| Part Ⅰ:Expression and localization of GDNF/RET on colonic mucosa of IBS paitents and their correlation with colonic sensitivityBackground and objectives:Irritable bowel syndrome(IBS)is a common functional bowel disorder characterized by abdominal pain with altered bowel habits.In most patients,IBS is a chronic relapsing disease which significantly reduces health-related quality of life.According to the Rome Ⅳ standard,there are four subtypes of IBS,namely,diarrhea type(IBS-D),constipation type(IBS-C),unshaped type(IBS-U)and mixed type(IBS-M).The pathophysiology and molecular mechanism of IBS are still unclear.Current studies have found that the changes of enteric neurotrophic factors on the intestinal epithelium may lead to IBS or IBS symptoms,and the neuroimmune-endocrine network of IBS has been gradually paid attention to.Glial cell-derived neurotrophic factor(GDNF)is primarily secreted by enteric glial cell(EGC).GDNF mainly binds to the co-receptor GFRal-RET and phosphorylates intracellular tyrosine kinase sites in RET for signal transduction.It is generally believed that GDNF is mainly related to the migration,proliferation and maturation of enteric nervous system(ENS)and the homeostasis of neural stem cells.In addition,studies have shown that GDNF expression is increased in duodenal mucosa of patients with functional dyspepsia.These studies suggest that GDNF may be associated with functional gastrointestinal disorder.RET is generally thought to be expressed in nerve cells.Recent studies have found that RET is specifically expressed in enteroendocrine cells in the intestinal epithelium of drosophila and mice.Enterochromaffin cells(EC cells)are one of the enteroendocrine cells in the intestinal epithelium.EC cells and serotonin(5-HT)play a key role in development of visceral hypersensitivity in IBS.IBS-D patients showed increased postprandial plasma 5-HT level,which can activate the 5-HT receptor in submucosal or intermuscular plexus,and increase gastrointestinal motility and visceral hypersensitivity.These studies suggest that RET not only exists in neurons,may be localized in the enteroendocrine cells of intestinal epithelium.However,the pathophysiological role RET in IBS remains unclear.The aim of this study is to investigate the expression and localization of GDNF and its receptor RET in the colon mucosa of IBS-D,and its correlation with anxiety score,depression score and disease symptoms.We first measured the protein expression level of GDNF in the colonic mucosa of IBS-D patients by ELISA,and analyzed its correlation with anxiety scores,depression scores and abdominal symptom scores.Immunohistochemical staining of 5-HT and ChgA was used to showed the number of EC cells and enteroendocrine cells in the colon mucosa.The correlation between GDNF and EC cells was analyzed.Finally,the localization of GDNF/RET in human colon epithelium was detected by immunofluorescence stainingMethods:(1)IBS-D patients and controls:11 patients with IBS-D and 12 normal controls were included according to the Rome Ⅳ criteria.Controls were selected from patients undergoing colonoscopy for cancer surveillance who received negative results.All specimens were taken from the rectosigmoid junction to standardize the site of sampling under colonoscopy.Hamilton Anxiety and Depression Scale(HADS)and IBS-SSS scale were collected for both patients and controls(2)Enzyme linked immunosorbent assay(ELISA)was used to detect the expression level of GDNF in intestinal mucosa of IBS-D patients and controls.(3)The correlation between GDNF level and HADS anxiety score,depression score and IBS-SSS abdominal pain score was calculated.(4)Immunohistochemical staining was used to detect the expressions of GDNF,ChgA and 5-HT in colon mucosa of IBS-D patients and control patients.The number of intestinal endocrine cells and EC cells were counted.The differences in EC cells between IBS-D patients and controls were calculated.We also calculated the correlation between the expression level of GDNF and the number of EC cells.(5)Expression and localization of RET and EC cells in colon mucosa of IBS-D patients and controls were detected by immunofluorescence stainingResults:(1)Demographics and Clinical Characteristics There was no significant difference in age,sex,or the body mass index(BMI)between the control group and IBS-D group.In IBS-D group,the anxiety subscale scores(control vs IBS-D:2.3 ± 0.8 vs.4.8 ± 1.7,P<.05)and depression subscale scores(control vs.IBS-D:2.6±1.2 vs.4.1±1.8,P<.05)were significantly higher.(2)The expression of GDNF was increased in IBS-D patients In IBS-D patients,GDNF protein was significantly increased in the colonic mucosa compared with controls(38.78 ± 1.25 vs.42.60 ± 1.22 pg/mg protein,respectively).Immunohistochemistry showed that GDNF was not only localized in interstitial cells but also enriched in colonic epithelial cells that scatter among colonic mucosa.The number of GDNF positive cells is about 1%of the total epithelial cells.We speculated that GDNF positive cells were endocrine cells.The number of GDNF-positive cells increased significantly in IBS-D patients.(3)Correlation analysis between GDNF level and anxiety and depression score There was no correlation between GDNF level and anxiety score in IBS-D patients(r=0.0017,P=0.8994),and no significant correlation between GDNF level and depression score in IBS-D patients(r=0.1003,P=0.3159).In healthy controls,there was no significant correlation between GDNF level and anxiety and depression scores(anxiety score correlation coefficient r=0.0833,P=0.3894;Correlation coefficient of depression score r=0.0033,P=0.8662).(4)Correlation analysis between GDNF level and symptom severity score There was no significant correlation between GDNF level and symptom severity(r=0.0029,P=0.9750).(5)The number of EC cells increased in the colonic mucosa of IBS-D patients Immunohistochemical staining showed that the number of ChgA-positive cells was significantly increased in the colon mucosa of IBS-D patients.The number of EC cells,which is 5-HT positive cells,was also significantly increased.Furthermore,the number of GDNF-producing epithelial cells and the number of 5-HT positive epithelial cells were positively correlated.(6)Distribution and localization of GDNF-RET in colonic mucosa Immunohistochemical staining showed that GDNF was mainly concentrated in EC cells in epithelium.Immunofluorescence showed that RET-positive epithelial cells corresponded to a subset of ChgA-positive cells,indicating that RET-positive cells were predominantly EC cells in human colonic mucosa.Conclusions:(1)GDNF was increased in IBS-D colonic mucosa.(2)The number of EC cells was increased in IBS-D patients,and was positively correlated with the number of GDNF-positive cells.(3)GDNF is enriched in EC cells in human colonic mucosa.RET is mainly located in EC cells in the intestinal epithelium.Part Ⅱ:GDNF/RET promotes proliferation and differentiation of intestinal stem cells to regulate irritable bowel syndromeBackground and objectives:Irritable bowel syndrome(IB S)is a common functional gastrointestinal disease of the digestive system,characterized by abdominal pain and changes in bowel habitat.The pathophysiological basis of IBS is mainly related to visceral hypersensitivity,mucosal barrier disorder and gastrointestinal dynamic abnormalities,etc.However,the specific pathogenesis of IBS is still unclear.Glial cell-derived neurotrophic factor(GDNF)is mainly secreted in the intestinal tract by EGC.GDNF binds to the co-receptor GFR-RET and activates the intracellular portion of RET for signal transduction.Current studies have shown that GDNF inhibits colonic epithelial cell apoptosis and alleviates experimental colitis.RET is generally thought to be expressed in nerve cells,but recent studies have shown that RET is specifically expressed in intestinal endocrine cells of drosophila and mice.Our preliminary results showed that the level of GDNF protein was significantly increased in the colonic mucosa of IBS-D patients than that of healthy controls,and enterochromaffin cells(EC)expressed GDNF-RET in the human colon.EC cells are terminally differentiated and are unable to replicate themselves.The Lgr5+stem cells under intestinal crypts have been shown to long-term self-renew and differentiate,giving rise to all cell lineages in the intestinal epithelium.Mouse atonal homolog 1(Mathl),Neurogenin3(Neurog3)and NeuroD,play prominent roles in ISC fate specification.Intestinal stem cells(ISC)and progenitors can switch fate choice to increase the proportion of EC cell progenitors,leading to increased EC cell number.However,the mechanisms may be occurring in patients with IBS-D is unknown.In this report,we demonstrated that stress induced visceral hypersensitivity by expanding of ISC and differentiation of EC cell via GDNF-RET,which was inhibited by treatment with RET inhibitor.Moreover,GDNF treatment amplified Wnt signal and increased serotonin levels in colonic organoids in a dose-dependent manner.Methods:(1)Wrap Restraint Stress(WRS)was used in this study.WRS is a hypersensitive model without ulcerative colon.After the mouse model was successfully constructed,RET inhibitor was administered orally.In addition,wild-type mice were intraperitoneal injection of GDNF.After that,AWR scores were recorded(2)The mRNA expression levels of Axin2,Lgr5,Sox9,Cd44,Ngn3,Tph1 and GDNF in the colon were detected by qPCR.Western blot was used to detect the protein expression levels of the genes above.(3)The number of EC in the colon of mice was detected by immunohistochemical staining,and the proliferation level of colonic stem cells was analyzed by immunofluorescence staining.(4)Small intestine and colonic organoids of mice were cultured in vitro and treated with GDNF and GDNF+RET inhibitors,respectively.Surface area and budding rate were analyzed.Immunofluorescence was used to detect the expression level of β-catenin.The expression level of 5-HT in the supernatant of organoids was detected by ELISA.QPCR was used to detect the proliferation and differentiation of stem cellsResults:(1)Acute adult stress induces proliferation of EC cells in mice colon.WRS treatment generated a sharper curve compared to control,and reached score 4 at small balloon volume,indicating a higher colorectal sensitivity.WRS significantly increased the expression of GDNF in the colon.The expression of RET in the colon was also upregulated by WRS.Compared with saline controls,GDNF treatment caused higher visceral sensitivity.Both WRS and GDNF stimulation increased EC cell expansion in the colon.TPH1 was upregulated by the WRS and GDNF treatment.Blocking GDNF-RET in WRS mice effectively reduced the visceral nociception.Moreover,inhibiting RET not only reduced serotonin-positive cells in the colon but also reduced the expression of TPH1 and GDNF.(2)GDNF-RET promotes intestinal stem cell proliferation and differentiation during acute adulthood stress.Both WRS and GDNF stimulation significantly promoted Lgr5 expression in mRNA and protein levels.Whereas inhibition of RET in WRS mice effectively suppressed Lgr5 expression.The expression of Axin2 and β-catenin was increased in WRS/GDNF-treatment group.Additionally,immunostaining revealed that the expression of β-catenin was increased in the colon mucosa from WRS and GDNF-treated mice.Inhibiting GDNF/RET by GSK3179106 suppressed the expression of β-catenin.The expression of Sox9 and CD44 were both upregulated by WRS/GDNF treatment.Furthermore,GSK3179106 administration reduced the expression levels of Axin2,β-catenin,Sox9,and CD44.The expression of Ngn3 was increased in WRS and GDNF-treated mice.The Ngn3 expression was inhibited by the treatment with GSK3179106.(3)GDNF-RET promotes maturation of ISC.The increased proliferation of ISC induced by GDNF/RET was evidenced by the increased branching efficiency and the size in GDNF-treated organoids,and their reduced branching and size in GSK3179106-treated organoids.GDNF-treated organoids have increased levels of β-catenin,while GSK3179106 prevented the increase in both small intestinal and colonic organoids.The increase in 5-HT was observed in 100nmol/L GDNF-treated colonic organoids and reduced by pre-incubation of GSK3179106.The secretion of serotonin by intestinal organoids did not respond to changes of GDNF.Consistently,mRNA level of THP1 was increased in GDNF-treated colonic organoids but not in GDNF-treated small intestinal organoids.Transcriptional analysis revealed an increase in Lgr5 and Sox9 in colonic organoids stimulated with 100nmol/L GDNF.Moreover,the expression of Wnt-related genes,such as Axin2 and Cd44,was also upregulated in 100nmol/L GDNF-treated colonic organoids.Pre-incubation of GSK3179106 significantly reduced the expression of Wnt-related genes to the levels similar to controls.GDNF upregulated the expression of Ngn3 and NeuroD at 100nmol/L.Conclusions:(1)The expression of GDNF/RET was significantly increased in the colon of WRS mice,the number of EC cells in the colon mucosa was significantly increased,the proliferation of ISC was increased,and the differentiation of EC cells was increased.RET inhibitors could antagonize the above phenotypes and reduce the visceral hypersensitivity of WRSmice.(2)The number of EC cells was significantly increased in GDNF treated mice,ISC proliferation was also increased,and the expression of key factors of ISC differentiation into EC cells was up-regulated,leading to high visceral sensitivity.(3)In vitro,GDNF increased the level of 5-HT secreted by colonic organoids in a dose-dependent manner.(4)GDNF/RET promoted the growth and maturation of colonic organoids and promoted the differentiation of stem cells into EC cells. |
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