| To determine the effects of 1,25-dihydroxyvitamin D3(1,25(OH)2D3)deficiency,exogenous calcium and phosphorus,as well as aging on the regulation of alveolar bone and periodontal tissues,we generated a mouse model with vitamin D-deficient mice(1?(OH)ase-/-),and the mice fed on normal or rescue diet.Their phenotypes were analyzed in alveolar bone volume,bone forming,bone resorption,and inflammatory response.We explored the phenotype differences between wild-type and 1?(OH)ase-/-at 10 weeks of age and 12 months of age fed on normal and rescue diet,respectively,and between wild-type and la(OH)ase-/-fed on rescue diet and normal diet at 10 weeks of age,as well as between wild-type and 1?(OH)ase-/-at 12 months of age and at 10 weeks of age fed on rescue diet.Compared with wild-type littermates,total collagen area,osteoblast number and osterix positive area were all reduced significantly in la(OH)ase-/-mice under normal diet or rescue diet.1,25(OH)2D3 deficiency has no significant influence on the osteocast number and bone resorption.It is shown that the reduction in the bone volume in 1?(OH)ase-/-mice arises from the decease in bone formation,and not theincrease of bone resorption.NF-?B p65 and CD3 positive cells were all greater in 1?(OH)ase-/-mice than in wild-type littermates under normal diet or rescue diet.Moreover,the mRNA of senescence-associated inflammatory cytokines,such as IL-1?,TNF-?,MMP-3 and MMP-8,were changed accordingly.Compared with wild-type and 1?(OH)ase-/-mice fed on normal diet at 10 weeks of age,total collagen area,osteoblast number and osterix positive area were all increased significantly in wild-typ and 1?(OH)ase-/-mice fed on rescue diet at 10 weeks of age.Exogenous calcium and phosphorus significantly inhibited the osteocast number and bone resorption.NF-?B p65 and CD3 positive cells were all less in wild-type and la(OH)ase-/-mice fed on rescue diet than in wild-type and 1?(OH)ase-/-littermates under normal diet at 10 weeks of age.Moreover,the mRNA of senescence-associated inflammatory cytokines,such as IL-1?,TNF-?,MMP-3 and MMP-8,were changed accordingly.Compared with 1?(OH)ase-/-mice fed on rescue diet at 10 weeks of age,total collagen area,osteoblast number and osterix positive area were all decreased significantly in 1?(OH)ase-/-mice fed on rescue diet at 12 months of age.In addition,aging could deteriorate the inflammation of gingival tissues via NF-?B signal pathway,and upregulate the level of senescence-associated inflammatory cytokines such as IL-1?,TNF-?,MMP-3 and MMP-8.1,25(OH)2D3 deficiency contributed to the greater reduction of alveolar bone volume and bone forming,the more bone resorption,as well as the more serious inflammation.To determine whether 1,25(OH)2D3 deficiency accelerated periodontitis,we generated a model in wild-typ and la(OH)ase-/-mice with exper-imental periodontitis induced by 5-0 silk thread ligature,and their phenotypes were analyzed by histology,histopathology,and Real-time RT-PCR methods.The mice at 10 weeks of age were fed on rescue diet.After two weeks of experimental periodontitis,their phenotypes were analyzed in alveolar bone volume,bone forming,bone resorption,and inflammatory response.We explored the phenotype differences between experimental periodontitis and sham groups,and between la(OH)ase-/-and wild-type mice in experimental periodontitis groups.Compared with littermates of the same genotype in sham groups,the alveolar bone loss,total collagen area,osteoblast number,ALP and COL-I positive area were all reduced significantly,and the osteoclast number was elevated significantly in wild-type and la(OH)ase-/-mice in experimental periodontitis groups,respectively.Moreover,NF-?B p65 and CD3 positive cells were all greater in mice in experimental periodontitis groups than in sham groups.The mRNA of senescence-associated inflammatory cytokines,such as IL-1(3,TNF-a,MMP-3 and MMP-8,were changed accordingly.Compared with wild-type mice in experimental periodontitis groups,the alveolar bone loss,total collagen area,osteoblast number,ALP and COL-I positive area were all further reduced significantly,and the osteoclast number was futher elevated significantly in 1?(OH)ase-/-mice in experimental periodontitis groups.Moreover,NF-?B p65 and CD3 positive cells were all greater in 1?(OH)ase-/-mice than wild-type mice in experimental periodontitis groups.The mRNA of senescence-associated inflammatory cytokines,such as IL-1?,TNF-?,MMP-3 and MMP-8,were changed accordingly.It is demonstrated that 1,25(OH)2D3 deficiency accelerates periodontitis via the increase of the inflammatory cytokines.To determine whether exogenous 1,25(OH)2D3 improved periodontitis,we generated experimental periodontitis model induced by 5-0 silk thread ligature in 8-week-old female C57BL/6 mice,and the mice were injected the 1,25(OH)2D3 every other day in experimental periodontitis groups.Their phenotypes were analyzed by histology,histopathology,and molecular biology methods.After four weeks of experimental periodontitis,their phenotypes were analyzed in alveolar bone volume,bone forming,bone resorption,and inflammatory response.We explored the phenotype differences between mice in experimental periodontitis groups treated with 1,25(OH)2D3 and vehicle and in sham groups,respetively.It was found that exogenous 1,25(OH)2D3 had no significant influence on the serum calcium and phosphorus levels,but significantly increased the serum 25(OH)D levels.Compared with experimental periodontitis group treated with vehicle,the alveolar bone loss,total collagen area,ALP positive area were all elevated significantly in experimental periodontitis group treated with 1,25(OH)2D3.Furthermore,the osteoclast number was declined significantly in mice treated with 1,25(OH)2D3 compared with vehicle in experimental periodontitis groups.It is shown that exogenous 1,25(OH)2D3 increased the bone volume in mice with experimental periodontitis through the greater bone formation and the lower bone resorption.Besides,NF-?B p65 and CD3 positive cells were all decreased in mice treated with 1,25(OH)2D3 compared with vehicle in experimental periodontitis groups.The mRNA of senescence-associated inflammatory cytokines,such as IL-1?,TNF-?,MMP-3 and MMP-8,were changed accordingly.However,exogenous 1,25(OH)2D3 failed to improve the phenotype of the periodontitis to the shame groups.It is shown that exogenous 1,25(OH)2D3 ameliorated experimental periodontitis via the decline of the inflammatory cytokines.The study dissected the mechanism of 1,25(OH)2D3 on the homeostasis of periodontal tissues and the prevention of periodotitis through the knockout of the 1?(OH)ase gene,the model of experimental periodontits and extrogenious supply of calcium and phosphate as well as 1,25(OH)2D3.Our results indicate that endogenous 1,25(OH)2D3 plays a critical role in the anti-aging of periodontal tissues through the increase of osteoblastic bone formation and the inhibition of NF-?B pathway and inflammatory cytokines,which was calcium and phosphate dependent or independent.In addition,endogenous and exogenous 1,25(OH)2D3 are important in the prevention of perodontitis through the augmentation of osteoblastic bone formation and the inhibition of NF-?B pathway and inflammatory cytokines as well as osteoclastic bone resorption.The study demonstrated the mechanism of 1,25(OH)2D3 on the homeostasis of periodontal tissues and the prevention of periodotitis,which provided the theoretical foundation for the prevetion of the aging of periodontal tissues and periodontitis. |
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