| Objection:Irritable bowel syndrome(IBS)is a group of clinical symptoms characterized by recurrent abdominal pain or(and)abdominal discomfort,changes in stool characteristics,disordered defecation habits,and mental symptoms such as anxiety,depression and somatoform disorders.The high incidence rate and the repeated variability of IBS symptoms result in IBS occupying a large amount of medical resources.Tong-xie-yao-fang(TXYF)has a history of nearly one thousand years in the treatment of IBS-D of liver depression and spleen deficiency syndrome.It has a good effect on the symptoms of "abdominal pain is diarrhea,and the pain after diarrhea is reduced",but the scientific principle behind its effectiveness is not completely clear.Basic research shows that the disorder of intestinal flora and metabolites may be the biological basis of IBS with liver depression and spleen deficiency syndrome,and the effect mechanism of TXYF may be related to the regulation of intestinal flora and metabolites.Therefore,through the classification of Western medicine and TCM syndrome differentiation of IBS,taking intestinal flora and metabolites as the starting point,combined with 16 S r RNA gene sequencing,macrogenomics,non-targeted metabolomics and targeted metabolomics technology,this study verified the effectiveness and safety of TXYF in the treatment of IBS-D patients with liver depression and spleen deficiency in Han population in Southwest China,and explored the underlying scientific principles of TXYF in the treatment of IBS-D with liver depression and spleen deficiency syndrome,so as to provide objective scientific basis for the clinical application of TXYF.Methods:1.120 IBS-D patients with liver depression and spleen deficiency syndrome were divided into TXYF group(IBS-T)and blank control group(IBS-CG),and 60 healthy controls(Health)were recruited at the same time.IBS-T group was given TXYF orally for 14 days,and IBS-CG group was treated with lifestyle regulation.The demographic data,diet,sleep,exercise and other baseline data of all subjects were collected.All subjects evaluated HAMA and HAMD scales.Patients with IBS-D took IBS-SSS scale,IBS-QOL scale and TCM syndrome efficacy evaluation form as efficacy indicators.2.The feces of healthy control group(Health),TXYF group,IBS-D group(IBS-TA)before treatment and TXYF group(IBS-TB)after treatment were collected and DNA was extracted.16 S r RNA gene sequencing was used to detect the diversity and structural differences of intestinal flora.3.Micropita supervised method distinct was used to screen 30 cases from TXYF group and healthy control group respectively as the research objects.The feces of the research objects were collected for macrogenomics research to explore the differences in the structure and function of intestinal flora.4.Collect the plasma of the subjects screened in the third step and detect the differences metabolites and significantly enriched KEGG pathway.The association analysis was carried out with the dominant bacteria screened by macrogenome.5.Collect the plasma of the subjects screened in the third step,detect the 33 kinds of bile acids in the plasma,screen the bile acids with significant differences,and analyze the association with the dominant bacteria screened by macrogenome.Result:1.The personal monthly income of IBS-D patients was significantly lower than that of health group,and the labor intensity was higher than that of health group(P<0.05);In terms of living habits,the length of exercise,the amount of vegetables and fruits consumed by IBS-D patients were significantly lower than those in health group(P<0.05),and the intake of meat and tobacco were higher than those in health group(P<0.05).IBS-D patients liked spicy food more,and those who liked sour food were significantly less than those in health group(P<0.05).The scores of HAMA and HAMD in IBS-D group were significantly higher than those in health group(P<0.05).2.After treatment with TXYF,the scores of HAMA and HAMD in IBS-TB group decreased significantly(P<0.05).After treatment with TXYF,the three single and total scores of "abdominal pain and discomfort","frequency of abdominal pain" and "degree of abdominal distension" of IBS-SSS decreased significantly(P<0.05).After treatment with TXYF,there were significant differences in the five single and total scores of "anxiety","behavior disorder","health anxiety","heterosexual relationship","social relationship" and "family relationship" of IBS-QOL(P<0.05).After treatment with TXYF,there were significant differences in TCM syndrome scores in main symptoms,secondary symptoms,tongue and pulse and total score.This shows that TXYF can significantly alleviate the emotional disorder of IBS-D patients,improve the clinical symptoms of abdominal distension and abdominal pain,improve the quality of life of patients,and has a good effect on the TCM syndrome of liver depression and spleen deficiency.3.Compared with the IBS-T group and the IBS-CG group after treatment,the three single and total scores of "abdominal pain and discomfort","frequency of abdominal pain" and "degree of abdominal distension" in the TXYF group after treatment were significantly lower than those in the blank control group after treatment(P<0.05).After treatment,the four single and total scores of "anxiety","health anxiety","social response" and "family response" of IBS-QOL in TXYF group were significantly higher than those in the blank control group(P<0.05).After treatment,the TCM syndrome score of the TXYF group was lower than that of the blank control group(P<0.05).This shows that the curative effect of TXYF on IBS-D is significantly better than that of lifestyle regulation.4.By comparing the 16 S r RNA gene sequencing results between IBS-TA group and health group,it was found that the uniformity and β-diversity of intestinal flora in IBS-TA group and health group.Akkermansia,Clostridium＿sensu＿stricto＿1,Roseburia＿inulinivorans and Romboutsia ilealis were the dominant species in IBS-TA group;Coprococcus,Veillonella,Bifidobacterium and Dialist are the dominant bacteria in health group.This shows that there is a significant difference in the structure of intestinal flora between IBS-D of liver depression and spleen deficiency syndrome and healthy control group;5.16 s r RNA gene sequencing results compared IBS-TA group and IBS-TB group,it was found that after receiving TXYF treatment,the intestinal flora did not change significantly in α-diversity and β-diversity.And the abundance of Faecalibacterium＿prausnitzii,Clostridia＿ucg-014,Oscillospiraceae and Agathobacter changed significantly,indicating that TXYF can change the intestinal flora of IBS-D patients with liver depression and spleen deficiency syndrome to a certain extent.6.By comparing the results of metagenomics among IBS-TA,IBS-TB and health groups,it was found that the structure and function of intestinal flora in the three groups were significantly different.In the health group,the dominant species are Enterobacteriaceae and Ochrobactrum＿Rhizophaerae,Ochrobactrum and Rhizobiales.In the IBS-TA group,the dominant species are Streptococcus,Roseburia＿inulinivorans,Roseburia＿hominis and Eubacterium＿sp＿cag＿251.In the IBS-TB group,the dominant species are Roseburia,Romboutsia＿timonensis,Streptococcus salivarius,Clostridium l2-50,Clostridium CAG-440,Clostridium CAG217,Romboutsia.MAPK signaling pathway,autophagy and T cell receptor signaling pathway were significantly enhanced in IBS-TA group;IBS-TB group significantly improved the function of arginine biosynthesis and fatty acid biosynthesis.The mainly resistance mechanism of intestinal flora in IBS-D patients is antibiotic efflux,which is closely related to Firmicutes and Proteus.7.The PLS-DA of IBS-TA and Health,IBS-TA and IBS-TB groups were compared through the results of non-targeted metabolomics.The results showed that the three groups were clustered respectively.The expressions of phenylalanine,sphingosine,glycine,arachidonic acid,cholic acid,7-ketcholesterol,docosahexaenoic acid and eicosapentaenoic acid were significantly up-regulated in IBS-TA group,the expressions of Cetamide,riboflavin,carbamate,ursodeoxycholic acid,arachidonic acid and docosanoic acid were significantly down regulated in IBS-TA group.Compared with IBS-TA group,the expression of adenine in IBS-TB group was significantly up-regulated.The KEGG pathway with significant enrichment of differential metabolites between IBS-TA and healthy control group is the biosynthesis of unsaturated fatty acids(map01040)and carbon metabolism(map01200);KEGG pathways with significant enrichment of differential metabolites between IBS-TA and IBS-TB groups are c GMP PKG signaling pathway(map04022),vascular smooth muscle contraction(map04270),renin secretion(map04924),c AMP signaling pathway(map04024)Regulation of adipocyte lipolysis(map04923),neuroactive ligand receptor interaction(map04080)and purine metabolism(map00230).In the results of non-targeted metabolomics,Streptococcus in IBS-TA group was positively correlated with 7-ketcholesterol and docosapentaenoic acid;The dominant bacteria in IBS-TB group was Romboutsia,which was positively correlated with citral;The dominant bacteria in IBS-TB group,Roseburia,were negatively correlated with acetoacetic acid,and positively correlated with sphingosine and cholic acid.In the results of non-targeted metabolomics,the content of cholic acid in IBS-TA group was positively correlated with the abdominal pain score(r=0.409,P=0.034)and abdominal distension score(r=0.4390,P=0.044)of IBS-SSS scale;Sphingosine was positively correlated with IBS-SSS abdominal distension score(r=0.409,P=0.031);Cholic acid was negatively correlated with behavioral disorder in IBS-QOL scale(r=-0.412,P=0.033);Ursodeoxycholic acid was negatively correlated with the frequency of abdominal pain in IBS-SSS scale(r=-0.462,P=0.015);In IBS-TB group,adenosine was negatively correlated with the heterosexual relationship in IBS-QOL scale(r=-0.466,P=0.014).This shows that there are significant differences in bile acids,unsaturated fatty acids,amino acids,adenosine and other metabolites between IBS-D with liver depression and spleen deficiency syndrome and healthy control group,especially bile acids.The therapeutic effect of TXYF on IBS-D with liver depression and spleen deficiency may be closely related to adenosine.8.The quantitative results of bile acids in IBS-TA and health,IBS-TA and IBS-TB groups were compared.Lithocholic acid and ursodeoxycholic acid in IBS-TA group were significantly lower than those in healthy controls(P<0.05).Deoxycholic acid,isolithic acid and sodium lithic acid-3-sulfate in IBS-TB group were significantly higher than those before treatment(P<0.05).In the results of targeted metabolomics,the dominant bacterium in IBS-TB group was Roseburia,which was positively correlated with sodium lithocholic acid-3-sulfate,cholic acid,glycine lithocholic acid,isolithocholic acid,taurine ursodeoxycholic acid dihydrate and chenodeoxycholic acid,and negatively correlated with 7-ketocholic acid;The dominant bacteria in IBS-TB group was Romboutsia,which was negatively correlated with deoxycholic acid.In IBS-TA group,chenodeoxycholic acid was negatively correlated with the heterosexual relationship in IBS-QOL scale(r=-0.679,P=0.000);Cholic acid was negatively correlated with the heterosexual relationship in IBS-QOL scale(r=-0.435,P=0.021);Ursodeoxycholic acid was positively correlated with health anxiety in IBS-QOL scale(r=0.385,P=0.043).In IBS-TB group,chenodeoxycholic acid was positively correlated with the frequency of abdominal pain in IBS-SSS scale(r=0.377,P=0.048);Lithocholic acid was negatively correlated with the frequency of abdominal pain in IBS-SSS scale(r=-0.439,P=0.019).This shows that the bile acids of IBS-D patients with liver depression and spleen deficiency syndrome are significantly abnormal,especially the production of lithocholic acid and ursodeoxycholic acid.TXYF can promote the production of secondary bile acids in IBS-D patients with liver depression and spleen deficiency syndrome.This promoting effect is related to the regulation of TXYF on Roseburia and Romboutsia.The regulation process may involve the production of primary bile acids αDecarboxylation.Conclusion:1.The IBS-D of the spleen deficiency syndrome and the healthy controls had significant difference in their living habits,and the incidence rate of emotional disorders increased.The TXYF could significantly relieve the emotional disorder of IBS-D patients,improve the clinical symptoms of abdominal distension and abdominal pain,and improve the quality of life of patients.2.There are significant differences in the structure and function of intestinal flora between IBS-D patients with liver depression and spleen deficiency syndrome and healthy controls.The dominant strain of IBS-D patients with liver depression and spleen deficiency syndrome is Roseburia＿inulinivorans.The recipe for painful diarrhea may be by increasing Roseburia,Romboutsia＿timonensis and Streptococcus-salivarius abundances in the treatment of IBS-D patients with liver depression and spleen deficiency syndrome;MAPK signaling pathway,autophagy and T cell receptor signaling pathway were significantly enhanced in the intestinal flora of IBS-D patients;After receiving TXYF,IBS-D patients significantly improved the function of arginine biosynthesis and fatty acid biosynthesis.3.There are significant differences in bile acids,unsaturated fatty acids,amino acids and other metabolites between IBS-D of liver depression and spleen deficiency syndrome and healthy control group,especially the production of lithocholic acid and ursodeoxycholic acid.TXYF can promote the production of secondary bile acids in IBS-D patients with liver depression and spleen deficiency syndrome,which is related to the regulation of TXYF on Roseburia and Romboutsia. |
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