The Study Of Different Type Of Diarrhea-predominant Irritable Bowel Syndrome Of TCM On Stress Level,Gene Expression And Ultrastructure

ObjectiveThe research was to analyze the level of psychological state,IBS symptom severity,quality of life in two type of TCM of Diarrhea-predominant Irritable Bowel Syndrome(IBS-D)patients and the healthy control.These two types of IBS-D of TCM were the type of Stagnation of Liver-QI with Deficiency of the Spleen(SLDS)and the type of Weakness of the Spleen and Stomach(WSS).Salivary amylase(sAA),salivary cortisol,sAA/saliva cortisol ratio and 10 types of gene expression by PCR array of IBS were detected,combined with immunohistochemical and ultrastructural observation.Our Purpose in this study was to explore the similarities and differences and the pathological essence of these two types of IBS-D of TCM from macro,micro and Ultra-microscopic view.Materials and Methods1 Subjects62 patients diagnosed with IBS by the Rome ? criteria,32 of them are the type of Stagnation of Liver-QI with Deficiency of the Spleen(SLDS),30 of them are the type of Weakness of the Spleen and Stomach(WSS),in addition to 30 healthy volunteers were selected.2 Methods2.1 Assessed the psychological state of the subjects by Symptom Checklist(SCL-90)2.2 Assessed the quality of life of the subjects by Gastroenterology disease PRO Scale-Irritable Bowel Syndrome Scale(GEDPRO-IBS)2.3 Assessed the Severity of symptoms of the subjects by IBS symptom severity scale(IBS-SSS)2.4 Assay the salivary amylase activity2.5 Assay the salivary amylase content.and saliva cortisol levels by Enzyme-linked Immunosorbent Assay(ELISA)2.6 Ten cases in each group were selected randomly to be checked with colonoscopy.Five pieces of intestinal mucosal biopsy specimens were clamped at 10cm above the rectosigmoid junction by colonoscopy.Two of which were immediately placed in Trizol solution and stored in-80? station.Six cases of the groups of SLDS and WSS of IBS-D and three cases of the healthy control group were selected randomly.Two pieces of these intestinal mucosa tissue were prepared for polymerase chain reaction(PCR)array;one pieces of intestinal mucosa tissue was stored in electron microscopy fixative at 4?for transmission electron microscopy to observe the ultrastructure;the last two pieces of intestinal mucosa tissue were stored in 10%neutral formalin solution for paraffin hematoxylin-eosin(HE)staining,also for immunohistochemical staining with brain-derived neurotrophic factor(BDNF)and transient receptor potential channel subtype 1(TRPV1).2.7 Assay the mRNA expression of the intestine mucosal with polymerase chain reaction(PCR)arrayPCR arrays of intestine mucosal were customized,according to the theory of Correlation of Liver and Spleen of IBS,88 gene loci were determined the to be detected include 10 types of gene locus:stress genes,brain-gut peptides and their receptors in series,TRP series,energy metabolism enzyme genes,inflammatory factors(pro-inflammat ory cytokines,anti-inflammatory cytokines),chemotactic factor,tight junction protein,Aquaporin,enzymes associated with smooth muscle Motion and cytoskeletal protein.Detect the gene expression in intestine mucosal2.8 Hematoxylin-eosin(HE)staining of intestinal mucosa2.9 Immunohistochemical staining with BDNF,TRPV1 to intestinal mucosa 2,10 Observe the ultrastructure of intestinal mucosa by transmission electron microscopyResults1 Basic Information of cases in Three groups62 patients with IBS-D were selected in this reseach,including 29 males,33 females,age from 18 to 60 years old,the average age(32.89 ± 11.32)years.The SLDS group included 32 patients(15 males and 17 females),aged from 18 to 60 years old,mean age(33.37 ± 12.39)years;The WSS group included 30 patients(14 male and 16 females),age from 21 to 60 years old,mean age(34.30± 12.29)years.The healthy control group included 30 cases,including 14 males and 16 females,mean age(30.97 ± 9.01)years old.The difference of age,education level,occupation,education,workload in three groups was not statistically significant(all P>0.05).2 Analysis of psychological state of subjects in each groupIn the SLDS group of IBS-D,SCL-90 Scale total score,the number of items and the number of positive factor scores were higher than the WSS group and the healthy control group.The difference was statistically significant(P<0.05).The difference in SCL-90 scores between the WSS group and the control group was not significant..The SCL-90 total score of SLDS group was most closely related to somatization,depression and terror.3 Analysis of quality of life of subjects in each groupIn the SLDS group of IBS-D,the self-rating total score of quality of life is lower than the WSS group;In patients of SLDS group worried about the extent of the disease.Their aspects of energy and complexion,pain and discomfort,psychological,social relations,etc.as well as the fields of severity of the psychological and the environmental were higher than the WSS group,the difference was statistically significant(P<0.05).The two groups of IBS-D patients psychological domain score with energy and complexion,pain and discomfort,digestive function and physiological field was positively correlated(P<0.05).The psychological domain score of SLDS group of IBS-D patients was positively correlated with the independence of the field.Multiple linear regression analysis showed that the self-rating total score of quality of life in the two groups of IBS-D patients are most closely related to the field of social relations and the environment.The self-rating total score of quality of life in SLDS group patients also had a particularly close relationship with the pain and discomfort.4 Analysis of symptom severity of IBS-DThe IBS-SSS scale score results showed that compared with the WSS group,the IBS-SSS Scale total score,abdominal pain score,abdominal pain frequency score of the SLDS group of IBS-D patients were significantly higher,the difference was statistically significant(P<0.05).The score of abdominal distension,satisfaction of bowel habits and extent of affect life were not statistically significant between the two groups(all P>0.05).Compared with the healthy control group,the degree of abdominal pain,abdominal pain frequency,abdominal distension,satisfaction of bowel habit Points,the extent of affect life,and total score were significantly higher in the SLDS group and the WSS group patients,the difference was statistically significant(P<0.01).5 Salivary amylase,salivary cortisol in each group5.1 Salivary amylase activity compared in each groupCompared with healthy controls,the salivary amylase(sAA)activity before acid stimulation and salivary amylase activity ratio(acid stimulation sAA activity/acid stimulation sAA activity)were decreased in two groups of IBS-D patients,the difference was statistically significant(both P<0.01).The salivary amylase(sAA)activity before and after acid stimulation,and salivary amylase activity ratios were not significantly different between the two groups of IBS-D(P>0.05).The sAA activity after acid stimulation among the three groups had no significant difference(both P>0.05).5.2 Salivary amylase content,saliva cortisol levels and sAA/salivary cortisol ratio compared in each groupCompared with healthy controls,the salivary amylase(sAA)content,sAA/cortisol ratio in two groups of IBS-D patients were significantly decreased,the difference was statistically significant(both P<0.01),the salivary cortisol levels was significantly increased in two groups of IBS-D patients,the difference was statistically significant(P<0.01).Compared with the WSS group,the salivary cortisol levels of SLDS group of IBS-D patients was significantly increased,the difference was statistically significant(P<0.01).The sAA content,sAA/cortisol ratio had no significant difference between the two groups of IBS-D patients(both P>0.05).6 Results of colonic mucosa tissue PCR arrayPCR array melting curve indicates the purity of each locus were high,there was no interference from other factors.Compared with healthy controls,34 genes were up-regulated more than 2 times in the SLDS group of IBS-D,mainly for stress genes,brain-gut peptide and the receptors and energy metabolism enzymes genes.There were 8 gene expression down-regulated more than 2 times,mainly were the pro-inflammatory cytokine a.nd tight junction protein genes.Compared with healthy controls,38 genes were up-regulated more than 2 times in the WSS group of IBS-D,mainly for the stress gene,receptors,chemokines and energy metabolism enzyme genes.There were six genes down-regulated more than 2 times,mainly for the suppression of inflammatory cytokines and tight junction protein genes.Compared with the WSS group,four genes of the SLDS group of IBS-D were up-regulated more than 2 times,including proto-oncogene C-FOS,motilin(MTL),vasoactive intestinal peptide(VIP)and tight junction proteins claudin-2.A total of 15 genes were down-regulated more than 2 times,including the glucocorticoid receptor(GR),CRF1 receptor,tyrosine aminotransferase gene(TAT),5-HT2,5-HT3,5-HT7,5-HT transporter(SERT),transient receptor potential channel subtypes TRPC4,TRPV4,IL-10,CCL16,CCL13,tight junction protein claudin-1 and smooth muscle enzymes Prokineticin 2.7 Results of mucosa HE staining in each groupsNo special exception emerged.8 Results of mucosal tissue immunohistochemical staining in each groupCompared with healthy controls,the brain-derived neurotrophic factor(BDNF)and transient receptor potential channel subtype 1(TRPV1)of intestinal tissue were significantly increased in two groups of IBS-D patients,the difference were statistically significant(P<0.01).The difference of BDNF and TRPVl of intestinal tissue between in two groups of IBS-D was not statistically significant(both P>0.05).9 Results of transmission electron microscopy of intestinal mucosa in each group9.1 Intestinal epithelial cell nuclei,intestinal microvilli,mitochondrial morphology in each groupIntestinal mucosa microvilli irregular shedding injury,goblet cell hyperplasia,exuberant,portion deformed nucleus,nucleolus change,mitochondrial hyperplasia,and vacuolar degeneration appearde in IBS-D patients.9.2 Intestinal mucosa mast cells and endocrine cells in two groups of IBS-D patientsCompared with the WSS group,there were more mast cells,neuroendocrine cells more and more active secretion in the SLDS group of IBS-D.Glycogen accumulation appeared in the WSS group of IBS-D.9.3 Intestinal mucosa tight junctions in two groups of IBS-D patientsNormal tight junctions arranged sparse.Intestinal mucosa tight junctions arranged in clusters in the WSS group of IBS-D.Conclusion1 Clinical study and experimental research findingsMental disorders and digestive dysfunction in patients with IBS-D reflected the overall macroeconomic performance of the dysfunction of Liver and Spleen in TCM.Because of "spleen in solution as saliva",results of salivary amylase and saliva cortisol confirmed that the HPA axis activity of SLDS group of IBS-D increased significantly,reflecting the Liver failing to maintain the normal flow of Qi.The capacity of sympathetic adrenal medullary system stress response decreased in two groups of patients of IBS-D,reflecting the severity of spleen deficiency and the mechanism of the dysfunction of Liver and Spleen in IBS-D.2 The overall analysis of results of intestinal tissue PCR array measurementIn this research,10 categories 88 genes were assayed by intestinal tissue PCR array,reflecting the essence of the dysfunction of Liver and Spleen of TCM in IBS-D.The elevated expression of stress gene reflected that stress levels increased in IBS-D.This related to Liver.The elevated expression of brain-gut peptides and their receptors reflected brain-gut axis changes.This related to Liver and Spleen.The change Immune level showed "nerve-endocrine immune network" disorders of IBS,underlying the dysfunction of Liver and Spleen in IBS-D.Disorders of energy metabolism enzymes and aquaporin,showed the disorders of transport of Mizutani and liquid water of Spleen.3 Brain-gut axis abnormalities and the dysfunction of Liver and Spleen in IBS-DObvious mental disorder,saliva cortisol elevated,elevated expression of stress gene,exuberant secreting of endocrine cells and mast cells,elevated expression of brain-gut peptides and their receptors,all of these may prompt that the whole stress levels and the activity of HPA axis increased in the SLDS group of IBS-D,resulting in visceral hypersensitivity,causing intestinal disorders,and causing abdominal pain,diarrhea and other symptoms ultimately.BDNF and TRPV1 abnormally elevated in two types of IBS-D,indicating the Liver failing to maintain the normal flow of Qi,result in.visceral hypersensitivity and visceral pain threshold reduced.4 Dysfunction of the Spleen in transport and IBS-D4.1 Abnormalities of transport Mizutani of Spleen and IBS-DDisorders of intracellular energy supplying and using in IBS-D patients,indicating abnormal of transport Mizutani of spleen,Qi and blood insufficiency.Immune dysfunction in IBS-D patients was caused by dysfunction of the Spleen in transport,insufficient in Qi and blood,leading to immune dysfunction ultimately.4.2 Anomalies of transport of liquid water of Spleen and IBS-DAquaporin 4,tight junction protein expression increased in IBS-D patients,hyperplasia of tight junctions in electron microscope,indicating anomalies of transport of liquid water of Spleen.In summary,this research focused on the results of 10 types of 88 loci of PCR array,combined with evaluation of psychological and physical symptoms and detection of stress-related indicators.This research explored the pathological essence of these two types of IBS-D of TCM from macro,micro and Ultra-microscopic view,including stress and the Liver failing to maintain the normal flow of Qi,abnormal brain-gut axis,abnormalities of transport Mizutani and water of Spleen,and insufficient in Qi and blood.