| Objective: Periodontitis is a chronic infectious disease of periodontal support tissue caused by microorganisms in plaque,which can lead to destruction of periodontal support tissues.Porphyromonas gingivalis(P.gingivalis)is the main pathogen of periodontitis and can escape the defense of the host’s natural immune system through a variety of pathways.Macrophages are important effector cells that can be activated into different phenotypes such as classical activated macrophages(M1)and selectively activated macrophages(M2).We constructed a P.gingivalis W83 sialidase gene mutant(ΔPG0352).The study found that the mutant strain is more easily cleared by macrophages,and this effect may be related to ΔPG0352 promoting M1 polarization of macrophages.The purpose of this study was to infect macrophages with P.gingivalis W83 and ΔPG0352,respectively,to examine their effects on macrophage polarization,antigen presentation and phagocytosis,and to establish a rat model of periodontitis to verify the results of in vitro experiments.The aim of this study is to explore the effects of sialidase gene deletion on macrophage polarization and immune clearance,and provide new research ideas and theoretical basis for the prevention and treatment of chronic periodontitis.Methods: Human monocytes U937 were differentiated into M0 macrophages by PMA,and CD68 expression was detected by flow cytometry.Macrophages were infected with P.gingivalis W83,ΔPG0352,comΔPG0352 and E.coli respectively.The phagocytic activity of macrophages was observed by transmission electron microscopy.ELISA was used to examine the levels of cytokines IL-12,iNOS,IL-10 and Arg-1,and flow cytometry was used to detect the expression of macrophage surface molecules CD80 and CD206,the phagocytic ability of macrophages to different bacteria and immunofluorescence detection of macrophage surface molecule MHC-II expression.The periodontitis model was established by infecting rat periodontal tissue with P.gingivalis W83,ΔPG0352 and comΔPG0352.The alveolar bone resorption was analyzed by stereomicroscope.The expression of iNOS and CD163 in rat periodontal tissue was analyzed by immunohistochemistry.Results: 1.PMA stimulate U937 to differentiate into M0 macrophage.The CD68 expression of U937-differentiated macrophages after was 95.4%,while the surface of U937 showed almost no CD68.At the same time,U937-differentiated macrophages don’t expresse CD80 and CD206.2.The morphology of macrophage phagocytic bacteria was observed by transmission electron microscope.The surface of macrophage membrane in control group was smooth and intact.However,there were microvilli and coated pits on the cell surface of macrophages infected by the four strains.The membrane was discontinuous.The phagocytized bacteria are visible in the cells and the intracellular bacteria were enclosed within endocytic vacuoles.3.The levels of IL-12,iNOS,IL-10 and Arg-1 were measured by ELISA,and the levels of IL-12,iNOS,IL-10 and Arg-1 were increased in the experimental group compared with the control group.The levels of IL-12 and iNOS in the E.coli and ΔPG0352 groups were higher than those in P.gingivalis W83 and comΔPG0352,and the E.coli group was higher than the ΔPG0352 group(P<0.05).The levels of IL-10 and Arg-1 in the E.coli and ΔPG0352 groups were lower than those in the P.gingivalis W83 and comΔPG0352 groups,and the E.coli group was lower than the ΔPG0352 group(P<0.05).There were no statistically significant differences between the four indicators in the P.gingivalis W83 and comΔPG0352 groups.4.Flow cytometry and immunofluorescence were used to detect the expression of CD80,CD206 and MHC-II on the surface of macrophages.The expression of CD80,CD206 and MHC-II in the experimental groups was higher than those in the control group(P<0.05).The expression of CD80 and MHC-II was the highest in the E.coli group,and the CD80 and MHC-II expression in the ΔPG0352 group was higher than that in the P.gingivalis W83 group(P<0.05).The expression of CD206 in P.gingivalis W83,ΔPG0352 and E.coli group decreased in turn(P<0.05).There were no significant differences in the three indicators between the P.gingivalis W83 and comΔPG0352 groups.5.The positive rate of macrophage phagocytose P.gingivalis W83 was about 52.5%,and ΔPG0352 was about 70.4%.The difference between the two was statistically significant(P<0.05).The positive rate of macrophage phagocytosis of E.coli was about 85.7%,which was statistically significant compared with the P.gingivalis W83 and ΔPG0352 groups(P<0.05).The difference between the P.gingivalis W83 and comΔPG0352 groups was not statistically significant.6.Analysis of rat periodontal soft and hard tissues revealed that group A was the position of alveolar crest in healthy rats,and there was no absorption of alveolar bone.The lowest absorption in group B was 585.40 ± 65.75 μm,and the absorption in group C and group E was the heaviest,which was 1680.40 ± 121.37 μm and 1694.20 ± 86.46 μm,respectively.There was no statistically significant difference between the two groups.Compared with group C and group E,the degree of alveolar bone resorption was reduced in group D,which was 1229.80 ± 99.55 μm(P<0.05).Immunohistochemical results of rat periodontal tissues showed that the expression levels of iNOS and CD163 in group C,D and E was higher than those in group A and B(P<0.05).Among them,the expression of iNOS and CD163 in group C and E was higher than that in group D(P<0.05).There was no statistically significant difference in the expression of iNOS and CD163 between groups C and E,and A and B.Conclusion: P.gingivalis W83 sialidase deficiency can promote M1 macrophage polarization and macrophage phagocytosis,and inhibit the escape of P.gingivalis W83 to the host immune system. |
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