Purinérgicos P2Y12/deficiência , Receptores Purinérgicos P2Y12/metabolismo , Vasodilatação/fisiologia
3.
Nat Biomed Eng ; 5(8): 847-863, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34385693

RESUMO

The therapeutic efficacy of stem cells transplanted into an ischaemic brain depends primarily on the responses of the neurovascular unit. Here, we report the development and applicability of a functional neurovascular unit on a microfluidic chip as a microphysiological model of ischaemic stroke that recapitulates the function of the blood-brain barrier as well as interactions between therapeutic stem cells and host cells (human brain microvascular endothelial cells, pericytes, astrocytes, microglia and neurons). We used the model to track the infiltration of a number of candidate stem cells and to characterize the expression levels of genes associated with post-stroke pathologies. We observed that each type of stem cell showed unique neurorestorative effects, primarily by supporting endogenous recovery rather than through direct cell replacement, and that the recovery of synaptic activities is correlated with the recovery of the structural and functional integrity of the neurovascular unit rather than with the regeneration of neurons.


Assuntos
AVC Isquêmico/terapia , Dispositivos Lab-On-A-Chip , Transplante de Células-Tronco , Astrócitos/citologia , Astrócitos/metabolismo , Barreira Hematoencefálica/química , Barreira Hematoencefálica/metabolismo , Técnicas de Cocultura , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Humanos , Microglia/citologia , Microglia/metabolismo , Microvasos/citologia , Modelos Biológicos , Neurônios/citologia , Neurônios/metabolismo , Pericitos/citologia , Pericitos/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo
4.
Phytomedicine ; 91: 153692, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34411834

RESUMO

PURPOSE: Magnolol (MA) exhibits anti-depressant effect by inhibiting inflammation. However, its effect on microglia polarization remains not fully understood. Herein, our study was performed to evaluate the effect of MA on microglia polarization in chronic unpredictable mild stress (CUMS)-induced depression and explore its potential mechanism. STUDY DESIGN: The CUMS procedure was conducted, and the mice were intragastrically treated with MA. BV2 cells were pretreated with MA prior to LPS/ATP challenge. METHODS: The levels of TNF-α, IL-1ß, IL-6 and IL-4, IL-10 in brain and BV2 cells were examined by ELISA. The mRNA expressions of Arg1, Ym1, Fizz1 and Klf4 in brains were measured. ROS content was determined using flow cytometry. Immunofluorescence was employed to evaluate Iba-1 level, Nrf2 nuclear translocation, Iba-1+CD16/32+ and Iba-1+CD206+ cell population. The protein expressions of Nrf2, HO-1, NLRP3, caspase-1 p20 and IL-1ß in brains and BV2 cells were investigated by western blot. Nrf2 siRNA was induced in experiments to explore the role of Nrf2 in MA-mediated microglia polarization. The ubiquitination of Nrf2 was visualized by Co-IP. RESULTS: The treatment with MA notably relieved depressive like behaviors, suppressed pro-inflammatory cytokines, promoted anti-inflammatory cytokines and the transcription of M2 phenotype microglia-specific indicators. MA upregulated the expression of Nrf2, HO-1, downregulated the expression of NLRP3, caspase-1 p20, IL-1ß both in vivo and in vitro. MA also reduced ROS concentration, promoted Nrf2 nucleus translocation and prevented Nrf2 ubiquitination. Nrf2 Knockdown by siRNA abolished the MA-mediated microglia polarization. CONCLUSION: The present research demonstrated that MA attenuated CUMS-stimulated depression by inhibiting M1 polarization and inducing M2 polarization via Nrf2/HO-1/NLRP3 signaling.


Assuntos
Compostos de Bifenilo/farmacologia , Depressão/tratamento farmacológico , Lignanas/farmacologia , Microglia , Transdução de Sinais/efeitos dos fármacos , Animais , Polaridade Celular , Heme Oxigenase-1 , Lipopolissacarídeos , Proteínas de Membrana , Camundongos , Microglia/citologia , Microglia/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR
5.
FASEB J ; 35(9): e21332, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34423867

RESUMO

Emerging research has highlighted the capacity of microRNA-23a-3p (miR-23a-3p) to alleviate inflammatory pain. However, the molecular mechanism by which miR-23a-3p attenuates inflammatory pain is yet to be fully understood. Hence, the current study aimed to elucidate the mechanism by which miR-23a-3p influences inflammatory pain. Bioinformatics was initially performed to predict the inflammatory pain related downstream targets of miR-23a-3p in macrophage-derived extracellular vesicles (EVs). An animal inflammatory pain model was established using Complete Freund's Adjuvant (CFA). The miR-23a-3p expression was downregulated in the microglia of CFA-induced mice, after which the inflammatory factors were determined by ELISA. FISH and immunofluorescence were performed to analyze the co-localization of miR-23a-3p and microglia. Interestingly, miR-23a-3p was transported to the microglia via M2 macrophage-EVs, which elevated the mechanical allodynia and the thermal hyperalgesia thresholds in mice model. The miR-23a-3p downstream target, USP5, was found to stabilize HDAC2 via deubiquitination to promote its expression while inhibiting the expression of NRF2. Taken together, the key findings of the current study demonstrate that macrophage-derived EVs containing miR-23a-3p regulates the HDAC2/NRF2 axis by decreasing USP5 expression to alleviate inflammatory pain, which may provide novel therapeutic targets for the treatment of inflammatory pain.


Assuntos
Vesículas Extracelulares/metabolismo , Histona Desacetilase 2/metabolismo , Inflamação/metabolismo , Macrófagos/citologia , Fator 2 Relacionado a NF-E2/metabolismo , Dor/metabolismo , Proteases Específicas de Ubiquitina/metabolismo , Animais , Linhagem Celular , Enzimas Desubiquitinantes/metabolismo , Modelos Animais de Doenças , Regulação para Baixo , Estabilidade Enzimática , Vesículas Extracelulares/genética , Inflamação/genética , Inflamação/terapia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/citologia , Microglia/metabolismo , Modelos Biológicos , Dor/genética , Manejo da Dor , Proteases Específicas de Ubiquitina/genética , Ubiquitinação
6.
Int J Mol Sci ; 22(15)2021 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-34360582

RESUMO

Although considered a rare retinal dystrophy, retinitis pigmentosa (RP) is the primary cause of hereditary blindness. Given its diverse genetic etiology (>3000 mutations in >60 genes), there is an urgent need for novel treatments that target common features of the disease. TLR2 is a key activator of innate immune response. To examine its role in RP progression we characterized the expression profile of Tlr2 and its adaptor molecules and the consequences of Tlr2 deletion in two genetically distinct models of RP: Pde6brd10/rd10 (rd10) and RhoP23H/+ (P23H/+) mice. In both models, expression levels of Tlr2 and its adaptor molecules increased in parallel with those of the proinflammatory cytokine Il1b. In rd10 mice, deletion of a single Tlr2 allele had no effect on visual function, as evaluated by electroretinography. However, in both RP models, complete elimination of Tlr2 attenuated the loss of visual function and mitigated the loss of photoreceptor cell numbers. In Tlr2 null rd10 mice, we observed decreases in the total number of microglial cells, assessed by flow cytometry, and in the number of microglia infiltrating the photoreceptor layers. Together, these results point to TLR2 as a mutation-independent therapeutic target for RP.


Assuntos
Modelos Animais de Doenças , Deleção de Genes , Microglia/metabolismo , Fármacos Neuroprotetores , Degeneração Retiniana/prevenção & controle , Retinite Pigmentosa/complicações , Receptor 2 Toll-Like/fisiologia , Animais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/citologia , Degeneração Retiniana/etiologia , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia
7.
Int J Mol Sci ; 22(16)2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34445510

RESUMO

Microglia are resident immune cells of the central nervous system that act as brain-specific macrophages and are also known to regulate the innate immune functions of astrocytes through secretory molecules. This communication plays an important role in brain functions and homeostasis as well as in neuropathologic disease. In this study, we aimed to elucidate whether astrocytes and microglia could crosstalk to induce microglial polarization and proliferation, which can be further regulated under a microenvironment mimicking that of brain stroke. Microglia in a mixed glial culture showed increased survival and proliferation and were altered to M2 microglia; CD11b-GFAP+ astrocytes resulted in an approximately tenfold increase in microglial cell proliferation after the reconstitution of astrocytes. Furthermore, GM-CSF stimulated microglial proliferation approximately tenfold and induced them to become CCR7+ M1 microglia, which have a phenotype that could be suppressed by anti-inflammatory cytokines such as IL-4, IL-10, and substance P. In addition, the astrocytes in the microglial co-culture showed an A2 phenotype; they could be activated to A1 astrocytes by TNF-α and IFN-γ under the stroke-mimicking condition. Altogether, astrocytes in the mixed glial culture stimulated the proliferation of the microglia and M2 polarization, possibly through the acquisition of the A2 phenotype; both could be converted to M1 microglia and A1 astrocytes under the inflammatory stroke-mimicking environment. This study demonstrated that microglia and astrocytes could be polarized to M2 microglia and A2 astrocytes, respectively, through crosstalk in vitro and provides a system with which to explore how microglia and astrocytes may behave in the inflammatory disease milieu after in vivo transplantation.


Assuntos
Astrócitos/citologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Macrófagos/citologia , Microglia/citologia , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/imunologia , Comunicação Celular , Polaridade Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Interleucina-10/metabolismo , Interleucina-4/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Microglia/efeitos dos fármacos , Microglia/imunologia , Ratos
8.
J Enzyme Inhib Med Chem ; 36(1): 1783-1797, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34340630

RESUMO

Carbonic Anhydrase Activators (CAAs) could represent a novel approach for the treatment of Alzheimer's disease, ageing, and other conditions that require remedial achievement of spatial learning and memory therapy. Within a research project aimed at developing novel CAAs selective for certain isoforms, three series of indole-based derivatives were investigated. Enzyme activation assay on human CA I, II, VA, and VII isoforms revealed several effective micromolar activators, with promising selectivity profiles towards the brain-associated cytosolic isoform hCA VII. Molecular modelling studies suggested a theoretical model of the complex between hCA VII and the new activators and provide a possible explanation for their modulating as well as selectivity properties. Preliminary biological evaluations demonstrated that one of the most potent CAA 7 is not cytotoxic and is able to increase the release of the brain-derived neurotrophic factor (BDNF) from human microglial cells, highlighting its possible application in the treatment of CNS-related disorders.


Assuntos
Anidrases Carbônicas/efeitos dos fármacos , Ativadores de Enzimas/farmacologia , Indóis/farmacologia , Isoenzimas/efeitos dos fármacos , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Anidrases Carbônicas/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Ativação Enzimática , Ativadores de Enzimas/química , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Indóis/química , Isoenzimas/metabolismo , Microglia/citologia , Microglia/efeitos dos fármacos , Modelos Moleculares , Espectroscopia de Prótons por Ressonância Magnética , Especificidade por Substrato
9.
Biomolecules ; 11(7)2021 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-34356682

RESUMO

Oligodendrocytes, the myelin-making cells of the CNS, regulate the complex process of myelination under physiological and pathological conditions, significantly aided by other glial cell types such as microglia, the brain-resident, macrophage-like innate immune cells. In this review, we summarize how oligodendrocytes orchestrate myelination, and especially myelin repair after damage, and present novel aspects of oligodendroglial functions. We emphasize the contribution of microglia in the generation and regeneration of myelin by discussing their beneficial and detrimental roles, especially in remyelination, underlining the cellular and molecular components involved. Finally, we present recent findings towards human stem cell-derived preclinical models for the study of microglia in human pathologies and on the role of microbiome on glial cell functions.


Assuntos
Doenças Desmielinizantes/patologia , Microglia/fisiologia , Bainha de Mielina/fisiologia , Oligodendroglia/fisiologia , Animais , Humanos , Microbiota , Microglia/citologia , Microglia/patologia , Bainha de Mielina/patologia , Regeneração , Remielinização/fisiologia , Células-Tronco/patologia
10.
Nat Commun ; 12(1): 4826, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34376696

RESUMO

Loss-of-function mutations in NEK1 gene, which encodes a serine/threonine kinase, are involved in human developmental disorders and ALS. Here we show that NEK1 regulates retromer-mediated endosomal trafficking by phosphorylating VPS26B. NEK1 deficiency disrupts endosomal trafficking of plasma membrane proteins and cerebral proteome homeostasis to promote mitochondrial and lysosomal dysfunction and aggregation of α-synuclein. The metabolic and proteomic defects of NEK1 deficiency disrupts the integrity of blood-brain barrier (BBB) by promoting lysosomal degradation of A20, a key modulator of RIPK1, thus sensitizing cerebrovascular endothelial cells to RIPK1-dependent apoptosis and necroptosis. Genetic inactivation of RIPK1 or metabolic rescue with ketogenic diet can prevent postnatal lethality and BBB damage in NEK1 deficient mice. Inhibition of RIPK1 reduces neuroinflammation and aggregation of α-synuclein in the brains of NEK1 deficient mice. Our study identifies a molecular mechanism by which retromer trafficking and metabolism regulates cerebrovascular integrity, cerebral proteome homeostasis and RIPK1-mediated neuroinflammation.


Assuntos
Barreira Hematoencefálica/metabolismo , Glucose/metabolismo , Complexos Multiproteicos/metabolismo , Quinase 1 Relacionada a NIMA/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Animais , Animais Recém-Nascidos , Linhagem Celular , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Ativação Enzimática , Células HEK293 , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/citologia , Microglia/metabolismo , Quinase 1 Relacionada a NIMA/genética , Necroptose/genética , Fosforilação , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo
11.
Nat Methods ; 18(8): 953-958, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34312564

RESUMO

Unbiased quantitative analysis of macroscopic biological samples demands fast imaging systems capable of maintaining high resolution across large volumes. Here we introduce RAPID (rapid autofocusing via pupil-split image phase detection), a real-time autofocus method applicable in every widefield-based microscope. RAPID-enabled light-sheet microscopy reliably reconstructs intact, cleared mouse brains with subcellular resolution, and allowed us to characterize the three-dimensional (3D) spatial clustering of somatostatin-positive neurons in the whole encephalon, including densely labeled areas. Furthermore, it enabled 3D morphological analysis of microglia across the entire brain. Beyond light-sheet microscopy, we demonstrate that RAPID maintains high image quality in various settings, from in vivo fluorescence imaging to 3D tracking of fast-moving organisms. RAPID thus provides a flexible autofocus solution that is suitable for traditional automated microscopy tasks as well as for quantitative analysis of large biological specimens.


Assuntos
Encéfalo/anatomia & histologia , Processamento de Imagem Assistida por Computador/métodos , Imageamento Tridimensional/métodos , Microglia/citologia , Microscopia de Fluorescência/métodos , Animais , Masculino , Camundongos
12.
DNA Cell Biol ; 40(9): 1125-1130, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34297618

RESUMO

In response to neuronal activity changes, the adult hippocampal circuits undergo continuous synaptic remodeling, which is essential for information processing, learning, and memory encoding. Glial cells, including astrocytes and microglia, actively regulate hippocampal synaptic plasticity by coordinating the neuronal activity-induced synaptic changes at the circuit level. Emerging evidence suggests that the crosstalk between neurons and glia in the adult hippocampus is region specific and that the mechanisms controlling this process are critically dependent on secreted factors. Interleukin-33 (IL-33), a cytokine of the IL-1 family, is a key factor that modulates such glia-driven neuromodulations in two distinct hippocampal circuits. The activation of IL-33 and its receptor complex is important for maintaining the excitatory synaptic activity in the cornu ammonis 1 subregion and the remodeling of dentate gyrus synapses through activity-dependent astrocyte-synapse and microglia-synapse interactions, respectively. Meanwhile, the dysregulation of this signaling is implicated in multiple neurological disorders, especially Alzheimer's disease. Further investigations of how IL-33/ST2 signaling is regulated in a region-specific manner as well as its diverse functions in glia-synapse communications in the adult hippocampal circuitry will provide insights into the nature of hippocampal synaptic plasticity and homeostasis in health and disease.


Assuntos
Doença de Alzheimer/metabolismo , Astrócitos/metabolismo , Interleucina-33/fisiologia , Microglia/metabolismo , Plasticidade Neuronal , Adulto , Animais , Astrócitos/citologia , Astrócitos/patologia , Hipocampo/metabolismo , Humanos , Camundongos , Microglia/citologia , Microglia/patologia , Sinapses/metabolismo
13.
Cell Prolif ; 54(8): e13094, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34312932

RESUMO

OBJECTIVES: Parkinson's disease (PD) is a common neurodegenerative disorder characterized by the progressive and selective degeneration of dopaminergic neurons. Microglial activation and neuroinflammation are associated with the pathogenesis of PD. However, the relationship between microglial activation and PD pathology remains to be explored. MATERIALS AND METHODS: An acute regimen of MPTP was administered to adult C57BL/6J mice with normal, much reduced or repopulated microglial population. Damages of the dopaminergic system were comprehensively assessed. Inflammation-related factors were assessed by quantitative PCR and Multiplex immunoassay. Behavioural tests were carried out to evaluate the motor deficits in MPTP-challenged mice. RESULTS: The receptor for colony-stimulating factor 1 inhibitor PLX3397 could effectively deplete microglia in the nigrostriatal pathway of mice via feeding a PLX3397-formulated diet for 21 days. Microglial depletion downregulated both pro-inflammatory and anti-inflammatory molecule expression at baseline and after MPTP administration. At 1d post-MPTP injection, dopaminergic neurons showed a significant reduction in PLX3397-fed mice, but not in control diet (CD)-fed mice. However, partial microglial depletion in mice exerted little effect on MPTP-induced dopaminergic injuries compared with CD mice at later time points. Interestingly, microglial repopulation brought about apparent resistance to MPTP intoxication. CONCLUSIONS: Microglia can inhibit PD development at a very early stage; partial microglial depletion has little effect in terms of the whole process of the disease; and microglial replenishment elicits neuroprotection in PD mice.


Assuntos
Intoxicação por MPTP/patologia , Microglia/metabolismo , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/administração & dosagem , Aminopiridinas/farmacologia , Animais , Comportamento Animal/efeitos dos fármacos , Ciclo-Oxigenase 2/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Mediadores da Inflamação/metabolismo , Intoxicação por MPTP/metabolismo , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microglia/citologia , Microglia/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Pirróis/farmacologia , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo
14.
Cell Prolif ; 54(8): e13093, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34231932

RESUMO

OBJECTIVES: The study aimed to determine whether dental pulp stem cell-derived exosomes (DPSC-Exos) exert protective effects against cerebral ischaemia-reperfusion (I/R) injury and explore its underlying mechanism. MATERIALS AND METHODS: Exosomes were isolated from the culture medium of human DPSC. Adult male C57BL/6 mice were subjected to 2 hours transient middle cerebral artery occlusion (tMCAO) injury followed by 2 hours reperfusion, after which singular injection of DPSC-Exos via tail vein was administrated. Brain oedema, cerebral infarction and neurological impairment were measured on day 7 after exosomes injection. Then, oxygen-glucose deprivation-reperfusion (OGD/R) induced BV2 cells were studied to analyse the therapeutic effects of DPSC-Exos on I/R injury in vitro. Protein levels of TLR4, MyD88, NF-κB p65, HMGB1, IL-6, IL-1ß and TNF-α were determined by western blot or enzyme-linked immunosorbent assay. The cytoplasmic translocation of HMGB1 was detected by immunofluorescence staining. RESULTS: DPSC-Exos alleviated brain oedema, cerebral infarction and neurological impairment in I/R mice. DPSC-Exos inhibited the I/R-mediated expression of TLR4, MyD88 and NF-κB significantly. DPSC-Exos also reduced the protein expression of IL-6, IL-1ß and TNF-α compared with those of the control both in vitro and in vivo. Meanwhile, DPSC-Exos markedly decreased the HMGB1 cytoplasmic translocation induced by I/R damage. CONCLUSIONS: DPSC-Exos can ameliorate I/R-induced cerebral injury in mice. Its anti-inflammatory mechanism might be related with the inhibition of the HMGB1/TLR4/MyD88/NF-κB pathway.


Assuntos
Citocinas/metabolismo , Exossomos/transplante , Traumatismo por Reperfusão/terapia , Animais , Sobrevivência Celular , Citoplasma/metabolismo , Polpa Dentária/citologia , Polpa Dentária/metabolismo , Modelos Animais de Doenças , Exossomos/metabolismo , Proteína HMGB1/metabolismo , Inflamação/metabolismo , Inflamação/terapia , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/citologia , Microglia/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , Traumatismo por Reperfusão/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Receptor 4 Toll-Like/metabolismo , Fator de Transcrição RelA/metabolismo
15.
Cell Physiol Biochem ; 55(S1): 171-184, 2021 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-34156175

RESUMO

BACKGROUND/AIMS: Trypan blue is routinely used in cell culture experiments to distinguish between dead cells, which are labelled by trypan blue, and viable cells, which are apparently free of any staining. The assumption that trypan blue labelling is restricted to dead cells derives from the observation that rupture of the plasma membrane correlates with intense trypan blue staining. However, decades ago, trypan blue has been used to trace fluid uptake by viable macrophage-like cells in animals. These studies contributed to the concept of the reticuloendothelial system in vertebrates. Trypan blue itself does not show a fluorescence signal, but trypan blue-labelled proteins do. Therefore, intracellular localization of trypan blue-labelled proteins could give a clue to the entrance pathway of the dye in viable cells. METHODS: We used fluorescence microscopy to visualize trypan blue positive structures and to evaluate whether the bactericide, silver, enhances cellular trypan blue uptake in the brain macrophage-like cell line, BV-2. The pattern of chromatin condensation, visualized by DAPI staining, was used to identify the cell death pathway. RESULTS: We observed that silver nitrate at elevated concentrations (≥ 10 µM) induced in most cells a necrotic cell death pathway. Necrotic cells, identified by pycnotic nuclei, showed an intense and homogenous trypan blue staining. Apoptotic cells, characterized by crescent-like nuclear chromatin condensations, were not labelled by trypan blue. At lower silver nitrate concentrations, most cells were viable, but they showed trypan blue labelling. Viable cells showed a cell-type specific distribution of heterochromatin and revealed a perinuclear accumulation of bright trypan blue-labelled vesicles and, occasionally, a faint homogenous trypan blue labelling of the cytoplasm and nucleus. Amiloride, which prevents macropinocytosis by blocking the Na+ / H+ exchange, suppressed perinuclear accumulation of dye-labelled vesicles. Swelling of cells in a hypotonic solution induced an intense intracellular accumulation of trypan blue. Cells exposed to a hypotonic solution in the presence of 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), which blocks volume-regulated ion channels, prevented labelling of the cytoplasm and nucleus but did not affect labelling of perinuclear vesicles. CONCLUSION: In viable cells trypan blue-labelled vesicles indicate trypan blue uptake by macropinocytosis and trypan blue-labelled cytosol could indicate a further entry pathway for the dye, like activated volume-regulated channels. Accordingly, fluorescence microscopic analysis of trypan blue-labelled cells allows not only a discrimination between necrotic and apoptotic cell death pathway but also a discrimination between the mode of trypan blue uptake in viable cells - via pinocytosis or via activated volume-regulated ion channels - in the same preparation at the single cell level.


Assuntos
Corantes/análise , Microglia/citologia , Pinocitose , Azul Tripano/análise , Animais , Morte Celular , Linhagem Celular , Sobrevivência Celular , Camundongos , Microscopia de Fluorescência/métodos , Coloração e Rotulagem/métodos
16.
Arch Biochem Biophys ; 706: 108918, 2021 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-33992596

RESUMO

Tripartite motif-containing 21 (TRIM21) has been confirmed to mediate the production of inflammatory mediators via NF-κB signaling. However, the function of TRIM21 in microglia-mediated neuroinflammation remains unclear. This study aimed to explore the effect of TRIM21 on LPS-activated BV2 microglia and its underlying mechanism. BV2 cells exposed to lipopolysaccharide (LPS) were used to simulated neuroinflammation in vitro. Loss-of-function and gain-of-function of TRIM21 in BV2 cells were used to assess the effect of TRIM21 on LPS-induced neuroinflammation. BV2 microglia and HT22 cells co-culture system were used to investigate whether TRIM21 regulated neuronal inflammation-mediated neuronal death. TRIM21 knockdown triggered the polarization of BV2 cells from M1 to M2 phenotype. Knockdown of TRIM21 reduced the secretion of TNF-α, IL-6, and IL-1ß, while increased the content of IL-4 in LPS-treated cells. Knockdown of TRIM21 inhibited the expression of p65 and the binding activity of NF-κB-DNA. Additionally, TRIM21 siRNA eliminated the increase in NLRP3 and cleaved caspase-1 proteins expression and caspase-1 activity induced by LPS. TRIM21 knockdown could resist cytotoxicity induced by activated microglia, including increasing the viability of co-cultured HT22 cells and reducing the emancipation of LDH. Moreover, the increased apoptosis and caspase-3 activity of HT22 neurons induced by activated BV2 cells were blocked by TRIM21 siRNA. Blocking of NF-κB abolished the effect of TRIM21 in promoting the expression of M1 phenotype marker genes. Similarly, the blockade of NF-κB pathway eliminated the promotion of TRIM21 on neurotoxicity induced by neuroinflammation. TRIM21 knockdown suppressed the M1 phenotype polarization of microglia and neuroinflammation-mediated neuronal damage via NF-κB/NLRP3 inflammasome pathway, which suggested that TRIM21 might be a potential therapeutic target for the therapy of central nervous system diseases.


Assuntos
Inflamassomos/efeitos dos fármacos , Lipopolissacarídeos/toxicidade , Microglia/efeitos dos fármacos , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Neurônios/efeitos dos fármacos , Ribonucleoproteínas/genética , Fator de Transcrição RelA/genética , Animais , Caspase 1/genética , Caspase 1/metabolismo , Diferenciação Celular , Linhagem Celular , Técnicas de Cocultura , Cultura em Câmaras de Difusão , Regulação da Expressão Gênica , Hipocampo/citologia , Hipocampo/metabolismo , Inflamassomos/genética , Inflamassomos/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-4/genética , Interleucina-4/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Camundongos , Microglia/citologia , Microglia/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Neurônios/citologia , Neurônios/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ribonucleoproteínas/antagonistas & inibidores , Ribonucleoproteínas/metabolismo , Transdução de Sinais , Fator de Transcrição RelA/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
17.
Aging (Albany NY) ; 13(9): 13108-13123, 2021 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-33971624

RESUMO

Evidence indicates that neutrophil has promoted inflammation in several central nervous system diseases. However, whether the peripheral blood levels of neutrophils are associated with the functional outcome after subarachnoid hemorrhage and its potential mechanism remain unclear. In this study, we showed that neutrophil levels in peripheral blood were higher in patients with subarachnoid hemorrhage (P < 0.001) than in healthy subjects. Neutrophil levels were positively associated with Hunt and Hess grade (P < 0.001) and modified Rankin Scale scores at 3 months after SAH (P = 0.008). In terms of the mechanism, neutrophil extracellular traps markedly increased the proinflammatory subtype transition of microglia. After treatment with DNAse I, the proinflammatory subtype transition of microglia involving CD16 positive and IL-1ß positive microglia was limited (P < 0.05). This mechanism was also verified in vitro. These results indicate that the existence of neutrophil extracellular traps, released from neutrophils after subarachnoid hemorrhage, can shift microglia toward a more proinflammatory phenotype and contribute to neuroinflammation and poor outcome in subarachnoid hemorrhage.


Assuntos
Inflamação/metabolismo , Microglia/citologia , Neutrófilos/citologia , Hemorragia Subaracnóidea/terapia , Adulto , Idoso , Idoso de 80 Anos ou mais , Armadilhas Extracelulares , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo
18.
J Med Chem ; 64(11): 7760-7777, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34019417

RESUMO

N-Phenylpropenoyl-l-amino acids (NPAs) are inducible nitric oxide synthase (iNOS) inhibitors possessing preventive effects for Parkinson's disease (PD). Here, structural modifications for improving the iNOS inhibitory activity and blood-brain barrier (BBB) permeability of NPAs were conducted, leading to 20 optimized NPA derivatives (1-20). Compound 18, with the most potent activity (IC50 = 74 nM), high BBB permeability (Pe = 19.1 × 10-6 cm/s), and high selectivity over other NOS isoforms, was selected as the lead compound. Further studies demonstrated that 18 directly binds to iNOS. In the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced acute PD model, the oral administration of 18 (1 and 2 mg/kg) exerted preventive effects by alleviating the loss of dopaminergic (DAergic) neurons. Notably, in the MPTP-/probenecid-induced chronic PD model, the same dose of 18 also displayed a therapeutic effect by repairing the damaged DAergic neurons. Finally, good pharmacokinetic properties and low toxicity made 18 a promising candidate for the treatment of PD.


Assuntos
Aminoácidos/química , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Propanóis/química , Aminoácidos/metabolismo , Aminoácidos/farmacologia , Animais , Sítios de Ligação , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Modelos Animais de Doenças , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/metabolismo , Desenho de Fármacos , Meia-Vida , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Intoxicação por MPTP/tratamento farmacológico , Intoxicação por MPTP/patologia , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Microglia/citologia , Microglia/efeitos dos fármacos , Microglia/metabolismo , Simulação de Acoplamento Molecular , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Relação Estrutura-Atividade
19.
Exp Eye Res ; 207: 108584, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33910034

RESUMO

Microglia are the resident immune cells in the retina. To investigate their properties and behaviour, a reliable and yielding procedure to culture them is necessary. We here describe a way of isolation of microglial cells from the porcine retina, as pig eyes are similar to human eyes in size, structure and vasculature, including similarities in proteins and pathways. Retina was isolated from fresh pig eyes, dissociated by a mixture of collagenase, hyaluronidase and DNAse, and passed through a cell strainer. After triple centrifugation with decreasing velocity and re-suspension, cells were seeded into poly-d-lysine coated culture flasks and cultured using DMEM and macrophage-colony stimulating factor (M-CSF). Number of cells increased gradually during the first 10-14 days, till they could be split and used for experiments. Identity of isolated cells as microglia was assessed by immunostaining against the microglia/macrophage markers Iba1, CD11b, CD68, CD45 and TMEM119. Phagocytic function of microglia could be demonstrated by phagocytosis of fluorescence beads and their response to lipopolysaccharide (LPS). As a conclusion, we developed a protocol for isolation and cultivation of pig retinal microglial cells that are suitable for research in the laboratory.


Assuntos
Separação Celular/métodos , Microglia/citologia , Retina/citologia , Animais , Biomarcadores/metabolismo , Contagem de Células , Técnicas de Cultura de Células , Imuno-Histoquímica , Microglia/metabolismo , Fagocitose/fisiologia , Retina/metabolismo , Sus scrofa
20.
Dev Cell ; 56(9): 1326-1345.e6, 2021 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-33887203

RESUMO

The interplay between hypothalamic neurons and microglia as they integrate stressors to regulate homeostasis is of growing interest. We asked if microglia in the embryonic hypothalamus were likewise stress responsive and, if so, whether their precocious activation perturbs nearby neural stem cell (NSC) programs. We performed single-cell transcriptomics to define embryonic hypothalamic microglia heterogeneity and identified four microglial subsets, including a subpopulation adjacent to NSCs that was responsive to gestational cold stress. Stress exposure elevated CCL3 and CCL4 secretion, but only in male brains, and ex vivo CCL4 treatment of hypothalamic NSCs altered proliferation and differentiation. Concomitantly, gestational stress decreased PVN oxytocin neurons only in male embryos, which was reversed by microglia depletion. Adult offspring exposed to gestational stress displayed altered social behaviors, which was likewise microglia dependent, but only in males. Collectively, immature hypothalamic microglia play an unappreciated role in translating maternal stressors to sexually dimorphic perturbation of neurodevelopmental programs.


Assuntos
Embrião de Mamíferos/citologia , Microglia/citologia , Células-Tronco Neurais/citologia , Estresse Fisiológico , Animais , Comportamento Animal , Contagem de Células , Diferenciação Celular/genética , Proliferação de Células/genética , Temperatura Baixa , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Hipotálamo/citologia , Masculino , Camundongos , Microglia/metabolismo , Células-Tronco Neurais/metabolismo , Neurônios/citologia , Oligodendroglia/citologia , Núcleo Hipotalâmico Paraventricular/citologia , Gravidez , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Caracteres Sexuais , Análise de Célula Única , Comportamento Social , Esferoides Celulares/citologia
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