Department of Pediatric Dentistry, Orthodontics and Public Health, Bauru School of Denistry, University of São Paulo, Brazil.
Department of Biological Sciences, Bauru School of Dentistry, University of São Paulo, Brazil.
Photodiagnosis Photodyn Ther. 2020 Mar;29:101654. doi: 10.1016/j.pdpdt.2020.101654. Epub 2020 Jan 7.
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 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 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.
抗菌光动力疗法 (aPDT) 已被用作口腔感染的辅助治疗方法，是一种微创临床方法。几项研究证明了其抗菌效果；然而，关于其对小鼠成纤维细胞 (NIH/3T3) 的细胞毒性作用的证据不足。本研究旨在评估亚甲蓝介导的 aPDT 对小鼠成纤维细胞的细胞毒性和凋亡途径。用 0.1 或 1.0 mg.L 亚甲蓝 (MB) 和 0.075 或 7.5 J.cm-² LED 630nm 处理细胞。通过 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐 (MTT) 和结晶紫 (CV) 测定法检查细胞活力，同时通过 qRT-PCR 评估 Bax、Bad、Bcl-2、VDAC-1、细胞色素 C 和 Fas-L 的 cDNA 表达 (1、3、6 和 24 h)。通过 Kruskal-Wallis 和事后 Dunn 检验检测 MTT 和 CV 测定法组间差异，通过 ANOVA 和事后 Tukey 检验检测 qPCR 组间差异 (P < 0.05)。1.0 mg.L MB 和 7.5 J.cm-² LED 的组合显着降低了细胞活力，而 MB 和 LED 单独使用对成纤维细胞无害。MB 介导的 aPDT 在 3 小时后增加了细胞色素 C 和 Fas-L 的表达，在 6 小时后增加了 Bax/Bcl-2、Bad/Bcl-2 和 VDAC-1 的表达。基于这些结果，MB 介导的 aPDT 诱导了小鼠成纤维细胞的细胞毒性，随后激活了 Bcl-2 凋亡信号通路。需要进一步的研究来确定 aPDT 的适当参数，以在不损伤成纤维细胞的情况下灭活微生物。
