Effect Of Rat Periodontitis On The Diversity Of Intestinal Flora

BACKGROUND:One hundred trillion microbes are colonized in the human intestines,and they are called intestinal flora.With the rapid development of sequencing technology,more and more intestinal flora research,many people think that intestinal flora is more like an organ,because intestinal flora can prevent intestinal pathogenic bacteria infection It can extract nutrients and energy from our diet and play a role in normal immune function.However,if there is a dysbiosis,some carcinogens may also be produced,or the metabolism and transformation of substances such as sugar,fat,protein,and inflammatory factors that affect the host may cause disease or discomfort,which will adversely affect human health.Periodontitis is a complex inflammatory disease related to the dysfunction of dental biofilms,which can cause long-term chronic inflammation of periodontal supporting tissues.There is a mechanism between periodontitis and other diseases,namely periodontal pathogens and their pathogenicity Factors can reach distant organs and then cause systemic inflammation and affect the host's immune defenses.In recent years,there have been more and more studies on periodontitis and systemic diseases,but there are few studies on the relationship between intestinal flora and periodontitis,and there is a lack of related basic research.OBJECTIVES: This study intends to explore the changes in the number and structure of the main flora in the intestine of the rat model of periodontitis using 16 S rDNA high-throughput sequencing technology through animal experiments.To explore the effect of periodontitis on rat intestinal flora from the composition and abundance of intestinal flora.METHODS: Twenty-two 8-week-old SPF SD rats were selected and randomly divided into periodontitis group and control group.After adaptive feeding,the rats in the periodontitis group were anesthetized by intraperitoneal injection,and the teeth were wrapped around the neck by the "8" ligation method.If any ligature wire is damaged or falls off,re-ligate it in time.Check the periodontal status once a week.All animals were sacrificed at 4 weeks after surgery.Gingival crevicular fluid from each rat was collected and stored in liquid nitrogen for cell and molecular biological detection.The maxillary bilateral alveolar bone was collected,one side retained intact periodontal tissue,and the other side removed the periodontal soft tissue on the surface of the alveolar bone for Micro-CT detection and hematoxylin-eosin staining.Fecal specimens from the small intestine of rats were collected and used to detect intestinal flora.After processing the feces of rats,the DNA of intestinal flora was extracted,and the qualified DNA was selected for PCR amplification,purification,library construction and sequencing.After filtering and connecting the sequenced data,we analyzed the OTU?Operational Taxonomic Units?,abundance and species composition between the two groups,and performed alpha diversity analysis and beta diversity analysis between the two groups.The statistical analysis of this study used both quantitative and qualitative methods.The data were recorded by Excel 2016 and processed by R software.Intestinal flora analysis mainly used USEARCH?v11.0.667?,UCHIME?v4.2.40?,Fast Tree?version 2.1.3?,R?v3.1.1?,FLASH?v1.2.11?and RDP classifier Bayesian algorithm Wait for statistical analysis and image drawing.RESULTS: According to the Micro-CT test,the alveolar bone of the control group without periodontal experiment was not significantly absorbed,while the proximal and distal alveolar crests and root bifurcations of the second molar in the periodontitis group were obviously absorbed.The results of hematoxylin-eosin staining showed that the gingival sulcus epithelium in the periodontitis group eroded and formed a network in the connective tissue.The main fibers were destroyed and broken,the epithelial adhesion was lost,and the alveolar bone was resorbed.In the control group,the gingival epithelium was intact,with no obvious pathological changes in the attached epithelium,the periodontal ligament fibers were neatly arranged,and the inherent alveolar bone morphology and structure were intact and without absorption damage.The results showed that the periodontitis group model was successfully constructed.The results of 16 S rDNA high-throughput sequencing showed that a total of 952 OTUs were obtained.Compared with the control group,the OTU of the periodontitis group decreased;the cumulative OTU curve shows that the number of experimental samples of our two groups is sufficient;The OTU PCA analysis and OTU PLS-DA analysis showed that there was a significant difference between the periodontitis group and the control group in the PC1 dimension and the x dimension.Based on the OTU data,this experiment analyzed the composition of the community at six levels: Phylum,Class,Order,Family,Genus,and Species.The results showed that the cyanobacteria,4C0d₂,Class YS2,Clostridiaceae,Clostridium butyricum,Bacteroides fragilis and butyric acid producing bacteria were statistically different?P <0.05?.Although other bacteria did not show statistical differences,the predominant species in the intestinal tract,Bacteroides phylum and Paecilomyces superior periodontitis group showed a downward trend,Proteobacteria and Actinomycetes show an upward trend.The results of the alpha diversity analysis show that the sample data is sufficient and reliable,and there is no statistical difference,but the trend of the decline of the alpha diversity analysis in the periodontitis group can be seen.The analysis between the species shows that the clustering of the samples between the two groups is not very good,but the difference between the two groups of samples can still be seen between the two.CONCLUSIONS: According to the results of 16 S rDNA high-throughput sequencing,there is a certain relationship between periodontitis and intestinal flora,and periodontitis will reduce the diversity of intestinal flora.