Resveratrol Nanoparticles Loaded With 20(S)-Protopanaxadiol For The Treatment Of Periodontitis By Regulating The Macrophage Phenotype

Background:Periodontitis is a chronic immune inflammatory disease caused by complex subgingival plaque biofilm,which is the main cause of adult tooth loss and the third killer threatening human health.About 743 million people in the world suffer from periodontitis,and its development process involves the interaction between periodontal pathogens in plaque biofilm and host immune response.Periodontal pathogens activate the host’s immune response,leading to excessive production of reactive oxygen species and oxidative stress,and ultimately damaging periodontal supporting tissues.Currently,the treatment of periodontitis mainly depends on mechanical debridement technology to eliminate periodontal bacteria.However,this method can not completely solve the problem,because the bacterial infection is only the initial factor of periodontitis progress,and the host immune response is the key.Therefore,it is very imessentialportant to regulate host immunity while eliminating periodontal pathogens.Macrophages are an important part of the body’s immune defense system,which are widely distributed in periodontal tissues and play a key role in periodontal occurrence and development.Macrophages are highly heterogeneous and plastic and can be divided into M1 and M2 subtypes according to different stimulation conditions.M1-type macrophages have a strong antimicrobial ability and can release a large number of pro-inflammatory mediators(such as IL-1β,IL-6,TNF-α,and O^2-)that promote oxidative stress and tissue damage.M2-type macrophages can release a large number of anti-inflammatory mediators（such as IL-10,Arg-1,and TGF-β）that inhibit oxidative stress and tissue damage,and participate in tissue repair and remodeling.Reducing the ratio of M1/M2 is of great significance for controlling periodontal tissue injury.Therefore,promoting the transformation of macrophages from M1 type to M2type or increasing the number of M2 type macrophages may be a new strategy to effectively cure periodontitis.Objective:In this study,resveratrol（RES）and 20（S）-protopanaxadiol（PPD）composite nanoparticles（RES@PPD NPs）were prepared,and the mechanism of regulating macrophage differentiation and its anti-inflammatory effect was discussed.Methods:1.RES NPs were synthesized by phenol-formaldehyde reaction,and RES@PPD NPsweres were prepared by adding PPD during the reaction.2.The materials were characterized by electron microscope（TEM）,scanning electron microscope（SEM）,nano-particle size and ZETA potential analyzer,ultraviolet-visible spectrophotometer（UV-vis）,Fourier infrared spectroscopy（FTIR）,X-ray diffractometer（XRD）and thermogravimetric analyzer（TGA）,and their reactive oxygen species（ROS）scavenging activity was determined.3.The vitro toxicity of the materials was evaluated by the CCK-8 method,living dying staining method,hemolysis test,and cell staining method.4.The effects of RES@PPD NPs on pro-inflammatory cytokines IL-1β,IL-6 and TNF-αand anti-inflammatory cytokines IL-10,Arg-1 and TGF-βwere detected by PCR.The effects of RES@PPD NPs on the phenotypic transformation of macrophage M1/M2 were analyzed by flow cytometry and immunofluorescence.And explore the mechanism of RES@PPD NPs on the NF-κB inflammatory pathway.5.Comparing the phenotypic transformation and anti-inflammatory effects of RES NPs and RES@PPD NPs on macrophages M1/M2 in vivo by H&E staining,Masson staining,immunohistochemical staining（IHC）and immunofluorescence staining（IF）.Results:1.RES@PPD NPs have good dispersibility and stability,and their average particle size is about 130 nm.2.RES@PPD NPs have good biocompatibility and no obvious cytotoxicity at a concentration below 50μg m L^-1.3.In vitro experiments,RES@PPD NPs can effectively remove ROS,inhibit the activation of M1-type macrophages,and promote the polarization of M2-type macrophages,thus playing an anti-inflammatory and repairing role.4.In vivo experiments,RES@PPD NPs can promote the transformation of macrophages in periodontal tissues from M1 type to M2 type more effectively than RES NPs,and reduce the degree of periodontitis.Conclusion:In this study,RES@PPD NPs were synthesized by loading PPD on RES NPs,and a nanocomposite with antioxidant and anti-inflammatory effects was constructed.Cytotoxicity test in vitro proved that RES@PPD NPs have no toxic or side effects on cells,and can regulate pro-inflammatory and anti-inflammatory cytokines secreted by macrophages to achieve two-way immune regulation.In addition,in periodontitis model rats,subgingival injection of RES@PPD NPs can effectively remove reactive oxygen species from periodontal tissues and alleviate the development of periodontitis.RES@PPD NPs play an immunomodulatory role by transforming macrophages from the M1 phenotype to the M2 phenotype,which may have great application potential in the treatment of periodontitis induced by other pathogens or other aspects.