Exploration Of The Novel Therapeutic Mechanism Of Tong-xie-yao-fang Against IBS Rats Based On The "Brain-gut-enteric Microbiota Axis"

Objective:Irritable bowel syndrome (IBS) is a typical and prevalent emotional disease, It has been given consensus on dysregulated brain gut axisin in the traditional Chinese and western academic field. While, it is lack of studies about specific mechanism of gut microbiota in IBS development and the curative process of the Chinese medicine. This article is based on Brain-gut-enteric microbiota axis(BGMA), which can reflect a higher and more complete research vision. On the one hand, more deeply reveals pathological biological basis of IBS, on the other hand,to observe the key link and regulating mode about traditional Chinese medicine formula represented by Tong-xie-yao-fang(TXYF) regulating the BGMA, explore the mechanisms of traditional Chinese medicine preventing and treating IBS.Methods:100 Sprague-Dawley rats aged 8 weeks, half of male and female, were applied to establish a chronic unpredictable mild stress and humid and hot type diarrhea IBS model by chronic unpredictable mild stress as well as high-sugar high-fat feed, hot and humidity environment and alcohol by gastric stimulation. Indexes of the rat body weight growth rate, Bristol fecal character score, AWR score and open field test(OFT) score, were applied to comprehensive evaluate and identify IBS model rats. The rats successful established IBS model were randomly divided into control group (M), TXYF treatment 7 days group (T7), TXYF treatment 14 days group (T7), bifidobacterium treatment 7 days group (B7), bifidobacterium treatment 14 days group (B14), and normal control group, a total of 6 groups. Lavage treatment respectively by TXYF and positive control drug bifidobacterium for 7 days,14 days, real-time observe general situation of rats, weight growth rate, bristol fecal character score, AWR score and OFT score,verify the therapeutic effect of the TXYF against IBS rats. After the treatment, feces was collected by sterile method, the brain and colon tissue were fetched after lavageing heart, immunohistochemical method was employed to detect nitric oxide synthase (NOS), acetylcholine transferase(CHAT), vasoactive intestinal peptide(VIP), Fos protein expression in the hippocampus and colon tissue of rats which were employed to explore the regulating mechanism of TXYF against IBS model based on brain-gut-axis disorder. Total RNA of gut microbiota were extracted, reverse transcription for cDNA, using real-time quantitative PCR to detect tryptophan synthetase(TRPA) gene expression, analysis on the key active material role of the gut microbiota, to explore TXYF influence on it. Extract the total DNA of gut microbiota, using Illumina high-throughput sequencing platform for metagenome sequencing, through Blast and other software to NR genebank gain species classification, COG, KEGG functional annotation, explore the influence of the TXYF on gut microbiota species,the function of protein and metabolic pathways.Results:1, Evaluation of chronic unpredictable mild stress and humid and hot type diarrhea IBS model:compared with N group, IBS model group rats with depression, irritability, back hunch up, burnout nuzzles, unresponsive, decreased activity, open box score significantly decreased(P>0.05). Appetite reduced, weight growth rate significantly decreased(P<0.05). Appeared different degree of loose stools, hair became yellow, filthy, the lack of gloss, around the anus covered in mucus,body hair was defiled by loose stools,Bristol fecal character score, AWR scores were significantly increased(P<0.05).2, Evaluation of effect of TXYF against IBS model rats:compared with the model group,the mental status and general status such as diet, hair, etc,which were obviously improved in T14 group rats, weight growth rate, open box test score were significantly increased (P<0.05), AWR score were significantly lower(P<0.05), Above index have no significant difference in T7 and B7 group(P>0.05).3, Effect of TXYF on CHAT, NOS,VIP, Fos in hippocampus and colon tissue in IBS model rats:compared with N group, expression of CHAT, c-Fos, VIP in hippocampus, colon tissue on M group rats were significantly increased(P<0.05), expression of NOS was significantly decreased(P<0.05). Compared with M group, expression of CHAT, Fos, VIP in hippocampus, colon tissue on T14, B14 group rats were significantly decreased (P<0.05), expression of NOS was significantly increased(P<0.05).Above index have no significant difference in T7 and B7 group(P>0.05).4, Effect of TXYF on expression level of TRPA gene of gut microbiota in IBS model rats. Compared with N group, expression level of TRPA gene of gut microbiota in M group rats obviously decreased(P<0.05). Compared with M group, expression level of TRPA gene of gut microbiota inT14 and B14 group waere obviously increased(P<0.05).Expression level of TRPA gene in T7 and B7 group have no statistical difference (P> 0.05).5, Effect of TXYF on structure clusters of gut microbiota in IBS model rats: Compared with N group, structure proportion of Bacteroidia in M group rats was lower, structure proportion of Clostridia was higher on phylum level;Compared with M group, structure proportion of Bacteroidia in T7 and T14 group, B7 and B14 group were increasing trend, structure proportion of Clostridia was decreasing trend. Comparison of genus structure clusters of gut microbiota with Heatmap, T7 group was similarity with B7 group, T14 group was similarity with B14 group, four treatment group were certain similarity to N group, M group was biggest difference with other groups.6, Effect of TXYF on COG clusters of gut microbiota in IBS model rats: compared with N group, COG clusters of protein function of gut microbiota in M group rats were decreased in energy generation and transformation, the transport and metabolism of amino acid, transport and metabolism of carbohydrates, transport and metabolism of enzyme, transport and metabolism of lipid.Compared with M group, in T7 and T14 group, B7 and B14, above COG clusters of function protein were rising trend. Comparison of the heatmap of COG Clusters in each groups, T7 group was similarity with B7 group, T14 group was similarities with B14 group, four treatment group have certain similarity to N group, M group was biggest difference with other groups.7, Effect of TXYF on KEGG clusters of metabolic pathways of gut microbiota in IBS model rats:Compared with N group, KEGG clusters of gut microbiotais in M group rats were reduced in ko00970 aminoacyl-tRNA biosynthesis, ko00230 purine metabolism, ko00240 pyrimidine metabolism, ko00640 propanoate metabolism, ko00030 pentose phosphate pathway, ko00400 phenylalanine, tyrosine, tryptophan biosynthesis,, ko03020 RNA polymerase.Compared with M group, T7 and T14 group, B7 and B14 were rising trend in KEGG clusters.Comparison the heatmap of COG clusters, T7 group was similarities with B7 group, and they were certain similarities with M group;T14 and B14 group have similarities, and they have certain similarities with N group.Conclusion:1, The pathogenesis of IBS is associated with brain gut axis disorders, at the same time, the structure of the gut microbiota, function of protein and metabolic pathways is one of the important pathogenesis, "brain-gut-enteric microbiota axis" disorder is the important pathologic basis of IBS.2, Tong-xie-yao-fang has a effective treatment of IBS, the mechanism may related to Brain-gut-enteric microbiota axis.