Establishment And Evaluation Of A Rat Model Of IBS-D With Liver Depression And Spleen Deficiency Syndrome

BACKGROUND:Irritable bowel syndrome(IBS) is a functional gastrointestinal disorder characterized by abdominal discomfort and altered bowel habits in the absence of structural or biochemical disorders to account for these symptoms. It’s prevalence rate is increasing every year, which is related to psychological factor and infectious factor. Diarrhea-predominant IBS is the major type of IBS in China, the pathogenetic mechanisms of IBS-D are complicated, including visceral hyperesthesia, enhanced colonic motility, brain-gut dysfunction, abnormal neuro-immuno-endocrine network and so on. Recently, some molecular mechanisms are studied. Liver depression and spleen deficiency is the basic pathogenesis of IBS-D in traditional Chinese medicine, liver depression and spleen deficiency syndrome is the main syndrome. There is no an effective drug to treatment all the symptoms of IBS-D in the western medicine, TCM has advantages in comprehensive treatment. Now, the research on the model of IBS-D is still at a groping stage. A reliable and repeatable animal model of IBS-D is needed to study it’s pathogenesis. The establishment of an animal model of IBS-D with liver depression and spleen deficiency syndrome is necessary for the research on mechanisms of Chinese herbal compound prescription, which will promote the development of new drugs.Part1:Study of methods of establishing an ideal rat model of IBS-DAims:To explore a reliable method of establishing a rat model of IBS-DMaterials and methods:The Sprague Dawley(SD) female rats were used in this part and were divided into7groups:normal group, high dose senna group, middle dose senna group, low dose senna group, lactose group, acetic acid group,5-HT group, there are10rats in each group. Pups were exposed to a3h period of daily maternal separation on postnatal day2-14except the normal group. The following treatments were performed at2month of age(>250g). The high dose senna group was given4.5g/kg Senna decoction by gavage for7days, the middle dose senna group was given3g/kg Senna decoction, low dose senna group was given2g/kg Senna decoction, the lactose group was given lactose enriched diet for7days, the acetic acid group was given4%acetic acid1ml by anus once, the5-HT group was given2.1mg/kg5-HT by intraperitoneal injection for7days. The diarrhea rate was evaluated during the process, the stool consistency was observed and the fecal score was calculated after the modeling for1week, score of stool consistency was classified as follows:normal1, soft2, watery3. Body weight was evaluated before and after the modeling, the histopathologic evaluation of the proximal colon was did after the modeling.Results:(1) Diarrhea rate:the diarrhea rate of the high dose senna group, lactose group and acetic acid group was100%, there was no diarrhea in the other groups.(2) Weight growth:Except the low dose senna group, the weight growth of all the other groups decreased significantly compared to the normal group(P<0.01).Negative growth was observed in the lactose group and acetic acid group.(3) Fecal score:the average fecal score of the high dose senna group and acetic acid group was significantly higher compared to the normal group after the modeling (P<0.05, P<0.01).(4) Histopathologic evaluation:the infiltration of lymphocytes, neutrophils, Eosnophils and interstitial edema were observed in colon of the lactose group, tissue adhesions and proliferation were observed in the acetic acid group. The other groups had no abnormal changes.Conclusions:(1) Maternal separation and senna by gavage were reliable to duplicate a rat model of IBS-D.(2) The character of stools and histopathologic evaluation of the colon were important to judge the rationality of methods to establish the animal model of IBS-D.Part2:Establishment and evaluation of a rat model of IBS-D with liver depression and spleen deficiency syndromeAims:To establish a rat model of IBS-D with liver depression and spleen deficiency syndromeMaterials and methods:The male SD rats were used in this part and were divided into5groups. The normal group (N), chronic restraint group(R), maternal separation group(S), maternal separation and chronic restraint (SR), Tongxieyaofang group (TX), each group has20rats. The establishment of IBS-D is based on the part 1.There were three steps in the process of modeling. Firstly, the pups in the S, SR and TX group were exposed to a3h period of daily maternal separation on postnatal day2-14, the N and R group didn’t exposed to maternal separation. Secondly, the R, SR and TX group were exposed to chronic restraint stress by specially designed restraint frame for3weeks at2month of ages, the N and S group received no treatment. Thirdly, from the second week of the restraint period, the TX group was given Tongxieyaofang decoction (3g/kg) by gavage on9:00am for2weeks, the other groups were given normal saline (10ml/kg) instead, on the last week of the restraint period, all the groups were given Senna decoction(4.5g/kg) by gavage on the4:00pm except the normal group. The models were evaluated by the features of IBS-D, liver depression and spleen deficiency syndrome, some biomarkers also were tested. The visceral sensitivity was tested before and after the restraint stress; the fecal scores were calculated during the restraint period and after the modeling. The liver depression syndrome was evaluated by open field test, sucrose preference test and tail suspension test. The spleen deficiency syndrome was evaluated by body weight and food consumption. The level of serum D-xylose,5-HT, BDNF, IgA, IgG were tested after the modeling. T lymphocyte subpopulation analysis of blood and thymus were did after the modeling by flow cytometry. The number of mast cells(MC) and enterochromaffin cell(EC) of colon and small intestinal was tested by immunohisto-chemistry. The histopathologic evaluation of the proximal colon and distal ileum was done after the modeling.Results:(1)Pain threshold:after the modeling, the pain threshold of the S group and SR group was lower compared to the N group(P<0.01),there was no significant difference between the R and N group,Tongxieyaofang could enhance the pain threshold of the model(P<0.01).(2)Fecal score:acute restraint stress could increase the number of fecal pellet output, chronic restraint stress didn’t influence the number of fecal pellet output, but could increase the water content. The average fecal score of the SR group was significantly higher than the N,R, and S group(P<0.01), Tongxieyaofang had no obvious effect on improving the diarrhea.(3)Weight growth:the weight growth of the R, S and SR was lower compared to the N group(P<0.01). Tongxieyaofang had no obvious effect on the weight growth.(4) Open field test:the horizontal and vertical motion of the rats in the R, S and SR group were decreased compared to the N group(P<0.05). Tongxieyaofang could improve this presentation of the rat model.(5) Sucrose preference test:the sucrose preference rate of the rats in the R, S and SR group was decreased compared to the N group(P<0.01, P<0.05, P<0.01). Tongxieyaofang could increase the sucrose preference rate of the rat model (P<0.05).(6) Tail suspension test:the still time in the R, S and SR group was longer compared to the N group(P<0.01, P<0.05, P<0.05).Tongxieyaofang could shorten the still time of the rat model(P<0.05).(7) Food consumption:There was no significant difference of the food consumption in the SR group compared to the normal group. Tongxieyaofang could increase the food consumption of the rat model(P<0.05).(8)Serum markers:The level of serum D-xylose decreased in the R,S and SR group compared to the N group(P<0.01).Tongxieyaofang had no effect on the serum D-xylose.The level of serum5-HT increased in the S and SR group compared to the N group(P<0.01).Tongxieyaofang could decrease the serum5-HT of the rat model(P<0.01). The level of serum IgA increased in the S and SR group compared to the N group(P<0.05). Tongxieyaofang had no effect on the serum IgA.There was no significant change in the level of serum BDNF, IgG in the SR group.(9)The proportion of T lymphocyte subpopulation:the proportion of blood CD3+CD4+CD8-increased, and the subpopulation CD3+CD4-CD8+decreased in the R,S and SR group compared to the N group(P<0.01). The proportion of thymus total T lymphocytes increased in the R,S and SR group compared to the N group(P<0.05). Tongxieyaofang had no significant effect on the proportion of T lymphocyte subpopulation.(10)Spleen index:the spleen index in the S and SR group decreased compared to the N group(P<0.01). Tongxieyaofang had no effect on the spleen index of the rat model.(11)The number of MC:the number of the MC increased significantly in the SR group compared to the N group(p<0.01), tongxieyaofang could decrease the number of MC of the rat model(P<0.01).(12) The number of EC:the number of EC in the R,S and SR group increased significantly compared to the N group (P<0.01). Tongxieyaofang had no effect on the number of EC of the rat model.(13)There was no obvious abnormal change of proximal colon and distal ileum in all the groups. Conclusions:(1)The maternal separation stress, chronic restraint stress and senna decoction by gavage could duplicate the IBS-D rat model with liver depression and spleen deficiency.(2)The combination of chronic restraint stress and maternal separation was superior to one factor.(3)The model was characterized by visceral hypersensitivity, increased permeability of intestinal epithelial cell, depression and immune dysfunction.(3) It’s scientific, reasonable and feasible to evaluate the rat model according to the character of IBS-D patients.Part3:Proteomic analysis of colon and brain in the rat model of IBS-D with liver depression and spleen deficiency syndromeAims:To screen the differential proteins in the colon and brain based on the rat models in the part2, contrastive analysis was did between the two tissues.Materials and methods:The colon and brain tissue of the part2rats were used to analysis. The differential proteins were screened based on the technology of isobaric tags for relative and absolute quantitation (iTRAQ). Protein identification was performed by mass spectrum. Mascot software was used to search the related information of the differential proteins based on the Uniprot and Gene Ontology database. The pathways and functional networks of the differential proteins were analysed by the ingenuity pathway analysis software(IPA).Selected proteins were validated by realtime RT-PCR.Results:(1) In the chronic restraint stress group(R),542proteins were identified in the colon tissue(ratio>1.2, P<0.05),309proteins were significantly up-regulated and233proteins were significantly down-regulated,1884proteins were identified in the brain tissue,764proteins were significantly up-regulated and1120proteins were significantly down-regulated. In the maternal separation group(S),809proteins were identified in the colon tissue,415proteins were significantly up-regulated and394proteins were significantly down-regulated,2386proteins were identified in the brain tissue,1080proteins were significantly up-regulated and1306proteins were significantly down-regulated. In the maternal separation and chronic restraint group (SR),731proteins were identified in the colon tissue,424proteins were significantly up-regulated and307proteins were significantly down-regulated,2567proteins were identified in the brain tissue,1187proteins were significantly up-regulated and1380 proteins were significantly down-regulated.(2) In the three groups,192proteins co-expressed in the colon tissue,1501proteins co-expressed in the brain tissue.(3)153proteins co-expressed in the colon and brain tissue in the R group,280proteins co-expressed in the colon and brain tissue in the S group, and239proteins co-expressed in the colon and brain tissue.in the SR group.55proteins of the colon and brain tissue co-expressed in the three groups.(4) In the SR group, there were6proteins differentially expressing with the the ratio>3in the colon tissue,4proteins were up-regulated:S-adenosylmethionine decarboxylase proenzyme, protein Itlnl, FH2domain-containing protein1, MHC class Ⅱ antigen;2proteins were down-regulated:PI-PLC X domain-containing protein3, Hemoglobin subunit alpha. They involved in the S-adenosylmethioninamine biosynthetic process, signal transduction, actin cytoskeleton organization, immune response, lipid metabolic process, oxygen transport. There were22proteins differentially expressing with the the ratio>3in the brain tissue.4proteins were up-regulated:peroxisomal membrane protein PEX14, NADH-ubiquinone oxidoreductase chain2, down syndrome critical region gene3, protein Lrba;18proteins were down-regulated:protein Hbb-bl, parathymosin, neurosecretory protein VGF, Cytochrome c oxidase subunit6B1,40S ribosomal protein S28, ATP synthase subunit d, mitochondrial import inner membrane translocase subunit Tim13, NADH dehydrogenase flavoprotein3, cytochrome c oxidase subunit5A, Pcp411protein, ATP synthase-coupling factor6, Acyl-CoA-binding protein, cytochrome c, metallothionein-3, AIP1, protein uqcrb, glyceraldehyde-3-phosphate dehydrogenase, thymosin beta4. They involved in protein transport, electron transport, vacuolar transport, oxygen transport, immunity, rRNA export from nucleus, hydrogen ion transport, protein import into mitochondrial inner membrane, cadmium ion binding, endosomal transport, actin cytoskeleton organization. Heat shock27kDa protein1and immunoglobulin joining chain were up-regulated, hemoglobin subunit alpha and protein Hbb-b1were down-regulated in the colon of the model rat, which were in accord with the clinical report.(5) The differential proteins in the colon tissue of the SR group mainly involved in cellular assembly and organization, cellular function and maintenance, cell death and survival, cell morphology, tissue development, cellular development, cellular growth and proliferation, nervous system development and function, cell to cell signaling and interaction, small molecule biochemistry.The differential proteins in the brain tissue of the SR group mainly involved in cellular assembly and organization, cellular function and maintenance, cell death and survival, cell morphology, small molecule biochemistry, nervous system development and function, molecular transport, cellular development, tissue development, nucleic acid metabolism.(6) The differential proteins in the colon tissue of the SR group could be assigned to27pathways, including integrin signaling, serotonin degradation, antigen presentation pathway, IL-4signaling, and so on. The differential proteins in the brain tissue involved26pathways, including gap junction siganling, epithelia adherens junction signling, mitochondrial dysfuncion, etc. The differential proteins in the colon tissue of the SR group included9interaction networks, and12interaction networks in the brain tissue.(7) The expression of AQP8mRNA and Na+/H+exchanger3(NHE3) mRNA in the colon tissue were in accordance with the expression of homologous proteins, while the expression of brain-derived neurotrophic factor (BDNF) mRNA in the colon and hippocampus tissue were different from the expression of homologous protein.Conclusions:(1) There were a large number of differential proteins in the colon and brain tissue of the rat model of IBS-D with liver depression and spleen deficiency syndrome, some of them were in accord with the clinical report.(2) The rat model of IBS-D had some proteins co-expressed in the colon and brain tissue, which indicated abnormal interaction between colon and brain.(3) The models induced by chronic restraint stress, maternal separation stress, and the combination of the two factors had the same molecule biological basis, mental stimulation mainly changed the proteins expression in the brain tissue.(4) The pathogenesis of IBS-D was related to changes of a number of proteins, studying the function of the differential proteins could gain a deeper understanding in the pathogenesis of IBS-D.