Cytotoxic effect and apoptosis pathways activated by methylene blue-mediated photodynamic therapy in fibroblasts
Giuliana Campos Chaves Lamarque1, Daniela Alejandra Cusicanqui Méndez1, Adriana Arruda Matos2
1Department of Pediatric Dentistry, Orthodontics and Public Health, Bauru School of Denistry, University of São Paulo, Brazil.
Abstract:
Antimicrobial photodynamic therapy (aPDT) has been used as an adjuvant treatment of oral infections as a minimal intervention clinical approach. Its antimicrobial efficacy was demonstrated in several studies; however, there is a lack of evidence on its cytotoxic effect on mouse fibroblasts (NIH/3T3). The aim of this study was to evaluate the cytotoxicity and apoptotic pathways of methylene blue-mediated aPDT on mouse fibroblasts. Cells were treated with 0.1 or 1.0 mg.L-1 methylene blue (MB), and 0.075 or 7.5 J.cm-² LED at 630 nm. Cell viability was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and crystal violet (CV) assays, while cDNA expression for Bax, Bad, Bcl-2, VDAC-1, cytochrome C and Fas-L was assessed by qRT-PCR (1, 3, 6 and 24 h). 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.L-1 MB and 7.5 J.cm-² LED significantly reduced the cellular viability, whereas MB and LED alone were innocuous to fibroblasts. 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 treatment. Based on these results, MB-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 fibroblasts.
Insights
Antimicrobial photodynamic therapy using methylene blue (MB-aPDT) can harm mouse fibroblasts. This study found MB-aPDT at higher doses induced cell death and activated apoptosis pathways in NIH/3T3 cells.
Area of Science:
- Biomedical Engineering
- Photochemistry
- Cell Biology
Background:
- Antimicrobial photodynamic therapy (aPDT) is a minimally invasive treatment for oral infections.
- While effective against microbes, its impact on host cells like mouse fibroblasts (NIH/3T3) needs more investigation.
- Methylene blue (MB) is a photosensitizer used in aPDT.
Purpose of the Study:
- To assess the cytotoxicity of MB-mediated aPDT on mouse fibroblasts.
- To investigate the apoptotic pathways activated by MB-mediated aPDT.
- To determine the effects of varying MB concentrations and LED light doses.
Main Methods:
- Mouse fibroblasts (NIH/3T3) were treated with different concentrations of methylene blue (MB) and LED light (630 nm).
- Cell viability was measured using MTT and crystal violet assays.
- Gene expression of apoptosis-related proteins (Bax, Bad, Bcl-2, VDAC-1, cytochrome C, Fas-L) was analyzed via qRT-PCR.
Main Results:
- A combination of 1.0 mg/L MB and 7.5 J/cm² LED significantly reduced fibroblast viability.
- MB or LED light alone did not harm the cells.
- MB-aPDT increased the expression of cytochrome C and Fas-L (3h), and Bax/Bcl-2, Bad/Bcl-2, and VDAC-1 (6h), indicating apoptosis activation.
Conclusions:
- Methylene blue-mediated aPDT induces cytotoxicity in mouse fibroblasts.
- The therapy activates Bcl-2 apoptosis signaling pathways.
- Further research is required to optimize aPDT parameters for microbial inactivation while preserving fibroblast viability.
