Hypoxia-simulated Self-assembled Nanoparticles Treat Periodontitis By Regulating Macrophage Polarization

Background:Periodontitis is a high incidence of oral disease affecting about 10%of adults and is a major public health problem and economic burden for society.Periodontitis,if not treated promptly,can eventually lead to the loss of connective tissue attachment and dental support structures such as alveolar bone,resulting in tooth loss that affects the quality of life of the individual.Currently,it is believed that the infectious mechanisms of periodontitis include the formation of plaque biofilm,bacterial infection into the circulatory system,and immune and inflammatory reactions caused by periodontal pathogens and their products.These infections trigger the host’s immune defense mechanism,and excess inflammatory factors will destroy periodontal tissue and increase inflammation load throughout the body.At present,the most commonly used treatment is mechanical therapy,including supragingival cleaning and root scraping.Mechanical plaque removal is not enough,instead,effective regulation of the host immune response is the key to the treatment of periodontitis.In periodontal tissues,macrophages make important contributions to tissue homeostasis and defense and play various roles in periodontitis such as regulation and phagocytosis.Induced by different factors,macrophages polarize and develop different functional phenotypes including M1-type macrophages with proinflammatory function and M2-type macrophages with anti-inflammatory function.They all regulate the immune system and are involved in the damage and repair stages of periodontitis.M1-type macrophages can transform with M2-type macrophages.M1-type macrophages are associated with tumor necrosis factor(TNF-α)and can increase periodontal tissue inflammation.They are considered to be the main driving factors of periodontitis and related tissue damage.M2-type macrophages release interleukin-10(IL-10)and transforming growth factor β(TGF-β),inhibiting the development of inflammation.Therefore,reducing the ratio of M1/M2 type macrophages is an effective method to inhibit the inflammatory response and prevent periodontal tissue destruction.Hypoxia is characteristic of the tissue microenvironment in an inflammatory state.Local hypoxia in chronic inflammatory tissue is related to the number of activated immune cells and the enhanced utilization of local oxygen supply by local inflammatory tissue.The central regulatory factor in an anoxic microenvironment is hypoxia-inducible factor 1α(HIF-1α).When the oxygen supply is sufficient,HIF-1α binds to Prolyl hydroxylase(PHD)for ubiquitination degradation.However,when oxygen supply is insufficient,HIF-1α does not degrade but enters the nucleus and activates the expression of downstream genes,including vascular endothelial growth factor(VEGF),glucose transporter(GLUT),and other factors,which promote angiogenesis and regulate metabolism.A large number of studies have shown that HIF is an important regulatory factor of the immune system.HIF-1α is responsible for controlling the activity of immune cells during inflammation and controlling the polarization of macrophages,which could be a potential therapeutic target to improve host defense.Targeting the HIF pathway may be an important therapeutic strategy for inflammation therapy.Objective:A PHD inhibitor FG-4592(Roxadustat)nanoparticles(GCS@FG-4592)containing epigallocatechin gallate(EGCG)oligomer GCS were prepared to simulate the hypoxia effect,and its anti-inflammatory effect and effect on macrophage differentiation were compared with that of EGCG.Methods:1.EGCG oligomer GCS and anoxic simulated self-assembled nanoparticles GCS@FG-4592 were prepared by stirring at room temperature according to the Mannich condensation reaction.2.The nanoparticles were characterized by transmission electron microscopy(TEM),ultraviolet-visible spectrophotometry,Fourier transform infrared spectroscopy(FTIR),ZETA potential,etc.,and the loading rate,encapsulation rate,and release curve were determined.3.The biocompatibility of GCS@FG-4592 was evaluated by CCK-8,staining of live and dead cells,and H&E staining of important tissues and organs of animals.4.In vitro experiments,antioxidant activities of GCS@FG-4592 and EGCG were detected and compared by measuring ROS levels.The expressions of the HIF-1α gene and protein were detected by PCR and Western Blot.The expression regulation of GCS@FG-4592 and EGCG on pro-inflammatory related cytokines such as TNF-α,IL-1βand nitric oxide synthase(iNOS)and anti-inflammatory related cytokines such as IL-10,TGF-β and arginase 1(Arg-1)were detected and compared by PCR.Immunofluorescence staining and flow cytometry were used to detect the anti-inflammatory effects of GCS@FG4592 and EGCG on macrophage differentiation at the protein level.5.In vivo experiments,H&E staining,Masson staining,immunohistochemical staining,and immunofluorescence staining were used to evaluate the anti-inflammatory effect of low-concentration group GCS@FG-4592 and high-concentration group GCS@FG-4592 ’as well as the effect of EGCG on macrophage differentiation.Results:1.The anoxic simulated self-assembled nanoparticles(GCS@FG-4592)showed a spheroidal structure with a diameter of about 60nm under an electron microscope,with a negative charge(-31mV)on the surface,drug loading rate of 32%and encapsulation rate of 40%.2.GCS@FG-4592 nanoparticles have good biocompatibility and no obvious cytotoxicity when the drug concentration is less than 20μM.3.Experiments in vitro have shown that GCS@FG-4592 has significant anti-inflammatory and antioxidant capacity compared with EGCG,which can effectively induce M2-type macrophages,which is conducive to eliminating tissue inflammation and repair and regeneration.4.Experiments in vivo have shown that GCS@FG-4592 has a stronger anti-inflammatory effect than EGCG,which can promote the healing of periodontitis in rats.Conclusion:In this study,the PHD inhibitor FG-4592 was self-assembled into nanoparticles using polyphenols as a carrier to simulate the hypoxia effect and up-regulate the expression of HIF-1α.It has the effect of clearing ROS,can significantly promote the polarization of M2-type macrophages,reduce the ratio of M1/M2 macrophages,regulate the immune response,and thus play a role in the treatment of rat periodontitis.The hypoxia-simulated nanoparticles have outstanding antioxidant effects and the regulation of macrophage polarization.FG-4592 has a wide application prospect in the treatment of periodontitis.