| Objective:This study focuses on the pathogenesis theory of "internal heat injuring yin" in Type 2 diabetes(Type 2 Diabetes Mellitus,T2DM)mediated by "Eryang knots are called elimination" in "Nei Jing",to explore the clinical characteristics of yin deficiency syndrome in T2DM(including prediabetes and diabetes),and based on the "bacteria" Group-metabolite"pathogenic theory,preliminary exploration of the biological basis of T2DM yin deficiency syndrome,in order to explain the scientific connotation of "internal heat damage yin" caused by "two yang knots are called elimination".details as follows:1.Based on Delphi method and AHP,item selection and weight assignment of"syndrome differentiation scale of yin deficiency syndrome of type 2 diabetes" were carried out.Through clinical cross-sectional study,the clinical phenotype characteristics of T2DM Yin deficiency syndrome were initially explored,and a prediction model was established to improve and verify the above scale.2.By detecting and analyzing the biological samples of T2DM patients with typical Yin deficiency syndrome/non Yin deficiency syndrome and healthy volunteers(nhv),explore the characteristic biomarkers of T2DM Yin deficiency syndrome and their correlation,and explore the effects of intestinal flora of T2DM patients with Yin deficiency syndrome on the clinical manifestations and intestinal microenvironment of db/db mice through fecal microbiota transplantation(FMT),Preliminarily verify the interaction between intestinal flora disorder and the formation of T2DM Yin deficiency syndrome.Methods:1.based on Delphi method,the results of expert questionnaire of TCM syndrome differentiation scale of type 2 diabetes mellitus were analyzed statistically,and the item was selected and weighted by AHP.2.based on the clinical cross-sectional study method,438 patients with Yin deficiency syndrome and 223 patients with non Yin deficiency syndrome were included in the T2DM.The DS01-G group was used to collect tongue and eye information,and the TCM syndrome differentiation scale of type 2 diabetes mellitus was used to record the general situation and physical and chemical indexes.The clinical characteristics of 2 groups were analyzed by logistic and single factor and multi factor analysis of logistic.Establish the prediction model of T2DM Yin deficiency syndrome.3.14 T2DM patients with typical Yin deficiency syndrome,8 patients with typical non Yin deficiency syndrome and 8 healthy volunteers were included.Biological samples were collected from each group.16S rDNA high-throughput sequencing technology was used to explore the differences of intestinal flora structure and abundance in each group through OTU cluster annotation,alpha diversity analysis,beta diversity analysis,lefse and stamp differential flora analysis;The differences of intestinal short chain fatty acids and serum metabolites in each group were detected and analyzed by targeted metabolomics based on gas chromatography mass spectrometry(GC-MS)and g350 targeted absolute quantitative detection technology based on ultra high performance liquid chromatography mass spectrometry(UPLC-MS);Spearman correlation analysis was used to jointly analyze the above characteristic biomarkers of T2DM Yin deficiency syndrome,explore the relationship between "host flora short chain fatty acids metabolites",and detect the expression difference of cytokines in each group by enzyme linked immunosorbent assay(ELISA),so as to evaluate the inflammatory state of T2DM Yin deficiency patients.4.One T2DM patient with typical Yin deficiency syndrome,one T2DM patient with typical non Yin deficiency syndrome and one healthy volunteer were included.Fecal samples from each group were collected to prepare fecal bacteria freeze-dried powder.Ordinary male db/db and db/m mice aged 3 weeks were taken as the research object,db/m mice were taken as the blank group and db/db mice were taken as the model group.Fecal bacteria transplantation experiments of 3 samples were carried out on the basis of the model group.After continuous intervention for 4 weeks,food intake,body weight,body temperature Fasting blood glucose and behavioral changes,and 16S rDNA high-throughput sequencing technology was used to detect the fecal samples of each group at week 0,2 and 4,so as to preliminarily explore the effect of intestinal flora of T2DM patients with typical Yin deficiency syndrome on db/db mice.Results:1.A total of 25 domestic famous experts in the field of traditional Chinese medicine in the prevention and treatment of endocrine and metabolic diseases were selected.The expert authority score was 18.3.The recovery rates of the three rounds of expert questionnaires were 84.00%,76.00%and 76.00%respectively.According to the coefficient of variation and mean value of each item in the second round of expert questionnaire,finally "red tongue and less moss(peeling/no moss)","five heart upset and heat","red tongue and less fluid","pulse count","night sweat","dry mouth and throat","dry eyes","thin body or getting thin","thirsty for cold drinks","dry stool","fear of heat" and "dry and itchy skin" "Upset" was included in the scale of TCM syndrome differentiation of yin deficiency syndrome of type 2 diabetes.Through the consistency test results of the third round of expert questionnaire,the weights and assignments of the above items are:0.1715(17 points),0.1297(13 points),0.1202(12 points),0.1006(10 points),0.0950(10 points),0.0687(7 points),0.0686(7 points),0.0583(6 points),0.0431(4 points),0.0417(4 points),0.0411(4 points),0.0328(3 points)and 0.0284(3 points).2.cross sectional study found that the proportion of women accounted for,hyperlipidemia,FBG,2hPG,HbAlc,TgAb,TG,SLDL,and the TCM syndrome differentiation scale of type 2 diabetes mellitus(yin deficiency)were related to the items(except body weight or thinning)scores,tongue-Lab-L in tongue,tongue-Lab-a in tongue,proportion of red spot in eye examination,and pink mist in scores of yin deficiency syndrome(T2DM<0.05).The proportion and score of ecchymosis,ecchymosis,white fur,dark gray spots and yellow fog were significantly lower in T2DM patients with Yin deficiency syndrome(P<0.05).The above differential indexes were analyzed by logistic single factor and multi factor analysis,and the prediction model equation of T2DM Yin deficiency syndrome was established:1.018 x "History of hyperlipidemia"+0.584 × "Red tongue with little moss(peeling/no moss)"+0.439 ×"Red tongue and less fluid"+0.437 x "Five heart upset heat"+0.288 × "Night sweat"+0.899 × "Fear of heat"+0.746× "Pulse number"+0.293 ×"Dry mouth and dry throat"+0.832 × "Dry stool",the area under the ROC curve of the equation is 0.977(95%CI:0.9167-0.9389,P<0.001),and the best score is 5.09 points.According to this value,it is divided into high-risk group(>5.09 points)and low-risk group(0-5.09 points).The probability of T2DM Yin deficiency syndrome in the two groups has obvious statistical difference(P<0.001),suggesting that the reliability and validity of the prediction model are good.3.The results of 16S rDNA showed that in terms of alpha diversity,the species diversity of T2DM Yin deficiency syndrome group was lower than that of non Yin deficiency syndrome group and healthy volunteers(P<0.1);In terms of beta diversity,there were significant differences in the classification and composition of intestinal microorganisms among the three groups(P<0.05);At the genus level,compared with healthy volunteers,the abundance of subdoligranulum,Ruminococcus 2,Eubacterium xylanophilum,Eubacterium coprostanogenes,holdemania,Pseudomonas,Desulfovibrio,collinella,alistipes and coprococcus 2 in T2DM Yin deficiency syndrome group decreased significantly(P<0.05),and the abundance of megamonas and gemmobacter increased significantly(P<0.05);The abundance of eggerthellaceae,nitrosomonadaceae,eisenbergiella,oscillibactereeggerthella,ellin6067,Clostridium innocum group,ruminococcaceae UCG 004 in T2DM Yin deficiency group was significantly lower than that in non Yin deficiency group(P<0.05),and the abundance of lachnospiraceae nk4a136,Ruminococcus 1,lachnospiraceae nd3007,Porphyromonas,gemmobacter and moryela was significantly higher(P<0.05).The results of GC-MS showed that the contents of acetic acid,butyric acid and total short chain fatty acids in T2DM Yin deficiency syndrome/non Yin deficiency syndrome group were significantly lower than those in healthy volunteers(P<0.05);Compared with non Yin deficiency group,the contents of butyric acid,isobutyric acid and isovaleric acid in T2DM Yin deficiency group tended to decrease(P<0.1),and the content of propionic acid tended to increase(P<0.1),UPLC-MS results showed that the expressions of leucyl glycine and D-2-hydroxyglutaric acid in T2DM Yin deficiency syndrome group were significantly higher than those in non Yin deficiency syndrome group and healthy volunteers(P<0.05)α-The expression of ketisovaleric acid,vitamin B2 and acetoin decreased significantly(P<0.05);The expression of D-lactic acid in T2DM Yin deficiency syndrome group was significantly higher than that in healthy volunteers(P<0.05),and the expression of tetradecanoic acid,ricinoleic acid,thdca and indole-3-formic acid were significantly lower(P<0.05);The expression of hydroxyproline in T2DM Yin deficiency syndrome group was significantly higher than that in non Yin deficiency syndrome group(P<0.05),and the expression of azelaic acid and lithocholic acid was significantly lower(P<0.05).Spearman correlation analysis showed that,compared with the healthy volunteers,the expression of butyric acid in T2DM Yin deficiency syndrome group was positively correlated with ruminal bacteria genus 2,Clostridium family XII UCG001 and butyric acid,rare coccobacterium genus was positively correlated with butyric acid,Clostridium XIII-UCG001,ruminal bacteria genus 2,Wolbachia,Corinthia,Eubacterium xylanophilum and maleic acid;Rumen 2,Trichospirillum UCG010,Wolbachia and vitamin B2 were positively correlated;There was a positive correlation between Cochrainia and tetracyclic acid,THDCA;Rumen 2 and Wolbachia were negatively correlated with 3-phosphoglycerate;Macromonas was negatively correlated with the maleic acid and tetracyclic acid;Christensen R7,Eubacterium xylanophilum,Eubacterium faecalis,Alternaria and a-Hydroxyisobutyric acid was negatively correlated with 2-hydroxybutyric acid;Trichospirillum UCG010,Wolbachia and glutaryl coenzyme A were negatively correlated;Trichospirillum UCG010 was negatively correlated with the guanylic acid;There was a negative correlation between D-2-hydroxyglutaric acid and rare coccidiomycetes;There was a negative correlation between Cochrainia and D-lactic acid;Compared with non Yin deficiency syndrome group,in T2DM Yin deficiency syndrome group,Oscillibacter was positively correlated with isobutyric acid and isovaleric acid,rumen bacteria UCG004 was positively correlated with isobutyric acid,isovaleric acid and caproic acid,and Oscillibacter was positively correlated with maleic acid;Clostridium harmlessness was positively correlated with vitamin B2;Rumen bacteria UCG004 and α-Ketoisovaleric acid was positively correlated;Trichospirillum NK4A136 and α-Ketoisovaleric acid was negatively correlated.ELISA results showed that compared with healthy volunteers,the expressions of GLP-1 and GLP-2 in T2DM Yin deficiency syndrome group were significantly lower(P<0.05),IL-1β、IL-6、TNF-α、NF-κβ、AMPK α、The expression of TLR4 and LPS increased significantly(P<0.05);The expression of IL-1 in non Yin deficiency syndrome group was significantly lower than that in non Yin deficiency syndrome group(P β、IL-6、TNF-α、NFκβ、AMPK α、The expression of TLR4 and LPS increased significantly(P<0.05).4.The results of animal experiments showed that c-t2dmyx group had the characteristics of sharp action,irritability,dry hair and red palm color compared with other groups in the same period,and its FBG,body weight,food intake and Anal temperature were significantly higher than those of other groups in the same period(P<0.05);At the 4th week,the species diversity of intestinal flora in c-t2dmyx group and d-t2dmfy group was significantly lower than that in e-hc group(P<0.05);At the genus level,the abundances of cupriavidus,muribaculaceae mouse gut metanome,mucispirillum,solibacteraceae subgroup 3 akiw659,Bacteroides and Eubacterium xylanophilum group in c-t2dmyx group were significantly higher than those in other groups at the same time(P<0.05);The abundances of alloprevotella,Helicobacter,lachnospiraceae UCG 001 and Roseburia in c-t2dmyx group were significantly lower than those in d-t2dmfy group(P<0.05);Compared with e-hc group in the same period,c-t2dmfy group included Eubacterium brachy group,acetitomaculum,harryflintia,papillibacter,clostridia XIII UCG 001,lachnoclostridium,Eubacterium coprostanogenes group,ruminococcaceae UCG 005,ruminiclostridium 6,anaerotruncus,ruminiclostridium 5,lachnospiraceae UCG 006,blautia,intetinimonas The abundance of ruminiclostridium,Ruminococcus 1 and ruminococcaceae UCG 014 decreased significantly(P<0.05).Conclusions:Around the "Er Yang Jie Wei Zhi Xiao",the TCM syndrome theory of T2DM,established by the theory of TCM pathogenesis,can significantly reflect the clinical phenotype characteristics of T2DM Yin deficiency syndrome.The prediction model of T2DM Yin deficiency syndrome based on the relevant items of the scale and the level of glucose and lipid has high reliability and validity.It can be used for the preliminary screening of patients with clinical T2DM Yin deficiency syndrome,is conducive to the prevention and treatment of clinical T2DM Yin deficiency syndrome in traditional Chinese medicine,and provides a relatively objective quantitative tool for the establishment of the universal judgment standard of yin deficiency syndrome in traditional Chinese medicine.In addition,the formation of T2DM Yin deficiency syndrome is closely related to the multi-channel cascade reactions such as SCFAs metabolism abnormality,bile acid metabolism abnormality,amino acid metabolism abnormality and immune inflammation mediated by intestinal flora disorder.Its characteristic intestinal microorganisms can aggravate the development of T2DM and the occurrence of some clinical manifestations of yin deficiency syndrome in db/db mice,indicating that T2DM Yin deficiency syndrome may be formed by the influence of "flora metabolite" disorder,To some extent,this explains the scientific connotation of "Yang Ming knot heat" leading to "internal heat injuring Yin" in"two Yang knot is called Zhixiao",and provides some new targets for the clinical treatment of T2DM Yin deficiency syndrome. |
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