| Background: Periodontitis is a chronic inflammatory destructive disease that occurs on the supporting tissues of the teeth,which is difficult to reverse and often leads to tooth loss if left untreated.The current conventional treatment strategy aims at eliminating the microbial trigger and removing the infection to halt the progression of the disease.However,the damaged periodontal tissue is difficult to restore,and even regenerative surgical treatments such as bone grafting and guided tissue regeneration have limitations,including strict indications,limited ideal periodontal tissue regeneration,and uncertain efficacy.With the development of tissue engineering and regenerative medicine,a growth factor-based delivery technology provides a new approach for the treatment of periodontitis.Glucagon-like peptide-1(GLP-1)is an intestinal hormone secreted by L cells,which exerts physiological effects by binding to its specific receptor(GLP-1R).Evidence-based medicine has confirmed its protective effects on the cardiovascular system,kidneys,and central nervous system.GLP-1R is widely distributed in multiple tissues,including periodontal tissue.Our previous studies have shown that the GLP-1R agonist has antiinflammatory and periodontal bone regeneration effects in cell experiments and systemic administration in animals.However,systemic administration has drawbacks such as significant side effects,low local effective concentration,and short duration of action.Therefore,based on the above strategy,we constructed a sustained-release system to deliver GLP-1R agonist to the periodontal site and investigated its therapeutic effect and anti-inflammatory mechanism on rat periodontitis.Liraglutide(LIRA)is currently the most commonly used GLP-1R agonist in clinical practice.Objective: We aim to to develop a novel periodontal sustained-release system that loads LIRA(LIRA@PLGA/HA)through the dual encapsulation of poly(lacticco-glycolic acid,PLGA)and hyaluronic acid(HA),and to investigate the therapeutic effect and anti-inflammatory mechanism of this system on rat periodontitis,providing a new theory and approach for clinical treatment of periodontitis.Methods:(1)PLGA nanoparticles loaded with LIRA were prepared using a modified emulsion solvent evaporation method.The morphology and size distribution of the nanoparticles were characterized by scanning electron microscopy(SEM)and dynamic light scattering(DLS),respectively.The encapsulation efficiency,drug loading,drug release kinetics,and secondary conformational stability of LIRA released from PLGA nanoparticles were determined by high-performance liquid chromatography(HPLC)and circular dichroism(CD).Finally,the PLGA nanoparticles were co-solubilized with HA powder to form an injectable gel system,which was then co-cultured with periodontal membrane cells to evaluate its potential cytotoxicity using the CCK-8 assay.(2)A rat experimental periodontitis model was established by ligature method combined with high-sugar viscous feed.After modeling,the effect of experimental periodontitis was validated by periodontal clinical indicators,HE staining and methylene blue staining.(3)After successful modeling,periodontitis rats were divided into negative control group with physiological saline(NaCl),blank system group with PLGA/HA,positive control group with minocycline hydrochloride ointment(PERIO),and experimental group with LIRA@PLGA/HA periodontal slow-release system.The drugs were injected into the periodontal pockets of the upper second molar of the rats once a week for four times.After treatment,the anti-inflammatory effect of LIRA@PLGA/HA system on periodontitis rats was explored by detecting the clinical indicators of periodontal disease,the expression of inflammatory factors(IL-1β,IL-18)in serum and gingival crevicular fluid of rats by enzyme-linked immunosorbent assay(ELISA),and HE staining.The bone protection and regeneration effect of the system were studied through Masson staining and Micro-CT.The expression of cell pyroptosis-related factors NLRP3 and Caspase-1 in periodontal tissue was detected by qRT-PCR and immunohistochemical experiments,preliminarily exploring the mechanism of LIRA@PLGA/HA system in inhibiting periodontal tissue inflammation.Results:(1)LIRA-loaded PLGA nanoparticles were successfully synthesized by modified emulsion solvent evaporation method.SEM and DLS analysis showed that the nanoparticles had intact morphology and good dispersibility.The nanoparticles had a loading efficiency of over 75% and a drug loading capacity of over 3.5%.They could sustainably release 60-70% of the drug within 8 days.During the encapsulation process,the secondary structure of LIRA’s peptide was not destroyed.Moreover,the synthesized LIRA@PLGA/HA system had no potential cytotoxicity.(2)A rat model of periodontitis was successfully established by ligature placement and high-sugar viscous diet feeding.The modeled rats exhibited typical symptoms of periodontitis such as increased probing depth,gingival bleeding,and tooth mobility.(3)After drug administration,compared with the NaCl group and the blank system PLGA/HA group,the LIRA@PLGA/HA group showed significantly reduced gingival bleeding index(BI),probing depth(PD),and expression levels of inflammatory factors IL-1β and IL-18(P<0.05).Although the corresponding indicators in the LIRA@PLGA/HA group were lower than those in the PERIO group,there was no statistical difference between the two groups.In addition,compared to the other three groups,HE staining,Masson staining,Micro CT three-dimensional reconstruction,and bone microparameter analysis further confirmed the results LIRA@PLGA /The HA group better suppressed inflammation of rat periodontal tissue,promoted alveolar bone calcification,maturation,and regeneration;The LIRA@PLGA/HA system downregulated the expression levels of NLRP3 and Caspase-1 m RNA and protein in the periodontal tissue of rats.Conclusion: The LIRA@PLGA/HA system had good sustained release effect and exhibited anti-inflammatory,bone-protective,and bone-regenerative effects in the treatment of periodontitis.The mechanism of inflammation inhibition may be related to the downregulation of protein expression of NLRP3 and Caspase-1,which are associated with cell pyroptosis. |
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