16S RRNA Sequence Analysis To Explore The Relationship Between Intestinal Flora And TCM Syndromes In Patients With Depression And Digestive Tract Symptoms

Major Depressive Disorder(MDD),a type of syndrome characterized by mood disorders.The World Health Organization predicts that depression will become the second leading cause of human death and disability in 2020,and it will become one of the biggest problems to be solved in the global healthcare system.So far,the pathogenesis of depression is still unclear.In recent years,more and more studies have shown that intestinal microbes,as the largest and most direct external environment of the human body,have about 10 trillion bacteria,which is equivalent to 9 times the number of human cells and 150 times the number of genes.The"microbial-intestinal-brain" axis affects brain function and behavior,leading to depression.Therefore,research to prevent the onset of depression,discuss the pathogenesis,and explore effective treatment options is essential and urgent.The aim of the research:Taking patients with depression and gastrointestinal symptoms as the research object,16S rRNA gene sequencing method was used to compare the composition of intestinal microflora in the feces of patients with different syndrome types(liver stagnation and spleen deficiency syndrome and liver and gallbladder dampness syndrome),and to evaluate whether there was significant change.The characteristics and relationships of different kinds of bacteria,thus providing a new perspective of TCM syndrome differentiation for depression,improving the level of syndrome differentiation and treatment,and improving the relationship between the "microbial-intestinal-brain" axis and traditional Chinese medicine theory,in order to further explore " syndrome-Chinese medicine-Microorganisms and individualized intestinal management lay the foundation.Method:Selected subjects:Depressed patients and healthy people who met the inclusion and exclusion criteria for the encephalopathy department of the Oriental Hospital of Beijing University of Traditional Chinese Medicine from January 20 to December 1818.The syndromes of depression patients were liver depression.15 cases of spleen deficiency,15 cases of liver and gallbladder dampness;Fill in the questionnaire:Sign the relevant informed consent form,collect the basic information of the three groups of subjects,and conduct the Hamilton Depression Scale and the TCM Syndrome Observation Table for Depression;Specimen collection:Collect the fecal specimens of the study subjects and quickly freeze the specimens in a refrigerator at-80℃ for subsequent experiments.Sequencing experiments:The extracted 16S rRNA genomic DNA fragments were synthesized by the Miseq sequencing method with specific primers for barcode amplification,and the PCR products were quantified by QuantiFluorTM-ST blue fluorescence quantitative system.Finally,the Miseq library was constructed.And sequencing.Result:(1)General information:The age distribution of the subjects is mainly between 31-50 years old,and the ratio of male to female is about 1:1.Most of the occupations are white-collar workers,students and teachers.The education level is mostly undergraduate and above.(2)Chief complaints,other symptoms and course of disease:Most patients have a course of 1-2 years;90%of patients complain of insomnia;100%of patients have pessimistic disappointment,thoughtfulness,inferiority and despair,and anorexia(70%,liver 50%spleen deficiency,20%liver and gallbladder dampness,good hunger(30%),sticky sputum(40%,liver spleen spleen 40%),constipation(50%,liver spleen spleen about 3%,liver and gallbladder damp heat about 47%).(3)Scale situation:all patients were mild to moderate depression,Hamilton depression scale scores were greater than 20 points,less than 35 points;4 cases of liver depression and spleen deficiency syndrome Hamilton anxiety scale score greater than 13 points less than 21 points,8 Patients with liver stagnation and spleen deficiency syndrome and 6 patients with hepatobiliary damp-heat syndrome scored greater than 7 points and less than 13 points.The average score of the Hamilton Anxiety Scale in patients with liver stagnation and spleen deficiency was significantly higher than that in patients with liver and gallbladder dampness syndrome.(4)Tongue pulse condition:patients with liver stagnation and spleen deficiency syndrome are mostly reddish tongue,thin white fur,moderate tongue shape,scalloped teeth,and smooth pulse line;patients with liver and gallbladder dampness syndrome are mostly red tongue,yellow greasy moss,or thin Or thick,moderate tongue shape,edge marks or cracks,slippery pulse.(5)Microbial structure:DNA was extracted from stool samples from 35 patients,and we obtained a total of 248,980 high-quality 16S rRNA gene sequences(7064±2350 reads/fecal samples),followed by similarity at 97%threshold.Horizontally divided into operational classification units(OTUs).These operational classification units(OTUs)are all attributed to the bacterial community.At the level of Phylum,the vast majority belong to the following two doors:Bacteroidetes and Fimicutes.The abundance of Bacteroides in the blank control group was about 54.94%±6.12%(x±s),the abundance of thick-walled bacteria was about 43.95%±6.53%,and the abundance of Bacteroides was about 50.05%±7.01%in patients with liver and gallbladder damp-heat syndrome.,the abundance of thick-walled bacteria is about 47.45%±8.63%,the abundance of Proteobacteria is about 1.48%±0.12%,and the abundance of Bacteroides is about 53.56%±6.65%(x±s)in patients with liver stagnation and spleen deficiency syndrome.The abundance of thick-walled bacteria is about 44.96%±7.81%,and the abundance of proteobacteria is about 1.46%±0.13%.At the level of family,the liver and gallbladder damp-heat syndrome patients’ milk Lactobacillaceae,Eubacteriaceae,Streptococcaceae,Veillonellaceae,Rikenellaceae,Alcaligenaceae,Burkholderia The proportion of Burkholderiaceae and Enterobacteriaceae is higher than that of patients with liver stagnation and spleen deficiency,while Lachnospiraceae,Bacteroides,Porphyromonadaceae,and Prevoaceae is lower than this type of patient.Ping Shang,liver and gallbladder damp-heat syndrome patients Lactobacillus,Eubacterium,Streptococcus,Veillonella,Acidaminococcus,Phascolarctobacterium,Rikenella,Burkholderia,Escherichia,Sutterella,Megamonas are higher than patients with liver spleen deficiency syndrome,while Bacteroides,porphyrin The proportion of the genus Porphyromonas,Prevotella,Coprococcus,and Ruminococcus is lower than that of such patients.(6)Microbial diversity:The Shan diversity index and Simpson index of the a diversity of the flora were higher than those of the liver and spleen dampness syndrome(but P>0.05),which was not statistically significant.Further,the Unweighted Unifrac distance matrix was used to study the β diversity of intestinal flora,and it was found that there were differences in the main components between the two groups.Further analysis of gender,age,disease duration and other categorical variables did not have a significant impact.(7)Analysis of the correlation between the abundance of intestinal flora and tongue image,and found that patients with thick red tongue and thick yellow fur are different from those with pale tongue and thin white fur;the abundance of intestinal flora is related to pulse Sexual analysis found that patients with sputum veins were higher than patients with non-chords,while Escherichia was lower;patients with Escherichia genus,Escherichia,and A.faecalis were higher than patients with non-slip veinsConclusion:(1)Most of the patients with depression are mental workers,with a high level of education and a younger trend;the main complaints at the time of treatment are mostly insomnia,accompanied by pessimistic disappointment,thoughtfulness,inferiority and despair,and the course of disease is generally 1-2 years.Most of the illness is mild to moderate.(2)The biggest difference between the symptoms of liver and gallbladder dampness syndrome and patients with liver stagnation and spleen deficiency syndrome is the nature of stool,tongue and depression.(3)Compared with patients with liver stagnation and spleen deficiency syndrome,there was no difference in a diversity,and there were significant differences in β diversity.The structural differences were mainly Bacteroides,thick-walled bacteria and proteobacteria.The degree of degree of change,and summed up the presence or absence of clinical symptoms of depression,and the relationship between the abundance and diversity of intestinal flora,and the main pathological changes of intestinal flora in patients with different syndromes were almost the same.But the degree of change is different.(4)Different tongue images and different pulse images have different abundances of intestinal flora,which means different meanings,and indirectly verify the theory of tongue and heart,moss and stomach in Chinese medicine.