| Periodontitis is one of the six most chronic inflammatory diseases in the world,resulting in persistent inflammation and consequent loss of alveolar bone due to micro-ecological imbalances in the mouth.The prevalence of periodontitis among middle-aged people aged 35-44 in China is about 33%,which seriously affects the oral health and daily life of the affected population.Removal of pathogenic bacteria and elimination of excessive inflammation is the key to periodontitis treatment.Limosilactobacillus fermentum CCFM1139 with the potential to alleviate periodontal disease was screened in our laboratory.In vitro,it significantly reduced the amount of mixed biofilm of periodontal pathogenic bacteria and could regulate the expression of inflammatory factors and tight junction proteins in cells.In vivo,it significantly reduced the amount of periodontal pathogenic bacteria colonization and alveolar bone resorption in rats,inhibited excessive host immune regulation.The present study was designed to investigate the clinical effects of Limosilactobacillus fermentum CCFM1139 as an adjunct to basic periodontal therapy and to investigate its mechanism of action in alleviating periodontitis.The main study components and results are as follows:(1)The correlation of clinical parameters of periodontitis with oral flora and metabolites was first analyzed to identify subgingival flora and salivary metabolites associated with the progression in patients with stage II periodontitis.Bacteria of the genera Porphyromonas,Treponema 2 and Prevotella were detected more frequently in the focal areas of periodontitis,and Saccharibacteria bacterium also played an important role in the development of periodontitis.Metabolites that showed a significant positive correlation with a number of periodontal clinical parameters were 5-Aminovaleric acid,Methyl indole-3-acetate,N6-acetyl-L-lysine and Ethyl palmitoleate,etc.Metabolites that promoted the development of periodontitis also showed a strong positive correlation with periodontal pathogens,and these genera and metabolites can be used as biomarkers to predict the development of stage II periodontitis.(2)Patients with stage II periodontitis were subsequently recruited for a 30 day randomized,double-blind,placebo-controlled clinical trial.Compared to placebo,subjects who took oral CCFM1139 live strain chewable tablets showed significant improvements in probing depth and clinical attachment loss related indicators.CCFM1139 inactivated strain significantly reduced the secretion of pro-inflammatory factors(MMP8 and IL-1β)by 40.8%and 16.9% respectively.The oral flora in the CCFM1139 live and inactivated strain group was significantly different compared to pre-intervention.CCFM1139 live and inactivated strain antagonized the growth of periodontal pathogens and maintained the ecological balance of the subgingival flora by increasing the relative abundance of oral commensal bacteria.CCFM1139 inactivated strain showed significant inhibition of a number of periodontal pathogens(Porphyromonas spp.,Treponema spp.and Candidatus Saccharibacteria bacterium spp.).The results of the non-targeted metabolomics showed that the composition and content of salivary metabolites in the subjects after the CCFM1139 live and inactivated strain interventions were significantly different from those before the interventions,and that the metabolic pathways enriched by the CCFM1139 live and inactivated strain largely overlapped,including the Arginine synthesis pathway,Alanine,Aspartate and Glutamate metabolism,and D-glutamine and D-glutamate metabolism.The multidimensional index evaluation of the clinical effects of Limosilactobacillus fermentum CCFM1139 on periodontitis showed that live CCFM1139 strain achieved relief of periodontitis by maintaining oral flora homeostasis and regulating the oral metabolic environment,while CCFM1139 inactivated strain slowed down the periodontitis process by regulating host immunity,thereby inhibiting the growth of periodontal pathogens and altering the composition of salivary metabolites.Also,both CCFM1139 live and inactivated strain reduced the levels of biomarkers associated with periodontitis.The abundance of periodontal pathogens was reduced after CCFM1139 live strain intervention,with significant differences in Prevotella spp.CCFM1139 inactivated strain significantly reduced the abundance of five periodontal pathogens.The metabolites of microbial catabolism of amino acids were significantly reduced after Limosilactobacillus fermentum CCFM1139 intervention,with the metabolites inhibited by CCFM1139 live and inactivated strain being 5-Aminovaleric acid and Methyl indole-3-acetate,respectively.(3)Finally,animal and cellular models of periodontitis were constructed to investigate the mechanism of action of Limosilactobacillus fermentum CCFM1139 in alleviating periodontitis.The results showed that the live strain could alleviate periodontitis in rats in three aspects: pathogenic bacteria colonization,host immune response and alveolar bone changes.The live strain reduced colonization of Fusobacterium nucleatum by 1-2 orders of magnitude in the rat oral cavity,competed cellularly with Porphyromonas gingivalis for adhesion sites and inhibited virulence gene expression,significantly down-regulated the expression of the pro-inflammatory factor IL-1β in gingival tissue and serum,reduced the recruitment of inflammatory factors at periodontal tissue and reduced alveolar bone loss by25.6% in rats.The effect of the inactivated strain on periodontitis was mainly focused on the modulation of excessive host immune response,reducing IL-1β levels in gingival tissue from39.54 pg/mg to 17.95 pg/mg and in serum from 28.80 pg/m L to 12.50 pg/m L.The inactivated strain also significantly reduced bone loss,resulting in a 15.9% reduction in alveolar bone loss in rats.In conclusion,Limosilactobacillus fermentum CCFM1139 can effectively relieve periodontitis.Its live strain competes with pathogenic bacteria for adhesion sites,increases the abundance of oral commensal bacteria and regulates oral flora homeostasis;At the same time,the inactivated strain regulates excessive host immune and reduces the abundance of pathogenic bacteria,thereby alleviating periodontitis.This study provides a theoretical basis and support for the development of Limosilactobacillus fermentum CCFM1139 as a probiotic product for the relief of periodontitis. |
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