The Change Of Intestinal Mucous Membrane Aquaporins Of Diarrhea-predominant Irritable Bowel Syndrome And Its Molecular Mechanism Study

Research background:Irritable bowel syndrome(IBS)is one of the most common chronic functional diseases of digestive system.The prevalence rate in the world is 1.1%-35.5%,among which 9.6%is in Asia.Due to the large population in our country,it is hypothesized that China is one of the most IBS patients populous country in the world,so the preventive task is very hard.According to the Rome IV standard,IBS can be divided into four subtypes:diarrhea type(IBS-D),constipation type(IBS-C),indeterminate type(IBS-U)and mixed type(IBS-M).Etiology and pathogenesis of IBS are unknown,the lack of effective means of prevention and treatment leads to seriously affect the patient's study,work and life.So IBS is not just a clinical problem,it is also a social problem remaining to be solved.Traditional idea is considered that IBS may be the result of various factors influencing each other,including abnormal gastrointestinal dynamics,visceral high sensitive,intestinal mucosal inflammation,nerve endocrine disorders,intestinal food allergies,mental abnormalities,and so on.However,we are still not able to clearly explain the pathogenesis,physiological basis,especially the molecular mechanism.Aquaporins(AQPs)which are distributed in the intestinal mucosa are considered to be mediated factor of the liquid water transport across the membrane,therefore,AQPs can maintain the stability of intestinal cells inside and outside environment,and AQPs can also adjust the nerve function of intestinal tract.Current view is that in the physical state,AQPs can maintain the balance of the intestinal absorption of water molecules and secretion.However,although it is not uncommon to study the metabolic mechanisms of AQPs,further research needs to be improved,the role and status of its molecular mechanisms need to be further clarified.Currently the correlation of the research on miRNAs and intestinal mucosal permeability in IBS has also become a hot.However,the research on the relationship between AQPs and miRNAs is not numerous.This study contains the establishment of the IBS-D rats model,water channel protein and ion channel analysis,permeable molecular signaling pathway and related genes and miRNAs analysis and screen,which is hoped to clarify the pathologic relationship between IBS-D and intestinal mucosa AQPs,and attempt to clarify its molecular pathologic mechanism,so as to provide new ideas and targets for clinical treatment of IBS-D.Part ? The research on abnormal expression of IBS-D rat colonicmucosa AQPs and ion channelsBackground and objective:The aquaporins(AQPs)family is the membrane channel protein with highly selective water,and they are widely distributed which are connected with the transmembrane transport of water and the regulation of water balance.Ion channels are a special class of hydrophilic proteins on the cell membrane microchannel,are the material basis of nerve and muscle activity.With the development of molecular biology,the molecular structure and characteristics of water channel proteins and ion channels have a deeper understanding,and we also found that their functional and structural abnormalities were associated with the initiation and progression of many gastrointestinal diseases.Recent studies have confirmed that the mechanism of IBS-D is related to the permeability of intestinal mucosa.Whether the configuration,expression or channel current of AQPs in the intestinal mucosal epithelial cells and ion channels on the nerve and smooth muscles are changed,and whether these changes participate in the occurrence of IBS-D are not clear.It has been studied that D-lactic acid is an indicator that reflects the increased permeability of intestinal mucosa.Our study was to establish the IBS-D rat models,with the detection of D-lactic acid,AQP1,AQP3,AQP8 and ion transporter SGLT1,NHE1,CFTR in colonic mucosa of rats,so as to further clarify the correlation between IBS-D and intestinal mucosal permeability,and further clarify its mechanism,laying the groundwork for the further study of mechanism net of IBS-D.Methods:IBS-D rat models were set up through the stress and rectum stimulus according to literature and preliminary research,and then water content and small intestinal propulsion rates were checked.D-lactic acid in blood,AQP1,AQP3 and AQP8 in the intestinal tissue were tested by ELISA method.The expression of AQP1,AQP3,AQP8 and ion transporter SGLT1,NHE1,CFTR were detected by immunohistochemistry and RT-PCR in intestinal mucosal tissue.Results:IBS-D rat models were established successfully.Compared with the control group(1.060±0.105)ml,the water content of IBS-D group rats(1.807±0.294)ml significantly increased(p<0.05).The small intestine propulsion rate(67.65±5.142)%of IBS-D group significantly increased compared with the control group(46.60±8.288)%(p<0.05).Compared with the control group(22.44±3.13)ng/ml,D-lactic acid content(49.79±3.55)ng/ml increased in IBS-D rats(p<0.05).The expression of AQP1,AQP3 and AQP8 in IBS-D rats' intestinal mucosa was less than the control group(p<0.05).The expression of SGLT1 and NHE 1 was reduced in IBS-D model group,and CFTR was increased in IBS-D model group(p<0.05).Conclusions:IBS-D rats showed increased intestinal motility,increased intestinal mucosa permeability,decreased expression of colonic mucosa AQP1,AQP3,AQP8,SGLT1 and NHE1,and increased expression of CFTR,suggesting that water-liquid metabolic abnormality of IBS-D is related to the expression of colonic mucosa AQPs and ion channels.Part II Study on the effect and mechanism of AQPs expression in intestinal mucosa of IBS-D via Classical cAMP/PKA pathwayand NF-?B pathwayBackground and objective:Nuclear transcription factors-?B(NF-?B)is a kind of multifunctional transcription factor family,which widely exists in organism tissues and cells,and is involved in physiological and pathological processes of organism inflammatory response,immune response,oxidative reaction,apoptosis.cAMP/PKA pathway is a classic of G protein coupled cell signal transduction pathways,which involved in various cellular physiological activities.The second part of this study was to establish IBS-D rat models,and to detect the expression of AQP1,AQP3,AQP8,NF-?B p65 and p-CREB with the intervention of the NF-?B inhibitor PDTC,PKA inhibitor H89 and cAMP acti-vator forskolin,so as to research the pathogenesis process of IBS-D via NF-?B pathway and cAMP/PKA pathway by the effect of AQP1,AQP3 and AQP8 and to explore the cross-talk between the two signal pathways.Methods:IBS-D rat models were set up through the stress and rectum stimulus.After the intervention of the NF-?B inhibitor PDTC,PKA inhibitor H89 and cAMP activator forskolin,the expression of AQP1,AQP3,AQP8,p-CREB and NF-?B p65 were detected by RT-PCR,Western blot and immunohistochemical detection.And the expression of IL-1?,TGF-? and TNF-a mRNA were detected by RT-PCR.Results:The expression of AQP1 AQP3,AQP8 and p-CREB in colonic mucosa of IBS-D model group were lower than the control group(p<0.05),the expression of NF-?B p65,IL-1?,TGF-? and TNF-a were higher than the control group(p<0.05).After the intervention of the cAMP activator forskolin,the expression of AQP1,AQP3,AQP8.p-CREB increased(p<0.05),and the expression of NF-?B p65,IL-1?,TGF-?and TNF-a decreased(p<0.05).After the intervention of the NF-?B inhibitor PDTC,the expression of AQP1,AQP3,AQP8,p-CREB increased(p<0.05),and the expression of NF-?B p65,IL-1?,TGF-? and TNF-a decreased(p<0.05).Conclusions:Both the cAMP/PKA signal pathway and the NF-?B signal pathway were involved in the occurrence of IBS-D.When IBS-D occurred,the cAMP/PKA signal pathway was inhibited,the NF-?B signal pathway was activated,the expression of AQP1,AQP3 and AQP8 decreased,and the expression of IL-1?,TGF-? and TNF-a increased.Activation of cAMP/PKA signal pathway and inhibition of NF-?B signal pathway can up-regulate the expression of AQP1,AQP3 and AQP8,and down-regulate the expression of IL-1?,TGF-? and TNF-? in intestinal mucosa of IBS-D.Part III The expression of genes and miRNA expression profile in colon tissue of IBS-D ratsBackground and objective:miRNAs have been shown to participate in and control a variety of physiological processes including apoptosis and differentiation,lipid metabolism,neuronal development,hormone secretion and so on.More studies of miRNAs focus on cancer,and there are few studies on miRNA in patients with functional gastrointestinal disorders,especially irritable bowel syndrome(IBS).The third part of this study was to build IBS-D rat models by stress and rectum stimulus,and to screen the differentially expressed genes and miRNAs in the colon of IBS-D rats by the liquid chip technology.Furthermore,the difference in genes and miRNAs expression between the IBS-D group and the control group were further compared,laying a foundation for further study on the molecular pathogenesis of IBS-D.Methods:IBS-D rat models were built by stress and rectum stimulus.Then we screened differential expression of genes and miRNAs by the liquid chip technology and verified by RT-PCR technology,and the expression differences of genes and miRNAs between the IBS-D rats and normal rats were further compared.Results:The amount of rectal injected water in the control group and the model group were respectively(1.52 + 0.09)ml and(0.77 + 0.08)ml,showing statistical difference(p<0.05),and the IBS models were built successfully.Among the selected genes(htr4,htr1a,2r13,nosl,Calca,npy,crhr2,illb,p2rx3,nos2,tphl,crhrl,hmox1,trpv1,Vip,f2rl,tgfbl,htr3 a,slc6a4,tff2),the expressions of nos 1,ill b and htr3 a were up-regulated in the colon tissues of the model group through PCR verification.Among the selected miRNAs(let-7f,let-7i,miR-130b,miR-29a,miR-132,miR-215 miR-375,miR-24,miR-31a,miR-192,miR-221,miR-223,miR-23a,miR-23b,miR-122-5p,miR-143,miR-145,miR-146a,miR-199a,miR-200b,miR-217),We found that the expressions of miR-29a,let-7f,let-7i,miR-130b,miR-132,miR-21,miR-375 in model group were up-regulated(p<0.05).However the expressions of miR-24,miR-31a,miR-192,miR-221,miR-223 in model group were down-regulated(p<0.05).The expressions of miR-23a,miR-23b>miR-122-5p,miR-143>miR-145,miR-146a,miR-199a,miR-200b,miR-217 showed no significant differences(p>0.05).Conclusions:The genes nosl,illb and htr3a express abnormally in the colon of IBS-D rats.There were a number of miRNAs with up-regulation such as miR-29a,let-7f and so on,and with down-regulation such as miR-24,miR-31a and so on in the colon tissues of IBS-D rats.Part IV The effect of AQPs on intestinal mucosa barrier dysfunctionin in IBS-D baesd on the regulation of miR-29aBackground and objective:Aquaporins(AQPs)have been studied to play an important role in water and fluid balance of the intestinal barrier.In the third part of the study,we have found the occurrence of IBS-D was related to the changes of multiple miRNAs,in which the expression of miR-29a was significantly up-regulated in the colon mucosa of the IBS-D rat models.Recent studies found that miR-29a was involved in the pathogenesis of IBS,and its mechanism may be related to intestinal mucosa permeability,while the specific mechanism is still unclear.The fourth part of this study aims to cultivate IBS-D rats colon epithelial cells,and to detect the expression of K+ and LDH by RT-PCR,and to detect the expression of AQP1,AQP3 and AQP8 after the inhibition of miR-29a by Western blot.Thus,the molecular mechanism of miR-29a affecting intestinal mucosa permeability and the effect on water channel proteins associated with IBS-D pathogenesis were revealed.Methods:IBS-D rat models were set up through the stress and rectum stimulus,and then the original generation of colon epithelial cells were cultivated and verified.The miR-29a inhibitor was used as the intervention.The experimental rats were divided into normal cells control group,IBS-D cell group,miR-29a inhibitor control group and miR-29a inhibitor group.We detected the expression of miR-29a,K+ and LDH by RT-PCR and the expression of AQP1,AQP3 and AQP8 by Western blot.Results:Compared with the control group,the expression of rmiR-29a of IBS-D cell group was much higher(p<0.001),the expression of K+ was lower(p<0.05),and the expression of LDH was much higher(p<0.001),and the expression of AQP1,AQP3,AQP8 was lower(p<0.05).Compared with miR-29a inhibitor control group,the expression of miR-29a in miR-29a inhibitor group decreased(p<0.001),the expression of K+ increased(p<0.05),and the expression of LDH decreased(p<0.01),and the expression of AQPl,AQP3,AQP8 increased(p<0.001).Conclusions:The expression of miR-29a in primary colon epithelial cells of IBS-D rats increased,the expression of AQP1,AQP3 and AQP8 decreased,and the intestinal epithelial permeability increased.Inhibition of miR-29a expression in intestinal epithelial cells can significantly increase AQPs expression and improve intestinal epithelial permeability.Total conclusions:IBS-D rats showed increased intestinal motility,increased intestinal mucosa permeability,decreased expression of colonic mucosa AQP1,AQP3,AQP8,SGLT1 and NHE1,and increased expression of CFTR,suggesting that water-liquid metabolic abnormality of IBS-D is related to the expression of colonic mucosa AQPs and ion channels.Both the cAMP/PKA signal pathway and the NF-?B signal pathway were involved in the occurrence of IBS-D.When IBS-D occurred,the cAMP/PKA signal pathway was inhibited,the NF-?B signal pathway was activated,the expression of AQP1,AQP3 and AQP8 decreased,and the expression of IL-1?,TGF-?and TNF-a increased.Activation of cAMP/PKA signal pathway and inhibition of NF-?B signal pathway can up-regulate the expression of AQP1,AQP3 and AQP8,and down-regulate the expression of IL-1?,TGF-? and TNF-? in intestinal mucosa of IBS-D.The genes nosl,illb and htr3a express abnormally in the colon of IBS-D rats.There were a number of miRNAs with up-regulation and down-regulation in the colon tissues of IBS-D rats.The expression of miR-29a in primary colon epithelial cells of IBS-D rats increased,the expression of AQP1,AQP3 and AQP8 decreased,and the intestinal epithelial permeability increased.Inhibition of miR-29a expression in intestinal epithelial cells can significantly increase AQPs expression and improve intestinal epithelial permeability.