The Role And Mechanism Of Clostridium Butyricum MIYAIRI 588 On Alveolar Bone Resorption In Periodontitis Mice With Diabetes Mellitus

Background:Periodontitis is a chronic inflammatory disease whose progression and severity are influenced by several systemic diseases,such as: obesity,diabetes and inflammatory bowel disease.There is a bidirectional relationship between periodontitis and diabetes mellitus.Diabetes increases the prevalence and severity of periodontitis and predisposes to clinical symptoms,such as severe alveolar bone resorption,loose and displaced teeth and periodontal abscesses.Patients with diabetes mellitus-associated periodontitis(DMP)have more severe periodontal symptoms and are more prone to recurrence,making it more difficult to manage oral health in clinic.How to effectively maintain good periodontal status in patients with DMP remains an urgent clinical and scientific problem.Antibiotics,a common adjuvant,have disadvantages such as drug resistance,disrupted intestinal flora and bone loss.Therefore,it is urgent to explore new adjuvant treatment in clinic.There is growing evidence that gut microbes play an important role in the progression and treatment of periodontitis accompanied with systemic disease as well as intestinal disease.Gut metabolites enter the circulation through the intestinal epithelial barrier and have a key role in the actual function of the host phenotype-microbiome,acting as a bridge between the gut and distal parenteral organs.Clostridium butyricum MIYAIRI 588(CBM588)is a safe,non-toxic,non-pathogenic over-the-counter probiotic used clinically for the treatment of gut diseases and gut dysbiosis,such as childhood diarrhoea.CBM588 regulates host gut microecology and metabolism,exerting a protective effect on distal parenteral organs.However,no studies have been reported on the use of CBM588 in the treatment of alveolar bone destruction in DMP.Therefore,it is of great scientific and clinical importance to investigate the role and mechanism of CBM588 in the treatment of DMP.Purpose:A model of type 2 DMP was constructed and supplemented with CBM588 to explore its effect on alveolar bone destruction;then,the mechanism of the protective effect of CBM588 was investigated based on changes in gut microbiology and serum metabolism;on the basis of this,the role and mechanism of serum metabolites in alleviating alveolar bone resorption in DMP were verified,further providing new strategies for the clinical adjunctive treatment of DMP.Methods:1.The preliminary study on the effect of CBM588 on alveolar bone resorption in mice with DMP:(1)High-fat diet combined with streptozotocin(STZ)injection was used to establish a type 2 diabetic mouse model,on the basis of which a periodontitis model was constructed and its effect on DMP was explored by oral gavage with CBM588;(2)The effect of CBM588 on blood glucose was evaluated by fasting blood glucose(FBG),oral glucose tolerance test(OGTT),and insulin tolerance test(ITT)assays;(3)Micro-CT analysis,H&E,and TRAP staining were performed to analyze the alveolar bone destruction and osteoclast activity.2.Influence of CBM588 on the gut microecology of mice with DMP:(1)Fecal 16 S r RNA sequencing was conducted to explore the effect of CBM588 on the microbial composition of the gut;(2)H&E staining,q RTPCR,and immunohistochemical staining in colonic tissues were used to explore the effect of CBM588 on intestinal inflammation and gut barrier.3.Effect of CBM588 on serum metabolism in mice with DMP:(1)Untargeted metabolomics was carried out to analyze the effect of CBM588 on serum metabolism,and screening for key differentially expressed metabolites;(2)Based on the results of non-target metabolomics,total antioxidant capacity(T-AOC)kit and malondialdehyde(MDA)kit were used to investigate the effect of CBM588 on oxidative stress in DMP.4.Validation of the serum metabolite 4-HBA to alleviate alveolar bone destruction in mice with DMP:(1)A mouse model of type 2 DMP was constructed and its effect on alveolar bone loss in DMP was investigated by intraperitoneal injection of key differentially expressed metabolite 4-Hydroxybenzenemethanol(4-HBA)in serum;(2)T-AOC kit and MDA kit were used to explore the effect of 4-HBA on oxidative stress in mice with DMP.5.The preliminary study on the mechanism of the serum metabolite4-HBA in inhibiting the alveolar bone destruction of mice with DMP:(1)CCK-8 assay was used to detect the effect of different concentrations of 4-HBA on the activity of bone marrow-derived macrophages(BMDMs);(2)Reactive oxygen species(ROS),q RT-PCR,WB as well as immunohistochemical staining were used to investigate whether 4-HBA regulates Nrf2 signaling to promote the expression of antioxidant proteins to alleviate oxidative stress;(3)q RT-PCR,WB as well as immunohistochemical and immunofluorescence staining were performed to examine whether 4-HBA regulated the polarization of BMDMs and inflammatory factors.Results:1.A type 2 diabetic model was successfully constructed by high-fat diet combined with STZ injection,exhibiting FBG ≥ 11.1 mmol/L.FBG was reduced in DMP mice after 2 weeks of CBM588 treatment.OGTT and ITT tests indicated that CBM588 could improve glucose metabolism capacity and insulin resistance of the DMP mice.Micro-CT and H&E staining further showed that the alveolar bone resorption was worsen in the DMP group than in the periodontitis group,and that CBM588 alleviated alveolar bone resorption in the DMP mice.TRAP staining showed an increase in osteoclasts in the DMP group compared to the periodontitis group,which was reduced by supplement with CBM588.2.The results of fecal 16 S r RNA sequencing showed that diabetes aggravated gut dysbiosis in periodontitis and CBM588 treatment restored intestinal microbial homeostasis in mice with DMP.q RT-PCR and colonic tissue staining showed that diabetes aggravated intestinal inflammation and reduced intestinal TJPs expression in periodontitis,while CBM588 treatment alleviated intestinal inflammation and increased the expression of TJPs in mice with DMP.3.Untargeted metabolomic assays revealed that CBM588 supplementation altered serum metabolism in mice with DMP,significantly elevating the concentration of 4-HBA and decreasing levels of oxidized glutathione in serum.Based on the results of non-target metabolomics,it was further found that CBM588 treatment increased T-AOC as well as reduced MDA levels in mice with DMP.4.Micro-CT and H&E staining showed that 4-HBA treatment mitigated alveolar bone resorption in mice with DMP.TRAP staining showed that 4-HBA reduced osteoclasts in the alveolar bone of mice with DMP.In addition,4-HBA treatment increased T-AOC and decreased MDA in mice with DMP.5.4-HBA(0-1000 μM)had no effect on the activity of BMDMs.Hyperglycemic inflammatory conditions promoted ROS production in BMDMs,whereas 4-HBA treatment reduced ROS production in BMDMs.q RT-PCR and WB results showed that 4-HBA promoted the expression of Nrf2 and its downstream antioxidant proteins HO-1 and NQO-1 in BMDMs.Furthermore,hyperglycemic inflammatory conditions promoted the expression of inflammatory factors and the polarization of M1 macrophages,which were reversed by 4-HBA.Moreover,4-HBA promoted the polarization of M2 macrophages under hyperglycemic inflammatory conditions.Conclusions:CBM588 could alleviate alveolar bone loss in DMP by elevating 4-HBA in serum through regulating intestinal microecology;the serum metabolite 4-HBA could inhibit oxidative stress by activating the Nrf2 signaling cascade,regulate the polarization of the BMDMs and exert antiinflammatory effects in vitro and in vivo.This study identified new roles for CBM588 in modulating gut microbes and 4-HBA as a beneficial metabolite to improve DMP,providing new insights and strategies to explore adjuvant treatment modalities and their mechanisms for DMP.