| Background:Periodontal disease is a chronic infectious disease,of which dental plaque is the initial factor.Periodontal disease is one of the most prevalent diseases in the population.Fourth national oral health epidemiological survey showed that the periodontal health rate was below 10%in over 35 years-old groups.Periodontal disease is the main cause of tooth loss in adults and seriously affects the oral health and systemic health of patients.A growing number of studies have shown that vitamin D is closely connected to periodontitis.Moreover,vitamin D pathway exists in periodontal connective tissue cells.25(OH)D3 is catalyzed by CYP27B1 to form 1,25(OH)2D3,which further binds with VDR to induce the expression of hCAP-18.hCAP-18 is cleaved by proteinases to form LL-37 after release from cells.Pg-LPS shows a synergistic effect with 25(OH)D3 on the production of LL37,which can decrease the expression of IL-8 and MCP-1.In addition,it has been found that vitamin D3 needs to be metabolized by two-step hydroxylations to play its biological function.First,vitamin D3 is hydroxylated by 25-hydroxylase CYP27A1,to form 25(OH)D3.Second,25(OH)D3 is catalyzed by 1?-hydroxylase CYP27B1 to form 1,25(OH)2D3.25(OH)D3 is synthesized by vitamin D3 through CYP27A1,so that whether vitamin D3 and CYP27A1 participate in vitamin D pathway remains to be proved.In our previous study,it was indicated that the plasmatic 25(OH)D3 levels of patients with aggressive periodontitis were significantly higher than those of healthy controls.Further studies showed that the plasmatic 25(OH)D3 levels of patients with aggressive periodontitis significantly decreased after periodontal initial therapy.25(OH)D3 may be closely related to periodontitis.25(OH)D3 is produced by vitamin D3 through CYP27A1.so that the function and mechanism vitamin D3 and CYP27A1 playing in periodontal connective tissue immune defense remain to be researched.Objective:To study whether vitamin D3 and CYP27A1 participant in the vitamin D pathway in periodontal connective tissue cells.To research the anti-bacterial and antiinflammatory mechanism of vitamin D3 and CYP 27A1 in periodontal connective tissues.To evaluate the role of vitamin D3 and CYP27A1 in the immune defense of periodontal connective tissues,in order that we can deeply understand the relationship between vitamin D3 and periodontitis.And to provide theoretical support for clinical periodontal vitamin D supplementation.Materials and methods:The fi rst part:primarily culture periodontal ligament cells from three donors.The fourth or fifth generation of cells was used in the following experiments with different stimulations(vitamin D3,25(OH)D3,Pg-LPS,Pg-LPS+vitamin D3,Pg-LPS+25(OH)D3).Real-time PCR was used to detect the mRNA expression of hCAP-18,CYP27A1.IL-8 and MCP-1.ELISA was used to detect the protein levels of LL-37,IL-8 and MCP-1.CYP27A1 siRNA was used to inhibit CYP27A1 expression to observe its function.The second part:primarily culture human gingival fibroblasts from three donors.The fourth or fifth generation of cells was used in the following experiments with different stimulations(vitamin D3,TNF-?,TNF-?+vitamin D3).Real-time PCR was used to detect the mRNA expression of HBD-2,HBD-3,CYP27A1,IL-8,MCP-1,p65,VDR and TLR2.ELISA was used to detect the protein levels of HBD-2,HBD-3,IL-8 and MCP-1.Western blot was used to detect the protein levels of p65,pp65,VDR and TLR2,CYP27A1 siRNA was used to decrease CYP27A1 expression to observe its function.Results:The results of experiments of h PDLCs:compared with the control group,vitamin D3 and 25(OH)D3 could significantly increase the expression of hCAP-18/LL-37,and the effect of vitamin D3 was weaker than 25(OH)D3;With the prolonged vitamin D3 stimulation.the expression of hCAP-18/LL-37 increased.Knockdown of the expression of CYP27A1 could significantly reduce the expression of hCAP-18/LL-37 induced by vitamin D3.Vitamin D3 and Pg-LPS had no significant effects on the mRNA expression of CYP27A1.Vitamin D3 could significantly inhibit the expression of IL-8 and MCP-1 induced by Pg-LPS.The results of experiments of hGFs:vitamin D3 could significantly up-regulate the expression of HBD-2 and HBD-3.With prolonged vitamin D3 stimulation,the expression of HBD-2 and HBD-3 increased.Knockdown of C YP27A1 expression could significantly reduce the expression of HBD-2 and HBD-3 induced by vitamin D3.TNF-? could significantly increase the expression of HBD-2,HBD-3,IL-8,MCP-1,VDR,TLR2 and p65.Furtherly,vitamin D3 could significantly decrease the expressions of HBD-2,HBD-3,IL-8,MCP-1,VDR,TLR2 and phosphorylation of p65 induced by TNF-?.Conclusion:The results indicated that in periodontal connective tissue cells,vitamin D3 and CYP27A1 participated in the vitamin D pathway.Vitamin D3 could up-regulate the expression of LL-37,HBD-2 and HBD-3,and decrease the expression of IL-8 and MCP-1 induced by pro-inflammatory cytokines.Vitamin D3 may play an important role in immune defense in periodontal connective tissue. |
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