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Abstract
Aim To investigate whether methylene blue-mediated photodynamic therapy (MB-PDT) can affect the “fate” of macrophages in vitro or in periodontitis tissues and to explore the potential mechanism. Methods For in vitro treatments, THP-1 macrophages were divided into three experimental groups: C/control, no treatment; MB, methylene blue treatment; and MB-PDT, MB and laser irradiation treatment. Then, apoptosis and apoptosis-related proteins were detected in each group. For in vivo treatments, periodontitis was ligature-induced in the first molars of the bilateral maxilla in 12 Sprague Dawley (SD) rats. After six weeks, the ligatures were removed and all the induced molars underwent scaling and root planning (SRP). Then, the rats were divided into three groups according to the following treatments: SRP, saline solution; MB, phenothiazinium dye; and MB-PDT, MB and laser irradiation. Apoptotic macrophages, inflammation levels, and alveolar bone resorption in the periodontal tissues of rats were analyzed in each group. Results In vitro, flow cytometry analysis demonstrated that 10 μM MB and 40 J/cm2 laser irradiation maximized the apoptosis rate (34.74%) in macrophages. Fluorescence probe and Western blot analyses showed that MB-PDT induced macrophage apoptosis via reactive oxygen species (ROS) and the mitochondrial-dependent apoptotic pathway. Conversely, the addition of exogenous antioxidant glutathione (GSH) and the pan-caspase inhibitor Z-VAD-FMK markedly reduced the apoptotic response in macrophages. In vivo, immunohistochemistry, histology, radiographic, and molecular biology experiments revealed fewer infiltrated macrophages, less bone loss, and lower IL-1β and TNF-α levels in the MB-PDT group than in the SRP and MB groups (P < 0.05). Immunohistochemistry analysis also detected apoptotic macrophages in the MB-PDT group. Conclusion MB-PDT could induce macrophage apoptosis in vitro and in rats with periodontitis. This may be another way for MB-PDT to relieve periodontitis in addition to its antimicrobial effect. Meanwhile, MB-PDT induced apoptosis in THP-1 macrophages via the mitochondrial caspase pathway.
Highlights
Periodontal disease is the result of the collapse of toothsupporting structures by the local action of periodontopathogenic microorganisms, which release inflammatory substances that severely injure periodontal tissues
No significant difference (P > 0 05) in the apoptosis rate was observed between doses of 10 and 15 μM in the methylene blue (MB)-photodynamic therapy (PDT) group, and 10 μM MB achieved the peak effect
When the concentration of MB was greater than 10 μM, MB-PDT did not cause a larger number of macrophages to undergo apoptosis
Summary
Periodontal disease is the result of the collapse of toothsupporting structures by the local action of periodontopathogenic microorganisms, which release inflammatory substances that severely injure periodontal tissues. Macrophages play an important role during the occurrence and development of periodontitis. IL-1, IL-6, TNF-α, PGE2, and other cytokines, resulting in inflammatory lesions in periodontal tissue and absorption of alveolar bone [3, 4]. Gemmell found that the number of macrophages in gingival tissues of patients with chronic periodontitis was considerably increased compared with that in normal gingival tissues [5]. Jagannathan [6] detected extensive macrophage infiltration in the gingival crevicular fluid of patients and animals with periodontitis and investigated several proinflammatory factors and bone resorption factors, such as IL-1β and TNF-α. In addition to eliminating bacteria, reducing the number of overinvasive macrophages is critical for periodontitis treatment
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