| Background:Periodontitis is the result of interaction between dental plaque biofilms and the host immune response,which can cause destruction of the soft and hard tissues around the teeth,even lead to tooth loss with the inflammation progresses.The commonly used method in the treatment of periodontitis is mechanically remove local irritants to control inflammation.Due to the complexity and difference of tooth structure,there are many infected parts that are difficult to reach with instruments during mechanical treatment,such as root bifurcation and deep periodontal pockets.Mechanical treatment sometimes requires the combination of antibiotics to further control subgingival plaque.However,prolonged use of systemic antibiotics can increase the production of drug-resistant strains and the risk of superinfection.Photodynamic therapy（PDT）has become a new adjuvant method for the treatment of periodontitis in recent years due to its non-invasiveness,localization and no drug resistance.A large number of clinical studies have shown that PDT can achieve stable clinical effects in the adjuvant treatment of periodontitis.At the same time,basic research has also confirmed that PDT can effectively prevent periodontal dominant pathogens and PDT composed of suitable concentration of photosensitizer and suitable light source has no toxicity to periodontal tissue-related cells.Nevertheless,the mechanism of PDT in the treatment of periodontitis is still unclear.Macrophage infiltration in the process of periodontitis happen,give priority to with M1 type,destructive secretion of proinflammatory factor,triggered a series of inflammatory reaction.Some literatures suggest that the number of macrophages in the periodontal tissue is significantly reduced during the PDT adjuvant treatment of periodontitis.At the same time,studies have shown that PDT can inhibit tumor growth by regulating the polarization of tumor-associated macrophages.Therefore,PDT may control periodontitis by regulating the polarization of macrophages.In view of this,this study took macrophages RAW264.7 stimulated by lipopolysaccharide of Porphyromonas gingivlis（P.g-LPS）,the main pathogen of periodontitis,as the research object to explore the methylene blue mediated PDT（MB-PDT）regulation function of macrophage inflammatory and its mechanism,so as to explore the mechanism of PDT in the treatment of periodontitis from the aspect of regulating macrophage polarization.Methods:Firstly,macrophages were induced to polarize to M1 type by P.g-LPS.The mouse macrophage cell line RAW264.7 was cultured,and RAW264.7 was stimulated with 1 μg/m L and 10 μg/m L P.g-LPS for 1 h and 24 h,and the m RNA expression level of interleukin-1β（IL-1β）,tumor necrosis factor-α（TNF-α）,interleukin-10（IL-10）were detected by q RT-PCR,selecting P.g-LPS induced suitable conditions for macrophage RAW264.7 polarization to M1 type.Secondly,we explored the regulatory effect and mechanism of PDT on macrophages in inflammatory state.The effect of different light time（2 s,5 s,10 s）on the proliferation activity of RAW264.7 cells induced by P.g-LPS was detected by CCK-8 assay,and selected the appropriate light time.Then further clarify the effect of5 μM MB-mediated PDT on the proliferative activity of P.g-LPS-induced RAW264.7under appropriate light time.The effect of MB-PDT on the migration ability of P.g-LPS stimulated RAW264.7 cells was detected by Transwell migration assay;the m RNA expression of the pro-inflammatory factors IL-1β,TNF-α,inducible nitric oxide synthase（i NOS）,interleukin-6（IL-6）and the anti-inflammatory factors IL-10 and M2 macrophage polarization markers arginase-1（Arg-1）in P.g-LPS stimulated macrophage were detected by q RT-PCR and evaluated the regulatory effects of MB-PDT on macrophage inflammation and its effect on macrophage polarization;the effect of MB-PDT on mitochondrial energy metabolism in P.g-LPS stimulated RAW264.7 cells was detected by Seahorse XF mitochondrial stress test.Result:Different concentrations（1 μg/m L,10 μg/m L）of P.g-LPS stimulated RAW264.7cells for 1 h,up-regulated the m RNA expression of IL-1β and TNF-α,and inhibited the m RNA expression of IL-10 in RAW264.7 cells;the suitable light time was 2 s;RAW264.7 cells stimulated with 1 μg/m L P.g-LPS for 1 h and treated with MB-PDT（5 μM MB,light for 2 s）had no significant difference in cell proliferation at 24 h and72 h,but significantly increased cell proliferation at 48 h;MB-PDT could promote the migration of P.g-LPS-stimulated RAW264.7 cells,reduce the m RNA expressions of intracellular inflammatory factors IL-1β,TNF-α,i NOS,IL-6,and promote the m RNA expression of anti-inflammatory factors IL-10 and M2 polarization markers Arg-1;the mitochondrial basal respiration,ATP production,maximal respiration,spare respiration were significantly increased in P.g-LPS stimulated RAW264.7 cells under MB-PDT intervention.Conclusion:1.1 μg/m L P.g-LPS stimulated RAW264.7 cells for 1 h,which could induce the polarization of the macrophage cell line RAW264.7 to M1 type.2.5 μM MB mediated PDT had no significant effect on the viability of RAW264.7cells stimulated by P.g-LPS under light for 2 s.3.MB-PDT promote the migration of P.g-LPS-stimulated RAW264.7 cells.4.MB-PDT promote M1-type macrophage polarization to M2-type.5.The promotion of M2-type polarization of RAW264.7 by MB-PDT may be achieved by enhancing the cell mitochondrial energy metabolism. |
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