| Objective: Chronic periodontitis is the sixth major complication of diabetes mellitus.A large number of studies have shown that periodontitis and diabetes mellitus promote and interact with each other.At present,research at home and abroad has found that saliva is a biological body fluid with unique diagnostic ability,which contains many biological molecules,including DNA,m RNA,protein,metabolites and microbiota,etc.Saliva research is a non-invasive method,the components in the blood can also be detected in saliva,saliva can be used together with blood to study the pathogenesis of diseases,etc.So this experiment collected through clinical health group,purity of periodontitis,simple type 2 diabetes mellitus,type 2 diabetes patients with periodontitis with saliva and blood,the application of metabolism,transcriptional group multiple omics conjoint analysis,the method of screening and identification of four groups of patients with the metabolic markers in the blood,saliva,and related differences in gene,explore disease may involve related to metabolism and signaling pathways,To provide a preliminary experimental basis for whether saliva can be used as a biological diagnostic tool for early diagnosis,monitoring and prognosis of diseases.Methods: In this study,blood and saliva were collected in vitro from healthy subjects,patients with type 2 diabetes alone,patients with periodontitis alone,and patients with type 2 diabetes with periodontitis.LC-MS/MS and RNA-seq research methods were used for detection.The experimental groups were as follows: healthy group(Z),periodontitis alone group(Y),diabetes alone group(T),diabetes with periodontitis group(TY);1.The metabolites of plasma and saliva were extracted from the four groups of samples,and the saliva samples and plasma samples were detected in a random order on the machine based on the research method of liquid chromatography tandem mass spectrometry(LC-MS/MS).The obtained data were processed,the relationships among samples and between metabolites were analyzed,and the biological significance of metabolites was explained through functional analysis such as metabolic pathways.2.Combined analysis of saliva metabolome data and plasma metabolome data.3.Total RNA was extracted from the blood of the 4 groups of patients,and GO and KEGG enrichment analysis was performed on the differentially expressed genes in the 4 groups of samples to explore the functional and metabolic pathways involved in the differentially expressed genes.4.Correlate the whole blood transcriptome with the joint analysis results of salivary metabolome data and plasma metabolome data,and search for common metabolic pathways,common metabolites and marker genes of upstream regulation.5.Use q RT-PCR to verify the transcriptome expression of marker genes.Results: 1.Saliva metabolic samples: healthy group(Z＿T),single diabetes group(T＿T),single periodontitis group(Y＿T),diabetes with periodontitis group(TY＿T)test results showed that in the PLS-DA model,the groups were significantly differentiated.In the comparison of T＿T and Z＿T,a total of 51 different metabolites were detected under the positive and negative modes,which were related to the disorder of multiple metabolic pathways,such as glycolysis/gluconeogenesis,thiamine metabolism,arginine and proline metabolism.In the comparison of Y＿t and Z＿t,a total of 55 different metabolites were detected,which were related to the metabolic pathway disorders of bile acid metabolism,glycerolipid metabolism,linolenic acid metabolism,etc.In the comparison of TY＿T and Z＿T,a total of 124 different metabolites were detected,which were related to the metabolic pathway disorders such as glycerolipid metabolism,sphingolipid metabolism and linolenic acid metabolism.2.Plasma metabolic samples: The results of plasma samples in the healthy group(Z＿x),the simple diabetic group(T＿x),the simple periodontitis group(Y＿x),and the diabetic group with periodontitis(TY＿x)showed that in the PLS-DA model,there were obvious differences among the groups.In the comparison of T＿x and Z＿x,a total of 152 different metabolites were detected under the positive and negative modes,which were related to the metabolic pathway disorders such as histidine metabolism,linoleic acid metabolism and tyrosine metabolism.In the comparison of Y＿x and Z＿x,a total of 99 different metabolites were detected,which were related to the disorder of arachidonic acid metabolism and phospholipase D signaling pathway.In the comparison between Ty＿x and Z＿x,a total of 57 different metabolites were detected,which were involved in the metabolic pathway disorders such as arachidonic acid metabolism and the biosynthesis of unsaturated fatty acids.3.Combined analysis of metabolic data of saliva samples and plasma samples: Saliva and plasma samples from patients with diabetes and periodontitis were enriched in 5pathways,including glycerol phospholipid metabolism,unsaturated fatty acid biosynthesis,choline metabolism in cancer,vitamin digestion and absorption,arginine and proline metabolism.Among them,the common characteristic metabolites were docosahexaenoic acid,arachidonic acid,neuroic acid,LPE 22:4 and LPC18:1;4.Transcriptome analysis of whole blood showed 130 up-regulated genes and 248down-regulated genes in the pure periodontitis group compared with the healthy control.Compared with the healthy controls,there were 195 up-regulated genes and726 down-regulated genes in the single diabetic group.Diabetes mellitus associated with periodontitis group compared with healthy controls,raised 1120 genes,cut 2323genes(threshold pvalue < 0.05,| log2 foldchange | > 1);The three groups shared 62 different genes.5.Through the combined analysis of whole blood transcriptome and plasma metabolome,the characteristic metabolites docosahexaenoic acid and arachidonic acid,as well as the marker gene ACTO2,were found to be highly related to diabetes mellitus with periodontitis.6.The results of q RT-PCR showed that ACOT2 had a downward trend in periodontitis and diabetes mellitus,but did not reach a significant level.It was significantly decreased in the diabetic periodontitis group,and the results of transcriptome and q RT-PCR were in good agreement.Conclusion: 1.Salivary and plasma of the 4 groups of patients were collected for non-target metabolomic detection,and the common metabolites of docosahexaenoic acid,arachidonic acid,neuronic acid,LPE 22:4 and LPC18:1 were screened out finally.The above 5 metabolites may be used as biomarkers for the diagnosis of periodontitis by saliva detection in clinical practice.Among them,neuric acid,LPE22:4 and LPC18:1 are characteristic metabolites of diabetes mellitus with periodontitis,and these three substances can be used as specific substances for the diagnosis of diabetes mellitus with periodontitis using saliva test instead of blood test.2.Salivary and plasma of the 4 groups of patients were collected for non-target metabolomics detection,and the common pathways were finally screened out,including glycerol phospholipid metabolism,unsaturated fatty acid biosynthesis,choline metabolism in cancer,vitamin digestion and absorption,arginine and proline metabolism,which laid a foundation for the follow-up study on the pathogenesis of the disease.3.Marker gene ACTO2 was found through whole blood transcriptome.ACTO2 serves as a genetic marker in the blood.These characteristic metabolites and marker genes have potential application value in early prevention,clinical diagnosis,treatment and prognosis of related diseases.Aiming at the screened common pathway and the upstream regulatory genes,this paper lays a foundation for the follow-up research on its specific regulatory mechanism.4.The results of this experiment provide a preliminary experimental basis for early auxiliary diagnosis,monitoring and prognosis of diseases with saliva samples as the research object and using multiomics research methods in the future. |
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