An Exploratory Study On The Microecology Of Tongue Coating In Patients With Dampness Syndrome Of Eczema

Objective1.Through the inclusion of healthy people and patients with eczema in various syndrome types,and the traditional Chinese medicine compound "Longdan Xiegan Decoction" and "Chushi Weiling Decoction" were used to intervene the eczema patients with the syndrome of dampness-heat immersion and the syndrome of spleen deficiency and dampness respectively to analyze the clinical efficacy.2.16S rDNA sequencing technology and targeted metabolome technology were used to jointly detect the tongue coating flora of patients with various types of eczema and healthy people,and the construction of multi-dimensional efficacy evaluation index of traditional Chinese medicine in the treatment of eczema with the syndrome of dampness-heat immersion and the syndrome of spleen deficiency and dampness was preliminarily discussed,so as to understand the internal relationship between micro ecological characteristics of the flora and metabolites on the tongue coating of eczema with dampness syndrome.3.To explore the specific biological mechanism of dampness syndrome in eczema patients based on microflora,and to further analyze the internal relationship among disease,syndrome and tongue coating microecology in eczema patients.Methods1.An exploratory study scheme was adopted,in which a total of 36 patients with dampness and heat immersion syndrome,105 patients with the syndrome of spleen deficiency and dampness(total 3 group),(40 patients in the syndrome group of Chushi Weiling decoction;18 patients in the syndrome group of Shenling Baizhu powder;47 patients in the syndrome group of Guben Huashi decoction);16 cases of eczema without dampness syndrome,meeting the diagnostic criteria of eczema were included in cross section;Patients in different groups were evaluated respectively according to different clinical indicators such as Investigator's Global Assessment(IGA),Eczema Area and Severity Index(EASI),Patient Oriented Eczema Measure(POEM),dermatology life quality index(DLQI/CDLQI),VAS and insomnia index scores,TCM dampness syndrome evaluation score,and filled in the sample collection questionnaire;22 cases of healthy dampness people and 28 cases of healthy non-dampness people who have been excluded related systemic diseases and allergic diseases were included in the study,and the sample collection questionnaire was filled in.Bacteria microecological detection and analysis:for above of each group(0 weeks)before treatment patients and healthy people to collect the tongue coating samples,sent to Wuhan metware Biotechnology Co.,Ltd.Carry on the tongue coating of 16 s rDNA flora sequencing and metabonomics detection,and then correlation analysis,simultaneously compared the diversity of bacteria and different metabolites between eczema and healthy people as well as between dampness syndrome and non-dampness syndrome eczema.2.Single arm clinical trial study:2 groups were selected:syndrome differentiation was conducted according to the consensus of TCM diagnostics experts on eczema(dampness syndrome sores)issued by the Dermatology Branch of China Association of Traditional Chinese Medicine(2018):1.Longdan Xiegan Decoction was taken orally for treatment of dampness-heat immersion syndrome for 8 weeks.2.Chushi Weiling Decoction was taken orally for the treatment of spleen deficiency and dampness syndrome for 8 weeks.Patients in different groups were evaluated Investigator's Global Assessment(IGA),Eczema Area and Severity Index(EASI),Patient Oriented Eczema Measure(POEM),dermatology life quality index(DLQI),VAS and insomnia index scores,TCM dampness syndrome evaluation score and filled in the sample collection questionnaire respectively before treatment and treatment of 2,4,6,8 weeks.Bacterial microecological detection and analysis:All the collected tongue coating samples were sent to Wuhan metware Biotechnology Co.,Ltd.for 16S rDNA and metabonomics detection,and correlation analysis was conducted.Combining the clinical data with the detected data,the characteristics of the microflora of patients with dampness syndrome of eczema were analyzed,and the effects of traditional Chinese medicine classic compounds "Chushi Weiling Decoction" and "Longdan Xiegan Decoction" on the bacterial microecology of patients with dampness accumulation syndrome of spleen deficiency and dampness-heat immersion syndrome were analyzed.Results1.Clinical study results(1)Exploratory research results:there were significant differences in Investigator's Global Assessment(IGA)and TCM dampness syndrome evaluation score among eczema patients with different syndrome types(P<0.05).There is no difference in other indicators;(2)Results of a single-arm clinical trial:40 patients with eczema with spleen deficiency and dampness syndrome were intervened with Chushi Weiling decoction,and there were statistically significant differences between before and after treatment in Investigator's Global Assessment(IGA),Eczema Area and Severity Index(EASI),Patient Oriented Eczema Measure(POEM),dermatology life quality index(DLQI),Pruritus Index(VAS)and TCM dampness syndrome evaluation score(P<0.05),but insomnia index scores were not statistically significant(P>0.05).36 patients with dampness-heat immersion syndrome were intervened with Longdan Xiegan decoction.Among them,11 cases were improved after 4 weeks of treatment(reaching EASI 50),and then stopped taking it,which reflected the characteristics of "Discontinue medication as soon as get effect" in traditional Chinese medicine.and there were statistically significant differents between before and after treatment in Investigator's Global Assessment(IGA),Eczema Area and Severity Index(EASI),Patient Oriented Eczema Measure(POEM),dermatology life quality index(DLQI)and insomnia index scores(P<0.05),whereas TCM dampness syndrome evaluation score were not statistically significant(P>0.05).However,after 4 weeks of significant improvement of the lesions,(at the point of 8 weeks of treatment),19 cases have statistically significant improvement in the TCM dampness syndrome evaluation score compared with that before treatment(P<0.05).2.Results of 16S rDNA analysis(1)The dominant species in the phylogenetic status of eczema patients and healthy people at the phylum level were mainly p_Firmicutes,p_Proteobacteria,p_Bacteroidetes,p_Actinobacteria,p_Fusobacteria,etc.The taxonomic units in the eczema patients group with higher abundance of tongue coating flora significantly different from phylum to genus level were:p_Actinobacteria),c_Actinobacteria,o_Actinomycetales,f_Actinomycetaceae,g_Actinomyces,c_Clostridia,o_Clostridiales,f_Veillonellaceae5 g-Veillonella,o_Bifidobacteriales,f_Bifidobacteriaceae,f_Lactobacillaceae,g_Lacto'bacillus,g_Peptostreptococcus;species with significant differences in the abundance of tongrye coating flora in the healthy group from the phylum to the genus level,respectively:p_Bacteroidetes,c_Bacteroidia,o_Bacteroidales,f_Prevotellaceae,g_Prevotella,p_Fusobacteria,c_Fusobacteriia,o_Fusobacteriales,f_Fusobacteriaceae,g_Fusobacterium,p_Chloroflexi,c_Ktedonobacteria,g_Moraxella,g_Actinobacillus,f_Camobacteriaceae,g_Granulicateila.differences between the two groups were statistically significant in richness and Good's coverage(P<0.05),while the differences between two groups of the Shannon and Simpson as well as Pielou's evenness,were not statistically significant(P>0.05).(2)The dominant species at the phylogenetic status at the phylum level between patients with dampness syndrome and without dampness syndrome were the same as above,and the species with significant differences in the abundance of tongue coating flora in the taxonomic unit in the patients with dampness syndrome group from the phylum to the genus level were:f_Lachnospiraceae,c_Coriobacteriia,o_Coriobacteriales,respectively.f_Coriobacteriaceae,g_Atopobium,g_Megasphaera,g_Butyrivibrio,;species with significant differences in the abundance of tongue coating flora from phylum to genus level for taxonomic units with higher abundance in the group of patients without dampness syndrome are:g_Actinobacillus,g_naerofilum,g_Anaerofustis,f_Dehalobacteriaceae,g_Dehalobacterium,f_Eubacteriaceae,g-Clostridium.Simpson was significantly higher in the eczema with dampness syndrome group than without dampness syndrome group(P<0.05),and there were no significant differences in richness and Good's coverage(P>0.05).3.Results of metabolomics analysis(1)After validation by OPLC-DA,it showed that there were significant differences in metabolites between patients with each syndrome group of eczema and healthy individuals,and the model was validated;there were significant differences in metabolites between spleen deficiency and dampness syndrome group of eczema and non-dampness syndrome group,and the model was validated;there were no significant differences in metabolites between dampness-heat immersion syndrome group of eczema and non-dampness syndrome group.(2)The application of untargeted metabolomics combined with target metabolomics assay revealed that the differential metabolites upregulated in the eczema group compared to the healthy population were mainly amino acids and their metabolites,benzene and its derivatives and organic acids and their derivatives,including N-Methyl-D-Aspartic Acid,Anisic acid,2-Aminobenzenesulfonic acid,Alanine tyrosine,2'-Deoxyinosine,Cis-4-Hydroxy-D-Proline,Creatine;the down-regulated differential metabolites were mainly organic acids and their derivatives,amino acids and their metabolites and glycerophospholipids,including Glycyl-L-valine,3-Ureidopropionate,2-Methylsuccinic Acid,Glutaric Acid,Ethylmalonate,10-Formyl-Thf,Folic acid,respectively.These Metabolic compounds mainly enriched for Purine Metabolism,Antifolate resistance,Pyrimidine metabolism,etc.The differential metabolites that were upregulated in the eczema patients with spleen deficiency and dampness syndrome group compared to the eczema patients without dampness syndrome group were mainly organic acids and their derivatives,amino acids and their metabolites,and benzene and its derivatives,including N-Methyl-D-Aspartic Acid,Glu-Met,Guanosine,Met-Glu,Alanine tyrosine,5'-Deoxyadenosine,Deoxyadenosine,4-Guanidinobutyric Acid;the differential metabolites that were downregulated were Carnitine C10:1,Undecanedioic acid,Acetyl-L-carnitine,Carnitine C5:0,L-Dihydroorotic Acid,Carnitine C5:0,L-Dihydroorotic Acid,LysoPC 20:2(2n isomer2),LysoPC 16:1(2n isomer)respectively.These Metabolic compounds mainly enriched pathways were ABC transporters,Pyrimidine metabolism,Purine metabolism,Galactose metabolism,Ascorbate and aldarate metabolism,etc.4.Results of combined 16S rDNA and metabolomics analysisIn the comparison between eczema and healthy populations,there was a negative correlation between most groups of bacteria and metabolites,with the highest correlation at the phylum classification level of p_Proteobacteria,with g_Burkholderia,g_Chelativoran,g_Devosia,g_Neisseria,g_Halomonas,g_Chelativorans,p_Firmicutes,f_Peptostreptococcaceae produced the highest number of compounds and the most relevant metabolites Tryptophan glutamic acid,adenosine-3'-phosphate,2-'deoxyguanosine 5'-monophosphate(DGMP),5-uracil nucleotide,L-homocysteine,thiamin mono phosphate,2'-O-methyladenosine,etc.The most relevant metabolites of Neisseria spp.,namely aminoglutamic acid and L-homocysteine,were significantly lower in the eczema population,and the most relevant metabolite of Streptococcaceae digestiveis,2-mercaptobenzothiazole,was significantly higher in the eczema population.In the comparison of eczema patients with dampness syndrome and non-dampness syndrome,there was a positive correlation between the majority of bacteria and metabolites,with the highest correlation at the phylum classification level being p_Firmicutes,g_Veillonella producing the highest number of compounds,followed by p_Firmicutes g?Lachnoanaerobaculum,p_Bacteroidetes,g_Prevotella,g_Firmicutes,f_Lachnospiraceae,g_Moryella.the metabolites of highest relevance were L-methionine-L-glutamate and glutamyl-methionine.The relative abundance of the p_Firmicutes g_Veillonella was significantly higher in dampness syndrome and showed positive correlations with both Met-Glu(|r|=0.60)and Glu-Met(|r|=0.59).In the comparison between post-treatment and pre-treatment of patients in the spleen deficiency and dampness syndrome group treated with the application of Chushi Weiling decoction,the relative abundance of p_Bacteroidetes,g_Prevotella and p_Firmicutes,g_Streptococcus was significantly higher,and both were positively correlated with Carnitine C10:1,respectively(|r|=0.68;|r|=0.64).In the comparison between post-treatment and pre-treatment of patients in the dampness-heat immersion syndrome group treated with the application of Longdan Xiegan decoction,the relative abundance of p_Firmicutes,g_Streptococcus was significantly higher,which is positively correlated with Guanosine 3',5'-Cyclic Monophosphate(|r|=0.62).ConclusionsIn this study,16SR DNA high-throughput sequencing combined" with flora metabonomics was used for the first time to study the correlation between the microecological structure of tongue coating and the metabolic phenotype of patients with various types of wet eczema syndrome,revealing the diversity characteristics of tongue coating flora and the characteristics of metabolic spectrum of patients with wet and non-wet syndrome,which were summarized as follows:Clinical study section:(1)The skin lesion score and self-conscious symptom score were improved significantly after the oral administration of Longdan Xiegan decoction for eczema patients in dampness-heat immersion syndrome group and Chushi Weiling decoction for spleen deficiency and dampness syndrome group,which indicates that the two classic compound decoction had definite effect on the improvement of eczema;After 4 weeks of treatment,patients who accepted oral administration of Longdan Xiegan decoction,the lesion scores and symptoms were significantly improved,especially the symptoms such as POEM score,VAS and insomnia index scores.And the improvement time was earlier(2 weeks),which may be related to the fact that patients with dampness-heat immersion syndrome were mostly in the acute phase of eczema;(2)The improvement time of TCM dampness syndrome score of the two groups was relatively lagging,and there was no significant difference in the metabolites of tongue coating samples of people with EASI50 or above.This may be related to the occurrence of dampness syndrome accompanied by different degrees of abnormal fluid metabolism.The macro and micro results are different,considering that the occurrence and development of syndrome is a long-term and chronic process,the appearance of the effective time point of the score is also after the effective time point of the disease.It is inferred that the time point of significant difference metabolites of tongue coating samples should correspond to the time point of improvement of damp syndrome.Tongue coating flora study section:(1)16S rDNA second-generation high-throughput sequencing technology was used to analyze the structure of tongue coating flora in patients with eczema with dampness syndrome.It was found that there were significant differences in abundance and diversity of tongue coating flora between eczema and healthy people,and between eczema with dampness syndrome and non-damp syndrome;(2)Through the correlation analysis between the microbial data and the metabolic spectrum of tongue coating tissue in eczema patients with dampness syndrome,it was found that there was a significant correlation between the changes of tongue coating flora and metabolic phenotype.Combined with clinical tongue diagnosis,it is of great significance to prompt early warning of the syndrome and intervene by correcting the flora.(3)After the application of two kinds of decoction to the patients with the syndrome of dampness-heat immersion and the syndrome of spleen deficiency and dampness,the abundance of p_Bacteroidetes g_Prevotella was significantly increased,whereas the change for abundance of p_Firmicutes is not obvious.This indicates that the ratio of Firmicutes to Bacteroidetes is significantly decreased,which may be one of the evidences to improve dampness syndrome of eczema,and it is of great significance to conduct follow-up study on flora from this point.Metabonomics Research Section:(1)There were significant differences in the metabolites of tongue coating samples between patients with eczema(including dampness syndrome and non-dampness syndrome)and healthy people;There were significant differences in the metabolites of tongue coating samples between eczema patients with spleen deficiency and dampness accumulation syndrome and non-dampness syndrome;It shows that disease and syndrome,namely eczema and dampness syndrome,are the common factors that lead to the pathological state of patients.(2)Respectively using UPLC-QTOFMS combined with multivariate statistical analysis to identify potential biomarkers of eczema patients with dampness syndrome.Through the analysis of biomarkers and their related metabolic function,it was found that the metabolic abnormality of patients with dampness eczema was mainly related to the Tricarboxylic acid cycle(TCA),the energy metabolism disorder of glycolysis pathway,and the metabolic disorder of tryptophane and lipopolysaccharide(LPS)mediated by tongue coating bacteria.