Methylene Blue-Mediated Photodynamic Therapy Induces Macrophage Apoptosis via ROS and Reduces Bone Resorption in Periodontitis

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.

<p>Enhanced Efficacy of Photodynamic Therapy by Coupling a Cell-Penetrating Peptide with Methylene Blue</p>

“…Release of cytochrome C activated caspase 9, which subsequently activated the caspase effector factor caspase 3. 30 Thus, we suggest that activation of MB by PDT causes cell destruction through the mitochondria, where release of cytochrome C from the mitochondria triggers caspase 9, caspase 3, and some poly (ADP-ribose) polymerase (PARP) proteins to induce cell apoptosis. Only a minute amount of selective lysosomal damage is associated with the induction of apoptosis.…”

“…12 So, MB induces ROS production by PDT cytoplasm or near the mitochondria, which has been shown to cause apoptosis. 20,21 CPPs (cell-penetrating peptides) can enter the cytoplasm of cells and have been used for the directed endocytosis of biomolecules using CPP-biomolecule conjugates. 22 Therefore, in this study, we synthesized MB with clinical-grade protamine, which belongs to the class of CPPs.…”

“…This implies that increasing the concentration of MB higher than 10 µM for MB-PDT does not increase the efficacy of MB-PDT. 30 Thus, the application of 10 µM MB-Pro with 60 J/cm 2 laser irradiation is optimal for causing cell death in PDT, as opposed to non-conjugated MB treatment.…”

<p>Pathological Mechanism of Photodynamic Therapy and Photothermal Therapy Based on Nanoparticles</p>

“…On the contrary, macrophage M2 synthesizes and secretes nourishing tumor cells and promotes angiogenic cytokines to inhibit T cell proliferation and activation. 51 However, the hypoxic microenvironment of the tumor itself polarizes macrophages to M2 phenotype, which seriously hinders tumor immunotherapy. 52 Therefore, the reversal principle of hypoxia in tumor microenvironment is helpful to improve the effect of macrophage immunotherapy.…”

“…On the contrary, macrophage M2 synthesizes and secretes nourishing tumor cells and promotes angiogenic cytokines to inhibit T cell proliferation and activation. 51 …”