Based On The Lab Color Model To Explore The Characteristics Of Different Tongue Microbes In Healthy People And Its Comparison With T2DM Patients

BackgroundAs the key content of inspection,tongue diagnosis is one of the important methods to obtain the objective indicators needed for clinical differentiation of symptoms and signs of traditional Chinese medicine(TCM).Under the background of modern science and technology,modern research in the field of microcosmic and objectification of TCM is in the ascendant,and tongue diagnosis is an important breakthrough.Traditional Chinese medicine emphasizes a holistic view,and guides theoretical research and clinical practice based on basic principles such as internal understanding and micro-knowledge.Microecology,as ecology at the cellular and molecular level,has similarities in basic theoretical viewpoints.With the gradual deepening of research in the field of tongue coating microecology and the use of newer and better microbial technologies,it will be possible to better discover and understand the relationship between diseases and the changes in the appearance of the tongue in traditional Chinese medicine,as well as the relationship and change laws of the tongue coating microecology,and continue to dig into TCM.The scientific connotation of tongue diagnosis enables TCM tongue diagnosis to better exert its advantages and functions in clinical practice and even modern medical diagnosis and treatment.However,the current researches on tongue coating microecology mostly focus on specific types of tongue coatings for specific diseases.Due to the interference of disease-specific factors,the reproducibility of research results is poor.In this study,eight tongue fur types of healthy people were the main research objects,and the microbiological characteristics of different tongue images and the relationship with diseases were explored through metagenomic sequencing.ObjectiveFrom the perspective of tongue fur microbes,we will study the scientific rules and formation mechanism of TCM tongue diagnosis and tongue picture,and then reveal the biological basis of TCM tongue picture under the background of modern medicine,and promote the modernization of TCM diagnosis and treatment.At the same time,modern scientific and technological means are used to explore the standardization of TCM tongue diagnosis,and establish reliable tongue diagnosis methods to lay a methodological foundation for further related research.MethodsOn the basis of assisted tongue diagnosis,we divide the tongue coating of healthy people into eight types according to color,thickness and moistness,including thin white,thin white greasy,white thick greasy,white thick dry,thin yellow,and thin yellow greasy,yellow thick greasy.First,the DS01-G tongue image acquisition instrument developed by Shanghai DAOSH company in China was used to take standardized photographs of the tongue coatings of healthy volunteers and diabetic patients.Secondly,after oral cleaning,use a sterile flocking cotton swab to collect the microbial population in the middle of the tongue according to a standardized operating procedure,and store it in a micro-based microbial specimen cryotube,and store it in a refrigerator at-80 degrees.Using proteinase K lysis combined with phenol-chloroform extraction method to extract total DNA from the sample.Run the sample test on a 1.2%agarose gel.The sequencing library was constructed by NEBNext(?)UltraTM DNA Library Prep Kit for Illumina(?)(New England Biolabs,USA).The genomic library was sequenced using Illumina Novaseq 6000,150-bp paired-end technology from Micro Biotechnology(Shanghai)Co.,Ltd.Cluster the predicted genes and use the CD-HIT package to construct a non-redundant gene catalog(parameters:identity=95%,coverage=90%).Use the genome calculation coverage function in BEDtools to quickly estimate the abundance of genes.Use DIAMOND software to classify Non-Redundant Database(NR).Use contest in the R language function to calculate and test the correlation coefficient,and use R's igraph software(version 1.2.5)to construct the symbiosis network.Perform principal component analysis using prcomp and taxonomic abundance information in R,and plot it with ggplot2 in R.The non-parametric rank sum test was used to analyze the microbial population results,and LEfSe v1.0 was used for LEfSe analysis.Statistics uses SPSS(version 24)and Microsoft Excel 2020 software,and a P value of<0.05 is considered statistically significant.ResultsResearch 1(1)Tongue image auxiliary syndrome differentiation.We analyzed the L value,A value and B value of the middle part of the tongue in 72 cases.The results show that there is a significant correlation between the LAB color quantification system and artificial tongue diagnosis.Among them,the L value,which reflects the color brightness,has no significant difference in each group.The B value is positively correlated with the color of the moss,the overall B value of white fur is low,and the overall B value of yellow fur is high.Under the conditions of using Daosheng hand-held tongue imager to shoot,b value lower than 8.5 can be judged as white fur,higher than 8.5 can be judged as yellow fur;A value is negatively correlated with the thickness of tongue coating,the overall A value of thin fur is high,and the whole thick fur A value at the end.White fur is lower than 21 as thick fur,higher than 21 is thin fur,yellow fur is lower than 17 is thick fur,and higher than 17 is thin fur.Research 2(1)Baseline characteristics.Among 180 people enrolled through case screening,94 tongue coating samples were finally used for tongue coating microbiota analysis.In the cohort,women accounted for 74.47%and men accounted for 25.53%.The age of each group is generally between 45 and 55 years old,and there is no significant difference between the groups.The most common tongue coatings are thin white,thin yellow greasy and thin white greasy,with more than 20 cases of each type of tongue coating.72.24%of the tongue coating can be defined as thin coating,while 27.76%belong to thick coating.The percentage of white tongue coating is 57.45%,which is higher than 42.55%of yellow tongue coating.The proportion of subjects with greasy coating is 69.15%.(2)Tongue coating microbial community characteristics.The most abundant genus in the research population is Prevotella,with an average abundance of 15.7%and Neiseria 13.1%.Followed by Strptococcus(Strptococcus)with 10.8%,Actinomyces(Actinomyces)with 8.8%,Veillonella(Veillonella)with 8.4%and so on.The most common phylum is Firmicutes with the highest abundance of 27.9%,followed by Bacteriodetes,Proteobacteria,Actinobacteria and Fusobacteria),the abundances are 21.9%,19.9%,18.9%,5.7%,respectively.According to the results of principal component analysis,it is found that there is no significant difference in the microbial ? diversity of different tongue coating types.(3)Characteristic microorganisms of different tongue coating types.Select bacteria with LDA higher than 3 as characteristic microorganisms.Among them,the thin white fur is Bacillales order;the white thick greasy tongue fur is Megasphaera micronuciformis,Vellonella and Streptococcus infantis;thin yellow fur is Proteobacteria;yellow thick The greasy tongue coating is Prevotella melaninogenica and Veillonella dispar.(4)Correspondence of clinical diseases of different tongue coating microorganisms.Veyronella with thick and greasy coating is a specific pathogenic bacteria of "cold" gastritis.Micrococcus is enriched in the tongue coating of gastric cancer patients,while Streptococcus infantis It indicates a precancerous cascade;Moraxella genus and Neisseria elongated in thin yellow moss are common bacterial flora in the oropharynx.Moraxella can increase the risk of causing opportunistic infections,while Neisseria slender has a chance to cause endocarditis,osteomyelitis and pancreatic cancer;Prevotella Melaninogenica and Veillonella dispar are related to esophageal squamous cell carcinoma and oral squamous cell carcinoma,Pobium parvulum can cause sepsis,etc.(5)The co-occurrence network of the tongue coating microbiota.The highly enriched Streptococcus infantis in the white thick and greasy tongue fur is positively correlated with Streptococcus peroris;the characteristic bacteria of thin yellow fur,Neisseria elongate.It is positively correlated with Capnocytophagas sputigena;in thick yellow tongue fur,Prevotella melaninogenic,Veillonella dispar and Atopobium parvulum There is a significant positive correlation,indicating the coexistence and symbiotic relationship of these bacteria in the yellow and greasy tongue coating.(6)Diabetic disease-specific bacterial markers.In the yellow and greasy tongue coating of diabetic patients,Capnocytophaga genus is highly enriched.This potentially pathogenic bacteria is related to periodontitis and type 1 diabetes.closely related Compared with diabetic patients,Prevotella melaninogenic is more significantly enriched in healthy people(7)Gene functional difference between diabetic and healthy yellow thick greasy coating.Compared with healthy people,T2DM patients have higher levels of drug-resistant gene expression,mainly macrolide antibiotics;T2DM patients have higher levels of drug resistance than healthy people.High-level expression of carbohydrate enzymes,mainly glycoside hydrolases;T2DM patients have higher levels of ABC transporter,AMPK signaling pathway,FoxO signaling pathway,insulin signaling pathway,peroxisome,prolactin signaling pathway,etc.Gene expression,healthy people have a higher level of apoptosis clustering gene expression,suggesting that cholesterol transport regulation,prolactin level,redox reaction and apoptosis may be the main differences in the physiological and pathological functions of the two.ConclusionBased on the Lab color system assisted TCM tongue picture syndrome differentiation research,the results show the subjectivity of TCM tongue picture syndrome differentiation.It is found that the color quantification of A and B value can standardize and assist TCM syndrome differentiation,and summarize the specific reference values of A and B for auxiliary syndrome differentiation,which is very strong Practicality.After the results of this study are verified by more tongue image dialectics and digitization,they can realize the wide application of color quantification assistance and standard tongue image dialectics.Subsequently,the microbial composition was studied by metagenomic sequencing,and the unique bacterial characteristics of different types of tongue coatings of healthy people were determined,and the symbiotic relationship between the tongue coating microorganisms was constructed.This provides a valuable reference for deciphering the formation mechanism of tongue coating and provides convenience for the modern diagnosis of traditional Chinese medicine in the future.In addition,we further studied the differentially labeled bacteria between healthy individuals and diabetic patients,proved the specific effect of the disease on the tongue coating microorganisms,and emphasized the necessity of identifying different tongue coating types in healthy people.