| Objective : To use high-throughput sequencing(HTS)technology to analyzed the characteristics of oral and intestinal microbial communities in a rat model of severe periodontitis with Alzheimer’s disease,Preliminarily explore the possible role of the microbial-gut-brain axis mechanism in the relationship between periodontitis and AD,in order to provide new ideas and basis for the mechanism and prevention of AD.Methods: Forty 8-week-old male SD rats were randomly divided into 4 groups: control group(H),simple periodontitis group(P),Alzheimer’s disease group(A),periodontitis combined with Alzheimer’s Murder disease group(AP);the bacteria can be divided into oral bacteria group(group o)and intestinal bacteria group(group f)according to different sampling locations.After one week of adaptive feeding in the AP and P groups,the bilateral maxillary first and second molars were ligated with silk thread on the neck of the teeth on the basis of separated the gums;45 days after the periodontal ligation,the A and AP groups were injected into the bilateral ventricle 25μL of μg/μL Aβ25-35 solution was used to establish AD model.After 14 days,the Morris water maze experiment was used to observe the behavioral changes of the rats;after the behavioral experiment,samples of oral flora and intestinal flora were collected and stored in the refrigerator at-80℃;the rats were sacrificed,and the brain and alveolar bone were taken;HE staining and stereo microscope observation of alveolar bone resorption;HE staining and Nissl(Nissl)staining to observe the morphology and number of hippocampal neurons.Each sample was subjected to high-throughput sequencing,used IMGT/High V-QUEST platform,QIIME,SPSS and other software to analyzed the structural characteristics and diversity of oral flora and intestinal microbial community and perform comparative analysis.Results: 1.Compared with the H group,the combined epithelium of the upper first and second molars of the rats in the P and AP groups receded toward the root,the alveolar crest was reduced,and the amount of bone loss was significantly increased.Behavioral results showed that the number of times the rats in the P,A,and AP groups crossed the platform(P<0.05,P <0.01,P <0.001)and the percentage stayed in the target quadrant(P <0.01,P<0.05,P <0.01)were both It was lower than that of group H,and although group AP has a downward trend compared with group A and group P,the difference was not statistically significant.The time for rats in the P,A,and AP groups to reach the platform for the first time was longer than that in the H group.The differences between group A,group AP and group H were statistically significant(P < 0.01,P < 0.01).Compared with the P group,the time was longer,but the difference was not statistically significant.HE staining and Nissl staining showed that the neuron structure in the hippocampus CA3 area of the rats in the A,P,and AP groups was damaged to different degrees,and the number of normal neurons was significantly reduced compared with the H group.The difference was statistically significant(P <0.05,P <0.001,P <0.001),the AP group had more serious neuronal damage than P and A groups,showed a downward trend,but the difference was not statistically significant.2.Oral flora and intestinal flora Through 4 groups of oral and intestinal microbial OTU venn diagrams,PCA diagrams and sample diversity analysis,it was found that the composition of oral and intestinal flora was similar.Compared with group H,when periodontitis occurs or exists,Firmicutes in the oral cavity and intestinal tract of P and A.P groups increase,Bacteroides decreases,and the ratio of Firmicutes/Bacteroides increases,which was similar to group A.The structural characteristics of the intestinal flora.The statistical analysis of species at the phylum level showed that: Proteus,Firmicutes,Bacteroides,and Actinomycetes were the oral and intestinal common dominant flora,and their shared flora accounted for more than half of the dominant flora.Species statistical analysis results of oral flora at the phylum level: Compared with the H.o group,the relative abundance of Proteus phylum showed an upward trend in the A.o group,while the P.o group and AP.o group showed a downward trend.Among of them,the A.o group was compared with the P.o group and AP.o was statistically significant(P<0.05;P<0.05).Compared with the H.o group,the relative abundance of Firmicutes was lower in the A.o group and the H.o group,while the AP.o group and the P.o group showed an upward trend.The difference between the A.o group and the P.o group and the AP.o group was statistically significant(P<0.01;P< 0.01);the relative abundance of Bacteroides in the A.o group was lower than that of the H.o group,P.o group,and AP.o group.There was no significant change in the P.o group,AP.o group and the H.o group.The difference between the A.o group and the H.o group was Statistically significant(P<0.05).The results of species statistical analysis at the level of intestinal flora: AP.f group,P.f group,A.f group Proteus phylum showed a downward trend compared with H.f group,but the difference was not statistically significant.Compared with the H.f group,the relative abundance of Firmicutes in the AP.f group,P.f group and A.f group showed an upward trend,but the difference was not statistically significant.Compared with the H.f group,the relative abundance of the AP.f group,P.f group,and A.f group Bacteroides showed a downward trend,but the difference was not statistically significant.The ratios of Firmicutes and Bacteroides in the AP.f group,P.f group and A.f group were higher than those in the H.f group.Subordinate level analysis of the top ten bacterial groups in each group: Fusobacterium exists in the P.o group and AP.o group,but not in the A.o group and H.o group,and further analyzed the periodontal pathogens in each group of Fusobacterium It was found that in the oral cavity,Fusobacterium nucleatum showed an upward trend in group P,group AP,and group A.The difference between group P and group AP and group H was statistically significant(P<0.01,P<0.05))The difference between group P and group A was statistically significant(P<0.05);in the intestine,Fusobacterium nucleatum showed an increasing trend in group P,group A,and group AP compared with group H.Among them,group A and group H The difference between the groups was statistically significant(P<0.01).The abundance values of the P and AP groups were similar,and there was no statistically significant difference from the H group.After being disturbed by periodontitis,the statistical analysis results of the top five species in each group at the level of oral flora: Compared with the H.o group,the relative abundance of E.coli Shigella was significantly lower in the A.o group,but the difference was not statistically significant;P.o The relative abundance levels of Streptococcus in the group,A.o group,AP.o group and H.o group were increased,and the P.o group,AP.o group and H.o group were statistically significant(P<0.05,P<0.05);P.o group,A.o group The relative abundance of Veillonella in the group,AP.o group and H.o group was increased,among which the H.o group was statistically significant with P.o and AP.o group(P<0.01,P<0.01);P.o group,A.o group,AP The relative abundance levels of Neisseria in the.o group and the Ho group were increased,among which the Ho group,the Po group and the AP.o group were statistically significant(P<0.01,P<0.05);the P.o group,A.o group,AP.o Compared with the H.o group,the relative level of Acinetobacter was lower in the H.o group,and the H.o group and the P.o group were statistically significant(P<0.05);the relative abundance of lactobacilli in the P.o group,A.o group,and AP.o group compared with the H.o group Compared with the decrease,the H.o group,A.o group and AP.o group have statistical significance(P<0.01,P<0.05).The statistical analysis results of the top five species in each group at the level of intestinal flora: P.f group,A.f group,AP.f group and H.f group compared with the relative abundance of rumen cocci,but there are differences No statistical significance;the relative abundance of Prevotella was significantly lower in the P.f group,A.f group,and AP.f group compared with the H.f group.Among them,the H.f group was different from the P.f group and the A.f group It is statistically significant(P<0.01,P<0.01);the relative levels of Escherichia coli Shigella are lower in P.f group,A.f group,AP.f group and H.f group,among which H.f group and P.f group The difference between the groups was statistically significant(P<0.05);the relative abundance of the NK4A136 group of Launospiraceae was not statistically significant in the P.f group,A.f group,AP.f group and the H.f group.Increasing trend,among which the values of P group and AP group are similar;P.f group,A.f group,AP.f group and H.f group compared with the relative abundance of Lactobacillus,but the difference was not statistically significant;P.f group,A.f group,AP.f group and H.f group Rombutzia relative levels decreased,H.f and P.f group,A.f group difference was statistically significant(P<0.01,P<0.05);P.f group,A Compared with the H.f group,the relative levels of strict Clostridium in the f group and AP.f group were lower,and the difference between the H.f group and the P.f group and the A.f group was statistically significant(P<0.05,P<0.05);P.f group Compared with the H.f group,the relative levels of Ekmania in the A.f group,AP.f group and the H.f group were significantly lower,and the difference between the H.f group and the A.f group was statistically significant(P<0.05).Conclusions: 1.Under the experimental conditions,experimental periodontitis can aggravate the characteristic brain pathological changes and cognitive impairment in AD model rats caused by Aβ25-35.2.When the oral cavity was disturbed by periodontitis,periodontal pathogens can affect the changes of oral flora and affect the intestinal tract,causing changes in the structure of the intestinal flora and AD-related flora such as Ekmania,Rumen cocci and other changes.3.In this experimental condition,experimental periodontitis aggravated the characteristic pathological changes and cognitive impairment of AD model rats caused by Aβ25-35,and the mechanism may be related to the microbe-gut-brain axis mechanism. |
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