Mecanismos neuroinflamatórios in vitro do alumínio em células microgliais: envolvimento da via de sinalização purinérgica
Autor(a) principal: | |
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Data de Publicação: | 2021 |
Tipo de documento: | Tese |
Idioma: | por |
Título da fonte: | Repositório Institucional Manancial UFSM |
Texto Completo: | http://repositorio.ufsm.br/handle/1/24435 |
Resumo: | Aluminum is a toxic metal to the nervous tissue and an etiological factor of neurodegenerative diseases, particularly Alzheimer's disease (AD). Furthermore, microglial cells and cellular signaling pathways, such as the purinergic system, are involved in neuroinflammation and neurodegeneration. However, aluminum effects and the participation of the purinergic system in the responses induced by this metal in microglial cells are poorly understood. Thus, the mechanisms associated with the neuroinflammatory profile of aluminum and the involvement of purinergic system components were investigated. The work was carried out, in vitro, with protocols exposing microglial cells to aluminum, in the form of aluminum chloride (AlCl3). The BV-2 cell line, commercially acquired, was grown under standard conditions. The AlCl3 concentration curve (1, 5, 10, 50, 100, 500 and 1000 μM) was used at different exposure times (6, 24 and 96 hours). Lipopolysaccharide (LPS, 1 μg/mL) was used as a pro-inflammatory control. The control group was not exposed to treatments. The uptake of aluminum, activities of the purinergic enzymes nucleoside triphosphate diphosphohydrolase (NTPDase) (substrates adenosine triphosphate (ATP) and adenosine diphosphate (ADP)), 5’-nucleotidase (5'-NT) (substrate adenosine monophosphate (AMP)) and adenosine deaminase (ADA) (substrate adenosine) and expression/protein levels of purinoreceptors (P2X7, A1 and A2A) were evaluated. Besides, cell viability/proliferation, propidium iodide (PI) uptake, oxidative stress parameters (reactive species, superoxide anion, nitric oxide), apoptosis, cell cycle regulation, genotoxicity, expression (NLRP3 inflammasome, interleukins IL-β, IL-6 and IL-10, interferon gamma (IFN-γ), and tumor necrosis factor alpha (TNF-α)) and protein density (NLRP3, nuclear factor kappa B (NF-ƙB), IL-β, and TNF-α) of inflammatory mediators were investigated. Initially, the presence of aluminum in microglial cells was verified. LPS reduced nucleotide hydrolysis (ATP, ADP and AMP), an effect also observed for AlCl3; AlCl3 500 and 1000 μM reduced the hydrolysis of ATP and AMP and AlCl3 1000 μM decreased the hydrolysis of ADP. For ADA, LPS and AlCl3 (500 and 1000 μM) increased adenosine hydrolysis. For purinoreceptors, LPS and AlCl3 1000 μM increased protein levels/expression of P2X7 and A2A, and decreased A1 expression/density. Moreover, AlCl3 decreased cell viability and proliferation, results supported by the increased incorporation of intracellular PI. For oxidative stress, AlCl3 increased levels of nitric oxide, superoxide anion (500 and 1000 μM) and reactive species (100, 500 and 1000 μM), also showing genotoxic potential, increasing DNA damage (500 and 1000 μM, Alkaline Comet assay) and double-stranded DNA (dsDNA) levels (1000 μM). Furthermore, AlCl3 increased the number of cells in the early stages of apoptosis (100, 500 and 1000 μM) and in late apoptosis or already dead (1-1000 μM) and decreased the number of cells in the S and G2/M phases (50-1000 μM) of the cell cycle. For the expression of inflammatory markers, LPS and AlCl3 1000 μM increased mediators such as NLRP3, TNF-α, IFN-γ and IL-1β; on the other hand, for protein density, LPS and AlCl3 1000 μM increased the levels of IL-1β and TNF-α. Thus, the compilation of results suggests that aluminum triggers oxidative-inflammatory responses in microglial cells, possibly involving components of the purinergic signaling pathway. |
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2022-05-24T13:20:05Z2022-05-24T13:20:05Z2021-11-09http://repositorio.ufsm.br/handle/1/24435Aluminum is a toxic metal to the nervous tissue and an etiological factor of neurodegenerative diseases, particularly Alzheimer's disease (AD). Furthermore, microglial cells and cellular signaling pathways, such as the purinergic system, are involved in neuroinflammation and neurodegeneration. However, aluminum effects and the participation of the purinergic system in the responses induced by this metal in microglial cells are poorly understood. Thus, the mechanisms associated with the neuroinflammatory profile of aluminum and the involvement of purinergic system components were investigated. The work was carried out, in vitro, with protocols exposing microglial cells to aluminum, in the form of aluminum chloride (AlCl3). The BV-2 cell line, commercially acquired, was grown under standard conditions. The AlCl3 concentration curve (1, 5, 10, 50, 100, 500 and 1000 μM) was used at different exposure times (6, 24 and 96 hours). Lipopolysaccharide (LPS, 1 μg/mL) was used as a pro-inflammatory control. The control group was not exposed to treatments. The uptake of aluminum, activities of the purinergic enzymes nucleoside triphosphate diphosphohydrolase (NTPDase) (substrates adenosine triphosphate (ATP) and adenosine diphosphate (ADP)), 5’-nucleotidase (5'-NT) (substrate adenosine monophosphate (AMP)) and adenosine deaminase (ADA) (substrate adenosine) and expression/protein levels of purinoreceptors (P2X7, A1 and A2A) were evaluated. Besides, cell viability/proliferation, propidium iodide (PI) uptake, oxidative stress parameters (reactive species, superoxide anion, nitric oxide), apoptosis, cell cycle regulation, genotoxicity, expression (NLRP3 inflammasome, interleukins IL-β, IL-6 and IL-10, interferon gamma (IFN-γ), and tumor necrosis factor alpha (TNF-α)) and protein density (NLRP3, nuclear factor kappa B (NF-ƙB), IL-β, and TNF-α) of inflammatory mediators were investigated. Initially, the presence of aluminum in microglial cells was verified. LPS reduced nucleotide hydrolysis (ATP, ADP and AMP), an effect also observed for AlCl3; AlCl3 500 and 1000 μM reduced the hydrolysis of ATP and AMP and AlCl3 1000 μM decreased the hydrolysis of ADP. For ADA, LPS and AlCl3 (500 and 1000 μM) increased adenosine hydrolysis. For purinoreceptors, LPS and AlCl3 1000 μM increased protein levels/expression of P2X7 and A2A, and decreased A1 expression/density. Moreover, AlCl3 decreased cell viability and proliferation, results supported by the increased incorporation of intracellular PI. For oxidative stress, AlCl3 increased levels of nitric oxide, superoxide anion (500 and 1000 μM) and reactive species (100, 500 and 1000 μM), also showing genotoxic potential, increasing DNA damage (500 and 1000 μM, Alkaline Comet assay) and double-stranded DNA (dsDNA) levels (1000 μM). Furthermore, AlCl3 increased the number of cells in the early stages of apoptosis (100, 500 and 1000 μM) and in late apoptosis or already dead (1-1000 μM) and decreased the number of cells in the S and G2/M phases (50-1000 μM) of the cell cycle. For the expression of inflammatory markers, LPS and AlCl3 1000 μM increased mediators such as NLRP3, TNF-α, IFN-γ and IL-1β; on the other hand, for protein density, LPS and AlCl3 1000 μM increased the levels of IL-1β and TNF-α. Thus, the compilation of results suggests that aluminum triggers oxidative-inflammatory responses in microglial cells, possibly involving components of the purinergic signaling pathway.O alumínio é um metal tóxico ao tecido nervoso e um fator etiológico de doenças neurodegenerativas, particularmente da doença de Alzheimer (DA). Ademais, células microgliais e vias de sinalização celular, como o sistema purinérgico, estão envolvidas na neuroinflamação e neurodegeneração. Todavia, o efeito do alumínio e participação do sistema purinérgico nas respostas induzidas por esse metal em células microgliais são pouco esclarecidos. Assim, investigaram-se mecanismos associados ao perfil neuroinflamatório do alumínio e envolvimento de componentes do sistema purinérgico. Os trabalhos foram realizados, in vitro, com protocolos de exposição de células microgliais ao alumínio, na forma de cloreto de alumínio (AlCl3). A linhagem BV-2, adquirida comercialmente, foi cultivada em condições-padrão. A curva de concentração de AlCl3 (1, 5, 10, 50, 100, 500 e 1000 μM) foi utilizada em diferentes tempos (6, 24 e 96 horas). Usou-se o lipopolissacarídeo (LPS, 1 μg/mL) como controle pró-inflamatório. O grupo controle não foi exposto aos tratamentos. Avaliaram-se a incorporação de alumínio, atividades das enzimas purinérgicas nucleotídeo trifosfato difosfohidrolase (NTPDase) (substratos adenosina trifosfato (ATP) e adenosina difosfato (ADP)), 5’-nucleotidase (5’-NT) (substrato adenosina monofosfato (AMP)) e adenosina desaminase (ADA) (substrato adenosina) e expressão/níveis proteicos de purinoreceptores (P2X7, A1 e A2A). Outrossim, investigaram-se viabilidade/proliferação celular, incorporação de iodeto de propídio (PI), parâmetros de estresse oxidativo (espécies reativas, ânion superóxido, óxido nítrico), apoptose, regulação do ciclo celular, genotoxicidade, expressão (inflamassoma NLRP3, interleucinas IL-β, IL-6 e IL-10, interferon gama (IFN-γ) e fator de necrose tumoral alfa (TNF-α)) e densidade proteica (NLRP3, fator nuclear kappa B (NF-ƙB), IL-β e TNF-α) de mediadores inflamatórios. Verificou-se, inicialmente, a presença de alumínio em células microgliais. O LPS reduziu a hidrólise de nucleotídeos (ATP, ADP e AMP), efeito observado também para o AlCl3; AlCl3 500 e 1000 μM reduziram a hidrólise de ATP e AMP e AlCl3 1000 μM diminuiu a hidrólise de ADP. Para a ADA, LPS e AlCl3 (500 e 1000 μM) aumentaram a hidrólise da adenosina. Para os purinoreceptores, LPS e AlCl3 1000 μM aumentaram níveis proteicos/expressão de P2X7 e A2A, e diminuíram a expressão/densidade de A1. Além disso, AlCl3 diminuiu a viabilidade e proliferação celulares, resultados corroborados pelo aumento da incorporação de PI intracelular. Para o estresse oxidativo, AlCl3 aumentou níveis de óxido nítrico, ânion superóxido (500 e 1000 μM) e espécies reativas (100, 500 e 1000 μM), além de demonstrar potencial genotóxico, aumentando dano de DNA (500 e 1000 μM, ensaio Cometa Alcalino) e níveis de DNA fita dupla (dsDNA) (1000 μM). Ademais, AlCl3 aumentou o número de células nos estágios iniciais de apoptose (100, 500 e 1000 μM) e apoptose tardia ou mortas (1-1000 μM) e diminuiu o número de células nas fases S e G2/M (50-1000 μM) do ciclo celular. Para a expressão de marcadores inflamatórios, LPS e AlCl3 1000 μM aumentaram mediadores como NLRP3, TNF-α, IFN-γ e IL-1β; já para a densidade proteica, LPS e AlCl3 1000 μM aumentaram os níveis de IL-1β e TNF-α. Destarte, a compilação de resultados sugere que o alumínio desencadeia respostas oxidativo-inflamatórias em células microgliais, possivelmente envolvendo componentes da sinalização purinérgica.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPESConselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPqFundação de Amparo à Pesquisa do Estado do Rio Grande do Sul - FAPERGSporUniversidade Federal de Santa MariaCentro de Ciências Naturais e ExatasPrograma de Pós-Graduação em Ciências Biológicas: Bioquímica ToxicológicaUFSMBrasilBioquímicaAttribution-NonCommercial-NoDerivatives 4.0 Internationalhttp://creativecommons.org/licenses/by-nc-nd/4.0/info:eu-repo/semantics/openAccessDoença de AlzheimerAl3+NeuroinflamaçãoSistema purinérgicoCitocinasAlzheimer's diseaseNeuroinflammationPurinergic systemCytokinesCNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICAMecanismos neuroinflamatórios in vitro do alumínio em células microgliais: envolvimento da via de sinalização purinérgicaNeuroinflammatory mechanisms in vitro of aluminum in microglial cells: involvement of the purinergic signaling pathwayinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/doctoralThesisMorsch, Vera Maria Melchiorshttp://lattes.cnpq.br/1519648219507868Cruz, Ivana Beatrice Mânica DaBattastini, Ana Maria OliveiraLeal, Daniela Bitencourt RosaSantos, Gabriela Trevisan dosSpanevello, Roselia Mariahttp://lattes.cnpq.br/5754493402648045Assmann, Charles Elias200800000002600600600600600600600600b9bc6912-392b-4d80-ac50-8a56c12e4902387cdf6a-2d2e-45e1-9ed0-7fbc82c5dab20c52547c-372f-4eed-89c6-3ef315588e0da57a4b61-d260-4f48-a43d-bd5a8c7151ffc557ce44-0763-4865-8372-1163d96ba82b621871da-e3cb-4a45-85b1-61ddfb6e070803c79a9b-51ab-4e2d-a595-8040766e0bd5reponame:Repositório Institucional Manancial UFSMinstname:Universidade Federal de Santa Maria (UFSM)instacron:UFSMORIGINALTES_PPGCBBT_2021_ASSMANN_CHARLES.pdfTES_PPGCBBT_2021_ASSMANN_CHARLES.pdfTese de Doutoradoapplication/pdf3125714http://repositorio.ufsm.br/bitstream/1/24435/1/TES_PPGCBBT_2021_ASSMANN_CHARLES.pdfc68fd952c558abbd25d9e5caffd49139MD51CC-LICENSElicense_rdflicense_rdfapplication/rdf+xml; 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dc.title.por.fl_str_mv |
Mecanismos neuroinflamatórios in vitro do alumínio em células microgliais: envolvimento da via de sinalização purinérgica |
dc.title.alternative.eng.fl_str_mv |
Neuroinflammatory mechanisms in vitro of aluminum in microglial cells: involvement of the purinergic signaling pathway |
title |
Mecanismos neuroinflamatórios in vitro do alumínio em células microgliais: envolvimento da via de sinalização purinérgica |
spellingShingle |
Mecanismos neuroinflamatórios in vitro do alumínio em células microgliais: envolvimento da via de sinalização purinérgica Assmann, Charles Elias Doença de Alzheimer Al3+ Neuroinflamação Sistema purinérgico Citocinas Alzheimer's disease Neuroinflammation Purinergic system Cytokines CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA |
title_short |
Mecanismos neuroinflamatórios in vitro do alumínio em células microgliais: envolvimento da via de sinalização purinérgica |
title_full |
Mecanismos neuroinflamatórios in vitro do alumínio em células microgliais: envolvimento da via de sinalização purinérgica |
title_fullStr |
Mecanismos neuroinflamatórios in vitro do alumínio em células microgliais: envolvimento da via de sinalização purinérgica |
title_full_unstemmed |
Mecanismos neuroinflamatórios in vitro do alumínio em células microgliais: envolvimento da via de sinalização purinérgica |
title_sort |
Mecanismos neuroinflamatórios in vitro do alumínio em células microgliais: envolvimento da via de sinalização purinérgica |
author |
Assmann, Charles Elias |
author_facet |
Assmann, Charles Elias |
author_role |
author |
dc.contributor.advisor1.fl_str_mv |
Morsch, Vera Maria Melchiors |
dc.contributor.advisor1Lattes.fl_str_mv |
http://lattes.cnpq.br/1519648219507868 |
dc.contributor.advisor-co1.fl_str_mv |
Cruz, Ivana Beatrice Mânica Da |
dc.contributor.referee1.fl_str_mv |
Battastini, Ana Maria Oliveira |
dc.contributor.referee2.fl_str_mv |
Leal, Daniela Bitencourt Rosa |
dc.contributor.referee3.fl_str_mv |
Santos, Gabriela Trevisan dos |
dc.contributor.referee4.fl_str_mv |
Spanevello, Roselia Maria |
dc.contributor.authorLattes.fl_str_mv |
http://lattes.cnpq.br/5754493402648045 |
dc.contributor.author.fl_str_mv |
Assmann, Charles Elias |
contributor_str_mv |
Morsch, Vera Maria Melchiors Cruz, Ivana Beatrice Mânica Da Battastini, Ana Maria Oliveira Leal, Daniela Bitencourt Rosa Santos, Gabriela Trevisan dos Spanevello, Roselia Maria |
dc.subject.por.fl_str_mv |
Doença de Alzheimer Al3+ Neuroinflamação Sistema purinérgico Citocinas |
topic |
Doença de Alzheimer Al3+ Neuroinflamação Sistema purinérgico Citocinas Alzheimer's disease Neuroinflammation Purinergic system Cytokines CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA |
dc.subject.eng.fl_str_mv |
Alzheimer's disease Neuroinflammation Purinergic system Cytokines |
dc.subject.cnpq.fl_str_mv |
CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA |
description |
Aluminum is a toxic metal to the nervous tissue and an etiological factor of neurodegenerative diseases, particularly Alzheimer's disease (AD). Furthermore, microglial cells and cellular signaling pathways, such as the purinergic system, are involved in neuroinflammation and neurodegeneration. However, aluminum effects and the participation of the purinergic system in the responses induced by this metal in microglial cells are poorly understood. Thus, the mechanisms associated with the neuroinflammatory profile of aluminum and the involvement of purinergic system components were investigated. The work was carried out, in vitro, with protocols exposing microglial cells to aluminum, in the form of aluminum chloride (AlCl3). The BV-2 cell line, commercially acquired, was grown under standard conditions. The AlCl3 concentration curve (1, 5, 10, 50, 100, 500 and 1000 μM) was used at different exposure times (6, 24 and 96 hours). Lipopolysaccharide (LPS, 1 μg/mL) was used as a pro-inflammatory control. The control group was not exposed to treatments. The uptake of aluminum, activities of the purinergic enzymes nucleoside triphosphate diphosphohydrolase (NTPDase) (substrates adenosine triphosphate (ATP) and adenosine diphosphate (ADP)), 5’-nucleotidase (5'-NT) (substrate adenosine monophosphate (AMP)) and adenosine deaminase (ADA) (substrate adenosine) and expression/protein levels of purinoreceptors (P2X7, A1 and A2A) were evaluated. Besides, cell viability/proliferation, propidium iodide (PI) uptake, oxidative stress parameters (reactive species, superoxide anion, nitric oxide), apoptosis, cell cycle regulation, genotoxicity, expression (NLRP3 inflammasome, interleukins IL-β, IL-6 and IL-10, interferon gamma (IFN-γ), and tumor necrosis factor alpha (TNF-α)) and protein density (NLRP3, nuclear factor kappa B (NF-ƙB), IL-β, and TNF-α) of inflammatory mediators were investigated. Initially, the presence of aluminum in microglial cells was verified. LPS reduced nucleotide hydrolysis (ATP, ADP and AMP), an effect also observed for AlCl3; AlCl3 500 and 1000 μM reduced the hydrolysis of ATP and AMP and AlCl3 1000 μM decreased the hydrolysis of ADP. For ADA, LPS and AlCl3 (500 and 1000 μM) increased adenosine hydrolysis. For purinoreceptors, LPS and AlCl3 1000 μM increased protein levels/expression of P2X7 and A2A, and decreased A1 expression/density. Moreover, AlCl3 decreased cell viability and proliferation, results supported by the increased incorporation of intracellular PI. For oxidative stress, AlCl3 increased levels of nitric oxide, superoxide anion (500 and 1000 μM) and reactive species (100, 500 and 1000 μM), also showing genotoxic potential, increasing DNA damage (500 and 1000 μM, Alkaline Comet assay) and double-stranded DNA (dsDNA) levels (1000 μM). Furthermore, AlCl3 increased the number of cells in the early stages of apoptosis (100, 500 and 1000 μM) and in late apoptosis or already dead (1-1000 μM) and decreased the number of cells in the S and G2/M phases (50-1000 μM) of the cell cycle. For the expression of inflammatory markers, LPS and AlCl3 1000 μM increased mediators such as NLRP3, TNF-α, IFN-γ and IL-1β; on the other hand, for protein density, LPS and AlCl3 1000 μM increased the levels of IL-1β and TNF-α. Thus, the compilation of results suggests that aluminum triggers oxidative-inflammatory responses in microglial cells, possibly involving components of the purinergic signaling pathway. |
publishDate |
2021 |
dc.date.issued.fl_str_mv |
2021-11-09 |
dc.date.accessioned.fl_str_mv |
2022-05-24T13:20:05Z |
dc.date.available.fl_str_mv |
2022-05-24T13:20:05Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/doctoralThesis |
format |
doctoralThesis |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://repositorio.ufsm.br/handle/1/24435 |
url |
http://repositorio.ufsm.br/handle/1/24435 |
dc.language.iso.fl_str_mv |
por |
language |
por |
dc.relation.cnpq.fl_str_mv |
200800000002 |
dc.relation.confidence.fl_str_mv |
600 600 600 600 600 600 600 600 |
dc.relation.authority.fl_str_mv |
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dc.rights.driver.fl_str_mv |
Attribution-NonCommercial-NoDerivatives 4.0 International http://creativecommons.org/licenses/by-nc-nd/4.0/ info:eu-repo/semantics/openAccess |
rights_invalid_str_mv |
Attribution-NonCommercial-NoDerivatives 4.0 International http://creativecommons.org/licenses/by-nc-nd/4.0/ |
eu_rights_str_mv |
openAccess |
dc.publisher.none.fl_str_mv |
Universidade Federal de Santa Maria Centro de Ciências Naturais e Exatas |
dc.publisher.program.fl_str_mv |
Programa de Pós-Graduação em Ciências Biológicas: Bioquímica Toxicológica |
dc.publisher.initials.fl_str_mv |
UFSM |
dc.publisher.country.fl_str_mv |
Brasil |
dc.publisher.department.fl_str_mv |
Bioquímica |
publisher.none.fl_str_mv |
Universidade Federal de Santa Maria Centro de Ciências Naturais e Exatas |
dc.source.none.fl_str_mv |
reponame:Repositório Institucional Manancial UFSM instname:Universidade Federal de Santa Maria (UFSM) instacron:UFSM |
instname_str |
Universidade Federal de Santa Maria (UFSM) |
instacron_str |
UFSM |
institution |
UFSM |
reponame_str |
Repositório Institucional Manancial UFSM |
collection |
Repositório Institucional Manancial UFSM |
bitstream.url.fl_str_mv |
http://repositorio.ufsm.br/bitstream/1/24435/1/TES_PPGCBBT_2021_ASSMANN_CHARLES.pdf http://repositorio.ufsm.br/bitstream/1/24435/2/license_rdf http://repositorio.ufsm.br/bitstream/1/24435/3/license.txt |
bitstream.checksum.fl_str_mv |
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bitstream.checksumAlgorithm.fl_str_mv |
MD5 MD5 MD5 |
repository.name.fl_str_mv |
Repositório Institucional Manancial UFSM - Universidade Federal de Santa Maria (UFSM) |
repository.mail.fl_str_mv |
ouvidoria@ufsm.br |
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1808854712530763776 |