Colonic Mucosal NR2B Contributes To Visceral Hypersensitivity Of IBS-D Through ProBDNF And MBDNF

BackgroundN-methyl-D-aspartate receptors(NMDARs)are heterotetramers that require assembly of two NMDA receptor 1(NR1)subunits together with two NR2 subunits(NR2A,NR2B,NR2C and NR2D).Previous studies have demonstrated that NMDARs increase in colonic mucosa of irritable bowel syndrome(IBS)and play an important role in visceral hypersensitivity in IBS.However,it is still unclear which subtype of NMDARs contributes to this biologic effect.This study aimed to investigate the effects of NMDAR subtypes on mechanisms implicated in visceral hypersensitivity in IBS with diarrhea(IBS-D).Brain-derived neurotrophic factor(BDNF)is a neurotrophin playing multiple biologic roles including neuronal survival,synaptic plasticity and pain in the nervous system.Previous studies have demonstrated that NMDARs increase production of mature BDNF(mBDNF).As an intermediate during the synthesis of mBDNF,the expression of precursor of BDNF(proBDNF)also induce by NMDARs.Recent studies reveal that proBDNF also expresses in the intestinal mucosa and exacerbates inflammatory,visceral and surgical pain in the peripheral nervous system.This study aimed to investigate the effects of proBDNF on mechanisms implicated in visceral hypersensitivity and if this subtype of NR2 contributes to visceral hypersensitivity in irritable bowel syndrome via proBDNF.Recent studies reveal that mucosal NMDARs in colon contributes to the visceral hypersensitivity in IBS by increasing production of mBDNF.However this study did not investigate the effects of NMDAR subtypes.In this study,we focused on if this subtype of NR2 contributes to visceral hypersensitivity in irritable bowel syndrome via mBDNF.Objective1.To investigate which subtypes of NMDARs increase in colonic mucosa of IBS-D and contributes to visceral hypersensitivity in IBS-D.2.To investigate the expression of proBDNF in colonic mucosa of IBS-D and the correlation between proBDNF and visceral hypersensitivity in IBS-D.3.To investigate if this subtype of NR2 contributes to visceral hypersensitivity in IBS-D via proBDNF4.To investigate if this subtype contributes to visceral hypersensitivity in IBS-D via mBDNFMethods1.Clinical studiesThe diagnosis of IBS-D were according to Rome ? criteria.Every participant was invited to complete an information collection and modified version of the bowel disease questionnaire to evaluate gastrointestinal symptoms.all participants underwent colonoscopy and we took 3 specimens from the rectosigmoid junction.two specimens were used for western blotting;One biopsy was used for immunohistochemistry.2.Cell studies Experiments were examined in HT-29,a human colonic epithelial cell line.MK-801 or Ro25-6981 has been used extensively to block NMDARs or NR2B subunit activation respectively.We respectively set up blank control group,agonist group,inhibitors' group.Western blot and real-time PCR detect the expression level of proBDNF;western blot and ELISA detect the protein expression of mBDNF.To investigate the effect of NMDARs on the expression of proBDNF andmBDNF,agonist group were divided into five groups,50?M NMDA and 10?MD-serine,100 P M NMDA and 10?M D-serine,500 u M NMDA and 10?M D-serine,100 ?M NMDA,10?M D-serine.Cells and culture medium were harvested for following experiments at 3h,6h,12h,24h and 48h.To investigate the effect of NR2B on the expression of BDNF,MK-801 or Ro25-6981 has been used extensively to block NMDARs or NR2B subunit activation respectively.the effects ofthem on inhibiting the expression of proBDNF and mBDNF were compared 2.Statistical analysisAll data were presented as mean values ± standard deviation.Mean values of quantitative variables from two groups were compared by the Student t-test;Mean values of classified variables were compared by Fisher exact probability test.Data from multiple groups were analyzed using one-way ANOVA,followed by multiple comparisons adjusted by Tukey tests.Correlation between NR2B and proBDNF protein expression were analyzed with the Pearson correlation coefficient.Correlation between proBDNF expression and abdominal pain/discomfort scores were assessed by Spearman rank correlation.Differences between means at a level of p<0.05 were considered to be significant.Results1.Clinical studies1)NR2 subunit was primarily expressed in intestinal epithelial cells.2)Only NR2B subunit expression increased in IBS-D patients and correlated with abdominal pain scores.3)The expression of mucosal proBDNF increased in IBS-D patients and correlated with abdominal pain scores.4)The expression of mucosal proBDNF correlated with NR2B level.5)The expression of mucosal mBDNF increased in IBS-D patients and correlated with abdominal pain scores.6)The expression of mucosal mBDNF correlated with NR2B level.2.Cell studiesActivation of NMDARs substantially induced proBDNF and mBDNF expression in HT-29 cells and his increase was in a dose-dependent manner.Meanwhile this effect was attenuated by NMDARs inhibitor MK-801 or NR2B subunit antagonist Ro25-6981 and there is no difference between MK-801 and Ro25-6981.Conclusionl.Only NR2B subunit expression increased in IBS-D patients and correlated with abdominal pain scores.2.NR2B contributes to visceral hypersensitivity in IBS-D via proBDNF.3.NR2B contributes to visceral hypersensitivity in IBS-D via mBDNF.