In Vitro And In Vivo Study Of Apoptotic Macrophages Induced By Methylene-blue-mediated Photodynamic Therapy In Periodontitis

Objective: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 its potential mechanism.Methods:For in vitro treatments,cells were divided into three experimental groups:control group,no treatment;MB group,methylene blue treatment;MB-PDT group,MB and laser irradiation treatment.Then apoptosis and apoptosis related genes were detected in each group.For in vivo treatments,periodontal disease in SD rats was orthodontic ligature and periodontal pathogen induced at the first maxillary molar.After6 weeks,the ligature was removed and all animals received scaling and root planning(SRP)and were divided according to the following treatments:SRP group,saline solution;MB group,phenothiazinium dye;and MB-PDT group,MB and laser irradiation,once a week.All animals in each treatment were killed after 3 weeks and detected the alveolar bone resorption,apoptotic macrophages and inflammation levels in periodontal tissues of rats in each group.Results:In vitro experiments showed that the combination of 10μM MB and 40J/cm~2 light dose could kill more than 50%macrophages,and the apoptosis of macrophages induced by MB-PDT in this mode was most obvious.Firstly,MB-PDT increased reactive oxygen species(ROS)level in mitochondria of macrophage as well as improved the expression ratio of proapoptotic gene Bax/antiapoptotic gene Bcl-2 in macrophages.Subsequently,increased cytoplasmic cytochrome C activated endogenous apoptotic pathway,and ultimately led to macrophage apoptosis.In vivo experiments showed less infiltrated macrophages in MB-PDT group than SRP and MB groups(P<0.05).And the macrophages infiltrated in tissues with periodontitis were apoptotic after MB-PDT treatment,while no obvious apoptosis of macrophages infiltrated in the periodontal tissues of SRP and MB rats was found.Micro CT,PCR and Elisa and analyses showed that the levels of alveolar bone resorption and inflammation in MB-PDT rats were less than that in SRP and MB rats(P<0.05),and there was no significant difference between SRP rats and MB rats.Conclusion:MB-PDT could reduce the excessive infiltration of macrophages in inflammatory periodontal tissues and induce the apoptosis of hyperinvasive macrophages while alleviating periodontitis symptoms.Compared with SRP,MB-PDT is an effective adjunctive treatment of periodontitis.