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Master's Dissertation
Full name
Giuliana de Campos Chaves Lamarque
Knowledge Area
Date of Defense
Bauru, 2019
Silva, Thiago Cruvinel da (President)
Monteiro, Douglas Roberto
Oliveira, Rodrigo Cardoso de
Silva, Francisco Wanderley Garcia de Paula e
Title in English
Evaluation of the cytotoxic effect of antimicrobial photodynamic therapy on fibroblasts (NIH/3T3) and expression of Bcl-2 family genes
Keywords in English
Cell viability
Photodynamic therapy
Abstract in English
Antimicrobial photodynamic therapy (aPDT) has been used as an adjuvant treatment of oral infections in a minimal intervention clinical approach. Its antimicrobial efficacy was demonstrated in several studies; however, there is a lack of evidence on its cytotoxic effects on eukaryotic cells. The aim of this study was to evaluate the cytotoxicity of aPDT mediated by two photosensitizing agents, methylene blue and curcumin, on mouse fibroblasts. Cells were treated with 0.1 or 1.0 mg.mL-1 methylene blue (MB) associated or not to LED at 630 nm, or 0.6 or 6 M curcumin combined or not with LED at 455 nm, with densities of 0.075 or 7.5 J.cm-2. Cellular viability was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and crystal violet (CV) assays. The expression of cDNA for Bax, Bad, Bcl-2, VDAC-1, cytochrome C and Fas-L genes related to apoptosis was assessed by quantitative PCR (qPCR) after 1, 3, 6 and 24 h from treatments. The differences between groups were detected by Kruskal-Wallis and post-hoc Dunn's tests for MTT and CV assays, and by ANOVA and post-hoc Tukey test for qPCR (P<0.05). The combination of 1.0 mg.mL-1 MB and 7.5 J.cm-² LED significantly reduced the cellular viability. The same was observed for the combinations of 6 M curcumin plus 0.075 and 7.5 J.cm-2 LED, which reduced viable cells in 47% and 99%, respectively. Also, the combination of 0.6 M curcumin plus 7.5 J.cm-2 LED reduced the viability of fibroblasts in 34%. MB-mediated aPDT increased the expression of cytochrome C and Fas-L after 3 h, and Bax/Bcl-2, Bad/Bcl-2, and VDAC-1 after 6 h from treatments. Curcumin-mediated aPDT increased significantly the relative expression of Bax/Bcl-2, cytochrome C, VDAC-1, and Fas-L genes. Therefore, MB- and curcumin-mediated aPDT induced cytotoxicity on mouse fibroblasts, with consequent activation of Bcl-2 apoptosis signaling pathways. Further studies are needed to determine the adequate parameters of aPDT to inactivate microorganisms without damaging eukaryotic cells.
Title in Portuguese
Efeito citotóxico da terapia fotodinâmica antimicrobiana sobre fibroblastos (NIH/3T3) e a expressão de genes da família Bcl-2
Keywords in Portuguese
Terapia fotodinâmica
Viabilidade celular
Abstract in Portuguese
A terapia fotodinâmica antimicrobiana (aPDT) tem sido utilizada como um tratamento coadjuvante de infecções bucais em abordagens de mínima intervenção clínica. Sua efetividade antimicrobiana foi demonstrada em diversos estudos. Entretanto, há uma falta de evidência sobre seu efeito citotóxico sobre células eucarióticas. O objetivo deste estudo foi avaliar o potencial citotóxico da terapia fotodinâmica antimicrobiana mediada por dois agentes fotossensibilizantes, azul de metileno e curcumina, sobre fibroblastos de camundongos. Células foram tratadas com 0,1 ou 1,0 mg.mL-1 de azul de metileno associado ou não a um LED 630 nm, ou 0.6 ou 6 M de curcumina associada ou não a um LED 455 nm, com densidades de energia de 0.075 ou 7.5 J.cm-². A viabilidade cellular foi determinada pelos ensaios de Brometo de 3-(4,5-dimetil-2-tiazolil)-2,5-difenil-2H-tetrazólio (MTT) e cristal violeta (CV). A expressão de cDNA para os genes ligados à apoptose Bax, Bad, Bcl-2, VDAC-1, citocromo C e Fas-L foi assessada por PCR quantitative (qPCR), após 1, 3, 6 e 24 h dos tratamentos. As diferenças entre os grupos foram detectadas pelos testes de Kruskal-Wallis e post-hoc de Dunn para os ensaios de MTT e CV, e pelos testes de ANOVA e post-hoc de Tukey para qPCR (P<0.05). A combinação de azul de metileno a 1,0 mg.mL-1 e LED a 7.5 J.cm-² reduziu significativamente a viabilidade celular, o mesmo sendo observado pelas combinações de curcumina a 6 M com LED a 0,075 e 7,5 J.cm-², que reduziram a viabilidade celular em 47% e 99%, respectivamente. Também, a associação de curcumina a 0,6 M com LED a 7,5 J.cm-² reduziu a viabilidade de fibroblastos em 34%. A aPDT mediada por azul de metileno aumentou a expressão de citocromo C e FAS-L (3 h), e Bax/Bcl-2, Bad/Bcl-2, e VDAC-1 (6 h). aPDT mediada por curcumina aumentou significativamente a expressão relativa de Bax/Bcl-2 e dos genes citocromo C, VDAC-1, e Fas-L. Portanto, aPDT mediada por azul de metileno e curcumina induziu citotoxicidade em fibroblastos de camundongo, com consequente ativação da via de sinalização apoptótica Bcl-2. Novos estudos são necessários para determinar parâmetros adequados de aPDT para inativar microrganismos com danos mínimos às células eucarióticas hospedeiras.
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