Effect Of Macrophage-derived Exosomes On Osteoclast Differentiation Under The Action Of Porphyromonas Gingivalis

Objective:Periodontitis is a complex inflammatory disease.Its clinical symptoms include gingival inflammation,alveolar bone resorption,loss of attachment,etc.In severe cases,it can lead to tooth loss.P.gingivalis is the main pathogen of periodontitis,which can locally invade periodontal tissues,escape the host’s defense mechanism,and secrete a variety of virulence factors to cause periodontal tissue destruction and alveolar bone resorption.Bacterial,fungal,and viral infections all cause the formation and release of exosomes from host cells.During extracellular circulation,exosomes can regulate the biological activity of recipient cells and play the role of regulating target cells by transporting lipids,proteins and nucleic acids from the source cells.Alveolar bone resorption is one of the main symptoms of periodontitis,and osteoclast is the main cells responsible for bone resorption.In this study,P.gingivalis-infected macrophage derived exosomes were used to stimulate osteoclast.The expressions of NFATc1,TRAF6 and RANK were detected by qRT-PCR and immunofluorescence to observe the effect of exosomes on osteoclast differentiation.Methods:1.U937 cells were cultured in vitro and induced into macrophages by adding PMA.2.P.gingivalis W83 was cultured in vitro,and the concentration of bacteria was detected after liquid proliferation.3.Macrophages were divided into the experimental group and the control group.In the experimental group,P.gingivalis W83was added at the rate of infection complex MOI=100:1,and the control group was added with culture medium of the same volume.4.After the samples were collected respectively,exosomes were extracted by ultra-high speed centrifugation method and were resuspended with PBS,and stored at-80℃for later use.Exosomes in the groups with and without P.gingivalis were expressed as EXO-P and EXO-N respectively.5.Exosomes were identified by TEM,Nanoparticle tracking analysis（NTA）and Western blot.6.U937 cells were cultured in vitro and induced into osteoclasts by the sequence of PMA and 1,25（OH）₂VD3.7.Osteoclasts were identified by tartrate-resistant acid phosphatase（TRAP）staining.8.The experiments were divided into control group,EXO-N group and EXO-P group.Osteoclasts were stimulated with EXO-N and EXO-P with concentration of 40μg/m L respectively.9.Total RNA was extracted from the control group、EXO-N group and EXO-P group,and Quantitative real-time PCR was used to detect the gene expression of NFATc1,TRAF6 and RANK.10.The protein expressions of NFATc1,TRAF6 and RANK in the control group、EXO-N group and EXO-P group were detected by immunofluorescence.Results:1.The exosomes of both the EXO-N group and the EXO-P group were observed with disk-like follicular structures of double membrane under TEM.NTA test results showed that the average diameter of exosomes of the EXO-N group was 128.6nm,and that of the EXO-P group was 133.9 nm.Western blot results showed that the exosomes of the EXO-N group and the EXO-P group were positively expressed in CD9,CD81 and Alix.2.U937 cells were induced into osteoclasts by PMA and 1,25（OH）₂VD3.3.Compared with the control group and the EXO-N group,the expressions of NFATc1,TRAF6 and RANK were increased in the EXO-P group,while there were no significant differences in the above indexes between the EXO-N group and the control group.Conclusion:Exosomes released by macrophages infected with P.gingivalis W83can promote osteoclast differentiation.It provides a new idea for the study of periodontal bone immunology.