| [Objective] In this study,the correlation between autophagy and periodontitis was preliminarily analyzed by detecting the expression of inflammation and autophagy-related genes between periodontitis and healthy controls.A rat model of periodontitis was established,and the infiltration of inflammatory cells and absorption of alveolar bone were evaluated by regulating the inflammation-activated autophagy after the inhibition of autophagy.To further investigate the role of autophagy in inflammatory bone resorption in periodontitis,the effect of autophagy inhibitors on osteoclast were also performed in vitro by using bone marrow–derived macrophages(BMMs).We hope this project can provide new strategies for the prevention and treatment of periondontitis.[Methods] This project was divided into three parts: clinical research,in vivo and in vitro experiments.For clinical research,in order to reveal the relationship between autophagy and periodontitis,gingival tissues of both peridontitis and healthy controls were collected,and the expression of inflammatory cells and autophagy-related proteins were assessed via hematoxylin and eosin(HE)staining and immunohistochemical(IHC)method.To further explore the role of autophagy on inflammation and bone metabolism in periodontitis,in vivo animal study was performed by using a rat model of periodontitis followed by the gingiva injection of the autophagy inhibitors 3-Methyladenine(3-MA)and Chloroquine(CQ).Rats were randomly divided into the following 6 groups: 1)no periodontitis group(NP);2)periodontitis group(P);3)periodontitis with 3-MA low-dose group(P+3-MA-L);4)periodontitis with 3-MA high-dose group(P+3-MA-H);5)periodontitis with CQ low-dose group(P+CQ-L);6)periodontitis with CQ high-dose group(P+CQ-H).Samples were collected at day 21,alveolar bone absorption was assessed by Micro-CT,and the infiltration of periodontal inflammatory cells was also evaluated.Among them,osteoclasts were assessed by tartrate-resistant acid phosphatase(TRAP)staining,and protein expression of autophagy-related genes and nuclear transcription factors were performed by IHC staining and Western blot(WB),which including homologue of yeast ATG6(Beclin-1),p62 / SQSTM1,microtubule-associated protein 1A / 1B light chain 3(LC3),nuclear factor-κB(NF-κB),and transcription factor EB(TFEB).Osteoclast(OC),is mainly responsible for bone resorption.In order to further observe the effects of autophagy inhibitors on OCs,mononuclear macrophages were acquired from mouse bone marrow and pre-osteoclasts(pre-OCs)were obtained by using macrophage colony-stimulating factors(M-CSF)and receptor activator of nuclear factor-kappa B ligand(RANKL).According to different treatments,all pre-OCs were allocated into the following 6 groups: 1)Control group;2)Lipopolysaccharide group(LPS);3)3-MA group;4)3-MA+ LPS group;5)CQ group;6)CQ+ LPS group.Osteoclast number was counted by TRAP staining,and autophagy-related proteins were detected by WB,and the autophagy status was evaluated by green fluorescent protein-marked LC3(GFP-LC3)spots in cell using a confocal fluorescence microscope.[Results] The inflammatory cell infiltration in gingival tissue of chronic periodontitis(CP)group was increased significantly compared with the periodontal healthy(PH)group.The protein expression of LC3 and p62 was also significantly upregulated in CP group compared to the PH group,which indicates that the level of autophagy in CP group is obviously higher than the PH group,and autophagy is involved in progress of periodontitis.A certain correlation exists between the level of autophagy and the degree of inflammation.In vivo experiment,a rat model of periodontitis was established successfully.Micro-CT results showed that 3-methyladenine and chloroquine inhibited inflammatory bone resorption,which reflected by a distance decrease of cemento-enamel junction to the alveolar bone crest(CEJ-ABC).There was a significant statistical difference between the P+CQ-H and P groups,and similar reduction of the CEJ-ABC distance were also observed in the P+3-MA-L,P+3-MA-H,and P+CQ-L groups but no statistic difference.In addition,a large amount of osteoclasts infiltration in the edge of the alveolar bone was also observed in the P group by using TRAP staining,while the number of osteoclasts decreased in all other autophagy inhibited groups.We speculate that autophagy inhibition may reduce alveolar bone absorption by preventing the formation of osteoclasts.Besides,the results of both IHC and WB showed that the infiltration of inflammatory cells was plenty,and the Beclin-1 and LC3 proteins increased significantly,while p62 decreased significantly in the P group compared with the NP group.The number of inflammatory cells and the proteins expression of NF-κB,Beclin-1,and LC3 also decreased significantly while p62 protein increased significantly in all the autophagy inhibited groups.In vitro study,osteoclast precursor cells were successfully acquired by bone marrow monocyte macrophages.LPS was used to stimulate the differentiation of the precursor osteoclasts into mature OCs,while autophagy inhibitors were used for the reduction of LPS-stimulated osteoclastogenesis.Moreover,a large number of green spots were generated in LPS-stimulated osteoclasts through adenovirus transfection of GFP-LC3 method,however,in all autophagy inhibitor groups,the status of green fluorescence scattered in the cytoplasm,suggesting autophagy participated the process of osteoclast formation.[Conclusions] Our study suggests that autophagy is associated with periodontitis.Autophagy pathway may be activated by periodontitis,and the using autophagy inhibitors can reduce the infiltration of inflammatory cells and the alveolar bone resorption by osteoclasts.Therefore,we conclude that the occurrence and development of periodontitis is regulated by autophagy mechanism.Using autophagy inhibitors can downregulate excessive level of autophagy in periodontitis,which may contribute to the cell homeostasis of autophagy,thereby reduce the inflammatory bone destruction in periodontitis. |
PDF Full Text Request