| Objective Periodontitis is a chronic inflammatory infectious disease initiated by plaque biofilm.If inflammation persists,it will eventually lead to alveolar bone destruction.It is known that Porphyromonas gingivalis(P.gingivalis)has a high detection rate in patients and is closely related to periodontitis.However,the pathological correlation between chronic periodontitis and P.gingivalis infection,especially in the inflammatory response,is still unclear.Understanding the molecular mechanism of inflammatory response induced by P.gingivalis can provide new ideas for the treatment of periodontitis.The cyclic GMP-AMP synthase(cGAS)-stimulator of interferon genes(STING)pathway is an important part of the body’s innate immune signal,which plays an important role in the host’s resistance to foreign pathogen infection or triggering its own inflammatory response.In this study,the effects of P.gingivalis on the activation of cGAS-STING pathway and the pathogenesis of periodontitis were analyzed at the cellular level and animal model to explore the possible mechanism of periodontitis caused by P.gingivalis infection.Methods(1)P.gingivalis was used to infect human gingival fibroblasts(HGFs).The effects of P.gingivalis on the survival rate of human gingival fibroblasts,the level of reactive oxygen species,the key molecules in the cGAS pathway(cGAS,STING),inflammatory factors(IL-6,IL-1β,TNF-α,IFN-β),and osteoclast-related molecules RANKL expression were detected.(2)A mouse model of experimental periodontitis infected with P.gingivalis was constructed.The morphological changes of alveolar bone and the positive rate of osteoclasts were observed.The osteoclast-related genes(receptor activator of nuclear factor kappa-B ligand(RANKL),matrix metalloprotein-9(MMP-9),cathepsin K(CTSK))were detected.The expression levels of interferon-β(IFN-β),tumor necrosis factor-a(TNF-α),interleukin-6(IL-6),interleukin-1β(IL-1β)and other inflammation-related genes were detected.(3)Animal models of periodontitis were established in WT and STING-deficient(StingGt)mice.WT periodontitis mice had a large number of inflammatory cell infiltration,while STING-deficient mice had only mild reaction.The level of inflammatory factors secreted by STING-deficient mice decreased;the expression level of IFN-β decreased by 1.6 times.F4/80+macrophage infiltration was significantly enhanced,CD206+(M2 marker)cells increased,and CD11c+(M1 marker)cells decreased.The levels of chemokines(CCR2 and CCL2)were significantly decreased.Alveolar bone resorption decreased;the expression of RANKL decreased.The above results indicate that in the cGAS-STING signaling pathway induced by P.gingivalis infection,the loss of STING function reduces the expression of type I interferon genes.(4)STING inhibitor(SN-011)or agonist(SR-717)was used to treat periodontitis mice.Compared with PBS group,SN-011 significantly reduced the production of inflammatory cytokines and osteoclast formation(P<0.01).The expression level of STING protein in SR-717 group was increased,while that in SN-011 group was decreased.The levels of inflammatory cytokines in the SR-717 treatment group were significantly increased(P<0.01),while the levels of inflammatory cytokines in the SN011 treatment group were significantly decreased(P<0.01).In addition,the number of inflammatory cells in the SN-011 treatment group decreased,while a large number of inflammatory cells infiltrated in the periodontal tissue of the SR-717 treatment group,and the histological scores were significantly different(P<0.01).The number of M1 macrophages(CD11c+F4/80+)in the SR-717 treatment group was significantly increased,and the number of M2 macrophages(CD206+F4/80+)was decreased,while the SN-011 treatment group showed inhibition of M1 macrophage polarization and promotion of M2 macrophage polarization.The levels of chemokines(CCR2 and CCL2)in the SR-717 treatment group were significantly increased,while the levels of chemokines in the SN-011 treatment group were significantly decreased.The above results indicate that STING inhibitor SN-011 can reduce P.gingivalis-induced alveolar bone resorption.Results(1)The results of cell level showed that the ROS level of HGFs infected with P.gingivalis at 6 h was significantly higher than that at 0 h and 4 h.After P.gingivalis infected HGFs,the expression levels of cGAS and STING genes and proteins were significantly up-regulated.Similarly,the expression levels of IL-6,IL-1β,TNF-α and IFN-β at 6 h after P.gingivalis infection were significantly higher than those at 0 h and 4 h.RANKL mRNA level was 1.3 times higher than that of untreated cells,but there was no significant difference.The expression of RANKL was further detected by Western blot,and the results showed that the expression level of RANKL protein was increased.The above results indicate that P.gingivalis can indeed promote the activation of cGASSTING pathway in HGFs and induce the expression of inflammatory cytokines.(2)In the animal model of periodontitis,inflammation occurred in the gingival tissue of periodontitis mice,and the high infiltration of inflammatory cells in the lamina propria,the decrease of alveolar bone height and the increase of absorption were obvious.In addition,the number of TRAP-positive osteoclasts in the alveolar bone on one side of periodontitis increased significantly.The expression of osteoclast-related genes(CTSK,MMP-9,RANKL)and inflammation-related genes(IL-1β,IL-6,TNF-α)was higher than that of the control side(P<0.05).The buccal side of periodontitis model mice showed increased alveolar bone resorption,cribriform mesh,exposed root,decreased height of the hemi-maxillae were determined based on the distance between the cementoenamel junction(CEJ)and the alveolar bone crest(ABC)(P<0.05).The mRNA expression levels of P.gingivalis virulence factors(RgpA and KGP)were significantly increased.The production of ROS in mouse gingival cells increased;the expression of cGAS and STING protein was significantly increased.The expression levels of pro-inflammatory cytokines(IL-6,IL-1β,TNF-α,IFN-β)in serum were significantly increased.The expression level of IFN-P was increased by 5 times.The level of RANKL mRNA was twice that of the control group.These results suggest that P.gingivalis infection activates the cGAS-STING pathway,leading to the production of type I IFN genes and a variety of inflammatory cytokines that contribute to osteoclast differentiation and activation.(3)An animal model of periodontitis was constructed in WT and STING-deficient(StingGt)mice.WT periodontitis mice had a large number of inflammatory cell infiltration,while STING-deficient mice had only mild reaction.The level of inflammatory factors secreted by STING-deficient mice decreased;the expression level of IFN-β decreased by 1.6 times.F4/80+macrophage infiltration was significantly enhanced,CD206+(M2 marker)cells increased,and CD11c+(M1 marker)cells decreased.The levels of chemokines(CCR2 and CCL2)were significantly decreased.Alveolar bone resorption decreased;the expression of RANKL decreased.The above results indicate that in the cGAS-STING signaling pathway induced by P.gingivalis infection,the loss of STING function reduces the expression of type Ⅰ interferon genes.(4)STING inhibitor(SN-011)or agonist(SR717)was used to treat periodontitis mice.Compared with PBS group,SN-011 significantly reduced the production of inflammatory cytokines and osteoclast formation(P<0.01).The expression level of STING protein in SR-717 group was increased,while that in SN-011 group was decreased.The levels of inflammatory cytokines in the SR-717 treatment group were significantly increased(P<0.01),while the levels of inflammatory cytokines in the SN-011 treatment group were significantly decreased(P<0.01).In addition,the number of inflammatory cells in the SN-011 treatment group decreased,while a large number of inflammatory cells infiltrated in the periodontal tissue of the SR717 treatment group,and the histological scores were significantly different(P<0.01).The number of M1 macrophages(CD11c+F4/80+)in the SR-717 treatment group was significantly increased,and the number of M2 macrophages(CD206+F4/80+)was decreased,while the SN-011 treatment group showed inhibition of M1 macrophage polarization and promotion of M2 macrophage polarization.The levels of chemokines(CCR2 and CCL2)in the SR-717 treatment group were significantly increased,while the levels of chemokines in the SN-011 treatment group were significantly decreased.The above results indicate that STING inhibitor SN-011 can reduce P.gingivalis-induced alveolar bone resorption.Conclusion These results together confirm our hypothesis that P.gingivalis activates the cGAS-STING signaling pathway,leading to the subsequent expression of proinflammatory cytokines and type I IFN genes.These pro-inflammatory cytokines enhance the activation of macrophages and the production of osteoclasts,thereby promoting the pathogenesis of periodontitis.The small molecule inhibitor SN-011 of STING can reduce the activation of macrophages and alleviate the destruction of alveolar bone.These findings may help us better understand the pathogenesis of P.gingivalisinduced periodontitis and provide clues for the prevention and treatment of periodontitis. |
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