A autofagia pode ser induzida por nanopartículas em diversas linhagens celulares (EIDI et al., 2012; STERN; ADISESHAIAH; CRIST, 2012), partindo desse princípio resolvemos investigar se a autofagia é desencadeada em macrófagos quando submetidos ao tratamento com as AgNP biossintéticas. Para este estudo utilizamos a Cloroquina (CLQ), agente lisotrópico que evita a acidificação endossomal (SMIT et al., 1987). O acúmulo da CLQ no interior de compartimentos ácidos da célula, incluindo os endossomos tardios e lisossomos, resulta na alteração do pH intraluminal e leva a uma inibição de enzimas lisossomais que necessitam de um pH ácido para o seu funcionamento. Como consequência, a fusão de endossomos e lisossomos fica impossibilitada. Sendo assim, a CLQ funciona como um inibidor do processo autofágico, pois a inibição da acidificação dos lisossomos e o impedimento da sua fusão com o autofagossomo são passos indispensáveis para a degradação de substratos autofágicos (EGGER et al., 2013; POOLE; OHKUMA, 1981; YOON et al., 2010).
Com base nessas informações, avaliamos a citotoxicidade das AgNP na presença de CLQ, na qual as concentrações testadas foram 12,5 μM, 25 μM e 50 μM. Nenhuma das concentrações de CLQ apresentou efeitos citotóxicos significativos em macrófagos peritoneais (fig. 23). Contudo, o tratamento com AgNP em presença de CLQ apresentou um perfil mais citotóxico, sendo significativamente potencializado quando associados à maior concentração de CLQ (*p<0,05 - 50 μM, 15,6% ±7,8%).
Esse resultado indica que as AgNP levam os macrófagos a uma situação de estresse celular, na qual teria estimulado o processo de autofagia. Além disso, o aumento na citotoxicidade observado no pré-tratamento com CLQ sugere que o fluxo autofágico estaria sendo utilizado pelas
51
células para a metabolização e possivelmente a neutralização do efeito citotóxico das AgNP biossintéticas.
Figura 23 – Estudo da autofagia na avaliação da citotoxicidade das AgNP em macrófagos peritoneais. As células
foram tratadas com o inibidor de autofagia Cloroquina e AgNP durante um período de 6h e um grupo foi incubado a 37 °C. A viabilidade celular foi avaliada pelo ensaio de MTT. Foi considerado como 100% as células que foram isentas de tratamento com as AgNP e inibidor de endocitose. Cada valor representa a média ± S.D. de três experiências independentes (n = 4). *p < 0,05 AgNP vs AgNP + CLQ. Análise estatística por ANOVA seguida por teste de Tukey.
Os resultados obtidos através da utilização do inibidor CLQ deixa claro que o ambiente ácido de vesículas intracelulares (endossomos tardios e lisossomos) tem papel fundamental na metabolização das AgNP, possivelmente essa análise se estenda para o processo autofágico. Entretanto, essa conclusão ainda precisa ser confirmada através de mais dados moleculares, como a quantificação da proteína p62 e a conversão da proteína LC3-I em LC3-II presentes no processo autofágico. Além disso, para a complementaridade de dados, utilizamos a técnica de MET para observar estruturas relacionadas à autofagia, entretanto os dados estão apresentados na forma de ANEXO 1, pois ainda não são conclusivos.
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5 CONCLUSÃO
A partir dos dados apresentados neste trabalho, concluímos que:
Macrófagos intraperitoneais tratados com AgNP biossintéticas apresentam uma citotoxicidade concentração- e tempo-dependentes.
As AgNP biossintéticas são capazes de promover alterações na integridade da membrana de macrófagos a partir de 3 h de tratamento.
A citotoxicidade das AgNP biossintéticas está diretamente relacionada com o estresse oxidativo, fato este evidenciado através da quantificação dos níveis de ROS e do ensaio de citotoxicidade frente ao pré-tratamento com N-Acetilcisteina, onde observou-se a reversão total da morte celular causada pelas AgNP.
O efeito citotóxico desencadeado pelas AgNP biossintéticas é dependente da via pela qual as partículas são endocitadas pelas células. A inibição da fagocitose e endocitose mediada por caveolina levou a uma redução do efeito citotóxico das nanopartículas em macrófagos peritoneais.
Possivelmente o tratamento de macrófagos intraperitoneais com AgNP biossintéticas estimula o fluxo autofágico, além disso, esse processo parece estar envolvido na metabolização das nanopartículas.
Desta forma concluímos que as AgNP biossintéticas apresentam um perfil citotóxico similar aos descritos em literatura com nanopartículas obtidas por métodos de síntese convencionais (químicas). Entretanto, ainda é necessária cautela e mais estudos para a aplicação dessas nanopartículas em seres vivos.
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Figura 24 –Citotoxicidade e processamento celular de nanopartículas biossintéticas de prata em macrófagos peritoneais. Citotoxicidade: 1- Internalização das AgNP por endocitose mediada por caveolina, na qual a inibição
desta via de endocitose proporcionou o aumento da viabilidade celular; 2- Internalização das AgNP por fagocitose, na qual a inibição desta via de endocitose proporcionou o aumento da viabilidade celular; 3- Danos à membrana celular através da produção e ROS. Processamento das AgNP: 4-Possível estímulo do fluxo autofágico pela indução das AgNP ou pela decorrência de eventos citotóxicos causados pelas nanopartículas.
54 6 PERPECTIVAS
Com os resultados iniciais dos mecanismos moleculares envolvidos na citotoxicidade das AgNP biossintéticas em macrófagos peritoneais, propomos dar continuidade nos seguintes assuntos para o encerramento deste trabalho:
i. Estresse Oxidativo: Avaliar a atividade mitocondrial para determinar se a produção de ROS está envolvida com danos mitocondriais em macrófagos induzidos pelas AgNP biossintéticas. Além disso, avaliar a peroxidação de lipídios para determinar se os danos em membrana são decorrentes do estresse oxidativo.
ii. Endocitose: Avaliar a toxicidade das AgNP utilizando outros inibidores de endocitose mediada por clatrina e macropinocitose, para confirmar se estas vias não influenciam na citotoxicidade das AgNP. Utilizar combinações entre inibidores de endocitose para determinar reduções ainda maiores da citotoxicidade, como por exemplo inibidores de fagocitose e caveolina juntos.
iii. Autofagia: Quantificar as proteínas envolvidas no processo autofágico, como por exemplo a p62 e a conversão da LC3-I em LC3-II, através de técnicas como Western Blotting e Microscopia Confocal;
iv. Avaliação da resposta imune inata dos níveis de expressão de RNAm de citocinas pró ou anti-inflamatórias, e depois fazer a confirmação através da quantificação dessas proteínas já expressas por ELISA.
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