The Role Of Enteric Glial Cells On Regulating Visceral Hypersensitivity In Irritable Bowel Syndrome

Part I:The role of nerve growth factor in the pathogenesis of visceral hypersensitivity in an IBS-like animal modelBackground and aims:Irritable bowel syndrome(IBS)is a common functional gastrointestinal disorder which could make a tremendous difference in the quality of patients’s life and burden the health-care system.In recent years,our understanding of the potential mechanisms involved in epithelial barrier,gut microbiota,abnormal immunity,visceral sensation and brain-gut axis is rapidly advancing.It’s widely accepted that visceral hypersensitivity is one of the most important pathophysiological mechanisms in IBS.However,the possible causes are far from understood.Some studies have found evidence for structural and functional changes of enteric nervous system(ENS)in the colon of IBS.ENS consists a large number of interneuron and enteric glail cells(EGCs).Recent studies have shown that EGCs could undergo profound change in response to inflammatory mediators or bacteria stimuli.Our previous study also proved that alterations of EGCs in structure and function may be a relevant factor in the pathogenesis of IBS.Nerve growth factor(NGF)is a key neurotrophic factor.It has been implicated in diverse effects including neuronal maintenance,regulation of axon sprouting,and modulating neuronal plasticity.Researches have suggested that NGF play a vital role in regulation of inflammatory pain.A previous study has reported the elevated level of NGF in intestinal mucosa of IBS patients participated in neuronal differentiation and neuroplasticity changes,thus promoting visceral hypersensitivity.However,the exact mechanism of NGF up-regulation has not yet been identified.Butyrate,which belongs to short chain fatty acids(SCFAs),is a small-molecule metabolite arising from symbiotic bacteria fermentation from insoluble dietary fibers in colon.In addition,animal studies have shown that rectal instillation of butyrate promoted visceral hypersensitivity and the mechanism through which is unclear.These results suggest that butyrate may be related to the pathogenesis of IBS.Of interest,in central nervous system,it has been demonstrated that sodium butyrate(NaB)as a histone deacetylase(HDAC)inhibitor,could up-regulate neurotrophins expression in cultured astrocytes cells which share morphological and physiological similarities with EGCs.This finding implies a possible link between butyrate and EGCs.Thus,we aim to further explore whether butyrate-EGC interaction exists and mediates IBS-like colonic hypersensitivity.Methods:1.After 5 mmol/L butyrate treatment for 6 hours,total RNA in CRL-2690 was extracted for RNA-seq analysis.2.Colonic hypersensitivity was induced by butyrate enemas in the distal colon.Rats received NaB solution(1 M,pH 6.9)or normal saline enemas,once daily,for 3 days.The colonic enemas were performed through a 2.7 mm Fogarty catheter positioned 7 cm from the anus.Every day each animal received 2 ml NaB solution or normal saline.Colorectal distention(CRD)test was performed to assess visceral sensitivity.The distention was applied using a 2.7 mm Fogarty catheter balloon inserted into the descending colon.Graded CRD was performed by rapidly injecting different volumes of normal saline(0.2 ml,0.4 ml,0.6 ml,0.8 ml,and 1.0 ml)into balloon over 1 s and maintaining the distention for 20 s.NGF expression levels were examined in rat colon.Immunofluorescence studies were used to evaluate the co-expression of glial fibrillary acidic protein(GFAP)and NGF in this animal model.NGF mRNA and protein expression in rat colon were also investigated.In vitro,rat enteric glial cells(CRL-2690)were stimulated with NaB at different final concentrations of 1 mmol/L,5 mmol/L and 10 mmol/L.Cells were incubated for 6 hours or 24 hours respectively.Since butyrate is a potent histone deacetylase(HDAC)inhibitor,we also used another well-characterized HDAC inhibitor,trichostatin A(TSA)at a concentration of 100 nmol/L and 500 nmol/L.After treatment,NGF expression at mRNA and protein levels was examined.Results:1.Cells were treated with or without NaB for 6 hours and then used for further analysis.In total,8375 differential expressed genes were generated.A value of 4346 genes was up-regulated while 4029 genes were down-regulated in NaB-treated group when compared with control group.The most-represented pathways were sorted by statistical significance in P value and false discovery rate(FDR),including Neurotrophin signaling pathway,Cell cycle,Proteoglycans in cancer and Spliceosome.52 of the 79 genes in the Neurotrophin signaling pathway were up-regulated and 27 were down-regulated.Four neurotrophin family members expression levels were normalized to control group after butyrate treatment,including NGF,BDNF,NT3 and NT4.Notably,NGF was found increased 3.2-fold in butyrate-treated group.2.NaB-treated rats showed significantly increased visceral sensitivity.Compared with control rats,graded distention(0.6 ml,0.8 ml)caused marked changes in NaB-treated rats,which was statistically significant at saline volumes of 0.6 ml(2.96±0.68 vs.1.92±0.63,P<0.05)and 0.8 ml(3.58±0.46 vs.2.46±0.73,P<0.01).An improved NGF mRNA or protein expression levels was observed in NaB-treated rats(P<0.01).NGF mRNA expression was significantly correlated with AWR scores at the balloon volumes of 0.6 ml(r=0.6194,P=0.0105)and 0.8 ml(r=0.8840,P<0.001)and 1.0 ml(r=0.7466,P<0.001),respectively.NGF protein expression also correlated with AWR scores at the balloon volumes of 0.6 ml(r=0.5739,P=0.0201),0.8 ml(r=0.8170,P<0.001)and 1.0 ml(r=0.65 65,P=0.0057),respectively.3.Double immunofluorescence showed 2.1-fold increase in co-expression of GFAP and NGF in rats received NaB enemas when compared with saline-treated group(P<0.05).There was no obvious difference of nerve fiber density between butyrate administration group and saline-treated group.4.In vitro cultured cells,NaB or TSA stimulation caused marked NGF mRNA expression and protein expression(P<0.05).Conclusions:1.Butyrate instillation could induce visceral hypersensitivity in animal model.The elevated expression of NGF in gut contributed to the development of butyrate-induced colonic hypersensitivity.2.The increased expression of NGF may derive from EGCs within the lamina propria in gut.In vitro,butyrate could promote the secretion of NGF via histone deacetylase inhibition.Part II:Alterations of miR-204-5p expression in colonic mucosa of IBS-D patients and their correlation with abdominal pain:in vivo and in vitro studiesBackground and aims:Irritable bowel syndrome(IBS)is a functional bowel disorder characterized by abdominal pain.It is widely accepted that alteration of visceral sensation is a key pathophysiological mechanism correlated with pain perception in IBS patients.Achievement of a better understanding of the causes of visceral pain will provide a basis for future treatments for IBS.MicroRNAs(miRNAs)are a class of about 22 nucleotides,endogenously expressed small non-coding RNAs.Mature miRNAs function via base-pairing with 3’-untranslated region(UTR)of target mRNA molecules,leading to mRNA silence and translation blocking.MicroRNAs have emerged as regulators involved in many human disease,such as cardiovascular disease,neurological disorder and immune system disease.A previous study has reported that miR-29a was increased in IBS-D patients and regulated intestinal permeability by down-regulating a key tight junction protein claudin-1.Another study showed that decreased miR-199 was an important factor that could induce increased expression of the transient receptor potential vanilloid type 1(TRPV1)and resulte in visceral hypersensitivity in IBS patients.In our preliminary study,we defined a part of the miRNAs analysis in the serum of IBS-D patients and healthy controls and discovered a set of differentially expressed miRNAs.By using miRNAs target genes prediction tool,miR-204-5p was chosen as a promising candidate for inducing visceral pain via targeting EphB2 which was involved in axon guidance signaling pathway.Moreover,EphB2 is a known regulator of N-methyl-D-aspartate receptor(NMDAR)expression.The activation of NMDAR resulted in the subsequent calcium influx,increased phosphorylation of cAMP-response element binding(CREB)protein and induction of c-fos.These may promote the reconstruction of synapse and nociceptive information transmission.To test this notion,we aimed to evaluate miR-204-5p expression and the predicted target of miR-204-5p(EphB2)as a critical protein related to pain sensation in IBS-D patients and visceral hypersensitivity animal model.Methods:1.IBS-D patients were diagnosed using the Rome III criteria.Controls were enrolled from participants undergoing colonoscopy examination for polyps and cancer surveillance.Each subject received a workup for exclusion of organic diseases.Serum samples and biopsy specimens obtained from the rectosigmoid junction were collected for further study.The serum of 5 IBS-D patients and 5 healthy controls matched by sex and age was used for small RNA high-throughput sequencing analysis.After screening,miR-204-5p was chosen for the following experiments.The expression of miR-204-5p was also examined in colon biopsy samples to further conform the sequencing data.Target mRNAs of the miR-204-5p were determined using the TargetScan web tool.Meanwhile,EphB2 expression was also determined by immunohistochemical staining in the colon of IBS-D patients.Their correlation with abdominal pain was also evaluated by using Spearman rank correlation analysis.2.Fluorescence in situ hybridization(FISH)was done on colon tissues using a Cy3-labeled miR-204-5p probe.3.To verify that EphB2 is a target of miR-204-5p,luciferase reporter constructs containing the miR-204-5p recognition sequences from the 3’-UTR of EphB2 inserted downstream of the luciferase gene were performed.Another plasmid containing mutant sequences in the 3’-UTR of EphB2 was constructed for control group.HEK293T cell line was co-transfected by EphB2 wild type or mutant type constructs and miR-204-5p mimic or inhibitor.Relative luciferase reporter activity was determined.4.SH-SY5Y cells were transfected with miR-204-5p mimic or inhibitor.Western blots were used for detecting the expression of EphB2,NR1,CREB,p-CREB and c-fos.5.C57BL/6 mice received TNBS enemas to establish an IBS-like visceral hypersensitivity model.Colorectal distention(CRD)was used to evaluate the visceral sensation.The expression of miR-204-5p,EphB2 and c-fos were determined in colon tissues.6.The visceral hypersensitivity mice received miR-204-5p agomir treatment intraperitoneally every 2 days and each one received 5nM every time.Seven days later,after the CRD test,mice were sacrificed and the distal colon was removed for determining the expression of miR-204-5p,EphB2 and c-fos.Resuts:1.Analysis on small RNA sequencing sifted 129 differential expressed genes.Among them,71 were significantly up-regulated and 58 were significant down-regulated.We chose miR-204-5p as the promising candidate for inducing visceral pain.Meanwhile,we evaluated the expression of miR-204-5p in colonic biopsies in IBS patients using qPCR.When compared with healthy controls,miR-204-5p expression was significantly decreased in IBS-D patients.Then we explored the potential targets of miR-204-5p using TargetScan web tools and found that miR-204-5p may be a regulator of EphB2 genes.Immunohistochemistry quantification analysis revealed that the expression of EphB2 was higher in in colonic mucosa of IBS-D patients compared with controls(P<0.01).Spearman rank correlation analysis showed that a positive correlation was found between EphB2 levels and severity of abdominal pain/discomfort in all subjects(r=0.5765,P<0.001).The colonic EphB2 levels also significantly correlated with the frequency of abdominal pain/discomfort in all subjects(r=0.6177,P<0.001).2.FISH confirmed the expression of miR-204-5p in lamina propria within human colonic mucosa.Double immunofluorescence showed co-localization of GFAP and miR-204-5p in human colon.3.In HEK293T cell line,when the cells were co-transfected by EphB2-wild type plasmid and miR-204-5p mimic,the relative luciferase activity of cells was significantly lower than that co-transfected by EphB2-mutant type and miR-204-5p mimic.On the contrary,when the cells were co-transfected by EphB2-wild type plasmid and miR-204-5p inhibitor,the relative luciferase reporter activity of cells was higher than that co-transfected with EphB2-mutant type and miR-204-5p inhibitor.4.In vitro culture of SH-SY5Y cells,we found that miR-204-5p mimic markedly decreased the expression of EphB2,NR1,CREB,p-CREB and c-fos(Both P<0.05).In addition,miR-204-5p inhibitor increased the expression of EphB2,NR1,CREB,p-CREB and c-fos(Both P<0.05).5.TNBS enemas markedly decreased the pain threshold and contributed to colonic hypersensitivity in mice.MiR-204-5p level was significantly decreased in colon of TNBS mcie compared with control group.The expression of EphB2 and c-fos were increased by 4.27-fold and 1.50-fold in TNBS group respectively.6.Seven days after intraperitoneal injection of miR-204-5p agomir,there was significant increase in pain threshold in visceral hypersensitivity mice.Compared with control group,the balloon distention volume which can induce pain sensation in miR-204-5p agomir treatment group was higher when compared with control group(3.62± 0.06 ml vs.3.13±0.10 ml,P<0.001).Western blots analysis showed that the expression of EphB2 decreased by 1.96-fold and c-fos levels decreased by 4.28-fold(Both P<0.001).Conclusions:1.There were significant decreases of miR-204-5p levels in IBS-D patients.EphB2 may be a target of miR-204-5p.Increased levels of EphB2 in colonic mucosa of IBS-D patients were associated with severity and frequency of abdominal pain/discomfort.2.In a human neuronal cell line,miR-204-5p could negatively regulate the expression of EphB2 and downstream signaling proteins,impacting on neuronal activation.3.Decreased miR-204-5p levels were found in colon of TNBS-induced visceral hypersensitivity mice.There was a potential therapeutic role for miR-204-5p agomir,which may reduce colonic hypersensitivity in animal model via decreasing the expression of EphB2 and c-fos.