The Role Of C3a-C3aR Axis In Periodontitis

Objectives: In this study,wild-type and C3 ar gene knockout animal model were used to establish mouse periodontitis model and observe the differential phenotypes.Meanwhile,the possible reason of the differential phenotypes and downstream signaling pathways were verified in vitro,in order to clarify the role and mechanism of C3a-C3 a R axis in the occurrence and development of periodontitis.Methods: 1.Micro-CT 3D reconstruction results showed that: Compared with the alveolar bone of the control side,different degrees of alveolar bone resorption occurred in the periodontitis modeling side of the three different genotypes of mice.There was no statistical difference in alveolar bone resorption between the periodontitis mice with genotype C3ar+/+ and the periodontitis mice with genotype C3ar+/-littermate.Compared with C3ar+/+ and C3ar+/-periodontitis mice,the alveolar bone resorption of C3ar-/-mice was significantly reduced.HE staining results showed that: Compared with the periodontal tissue of the control side,the periodontal tissue of the periodontitis modeling side of the three genotypes of mice showed varying degrees of inflammatory cell infiltration,attachment loss and alveolar bone loss.Compared with the periodontitis mice of the genotypes of C3ar+/+ and C3ar+/-,there was no significant difference in inflammatory cell infiltration,attachment loss and alveolar bone resorption in periodontitis mice with C3ar+/+ and C3ar+/-genotypes,but the inflammatory cell infiltration,attachment loss and alveolar bone resorption in periodontitis mice with C3ar-/-genotypes were decreased to different degrees compared with those with C3ar+/+ and C3ar+/-genotypes.Tartrate-resistant acid phosphatase staining results showed that: Compared with the un-ligated side,the number of Trappositive cells in the periodontal tissues of the ligated side of the three genotypes of mice was increased,and there was no statistical difference in the number of Trap-positive cells in the periodontal tissues of the ligated side of the mice with genotype C3ar+/+ compared with the periodontitis mice with genotype C3ar+/-.However,compared with the periodontitis mice with C3ar+/+ and C3ar+/-genotypes,the number of TRAP positive cells in the periodontal tissues at the ligation side of the mice with C3ar-/-genotypes was significantly reduced.Immuno-histochemical staining results showed that the expression levels of inflammatory factors TNF-α and IL-1β in the periodontal tissues of the ligation side of the three genotypes mice were increased compared with the non-ligation side.There was no statistical difference in the expression levels of inflammatory factors in the periodontal tissues of the ligation side of the C3ar+/+ genotype mice compared with the C3ar+/-genotype mice.Compared with the periodontitis mice with C3ar+/+ and C3ar+/-genotypes,the expression levels of inflammatory factors in periodontal tissues at the ligation side of mice with C3ar-/-genotypes were significantly decreased.2.Compared with the blank control group,LPS,C3 a and LPS+C3a group macrophages were all extended pseudopodia and the cell morphology was finger like,dendritic and radial,etc.LPS+C3a group was more obvious than LPS group and C3 a group,LPS group was more obvious than C3 a group,and LPS+C3a group cells growth more gathered than LPS group,C3 a group and control group cell growth dispersed.RTq PCR results showed that: Compared with the blank control group,LPS group,C3 a group and LPS+C3a group could promote the m RNA expression of TNF-α,IL-1β and i NOS in macrophages,but the effect of C3 a group was significantly weaker than LPS group.The m RNA expression levels of TNF-α,IL-1β and i NOS in C3a+LPS group were higher than those in C3 a and LPS group.Enzyme-linked immunosorbent assay results showed that compared with blank control group,LPS group or C3 a group and LPS+C3a group could promote macrophages to secrete inflammatory cytokines TNF-α and IL-1β,but the effect of C3 a group was significantly weaker than LPS group,and the secretion of TNF-α and IL-1β in C3a+LPS group was higher than that in C3 a group and LPS group.Western blot results showed that: Compared with the blank control group,the protein expression levels of P-NF-κB,TNF-α,IL-1β and i NOS in macrophages in LPS or C3 a group and LPS+C3a group were increased,but the effect of C3 a group was significantly weaker than LPS group.The protein expression levels of P-NF-KB,TNF-α,IL-1β and i NOS in C3a+LPS group were higher than those in C3 a group and LPS group.DPAI nuclear staining was performed after cell migration experiment,and the results showed that complement C3 a had chemotactic effect on macrophages obviously.3.Tartrate-resistant acid phosphatase staining results showed that compared with the untreated C3 a group,different concentrations of C3 a were treated Osteoclast precursor cells can increase osteoclast differentiation.Alkaline phosphatase staining showed that different concentrations of C3 a did not increase osteoblast differentiation in osteoblast precursor cells compared with the untreated group.Conclusion: 1.C3a-C3aR axis mediated signaling can promote periodontal inflammation and alveolar bone loss in mice with periodontitis.2.C3 a chemotaxis macrophages and promotes macrophages to secrete inflammatory cytokines through the NF-KB signaling pathway.3.C3 a can promote osteoclast differentiation,but has no effect on osteoblast differentiation.