Study On The Microbes Of Greasy Tongue Coating In Coronary Heart Disease With Phlegm Syndrome Based On The Combination Of Disease And Syndrome

Objective:Using the combination of disease and syndrome method,taking coronary heart disease and phlegm syndrome as the research object,using phlegm syndrome scale of coronary heart disease,the tongue diagnosis instrument of objective measurement,collecting tongue coating and stool sample of coronary heart disease with phlegm syndrome of white greasy and yellow greasy and healthy people.The sample was used to study the correlation between the microbial markers specific to the coronary heart disease with phlegm syndrome,and to explore the value of the microbes on the coronary heart disease with phlegm syndrome.Method:1.High-throughput sequencing of microbial microbes:The 16S rDNA high-throughput sequencing technology was used to analyze the tongue coating and intestinal microbes of patients with phlegm syndrome of coronary heart disease?yellow greasy coating,white greasy coating?,and healthy people were established as a control group.To study the differences between the patients with phlegm syndrome of coronary heart disease.2.Macroscopic measurement of coronary heart disease with phlegm syndrome:scale survey was used to conduct a survey on patients with coronary heart disease with phlegm syndrome on the first,fourth,and seventh days after admission,and to collect general information,clinical symptoms,and diagnosis basis.3.Object measurement of typical greasy coating:The tongue image information of patients with phlegm syndrome of coronary heart disease was collected by tongue diagnosis instrument,including tongue color and tongue coating color,tongue coating area,tooth marks,ecchymoses,cracks and pricks.Result:1.General:A total of 41 eligible subjects were included in the study,including 24 cases of coronary heart disease,14 cases of white greasy coating,17 cases of yellow greasy coating,and 21 cases of healthy people.In the phlegm syndrome group,27 males,accounting for 65.8%,14 females,accounting for 34.2%,with an average age of 67.5?41-78?years,a course of 36?1-620?months,with 30 patients with hypertension,accounting for 73.2.%,5 patients with diabetes,accounting for 12.2%,9 patients with hyperlipidemia,accounting for 22.0%.2.Microbial high-throughput sequencing:?1?Sequencing quality? tongue coating sample quality evaluation:PE Reads?77292± 6270?,Raw Tags?68165 ± 7893?,Clean Tags?62086±8710?,AvgLen?bp??422±2?,GC?%??52.08± 0.64?,Q20?%??95.86 ±0.48?,Q30?%??92.2 ± 0.8?,suggesting that the quality of the tongue coating sample is qualified and can be analyzed downstream.? stool sample quality evaluation:PE Reads?80284 ± 5665?,Raw Tags?73765± 5349?,Clean Tags?68206±5276?,AvgLen?bp??419± 3?,GC?%??51.52±1.22?,Q20?%??96.28±1.02?,Q30?%??92.89±1.68?,indicating that the sample quality is qualified,and can be analyzed downstream.?2?OTU Analysis of the distribution of species? The number and species distribution of microbial OTU in tongue coating:The number of microbial OTUs in the tongue coating samples was 184±24,and the overall distribution was 10±1 gates,16±2 classes,25±3 orders,44±5 families,86±10 genera.? tongue coating dominant microbial species:The dominant group of the case group:A.The door level is Bacteroidetes,Firmicutes,Proteobacteria,Fusobacteria,Actinobacteria;B.The main levels are Bacteroidia,Bacilli,Gammaproteobacteria,Negativicutes,Clostridia;C.The main levels are Bacteroidales,Lactobacillales,Selenomonadales,Betaproteobacteriales,Clostridiales;D.is mainly Prevo Prevotellaceae,Streptococcaceae,Veillonellaceae,Neisseriaceae,Ruminococcaceae;E.genus is mainly Streptococcus,Neisseria,Veillonella,Prevotella₇,Rothia.The dominant group of healthy people in the control group:A.The level of the door is Bacteroidetes,Firmicutes,Proteobacteria,Fusobacteria,Actinobacteria;B.The main levels are Bacteroidia,Bacilli,Gammaproteobacteria,Negativicutes,Clostridia;C.The main levels are Bacteroidales,Lactobacillales,Selenomonadales,Betaproteobacteriales,Fusobacteriales;D.familv levels are mainlv Prevoaceae,Strentococcaceae,Veillonellaceae,Neisseriaceae.Lachnospiraceae;E.genus is mainly Streptococcus,Neisseria Genus,Veillonella,Prevotella₇,Fusobacterium.? Intestinal microbial OTU quantity and species distribution:The total number of OTUs in the stool sample was 156±37,and the overall distribution was 9±1 gates,15±2 classes,23±5 orders,41±9 families,and 91±17 genera.? intestinal microbial dominant species:The dominant group of the case group:A.The gate level is mainly Bacteroidetes,Firmicutes,Proteobacteria,Fusobacteria,Actinobacteria;B The main levels are Bacteroidia,Clostridia,Negativicutes,Gammaproteobacteria,Fusobacteriia;C.The main levels are Bacteroidales,Clostridiales,Selenomonadales,Enterobacteriales,Betaproteobacteriales The D.family level is mainly Bacteroidaceae,Lachnospiraceae,Ruminococcaceae,Veillonellaceae,and Leptotrichiaceae;E.is mainly of the genus Ciliida,Faecalibacterium,Prevotella 9,Streptococcus.The dominant group of healthy people in the control group:A.The gate level is mainly Bacteroidetes,Firmicutes,Proteobacteria,Fusobacteria,Actinobacteria.B.The main level is Bacteroidia,Clostridia,Negativicutes,Fusobacteriia,Actinobacteria;C.The main level is Bacteroidales,Clostridiales,Selenomonadales,Pasteurellales,Betaproteobacteriales;D.family levels are mainly Bacteroidaceae,Lachnospiraceae,Leptotrichiaceae,Veillonellaceae,Ruminococcaceae;E.genus is mainly of the genus Leptotrichia,Faecalibacterium,Neisseria,Veillonella,Bacteroides.?3?Alpha diversity?tongue microbial diversity Case group:Ace?203±28?,Chao?197±29?,Shannon?0.10±0.05?,Simpson?3.01±0.24?index and coverage?99.95±0.01?.healthy controls:Ace?181±41?,Chao?177±43?,Shannon?0.13±0.07?,Simpson?2.81±0.44?index and coverage?99.96±0.01?.?intestinal microbial diversity Case group:Ace?203±28?,Chao?197±29?,Shannon?0.10±0.05?,Simpson?3.01±0.24?index and coverage?99.95±0.01?.healthy controls:Ace?181±41?,Chao?177±43?,Shannon?0.13±0.07?,Simpson?2.81±0.44?index and coverage?99.96±0.01?.Compared with the healthy controls,the Ace,Chao,and Simpson indices of the tongue and intestinal microflora were increased,and the Shannon index was decreased.The difference was not statistically significant.?4?Beta diversity? tongue microbeThe difference in microbial Beta diversity of the tongue coating between the case group and the healthy control group was 12.5%;the difference in the microbial Beta diversity of the tongue coating between the white greasy coating and the yellow greasy coating group was 4.6%.? intestinal microbesThe intestinal microbial Beta diversity between the case group and the healthy control group was 38.9%;the intestinal microbial Beta diversity between the white greasy group and the yellow greasy group was 9.2%.There was a difference in the diversity of tongue and intestine microbes between the case group and the healthy control group.The white greasy and yellow greasy groups also had different differences in tongue and intestine microbial Beta diversity,and the intestinal tract.The microbial Beta diversity is higher than that of the tongue coating microorganism.?5?Functional prediction? Comparison of microbial species abundance difference of tongue coating:There were differences in species abundance between the case group and the healthy person control group,specifically the decrease in bacterial abundance of campylobacterales,campylobacteraceae,peptococcaceae,alloprevotella,campylobacter and lachnoanaerobaculum.? Comparison of microbial species abundance difference in intestinal:There were differences in species abundance between the case group and the healthy person control group,specifically at the genus level:Streptococcus,Neisseria,Veillonella,Prevotella₇,Actinomyces,Granulicatella,Prevotella,Solobacterium,Lachnoanaerobaculum,Oribacterium,Peptostreptococcus,Campylobacter,Catonella,Bergeyella.The abundance of Mogibacterium is relatively high,and the abundance is low in Erysipelotrichaceae UCG-003,Bilophila,Blautia,Lachnospiraceae_UCG-004,Lachnospiraceae_NK4A136_group,uncultured bacterium_fLachnospiraceae,Sutterella,Roseburia,Lachnospira,Bacteroides.?6?Functional prediction?Tongue coating microbial KEGG function analysis:There was a difference in the functional analysis of KEGG between the case group and the healthy control group,specifically:A.classl level,the case group had lower functional expression in genetic information processing and strong expression in metabolism.And the difference is significant.B.class2 level,the case group is higher in Amino acid metabolism,lower in translation,Nucleotide metabolism,Metabolism of terpenoids and polyketides,Replication and repair,Folding,sorting and degradation,Infectious diseases,Cell growth and death.And the difference is significant.? Tongue microbial COG analysis:There was a difference in the COG function analysis between the case group and the healthy person control group,specifically:A.classl level,the expression of metabolism in the case group was higher,and the difference was significant.B.class2 level,the case group has higher expression in amino acid transport and metabolism,lower in post-transcriptional modification,protein turnover and chaperones,replication,recombination and repair,cell wall/membrane/envelope biosynthesis.And the difference is statistically significant.? intestinal microbe KEGG analysisThere was a difference in the functional analysis of KEGG between the case group and the healthy control group,specifically:A.classl level,the case group had high expression in genetic information processing and human disease,in the organic system,Cellular Processes,and Metabolism are less expressed.B.class2 level,case group had high expression in translation,Nucleotide metabolism,Membrane transport,folding,sorting and degradation,Neurodegenerative diseases,Metabolism of other amino acids,Transcription,Endocrine and metabolic diseases,Infectious diseases,and low expression in the immune system,environmental adaptation,other secondary metabolites,cell motility expression.And the difference is significant.? intestinal microbe COG analysisThere was a difference in COG function analysis between the case group and the healthy control group,specifically:A.classl level,the case group had higher expression in cellular processes and signaling,and the difference was significant.B.class2 level,case group highly expressed in translation,ribosomal structure and biogenesis,post-translational modification,protein turnover,posttranslational modification,protein turnover,chaperones,nucleotide transport and metabolism,lipid transport and metabolism,secondary metabolites biosynthesis,RNA processing and modification,Chromatin structure and dynamics,and extracellular structures,and lowly expression in the cell motility defense mechanisms,carbohydrate transport and metabolism,Signal transduction mechanisms.And the difference is statistically significant.3.Macroscopic measurement results of the phlegm syndrome of coronary heart disease scale:?1?clinical symptomsThe main symptoms of patients with coronary heart disease and phlegm syndrome are chest tightness,greasy fur,slippery veins,body obesity,fatigue,yellow greasy fur,greasy fur,bruising lips,insomnia,dry mouth.Comparison of cold syndrome and heat syndrome group:chest tightness,chest pain,greasy greasy,pulse slip,body obesity are common symptoms,clinical manifestations of heating moss yellow greasy group:moss yellow,dry mouth,mouth bitterness,tongue red is hot Cold;white and white fur group:fatigue,insomnia,and phlegm are clinical manifestations of cold syndrome.?2?Measurement scores of the scalePatients with phlegm syndrome of coronary heart disease had higher scores in the three items of chest tightness,greasy greasy,and pulse slip,and scores lower in other items.Specifically:chest tightness?2.01±10.5?,chest pain?0.54±0.74?,sputum?0.62±0.83?,body weight?0.63 ±0.82?,distention and fullness?0.48±0.74?,body obesity?0.9±0.88?,head weight?0.44±0.77?,dizziness?0.42±0.67?,pale tongue?0.67±0.95?,greasy greasy?1.89±0.49?,pulse slip?1.47±0.77?,pulse sputum?0.07±0.26?,pulse string?0.74±0.95?,total score?10.89±2.94?.The comparison between the repeated measurements of the three scales showed that chest tightness,chest pain,phlegm,sputum,dizziness,pulse slip,and total score were seven,and the difference was statistically significant.4.Typical greasy objectification measurement:?1?White coating of Coronary heart disease with phlegm syndrome group?white coating group?:Tongue measurement:A.tongue R value?158±25?,tongue G value?116 ± 19?,tongue B value?112±21?;B.tongue coating R value?180 ± 26?,tongue coating G value?145 ± 19?,tongue B value?140±25?;C.tongue coating area ratio?0.38±0.22?;D.tooth marks:number of tooth marks 1?0-4?,tooth mark area 0.57?0-8.06?;E.crack:The number of cracks is 1.5?0-8?and the maximum crack length is 0.17?0-0.94?.?2?Yellow coating of Coronary heart disease with phlegm syndrome group?yellow coating group?:Tongue measurement:A.tongue R value?159±17?,tongue G value?108±13?,tongue B value?99±16?;B.tongue coating R value?188 ± 13?,tongue coating G value?141 ± 11?,tongue B value?126±13?;C.tongue coating area ratio?0.28±0.15?;D.crack:crack number 2?0-7?,maximum crack length 0.17?0-0.92?.?3?Healthy control group:Tongue measurement:A.tongue R value?147±17?,tongue G value?119 ± 16?,tongue B value?123±15?;B.tongue coating R value?169 ± 17?,tongue coating G value?153 ± 14?,tongue B value?158±16?;C.tongue coating area ratio?0.75±0.21?.Compared with the white coating group,the yellow coating group in the case group suggested that the B-value of the yellow-stained moss group and the B-value of the tongue coating were lower.Compared with the control group,the white coating group of the case group suggested that the B-value of the tongue coating of the white-scarred group of the coronary heart disease was lower,the largest tooth mark area was larger,the number of cracks was larger,the maximum crack length was longer,and the number of pricks was less..Compared with the control group,the yellow coating group in the case group showed that the B-value of the tongue-stained sputum was lower in the tongue,the R value of the tongue coating was higher,the B value and G value of the tongue coating were lower,and the number of cracks was larger.The crack length is longer,the number of pricks is less,the maximum puncture length is shorter,and the maximum puncture area is smaller.Conclusion:1.Tongue microbe and intestinal microbes of phlegm syndrome in Coronary heart disease,are differed from the healthy control group in terms of species diversity and species abundance.In the case group,the difference between tongue coating and intestinal microbes was not significant,and the abundance was consistent.2.Species and functional analysis of the tongue coating revealed that Streptococcus,Neisseria,Veillonella,Prevotella₇,and Actinomyces may be potential biomarkers for phlegm syndrome with coronary heart disease.3.The macroscopic measurement of the phlegm scale revealed that the chest tightness,greasy,and pulse slip weight were higher.4.Objective measurement of tongue image:the tongue B value,tongue coating B value and tongue G value decrease in the case group,which provides a reference for the diagnosis of coronary heart disease.