Lactobacillus Casei Attenuates Alveolar Bone Loss In Diabetic Periodontitis

Backgrounds:Periodontitis is a chronic inflammatory disease,which causes the destruction of periodontal tissue and tooth loss.Diabetes mellitus（DM）is a risk factor for periodontitis,which leads to hyperglycemia,over-expressed inflammatory cytokines,and increased oral microbiota pathogenicity.DM would accelerate the development and onset of periodontitis,as well as increase the difficulty of treatment for periodontitis.Conventional therapies for periodontitis mainly focused on the mechanical removal of calculus and plaque biofilm,which have limited efficacy against diabetic periodontitis（DP）.It is suggested that gut microbiota plays an important role in bone metabolism.Lactobacillus casei（L.casei）is a common probiotic and has a potent role in gut microbiota regulation.However,it is still unclear whether L.casei could alleviate alveolar bone loss in DP by regulating gut microbiota.Objectives:The present study aims to explore the role of L.casei on alveolar bone loss in DP by regulating gut microbiota and the absorption of metabolites and elucidate the mechanism.Methods:1.A DP mouse model was established,and L.casei or PBS was intragastrically administrated to mice for 4 weeks.Micro-computed tomography（Micro-CT）and hematoxylin and eosin（H&E）staining were performed to evaluate the effect of L.casei on alveolar bone loss in DP mice.Tartrate-resistant acid phosphatase（TRAP）staining was used to detect the number of TRAP⁺osteoclasts in the alveolar bone of DP mice.The expression levels of osteoprotegerin（OPG）and receptor activator of nuclear factor kappa B ligand（RANKL）in the periodontal tissue were determined by immunohistochemical（IHC）staining.An oral glucose tolerance test（OGTT）was conducted to investigate the role of L.casei in the glucose tolerance of DP mice.2.Fecal 16S r RNA sequencing was performed,andα-diversity analysis,β-diversity analysis,community diversity analysis,and differential species analysis were used to explore the effect of L.casei on the composition of gut microbiota.H&E staining and q RT-PCR were performed to detect the role of L.casei in intestinal structure and function.3.Untargeted metabolomics of serum and feces was performed.The differential metabolites that satisfied both variable importance in projection（VIP）>2.0 and P<0.05 were screened out.α-tocopherol acetate（α-TA）was chosen for further study for its highest increase in serum.4.A DP mouse model was established,and the mice received intraperitoneal injections ofα-TA or saline for three weeks.Micro-CT and H&E staining were performed to evaluate the effect ofα-TA on alveolar bone loss of DP mice.TRAP staining was used to detect the number of TRAP⁺osteoclasts in the alveolar bone of DP mice.The expression levels of OPG and RANKL in the periodontal tissue were determined by IHC staining.5.Human periodontal ligament cells（h PDLCs）and human gingival fibroblasts（HGFs）were isolated and the in vitro DP model was established,to detect the role ofα-tocopherol（α-T,the in vivo hydrolysate ofα-TA）in the osteogenic differentiation of h PDLCs and the inflammatory responses of HGFs under high glucose and lipopolysaccharide（LPS）stimulation.6.Transcriptomic analysis and IHC staining were performed to investigate the major affected signaling pathways upon L.casei andα-TA administration.The expression of p-JAK2,JAK2,STAT3,and p-STAT3 in high glucose-and LPS-stimulated HGFs was detected by western blot.Finally,STAT3 was over-activated in HGFs by transducing adenovirus with GFP–tagged STAT3^Y705D to confirm the involvement of the STAT3 signaling pathway in the effect ofα-T on the inflammatory response of high glucose-and LPS-stimulated HGFs.Results:1.Intragastric administration of L.casei can alleviate alveolar bone loss,decrease the number of TRAP⁺osteoclasts in the alveolar bone,increase OPG expression,and reduce the expression of RANKL in the periodontal ligament of DP mice.Moreover,L.casei can also increase the glucose tolerance of DP mice.2.Intragastric administration of L.casei can alter the gut microbiota composition and improve intestinal structure and functions.3.Intragastric administration of L.casei alters metabolic profiling and promotes the enrichment of serumα-TA.4.Administration ofα-TA alleviated alveolar bone loss,decreased the number of TRAP⁺osteoclasts in the alveolar bone,increased OPG expression,and reduced the expression of RANKL in the periodontal ligament of DP mice.5.α-T,the in vivo hydrolysate ofα-TA,can promote the osteogenic differentiation of h PDLCs,improve the migration and suppress the inflammatory responses of HGFs under high glucose and LPS stimulation.6.Administration of L.casei andα-TA can inhibit the STAT3 signaling pathway in the gingival tissue of DP mice.When STAT3 was over-expressed in HGFs,the expression of IL-1βand IL-6 was increased under high glucose and LSP conditions;however,this change could be attenuated byα-T.Conclusions:In the present study,it was found that administration of L.casei attenuated alveolar bone loss,positively modulated the composition of the gut microbiota,and improved the intestinal structure and function of PD mice.Additionally,L.casei mediated the increased enrichment of serumα-TA,which inhibited the STAT3 signaling pathway and contributed to the alleviation of alveolar bone loss in PD.Data from this study may provide new insights and ideas for the clinical treatment of periodontitis in diabetic patients in the future.