Effects Of Saliva Transplantation From Periodontitis Patients On Gut Microbiota And Hyperlipidemia In ApoE^-/- Mice

[Objective]The purpose of this study is to firstly explore the differences of microbiota in salivary and fecal samples from patients with severe generalized periodontitis,and then investigate whether the periopathogenic microbiota in saliva can enter and survive in the gastrointestinal tract of C57BL/6J mice via gavage,and further could affect the gut microbiota and gut barrier,and finally assess the effects of human periopathogenic microbiota on the gut microbiota and hyperlipidemia in ApoE^-/-mice[Methods]Salivary and fecal samples were collected from 18 periodontal healthy volunteers（PH group）and 21 patients with severe and generalized periodontitis（SP group）for this study.16S rRNA high-throughput sequencing technology was used to analyze the microbiota changes in both saliva and feces,which including microbiota diversity,microbial composition,linear discriminant analysis,and so onHuman periopathogenic microbiota was stained with carboxyfluorescin diacetate succinimidyl ester（CFSE）and then given to C57BL/6J mice by gavage.The mean of fluorescence intensity of contents in the stomach,small intestine,cecum and colon were measured after 2 h and 24 h respectively to analyze whether the human periopathogenic microbiota could survive in the intestine and record the site as wellTo further explore the effects of human periopathogenic microbiota on body weight,blood lipid,gut microbiota and gut barrier,human periopathogenic microbiota from both PH and SP group were given to C57BL/6J（C-PH group and C-SP group）and ApoE^-/-mice（A-PH group and A-SP group）by gavage.The body weight and blood lipids were measured after 2 weeks.Gut microbiota were analyzed by 16S rRNA high-throughput sequencing technology.Hematoxylin-Eosin（HE）staining,immunofluorescence staining and Quantitative real time polymerase chain reaction（qPCR）were used to analyze the changes of histological structure of gut barrier,expression of intestinal inflammatory factors and macrophage surface markers[Results]Periodontitis induced microbiota changes in both salivary and fecal samples Compared to the microbiota in salivary samples of the PH group,the species richness and diversity were significantly increased in the SP group.The periopathogenic microbiota（mainly red complex）were also significantly increased,while the periodontal health-related microbiota were significantly reduced as hypothesized in the SP group.The species richness and diversity of the fecal microbiota were slightly affected in the SP group,and there were differences between groups.For fecal microbiota,the abundance of Firmicutes was increased,and microbiota related to inflammation（such as Erysipelotrichaceae）and short chain fatty acids（SCFAs）synthesis（such as Lachnospiraceae）were also enriched in the SP group.The microbiota present in salivary samples were also found in the fecal samples among 11 out of 21 patients,which suggests that periopathogenic microbiota can pass through the intestine and be excreted through the fecesIt was found that human periopathogenic microbiota mainly survived in the cecum after gavage administration,which induced the weight gain and changes of gut microbial composition in the C-SP group,while no significant difference was found in blood lipids.The relative abundance of Firmicutes and microbiota related to inflammation（such as Porphyromonadaceae）increased,while microbiota related to health（such as Akkermansia）decreased in the C-SP group.The histological structure of gut barrier was changed and the expression of Zonula Occludens-1（ZO-1）in gut barrier decreased in the C-SP group.The expression of intestinal inflammatory factors,such as Tumor necrosis factor-α（TNF-α）and Interleukin-1β（IL-1β）increased significantly along with an increase of M1 macrophages surface marker（Nitric Oxide Synthetase 2）,while M2 macrophages surface marker（Arginase-1 and Mannose receptor C type-1）decreasedHowever,changes of gut microbiota and phenotype of ApoE^-/-mice were not the same as those of C57BL/6J mice.The body weight,blood total cholesterol（TC）,and low-density lipoprotein（LDL）all increased in the A-SP group.The relative abundance of Firmicutes decreased in the A-SP group,which was mainly caused by the decreased relative abundance of Lachnospiraceae.However,Bacteroidetes increased in the A-SP group.Changes of microbiota related to inflammation and health as well as gut histological structure,the expression of inflammatory cytokines and cell surface markers of macrophage in the ApoE^-/-mice were similar to those in the C-SP group.However,the expression of ZO-1 in the gut barrier increased.The function prediction analysis of intestinal microbiota found the oxidative phosphorylation and biosynthesis of lipopolysaccharide were enriched in the A-SP group[Conclusions]The relative abundance of inflammation related microbiota in saliva and fecal samples from patients with severe and generalized periodontitis increased significantly compared to periodontal healthy controls.After the gavage administration of human periopathogenic microbiota,it was found that the periopathogenic microbiota can survive in the cecum,resulting in the disorder of gut microbiota,changes to the histological structures of gut barrier,intestinal inflammation,and macrophage related immune system changes in C57BL/6J mice Similar intestinal structure changes and microbiota dysbiosis was also observed by using an ApoE^-/-mouse model,with increased weight and blood lipids,which indicates that the saliva microbiota of periodontitis have effects on gut microbiota diversity and hyperlipidemia.