| Objective:1.By establishing three groups of SD rats(Sprague-Dawley rats)of dental caries and performing different degrees of intestinal dysbacteriosis models on SD rats,this study analyzes whether intestinal dysbacteriosis of rats can accelerate the progress of caries.2.This study explores whether intestinal dysbacteriosis in rats affects the concentration of Slg A in the saliva for the oral immune system.3.In this study,the high-throughput sequencing-based approaches were used to collect the saliva,fecal secretions and faeces of caries-free children and severe-caries children and explore whether there are differences between the community structures and diversity of the saliva and intestinal of the two groups of children.The species that may have a potential impact on the progression of caries could be therefore identified at the level of phylum,genus,and species.Methods:Experiment one1.Animal selection and grouping: 24 male Sprague–Dawley(SD)rats,19 days old,were divided into three groups,Group 1: mild intestinal dysbacteriosis group;Group2: moderate intestinal dysbacteriosis group;Group 3: control group.Modeling was conducted after 3 days of adaptive feeding.2.Construction of caries model: a high-carbohydrate diet(Keyes 2000)and oral smeared Streptococcus mutans standard bacterial solution were used to feed the three groups of SD rats at the age of 21 days for 2 months to construct the SD rats’ caries model.3.Construction of intestinal flora imbalance model: When the rats reached the age of24 days,the intestinal flora imbalance model was established by intragastric administration of ceftriaxone sodium solutions with different concentrations.Dosage:0.5ml each time twice a day with an interval of more than 6 hours.Each round last 8consecutive days.Total dosage: 2 rounds at a three-week interval.The specific dosage is: Group 1: ceftriaxone sodium 200mg/m L;Group 2: ceftriaxone sodium 320mg/m L;Group 3: saline.4.Preliminary judgment on the modeling effect of intestinal flora imbalance of SD rats: After the first round of oral gavage,the standard plate count method was used to measure the number of Bifidobacterium and E.coli colonies in the feces,and then the mental status and fecal traits of the rats and the pathological examination of the small intestine were observed,which enabled a preliminary judgment on whether the modeling is successful.5.Final judgment on the modeling effect of intestinal flora imbalance of SD rats:After the first round of oral gavage,16s-r DNA sequence analysis was performed on the fecal flora of three groups of rats.6.Preliminary judgment of oral immune function: On day 1,day 3,day 7 and day 14 after the first round of oral gavage,the indirect ELISA method was used to detect the content of SIg A in saliva.7.Scoring of the caries degree: After feeding the rats for 2 months,the three groups of rats were sacrificed.The upper and lower jaw segments of the rats were taken for dental caries scoring by Keyes’ method.Experiment two1.The clinical experiment was conducted by performing epidemiological investigation on all children(around 1,000 people)aged 3-5 years old in four kindergartens in Zunyi City.The children’s gender,age and the total number of the decayed,missing,and filled teeth(dmft index)were recorded.2.According to the dmft index,children who met the inclusion criteria were divided into caries-free group(CF group,dmft=0)and the caries-active group(S-ECC group,dmft≥10).25 children was selected from each group to collect feces and 25 children to collect saliva.3.The 16s-r DNA high-throughput sequencing approach and bioinformatics analysis methods were used to analyze the flora differences in community richness,composition and diversity of saliva and feces of two groups of children,and to further identify the species with significant differences in average relative abundance at the level of phylum,genus,and species.Results:1.Caries models of three groups of rats and different degrees of intestinal flora imbalance models were successfully constructed.2.At the 4th week,the weight of Group 2 was greater than that of Group 1 and Group 3,and was statistically significant(p<0.05);At the 8th week,the weight of Group 2 was greater than that of Group 1,and it was statistically significant(p<0.05).3.On the 1st and 7th day after antibiotic gavage in SD rats,the salivary SIg A concentration in the moderate intestinal dysbacteriosis group was lower than that in the mild intestinal dysbacteriosis group and the control group.Different structures of gut microbiota in the test and control groups,and it is statistically significant(p<0.05);on the 3rd day,the salivay SIg A concentration of the two groups of different degrees of intestinal microbiota in the moderate intestinal dysbacteriosis group was lower than the control group,and it was statistically significant(p<0.05).From the longitudinal perspective,the saliva SIg A concentration of the three groups showed an upward trend on day 1,day 3 and day 7.4.The Keyes caries score of SD rats showed the frequency of occurrence of Dx in the moderate intestinal dysbiosis group is higher than that of the control group,and it was statistically significant(p<0.05);There was no significant difference in the number of occurrences of E,Ds,and Dm among the three groups,and the difference was not statistically significant(p>0.05).5.From the analysis results of the total number of OTUs,Alpha diversity,and Beta diversity,it can be seen that there is no significant difference in the community richness,diversity and composition of salivary flora between the caries-free group and the caries-active group(p>0.05).6.It was found that the salivary flora of the caries-free group and the caries-active group had significant differences at the genus level;the Peptostreptococcus of caries-free group was significantly higher in the caries-active group(p<0.05);In the caries-free group,Dialister,Kingella,Escherichia-Shigella and Treponema were identified significantly lower than those in the caries-active group(p<0.05).The salivary flora of the caries-free group and the caries-active group had significant differences at the species level: Rothia mucilaginosa and Fusobacterium periodonticum in the caries-free group were significantly higher than the caries-active group(p<0.05);Aggregatibacter aphrophilus,Escherichia coli,Prevotella denticola,Prevotella Ioescheii and Dialister pneumosintes in the caries-free group were significantly lower than those in the caries-active group(p<0.05).7.From the analysis results of the total number of OTUs,Alpha diversity,and Beta diversity,it showed that there was no significant difference in the species diversity and composition of the salivary flora of children in the caries-free group and the caries-active group(p>0.05).The species richness in the salivary flora of children in the caries-active group was significantly higher than that in the caries-free group(p<0.05).8.Through the T-test test of the average relative abundance of the species between the groups,it was found that the fecal flora of the caries-free group and the caries-active group had significant differences at the genus level: Blautia,Eubacterium hallii group,Eubacterium eligens group in the caries-free group were significantly higher than those in the caries-active group(p<0.05);Parasutterella and Christensenellaceae R-7 group in the caries-free group were significantly lower than those in the caries-active group(p<0.05).Conclusion:1.Intestinal dysbacteriosis in SD rats may indirectly cause the salivary SIg A to decrease.2.Intestinal dysbacteriosis in SD rats will accelerate the progress of dental caries.The severer level of the dysbacteriosis can result in the faster progress of dental caries,which may be related to the decrease in the content of Bifidobacterium in the intestine.3.Caries in young children will increase the richness of children’s intestinal flora and have little effect on species diversity and composition of children’s intestinal flora.The experimental results preliminarily suggest that in the fecal microbial community of caries-active children,the increase of 2 bacteria genus: Parasutterella,Christensenellaceae R-7 group,and 3 bacterial strains: Bacteroides vulgatus,Streptococcus mutans,and Streptococcus anginosus may be related to the progression of dental caries.The experimental results preliminarily suggest that in the salivary microbial community of carries-active children,the increase of the 4 bacteria genus including Dialister,Kingella,Escherichia-Shigella and Treponema,and the 5 bacteria strains including Aggregatibacter aphrophilus,Escherichia coli,Prevotella denticola,Prevotella loescheii and Prevotella loescheii may be related to the progression of dental caries. |
