| Objective:Irritable bowel syndrome with diarrhea(IBS-D)is a dysfunctional disease that consumes a large amount of medical resources due to its high incidence and recurrence symptoms.Although the name of IBS-D is not found in classical medical texts,modern medical practitioners have classified it under the names "abdominal pain" and "diarrhea" in Chinese medicine based on the characteristics of its clinical manifestations.Shen-Ling-Bai-Zhu-San(SLBZS)have good efficacy in treating IBS-D of spleen deficiency and dampness excess syndrome,but the mechanism behind its effectiveness are not fully understood.Basic research suggests that the biological basis of spleen deficiency and dampness excess syndrome in IBS-D may be related to gut microbiota and metabolite disorders,and the effect mechanisms of SLBZS may be related to the regulation of gut microbiota and metabolites.Therefore,in this study,16 S r RNA,metagenomics,non-targeted metabolomics and targeted metabolomics was used to explore the mechanisms of SLBZS for the treatment of spleen deficiency and dampness excess in IBS-D and to provide an objective scientific basis for the clinical application of SLBZS.Methods:1.Sixty patients with IBS-D of spleen deficiency and dampness excess syndrome were collected at the Affiliated Hospital of Chengdu University of Traditional Chinese Medicine.Sixty healthy controls(Health)with age,sex,and BMI matching the case group were also recruited.The IBS-D patients with spleen deficiency and dampness excess syndrome were treated with SLBZS for 14 days.Baseline information was collected from all study subjects.The HAMA and HAMD scales were assessed in all study subjects.The IBS-SSS scale,the IBS-QOL scale,and the Traditional Chinese Medicine Evidence-based Efficacy Rating Scale were used as efficacy indicators in IBS-D patients.2.The stools of the Health group,the IBS-D group before treatment with SLBZS(IBS-SA),and the IBS-D group after treatment with SLBZS(IBS-SB)were collected.DNA was extracted from these three groups.16 S r RNA gene sequencing technology was used to detect the diversity and structural differences of gut microbiota in the study subjects.3.Using the Micro PITA method,30 cases from each of the Health and IBS-D groups were screened as study subjects.Their stools were collected for metagenomics to explore differences in the structural and functional levels of the study subjects’ gut microbiota.The screened differential genera were correlated with the HAMA,HAMD,IBS-SSS,and IBS-QOL scales.4.Plasma from study subjects screened in in step 3 was collected,and the concentrations of metabolites in plasma were detected using non-targeted metabolomics to screen for differential metabolites and perform KEGG enrichment analysis.The screened differential metabolites were analyzed in association with the HAMA,HAMD,IBS-SSS,and IBS-QOL scales and with the differential genera screened by metagenomics.5.Plasma from the study subjects screened in step 3 was collected,and plasma concentrations of the 25 bile acids were measured.The 25 bile acids were analyzed in association with the HAMA,HAMD,IBS-SSS,and IBS-QOL scales and with the differential genera screened by the metagenomics.Results:1.The HAMA and HAMD scores were significantly higher in the IBS-SA group than in the Health group(P<0.05).The HAMA,HAMD,and IBS-SSS scores were significantly lower in the IBS-SB group compared with the IBS-SA group(P<0.05).The IBS-QOL scores were significantly higher in the IBS-SB group compared with the IBS-SA group(P<0.05).The Traditional Chinese Medicine Evidence-based Efficacy Rating Scale scores were significantly lower in the IBS-SB group compared with the IBS-SA group(P<0.05).2.The 16 S r RNA gene sequencing results showed that the PD＿whole＿tree indexes was significantly lower in the IBS-SA group than in the Health group(P<0.05).The gut microbiota of the IBS-SA group showed higher abundance of Agathobacter,Lachnospira,Sutterellaceae,Parasutterella,and Phascolarctobacterium.The gut microbiota of the Health group showed higher abundance of Coprococcus,Veillonella,Oscillospiraceae,and Bifidobacterium.3.The 16 S r RNA gene sequencing results revealed that the Shannon and Simpson indices of gut microbiota were significantly higher in the IBS-SB group compared with the IBS-SA group(P<0.05).The gut microbiota of the IBS-SB group showed higher abundance of Clostridia,Firmicutes,Oscillospirales,and Eubacterium＿hallii＿group.4.The metagenomics results revealed that the structural differences in gut microbiota between the Health,IBS-SA,and IBS-SB groups were significant.The Health group showed higher abundance of Sphingomonas,Blautia＿sp＿KLE＿l732,Ochrobactrum＿rhizosphaerae,and Ochrobactrum.The IBS-SA group showed higher abundance of Megamonas,Escherichia,and Escherichia＿coli.The IBS-SB group showed higher abundance of Roseburia,Roseburia＿inulinivorans,Faecalibacterium,Faecalibacterium＿prausnitzii,and Clostridium.Pearson correlation analysis revealed that Escherichia was positively correlated with HAMD and negatively correlated with the IBS-QOL total score.Pearson correlation analysis revealed that Roseburia was positively correlated with body image and interference with daily activities in IBS-QOL,and Faecalibacterium was positively correlated with body image,impact on relationships,and IBS-QOL total score.5.Metagenomics results showed significant differences in gut microbiota function in the Health group compared to the IBS-SA group,and in the IBS-SA group compared to the IBS-SB group.Flagellar assembly,benzoate degradation,and dioxin degradation signaling pathways were significantly increased in the gut microbiota of the IBS-SA group compared to the Health group.Compared to the IBS-SA group,the IBS-SB group showed a significant decrease in the lipopolysaccharide biosynthesis pathway and a significant increase in the fatty acid biosynthesis and glutamatergic synapse pathways.6.Compared the Healthy and IBS-SA groups,as well as compared the IBS-SA group with the IBS-SB group,by non-targeted metabolomics analysis.The findings revealed significant differences in metabolites among each of the paired groups.Kynurenic acid and sphingosine were significantly increased while nobiletin,wogonin,and taurine were significantly lower in the IBS-SA group compared to the Health group.Taurine,prostaglandin A2,cis-4-Hydroxy-D-proline,and adenosine were significantly increased in the IBS-SB group compared with the IBS-SA group.Fumaric acid expression was significantly reduced in the IBS-SB group compared to the IBS-SA.The KEGG pathways that were significantly differentially enriched for metabolites between IBS-SA and Health groups contained tryptophan metabolism,neuroactive ligand-receptor interaction,sulfur metabolism,primary bile acid biosynthesis,ABC transporters,and metabolic pathways.The KEGG pathways that were significantly differentially enriched for metabolites between the IBS-SA and IBS-SB groups contained c AMP signaling pathway,arachidonic acid metabolism,and neuroactive ligand-receptor interaction,morphine addiction,phenylalanine metabolism,oxidative phosphorylation,purine metabolism,arginine and proline metabolism,tyrosine metabolism,and primary bile acid biosynthesis pathways.Pearson correlation analysis showed that taurine was positively correlated with sexual function,while it was negatively correlated with Escherichia.7.Compared the Healthy and IBS-SA groups,as well as compared the IBS-SA group with the IBS-SB group,by targeted metabolomics analysis.The findings revealed significant differences in bile acids among each of the paired groups.Compared with the Health group,the IBS-SA group showed significant increased of glycochenodeoxycholic acid sodium salt,tauroursodeoxycholic acid dihydrate,chenodeoxycholic acid,and taurochenodeoxycholic acid sodium salt(P < 0.05).Compared with the Health group,the IBS-SA group showed significant decreased of ursodeoxycholic acid and lithocholic acid(P < 0.05).Glycochenodeoxycholic acid sodium salt,tauroursodeoxycholic acid dihydrate,and taurochenodeoxycholic acid sodium salt were significantly decreased in the IBS-SB group compared to the IBS-SA group(P < 0.05).Ursodeoxycholic acid and lithocholic acid were significantly increased in the IBS-SB group compared to the IBS-SA group(P < 0.05).Pearson correlation analysis showed a negative correlation between glycochenodeoxycholic acid sodium salt and health worries.Tauroursodeoxycholic acid dihydrate was positively correlated with abdominal pain intensity,abdominal distension,and IBS-SSS total score.Tauroursodeoxycholic acid dihydrate was negatively correlated with health worries.Chenodeoxycholic acid was negatively correlated with interference with quality of life.Taurochenodeoxycholic acid sodium salt was positively correlated with dissatisfaction with bowel habits.Lithocholic acid was negatively correlated with HAMD and positively correlated with the IBS-QOL total score.Ursodeoxycholic acid was negatively correlated with food avoidance,health worries,social reaction,impact on relationships,and the IBS-QOL total score.Conclusions:1.Patients with IBS-D of the spleen deficiency and dampness syndrome exhibit significantly higher scores of anxiety and depression compared to the healthy controls.SLBZS significantly alleviates emotional disorders in patients with IBS-D of the spleen deficiency and dampness syndrome,improves clinical symptoms,and enhances their quality of life.Moreover,SLBZS shows promising therapeutic effects for the corresponding traditional Chinese medicine syndrome of spleen deficiency and dampness excess syndrome of IBS-D.2.There were significant differences in the gut microbiota structure between spleen deficiency and dampness excess syndrome of IBS-D and healthy controls.The IBS-D group showed higher abundance of Agathobacter,Lachnospira,Sutterellaceae,Parasutterella,Phascolarctobacterium,Megamonas,Escherichia,and Escherichia＿coli.3.SLBZS may treat spleen deficiency and dampness excess syndrome of IBS-D by increasing the abundance of Roseburia,Roseburia＿inulinivorans,Faecalibacterium,Faecalibacterium＿prausnitzii,Clostridium,and Eubacterium＿hallii＿group.4.Significant differences in gut microbiota function were observed among each of the paired groups.Compared to the healthy controls,flagellar assembly,benzoate degradation,and dioxin degradation signaling pathways were significantly increased in the gut microbiota of the IBS-D group.Compared to before treatment,IBS-D patients with SLBZS exhibit a significant decrease in the lipopolysaccharide biosynthesis pathway and a significant increase in the fatty acid biosynthesis and glutamatergic synapse pathways.5.Compared to the healthy controls,kynurenic acid and sphingosine were significantly increased while nobiletin,wogonin,and taurine were significantly lower in the IBS-D group.Compared to before treatment,IBS-D patients with SLBZS exhibit a significant increase in taurine.The therapeutic effect of SLBZS on IBS-D may be related to the modulation of arachidonic acid metabolism,phenylalanine metabolism,purine metabolism,arginine and proline metabolism,and primary bile acid biosynthesis pathway.6.Compared with healthy controls,glycochenodeoxycholic acid sodium salt,tauroursodeoxycholic acid dihydrate,chenodeoxycholic acid,and taurochenodeoxycholic acid sodium salt were significantly increased in IBS-D patients,while ursodeoxycholic acid and lithocholic acid were significantly decreased in IBS-D patients.Compared to before treatment,IBS-D patients with SLBZS exhibit a significant decrease in glycochenodeoxycholic acid sodium salt,tauroursodeoxycholic acid dihydrate,chenodeoxycholic acid,and taurochenodeoxycholic acid sodium salt.Compared to before treatment,IBS-D patients with SLBZS exhibit a significant increase in ursodeoxycholic acid and lithocholic acid. |
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