The Effect Of 1,25（OH）₂D₃ On Porphyromonas Gingivalis-induced Inflammation And Bone Resorption

Periodontitis is a highly prevalent,chronic inflammatory disease that occurs in periodontal tissues.It causes chronic inflammation of the gingival tissue,periodontal ligament,and progressive absorption of adjacent supporting alveolar bone,which eventually results in loose teeth.Periodontitis has been defined by the medical community as the third major threat to human health after malignant tumors,cardiovascular and cerebrovascular diseases（Cao,2003）.The disease is initiated by specific bacteria within the plaque biofilm and progresses due to abnormal immune responses to these bacteria.Porphyromonas gingivalis,a gram-negative,anaerobic bacterium,is the one of the most important pathogens in the initiation and progression of chronic periodontitis,also in the lesions or active site of the periodontitis（Holt et al.,1999;Rautemaa et al.,2004）.Host cell-derived cytokines are the body’s immune response to Porphyromonas gingivalis infection,which not only plays a role in the immune defense of the host,but also causes periodontal tissue collapse during periodontal inflammation.Activated lymphocytes,macrophages and neutrophils infiltrate into the inflamed gingival tissue,while secreting pro-inflammatory cytokines,including IL-6,IL-1,TNF-a and prostaglandin E2 and so on.These cytokines can induce alveolar bone absorption（Eastcott et al.,1994;McKenna and O’Malley,2002;Froicu et al.,2003）.Finally,the typical clinical symptoms of periodontitis appear:chronic inflammation of the periodontal tissue and alveolar bone absorption,which is the main cause of adult tooth loss.With the deepening of research,more and more studies have found that vitamin D plays an important role in regulating the immune system and maintaining the balance of the immune system.Studies have shown that vitamin D,despite its traditional association with calcium and phosphate homeostasis,has been shown to exhibit immunological effects,influencing both innate and adaptive immune systems（Adams et al.,2007;Bikle,2008）.1,25（OH）₂D₃ is the final bioactive component of vitamin D in vivo,which binds to the vitamin D receptor and acts on the target genes,thus exerting its biological effect.We used Porphyromonas gingivalis-induce mouse calvarial model to investigate whether 1,25（OH）₂D₃ can reduce the damage of tissue and to explore the mechanism of 1,25（OH）₂D₃ as a adjuvant therapy for periodontitis from two aspects of anti-inflammatory and bone protection.The subject is divided into three parts:Part 1 The effect of 1,25（OH）₂D₃ on mouse calvarial modelObjective:To establish an animal model of Porphyromonas gingivalis infection and observe whether 1,25（OH）₂D₃ can protect Porphyromonas gingivalis-induced tissue damage.Materials and methods:Thirty-two male Balb/c mice were randomly divided into four groups,each group of eight:vehicle + PBS group,vehicle +Pg group,1,25（OH）₂D₃ + PBS group,1,25（OH）₂D₃ + Pg group.1,25（OH）₂D₃ was delivered in mice by oral gavage once a day.A certain concentration of Porphyromonas gingivalis was injected into the skull midline between the ears to establish a mouse calvarial infectious model,and the mice were sacrificed on the sixth day after modeling.Take the calvarial surface soft tissue photography.The complete calvarial bone was scanned with micro-CT to observe the bone destruction.The calvaria was decalcified and made into slices,and the inflammatory cell infiltration was observed by HE staining.TRAP staining was used to detect the content of osteoclasts in the calvaria.Results:Micro-CT results showed that 1,25（OH）₂D₃ reduced the degree of bone resorption of the calvaria,and the bone loss area,sagittal suture area and Tb.Sp in 1,25（OH）₂D₃+ Pg group were significantly lower than those in vehicle + Pg group.The BV/TV and Tb.N of 1,25（OH）₂D₃ + Pg group were significantly higher than those of vehicle + Pg group.In the vehicle + Pg group,there was obvious edema and abscess on the soft tissue surface in contact with the calvaria.The inflammatory reaction in the soft tissue of 1,25（OH）₂D₃ + Pg group was significantly alleviated,compared with the vehicle + PBS group and 1,25（OH）₂D₃ + PBS group.There were no significant differences between groups.HE staining results showed that inflammatory cell infiltration and bone destruction of 1,25（OH）₂D₃ + Pg group were significantly alleviated.TRAP staining results also showed that the number of positive osteoclasts in 1,25（OH）₂D₃ + Pg group was significantly lower than that in vehicle +Pg group.Conclusion:1,25（OH）₂D₃ can alleviate the bone resorption and inflammatory infiltration of mouse calvarial bone induced by Porphyromonas gingivalis.Part 2 Protective effects of 1,25（OH）₂D₃ on boneObjective:RANKL,OPG,c-Fos,NFATc1,CTSK,TRAP are key molecules that regulate osteoclast differentiation and bone destruction.The effects of 1,25（OH）₂D₃ on these molecules were detected by animal experiments,and the possible mechanism of 1,25（OH）₂D₃ on bone protective effects was discussed.Materials and methods:The expression levels of RANKL,OPG,c-Fos,NFATc1,CTSK and TRAP mRNA in the calvaria were detected by real-time PCR using the mouse calvarial infectious model.The expression of CTSK protein was detected by Western blot.The calvaria was decalcified to make slices,and the changes of CTSK were detected by immunohistochemical staining.Results:Real-time PCR results showed that 1,25（OH）₂D₃ could significantly inhibit the expression of RANKL induced by Porphyromonas gingivalis.The expression levels of c-Fos,NFATc1,CTSK and TRAP mRNA in 1,25（OH）₂D₃ + Pg group was significantly lower than those in the vehicle + Pg group.Western blot and immunohistochemistry results were consistent with real-time PCR.1,25（OH）₂D₃ could significantly decrease the expression of CTSK.Conclusion:1,25（OH）₂D₃ can regulate the expression of RANKL,c-Fos,NFATc1,CTSK and TRAP,which are the key molecule of osteoclast differentiation and activity,thus protect bone tissue.Part 3 Anti-inflammatory effects of 1,25（OH）₂D₃Objective:Proinflammatory cytokine IL-6 and anti-inflammatory cytokine IL-10 play an important role in the inflammatory process and are regulated by MAPK and NF-κB signaling pathways.The effects of 1,25（OH）₂D₃ on cytokines were examined in vivo and in vitro to evaluate whether 1,25（OH）₂D₃ has anti-inflammatory effects.Materials and methods:In vitro,Porphyromonas gingivalis-stimulated murine macrophage RAW264.7 was treated with 1,25（OH）₂D₃ and the expression of cytokines mRNA and protein was detected by real-time PCR and ELISA respectively.Western blot was used to detect the MAPK and NF-κB signal transduction.Specific inhibitors of MAPK and NF-κB signaling pathway were used to inhibit the pathways,and real-time PCR and ELISA were used to detect the effects of these inhibitors on cytokines.IL-6,IL-10,IL-1β,IL-12p40 and TNF-α mRNA expression in skin tissues were detected by real-time PCR in animal experiments.The protein expression of IL-6 and IL-10 was detected by Western blot.The expression of IL-6 and IL-10 in serum was detected by ELISA.Results:In cell experiments,1,25（OH）₂D₃ inhibited Porphyromonas gingivalis-stimulated IL-6 cytokine expression and up-regulated the expression of anti-inflammatory cytokine IL-10 in RAW264.7 cells.Further analyses showed that 1,25（OH）₂D₃ can inhibit the phosphorylation of p38,ERK1/2 and the activation of NF-κB in cells.In animal experiments,1,25（OH）₂D₃ can significantly reduce the skin tissue and serum IL-6 expression and increased IL-10 expression.Conclusion:1,25（OH）₂D₃ can regulate the expression of cytokines by blocking the expression of p38,ERK1/2 and NF-κB signaling pathways thus exerting a good anti-inflammatory effect.In conclusion,1,25（OH）₂D₃ can effectively inhibit Porphyromonas gingivalis-induced bone resorption and soft tissue destruction in the Porphyromonas gingivalis-induced mouse calvarial infectious model.The mechanism of action is derived from both bone protection and anti-inflammation of 1,25（OH）₂D₃.1,25（OH）₂D₃ can down-regulate the expression of RANKL and osteoclast-associated genes,regulate the expression of inflammatory related cytokines by blocking MAPK and NF-κB signaling pathways.