| ObjectiveIrritable bowel syndrome is a functional gastrointestinal disorder with clinical symptoms of recurrent abdominal pain,which related to defecation or accompany by changes with bowl evacuation habit.Abnormal defecation habits include constipation,diarrhea or alternation of constipation and diarrhea,which with symptom of abdominal distention.Patients with IBS may have clinical symptoms persistently or recurrently,and no histopathological lesion is founded in their guts.Irritable bowel syndrome with predominant diarrhea(IBS-D)is the most common subtype of IBS.It reduced the the quality of life and made a large burden on national medical system.IBS was considered to be caused by many factors with complex pathophy,siological mechanisms.The mainly treatment measures are symptomatic treatment,such as antispasmodic,antidiarrheal,drugs of regulating intestinal flora,drugs of regulating visceral sensation,tricyclic antidepressant,et.al.However,there are no ideal treatment methods and special drugs for IBS.In the view of traditional Chinese Medicine,liver,spleen and kidney is closely related to morbidity of IBS-D.Depression of liver,deficiency of spleen and kidney-yang is the main pathogenesis of IBS-D.Traditional Chinese Medicine specialized in the treatment of IBS-D,had great potential for development.The application of syndrome differentiation and treatment combined with traditional therapy such as acupuncture and moxibustion had good clinical effect in the comprehensive treatment of IBS-D.A large number of clinical literature and meta-analysis showed the characteristics and advantages of TCM for the treatment of IBS-D.Therefore,exploring the pathogenesis and therapeutic mechanism of IBS-D is one of the hotspots in functional gastrointestinal disorders,and that also the key point in clinical treatment,which had important scientific and social significance.The study includes two parts:literature review and experiment research.In the literature review,we summaried on two aspects about IBS-D:Firstly,in view of modern medicine,we reviewed the epidemiology,etiology,pathogenesis,diagnostic criteria and clinical treatment.Secondly,in view of TCM,we reviewed the TCM name of IBS,etiology and pathogenesis of TCM,syndrome classification,treatment of TCM,experience of experts,and modern clinical analysis on TCM.In the experiment research,we designed a rat model of IBS-D with spleen-kidney yang-deficiency and analyzed effects of Fuzi-Lizhong decoction on the index about model evaluation,gene expression and histology.Before and after treatment,we observed the changes on the general condition,symptom scores,histopathology of colonic mucosa on rats.After treatment,we analyzed the Digital Gene Expression Tag Profiling(DGE)of colon mucosa,the expression of NALP-3 inflammasome,CD4+,CD8+and the quantity of mast cell on the colon mucosa,the thymus index&spleen index.Methods30 healthy rats were randomly divided into three groups:treat,model and control.We established a rat model of IBS-D by senna intragastric administration combined with restraint stress.Subjects in the treat group received FuZi-LiZhong Decoction(1.5g/kg),once a day,for 14 days.Subjects in the model group and control group received physiological saline(20ml/kg/d).Aftre treatment,we detected the general condition,the Relative growth rate of weight,Grading-score of Bristol Stool From scale,Rate of diarrhea,Diarrhea index,Rectal temperature,TCM-syndrome score,Thymus&spleen index,the morphology of intestinal tissue.We extracted RNA by Trizol method,tested the Digital Gene Expression Tag Profiling by using high-throughput sequencing technology.We detected the expression of NALP-3 inflammasome,CD4+ and CD8+cells by immunohistochemical method and counted the number of mast cells aldehyde-fuchsin method in colonic mucosal tissues.Results1.Evaluation of IBS-D model in rats1.1 General condition:The control group had good mental state,bright hair color and normal feces.From the fifth day,the model group and the treat group were depression in spirit,which diet reduced and hair color became dark,defecation increased and had loose stools.From the fourth day of treatment,the treat gourp recovered good mental state,increased diet,reduced defecation and had no diarrhea.1.2 Relative growth rate of body weight:After model-making,the relative growth rate of body weight in three groups showed no difference(P>0.05).After treatment,it showed difference among three groups(P<0.05).Compared with model group,treat group showed a increase of relative growth rate of body weight(P<0.05).It showed no difference between other groups(P>0.05).1.3 Grading-score of Bristol Stool From scale:After model-making,it.showed significant difference among three groups on the grading-score of Bristol Stool From scale(P<0.01).Compared with control group,the model group and treat group showed a significant.increase(P<0.01).It showed no difference between treat group and model group(P>0.05).After treatment,it.showed significant difference among three groups on grading-score of Bristol Stool From scale(P<0.01).Compared with model group,treat group showed a significant decrease(P<0.01).Compared with control group,model group showed a significant increase(P<0.01).It showed no difference between treat.group and control group(P>0.05).1.4 Rate of diarrhea:After model-making,it showed significant difference among three groups on the rate of diarrhea(P<0.01).Compared with control group,model group and treat group showed a significant increase(P<0.01).It showed no difference between treat group and model group(P>0.05).After treatment,it showed significant difference among three groups on the rate of diarrhea(P<0.01).Compared with model group,treat group showed a significant decrease(P<0.01).Compared with control group,model group showed a significant increase(P<0.01).It showed no difference between the treat group and the control group(P>0.05).1.5 Diarrhea index:After model-making,it showed significant difference among three groups on the diarrhea index(P<0.01).Compared with control group,the model group and treat group showed a significant increase(P<0.01).It showed no difference between treat group and model group(P>0.05).After treatment,it showed significant difference among three groups on the diarrhea index(P<0.01).Compared with model group,treat group showed a decrease(P<0.05).Compared with control group,model group showed a significant increase(P<0.01).Compared with control group,treat group showed a increase(P<0.05).1.6 Rectal temperature:After model-making,it showed significant difference among three groups on the rectal temperature(P<0.01).Compared with control group,model group and treat group showed a significant decrease(P<0.01).It showed no difference between treat group and model group(P>0.05).After treatment,it showed significant difference among three groups on the rectal temperature(P<0.01).Compared with model group,treat group showed a significant increase(P<0.01).Compared with control group,model group showed a significant decrease(P<0.01).It showed no difference between treat group and control group(P>0.05).1.7 TCM-syndrome score:After model-making,both treat and model group showed spleen&kidney yang-deficiency syndrome.After model-making,it showed significant difference among three groups on the TCM-syndrome score(P<0.01).Compared with control group,model group and treat group showed a significant increase(P<0.01).It showed no difference between treat group and model group(P>0.05).After treatment,it showed significant difference among three groups on the TCM-syndrome score(P<0.01).Compared with model group,treat group showed a decrease(P<0.05).Compared with control group,model group showed a significant increase(P<0.01).Compared with control group,treat group showed a increase(P<0.05).Compared with after model-making,treat group showed a significant decrease on TCM-syndrome score of treat group(P<0.01),and other two groups showed no difference(P>0.05).1.8 Intestinal histopathology:We found no obvious ulcer,erosion,acute or chronic inflammation,intestinal tumor and other organic lesions on the intestinal mucosa in three groups.In the control group,the epithelial cells of intestinal tissue were arranged closely,and the glands in the lamina propria did not decrease or atrophy.In the model group,the tissue specimens were infiltrated with inflammatory cells and the submucosa structure was loosely edematous.In the treat group,a small amount of lymphocyte or neutrophils infiltration and submucosal structure were seen in a few specimens.2.DGE of rat colonic mucosa2.1 Sequencing quality statistics and charts showed that samples in three group all had a good Sequencing quality distribution and good randomness,which indicated the quality of sequencing was good.2.2 Clean reads of each group compared with the reference genome:There were 93.14%(treat group),91.77%(model group),95.66%(control group)clean reads respectively mapped the reference genome.The rate of multiple mapped resads was 14.06%(treat group),13.82%(model group)and 11.29%(control group).The rate of uniquely mapped reads was 79.08%(treat group),77.95%(model group)and 84.39%(control group).There was no difference among three groups on the total mapped,multiple mapped,uniquely mapped,reads map to '+' and reads map to '-'(P>0.05)?2.3 Monolithic level of gene expression:We test nine samples and found the total number of genes were from 20042 to 21560,which had conformance.The RPKM of treat.group is 0.0063-86485.80,of which RPKM is less than 100 of gene proportion 95.93%-96.64%.The RPKM of model group is 0.0057-72689.10,of which RPKM is less than 100 of gene proportion 96.39%-97.23%.The RPKM of control group is 0.0061-19715.30,of which RPKM is less than 100 of gene proportion 96.15%-96.64%.2.4 Analysis of difference in gene expression:In the analysis of model group vs treat group,30 genes up-regulated,75 genes dowen-regulated;In the analysis of control group vs model group,41 genes up-regulated,61 genes dowen-regulated;In the analysis of control group vs treat group,102 genes up-regulated,144 genes dowen-regulated.We found 29 differential genes appeared both treat.group vs model group and model group vs control group.For these 29 genes,we found that 13 genes up-regulated and 16 genes dowe-regulated in the analysis of control group vs model group.And we also found that 16 genes up-regulated and 13 genes down-regulated in the analysis of model group vs treat group.Gene Aqp8 was down-regulated and Gene LOC103692592 was up-regulated in two analysis.Other genes regulated oppositely in two analysis.2.5 "Gene Ontology" functional annotation and classification of differential genes:In total three analysis of differential genes,we got 1280 GO annotation which had statistical significance(P-value<0.05),146 GO annotation of molecular function,138 GO annotation of cellular component,996 GO annotation of biological process.In the analysis of treat group vs model group,we got total 448 GO annotation,52 GO annotation of molecular function,56 GO annotation of cellular component,340 GO annotation of biological process.GO annotation of molecular function mainly were:MHC class ? protein complex binding,MHC protein complex binding,antigen binding,water channel activity,water transmembrane transporter activity,Platelet-derived growth factor binding,growth factor binding,et al.GO annotation of cellular component 56 mainly were:extracellular vesicular exosome,extracellular organelle,extracellular membrane-bounded organelle,extracellular region,extracellular space,et al.GO annotation of biological process mainly were:immune response,immune system process,regulation of immune system process,regulation of immune response,production of molecular mediator of immune response,collagen fibril organization,cell adhesion,negative regulation of cell adhesion,cell-substrate adhesion,biological adhesion,et al.In the analysis of model group vs control group,we got total 355 GO annotation,44 GO annotation of molecular function,18 GO annotation of cellular component,293 GO annotation of biological process.GO annotation of molecular function mainly were:plasma membrane region,bile acid binding,phospholipase inhibitor activity,chemokine activity,chemokine receptor binding,secondary active sulfate transmembrane transporter activity,et al.GO annotation of cellular component mainly were:anion transport,hemoglobin complex,extracellular matrix,membrane region,extracellular space,endoplasmic reticulum,et al.GO annotation of biological process mainly were:dendritic cell chemotaxis,dendritic cell migration,fatty acid catabolic process,lipid metabolic process,cellular lipid catabolic process,positive regulation of lipid metabolic process,monocarboxylic acid catabolic process,response to estrogen,et al.In the analysis of treat group vs control group,we got total 477 GO annotation,50 GO annotation of molecular function,64 GO annotation of cellular component,363 GO annotation of biological process.GO annotation of molecular function mainly were:act in binding,cytoskeletal protein binding,chemokine activity,MHC class ? protein complex binding,MHC protein complex binding,chemokine receptor binding,growth factor binding,et al.GO annotation of cellular component 56 mainly were:adherens junction,anchoring junction,cell-substrate adherens junction,extracellular region,et al.GO annotation of biological process mainly were:T cell activation,regulation of T cell activation,cell activation,regulation of immune system process,regulation of cell migration,regulation of cell motility,regulation of cell adhesion,cell adhesion,biological adhesion,et al.2.6 KEGG biological pathway enrichment analysis of differential genes:In total three analysis of differential genes,we got 371 KEGG biological pathway,which had statistical significance(P<0.05)were 68 and extremely significant enrichment(Corrected P-value<0.05)were 38.In the analysis of treat group vs model group,we got total 106 KEGG biological pathway,28 pathway which had statistical significance,13 pathway which had extremely significant enrichment.They mainly were:Intestinal immune network for IgA production,Antigen processing and presentation,Allograft rejection,Graft-versus-hostdisease,Phagosome,Hematopoietic cell lineage,Cell adhesion molecules(CAMs),Asthma Inflammat.ory bowel disease(IBD),Autoimmune thyroid disease,Viral myocarditis,Staphylococcus aureus infection,Type I diabetes mellitus.Other pathway were:ECM-receptor interaction,Toxoplasmosis,Primary immunodeficiency,Systemic lupus erythematosus,B cell receptor signaling pathway,Vascular smooth muscle contraction,Vasopressin-regulated water reabsorption,et al.In the analysis of model vs control group,we gottotal 98 KEGG biological pathway,13 pathway which had statistical significance,3 pathway which had extremely significant enrichment.They mainly were:Aminoacyl-tRNA biosynthesis,Bile secretion,PPAR signaling pathway.Other pathway were:Ribosome biogenesis in eukaryotes,African trypanosomiasis,Steroid hormone biosynthesis,Fatty acid degradation,Fat digestion and absorption,et al.Gene Fabpl which in the PPAR signaling pathway and Gene Hmgcs2 which in Bile secretion were the overlapping differential genes both in analysis of treat group vs model group and analysis of model group vs control group.In the analysis of treat vs control group,we got total 167 KEGG biological pathway,27 pathway which had statistical significance,12 pathway which had extremely significant enrichment.They mainly were:Aminoacyl-tRNA biosynthesis,Vascular smooth muscle contraction,Cell adhesion molecules(CAMs),Intestinal immune network for IgA production,Graft-versus-host disease,Allograft rejection,Asthma,Inflammatory bowel disease(IBD),Autoimmune thyroid disease,et al.3.Immune index of histology on rats3.1 Thymus index and spleen index:It showed no difference among three groups on thymus index&spleen index(P>0.05).3.2 Mast cell count on colon mucosa:Compare with control group,model group showed a significant increase(P<0.01);Compare with model group,treat group showed a decrease(P<0.05);Compare with control group,treat group showed a significant increase(P<0.01).3.3 Expression of NALP-3 on colon mucosa:Compare with control group,model group showed a significant increase(P<0.01);Compare with the model group,the treat group showed a significant decrease(P<0.01);Compare with control group,treat group showed a increase(P<0.05).3.4 Expression of CD4+&CD8+on colon mucosa and the CD4+/CD8+ cell ratio:Compare with control group,model group showed a significant increase of expression of CD4+(P<0.01);Compare with model group,treat group showed a increase of expression of CD4+(P<0.05);It showed no difference among three groups on the expression of CD8+.Compare with control group,model group showed a significant increase of CD4+/CD8+ cell ratio(P<0.01);Compare with model group,treat group showed a decrease of CD4+/CD8+ cell ratio(P<0.05);It showed no difference between treat group and control group of CD4+/CD8+cell ratio.Conclusion1.Model-making of IBS-D in rats were successful,and the results of the model evaluation were reliable.Fuzi-Lizhong decoction induced the relative growth rate of body weight,reduced the frequency of defecation and improve stool traits,decreased the Grading-score of Bristol Stool From scale&rate of diarrhea&diarrhea index.Fuzi-Lizhong decoction might improve the clinical symptoms on the rats with IBS-D.2.The DGE sequencing data was qualified.The quality of the sequencing was good and the result was reliable.The percentage of genes with RPKM less than 100 was above 95%.Some high expression genes may play a key role in the pathogenesis of IBS-D or in the treatment of Fuzi-Lizhong decoction.3.The analysis between the control group and model group showed that 102 differential genes might be associated with the pathogenesis of IBS-D.The analysis between the treat group and model group showed that 105 differential genes might be associated with therapeutic mechanism of IBS-D.Up or down regulation of 29 overlapping genes which appeared in two analysis might simultaneous correlate with molecular pathogenesis and therapeutic mechanism.4.It suggested that the pathogenesis of IBS-D might associate with immune inflammatory reaction mediated by dendritic cells or abnormal PPAR signaling pathway.5.It suggested that Fuzi-Lizhong decoction could improve the dysfunction of immune system and keep immune balance in the intestine through regulating the molecular pathway or biological signal pathway which relating to immue system and immunologic process.6.The pathogenesis of IBS-D maybe relate to the intestinal low-grade immue inflammation which caused by increase of NALP-3 inflammasome and mast.cell on colon mucosal,and also relate to the imbalance of CD4+/CD8+T lymphocytes.7.Fuzi-lizhong decoction might.reduce the release of intestinal inflammatory substances to alleviate immune response through regulating the expression of NALP-3 inf lammasome&CD4+,CD4+/CD8+ ratio and the number of mast,cell. |
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