Análise in vitro da N-Acetilcisteína em fibroblastos do ligamento periodontal estimulados por LPS bacteriano
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Data
2018-02-16
Autores
Bittencourt, Tatiane Sampaio
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Universidade Estadual Paulista (Unesp)
Resumo
O objetivo deste estudo foi avaliar in vitro o comportamento biológico celular da N-Acetilcisteína (NAC), diante da estimulação ou não pelo LPS bacteriano. Foram utilizados fibroblastos do ligamento periodontal, que ficaram em contato por 48 horas com as substâncias testadas: NAC, Hidróxido de cálcio p.a. (HC), Lipopolissacarídeo de Escheria Coli (LPS), NAC + LPS e HC + LPS. Para os grupos NAC + LPS, HC + LPS e LPS, as células foram estimuladas com 2µg/mL de LPS por 24 horas, previamente aos tratamentos descritos. Foram realizados os testes biológicos de viabilidade celular (XTT), Espécies Reativas de Oxigênio (ROS), Elisa (para as citocinas IL-6, IL-8, IL-10, IL-1β e TNF-α) e Micronúcleo (MNT). Os dados foram analisados estatisticamente pela análise descritiva e pelos testes ANOVA e Kruskall Wallis, seguido do teste Dunn (p˂0.05) para os testes de XTT, ROS e MNT, e para o teste Elisa foi realizado cálculo das médias e desvio padrão. Os resultados obtidos mostraram que NAC teve um bom comportamento, frente à agressão provocada pelo LPS, quanto à produção de ROS. HC apresentou maior viabilidade celular que NAC, embora NAC não tenha apresentado citotoxicidade. Em relação à expressão das citocinas, NAC foi capaz de reduzir o potencial inflamatório do LPS quando da análise do TNF- α e IL-1β. NAC foi capaz de reduzir a genotoxicidade do LPS, como mostrado pelo teste MNT. Concluiu-se que NAC apresentou um comportamento biológico satisfatório, pela viabilidade celular dos fibroblastos, pela redução da geração de ROS e do potencial inflamatório provocado pelo LPS, e por reduzir a sua genotoxicidade.
The purpose of this study was to evaluate in vitro the action of N-Acetylcysteine (NAC) and calcium hydroxide (CH) on biological activity, either without or with LPS stimulation in periodontal ligament fibroblasts (PDLF) cells. PDLF were placed in contact with NAC, CH, NAC + LPS, LPS and CH + LPS for 48 hours. PDLF were stimulated by bacterial LPS for 24 hours in NAC + LPS, LPS and CH + LPS groups, before the substances described above are applied. The LPS and NAC effect on cell viability was measured using a XTT test. Reactive oxygen species (ROS) production was evaluated using ROS/superoxide detection kit. Inflammatory cytokines (IL-6, IL-8, IL-10, IL-1β and TNF-α) were evaluated by enzyme-linked immunosorbent (ELISA) assay. Genotoxicity was measured using micronucleus test (MNT). The means and standard deviation for all tests were calculated. Data were analyzed statistically by descriptive analysis, ANOVA and Kruskall Wallis tests, followed by Dunn test (p˂.05). CH was superior to NAC on cellular viability, although NAC was not cytotoxic. The results showed that NAC was able to reduce ROS production of LPS. NAC was able to reduce the inflammatory potential of LPS by decreasing the TNF-α and IL-1β release. NAC was able to reduce the genotoxicity of LPS. It was concluded that NAC showed a satisfactory biological activity, presenting a minimal effect on cell viability of PDLF cells and reducing ROS production and inflammatory potential provoked by LPS, and to decrease its genotoxicity.
The purpose of this study was to evaluate in vitro the action of N-Acetylcysteine (NAC) and calcium hydroxide (CH) on biological activity, either without or with LPS stimulation in periodontal ligament fibroblasts (PDLF) cells. PDLF were placed in contact with NAC, CH, NAC + LPS, LPS and CH + LPS for 48 hours. PDLF were stimulated by bacterial LPS for 24 hours in NAC + LPS, LPS and CH + LPS groups, before the substances described above are applied. The LPS and NAC effect on cell viability was measured using a XTT test. Reactive oxygen species (ROS) production was evaluated using ROS/superoxide detection kit. Inflammatory cytokines (IL-6, IL-8, IL-10, IL-1β and TNF-α) were evaluated by enzyme-linked immunosorbent (ELISA) assay. Genotoxicity was measured using micronucleus test (MNT). The means and standard deviation for all tests were calculated. Data were analyzed statistically by descriptive analysis, ANOVA and Kruskall Wallis tests, followed by Dunn test (p˂.05). CH was superior to NAC on cellular viability, although NAC was not cytotoxic. The results showed that NAC was able to reduce ROS production of LPS. NAC was able to reduce the inflammatory potential of LPS by decreasing the TNF-α and IL-1β release. NAC was able to reduce the genotoxicity of LPS. It was concluded that NAC showed a satisfactory biological activity, presenting a minimal effect on cell viability of PDLF cells and reducing ROS production and inflammatory potential provoked by LPS, and to decrease its genotoxicity.
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Palavras-chave
N-Acetilcisteína, Fibroblastos, Lipopolissacarídeo, Viabilidade celular, Espécies reativas de oxigênio, N-Acetylcysteine, Fibroblasts, Lipopolysaccharide, Cell viability, Oxigen-reactive species