EphrinB2/ephB2 Promoted Synaptic Plasticity Of ENS And Contributed To Visceral Hypersensitivity In Irritable Bowel Syndrome

Background:Visceral hypersensitivity is the most important biomarker of irritable bowel syndrome(IBS)and associated with the recurrent abdominal pain.However,the specific molecular mechanisms are not fully understood.In our previous research,the spontaneous and mechanical/chemical induced firing activity of the mesenteric afferent nerve from the post-infectious IBS rats were enhanced,which indicated hypersensitive state of enteric nervous system(ENS).The latter is the key of sustained visceral hypersensitivity.Synaptic plasticity of hippocampal neurons is a marker of central memory formation,which is related with central sensitization to pain.Does ENS have a similar foundation of "memory"formation,and lead to sustained hypersensitivity?EphrinB2/EphB2 receptor tyrosine kinase is located in the presynaptic and postsynaptic membrane,especially the excitatory synapses,which plays an important role in synapse formation and functional maintenance,andparticipates in the regulation of synaptic plasticity in the central nervous system(CNS).Since there is a common relationship between ENS and CNS,whether the regulation of synaptic plasticity may have the same pathway?Therefore,we speculated that EphrinB2/EphB2 may be involved in the regulation of ENS synaptic plasticity and the formation of visceral hypersensitivity,resulting in ENS hypersensitivity and peripheral"memory".Methods:(1)The distribution and expression of ephrinB2/ephB2 in human colonic ENS of biopsy mucosa fromIBS-D and IBS-C patients were detected by immunofluorescence and immunoblotting.The expression of synaptic plasticity-related immediate early genes(c-fos and arc),synapse-associated proteins(PSD95 and Synaptophysin),excitatory glutamate transporters VGluT and receptors NMDAR were also detected.The relationship between ephrinB2/ephB2 and abdominal pain was analysed.(2)The PI-IBS model was induced by Trichinella spiralis infection,and randomly divided into normal group(NC),PI-IBS group(VH)and PI-IBS intervention group(VH+EphB2Fc).HE staining was used to evaluate the level of intestinal inflammation.AWR score and mesenteric afferent firing record was used to evaluate the visceral hypersensitivity.The distribution and expression of ephrinB2/ephB2 in ENS and the relationship with visceral hypersensitivity were detected,as well as the synaptic plasticity-related immediate early genes(c-fos,arc,bdn,ptgs2,creb,creme and icer).To evaluate the structural synaptic plasticity,synaptic density was detected by immunofluorescence,synaptic vesicle density was detected by transmission electron microscope,synaptic proteins PSD95,Synaptophysin and Synaptobrevin were detected by immunob lotting.To evaluate the functional synaptic plasticity,the expression of VGluT and NMDAR,and the phosphorylation of NR2B were detected.(3)Trough in vitro culture of LMMP tissue,administration of ephrinB2-Fc(eB2Fc)to activate ephB2 receptor and addition of PD98059 and U0126(Erk/MAPK inhibitor),LY294002(PI3K/Akt inhibitor),U73122(PLC? inhibitor),Chelerytrine(PKC inhibitor)and KN93(CaMKIIinhibitor)respectively,to investigated the structural synaptic plasticity of ENS.Via primary ENS neuron culture,to investigate the neural nerves regeneration,neuronal differentiation and maturation,and synaptic sprouting of ENS induced by eB2Fc.(4)Trough in vitro culture of LMMP tissue,administration of ephrinB2-Fc(eB2Fc)to activate ephB2 receptor and addition of PP2(Src kinase inhibitor),MK801(nonspecific NR inhibitor)and ifenprodil(selective NR2B blocker),to investigated the functional synaptic plasticity of ENS.Via primary ENS neuron culture,to investigate the affect of eB2Fc on the expression of synaptic plasticity factor c-fos,and glutamate induced[Ca2+]i.Results:(1)EphrinB2/ephB2 was expressed specifically in human colonic mucosal ENS,and enhanced in IBS-D and IBS-C patients.There were significant changes in the synaptic plasticity of ENS in colonic mucosa of patients with IBS.The expression of EphrinB2/ephB2 was positively correlated with the degree of abdominal pain,and positively correlated with c-fos level in IBS patients.(2)EphrinB2/ephB2 expressed in rat colonic myenteric and submucousal plexuses and its nerve fibers.The AWR score were increased and the pain threshold were decreased,the spontaneous and stimulated mesenteric afferent discharge were increased in the VH rats.The level of EphrinB2 expression and ephB2 phosphorylation was significantly increased,which was negatively correlated with pain.The immediate early factors c-fos and arc were increased in the VH rats.There was obviously structural synaptic plasticity in VH rats,including increased synaptic density,increased synaptic vesicle density,and increased expression of synaptic proteins PSD95,Synaptophysin and Synaptobrevin.There was also obviously structural synaptic plasticity in VH rats,including increased expression of NR1,NR2B,VGluT1 and VGluT2,as well as phosphorylation of NR28(Y1472).EphB2Fc significantly blocked the synaptic structure and functional plasticity of ENS in VH rats,and improved the visceral sensitivity of PI-IBS rats.(3)EphrinB2Fc promoted ENS synaptic structural plasticity in LMMP tissue,which could be obviously blocked by LY294002 and partially blocked by PD98059 and U0126,but PLC?/PKC and CaMK? blocking had no effect.EphrinB2Fc increased the number of primary,secondary and neurite branches promoting nerves regeneration,increased the proportion of neurons with neurite outgrowth promoting neuronal differentiation and maturation,and increased synaptic sprouting of ENS.(4)EphrinB2Fc promoted ENS synaptic functional plasticity in LMMP tissue,with increased phosphorylation of NR28(Y1472)and CREB(S133),and expression of c-fos and arc.EphrinB2Fc promoted c-fos mRNA expression in ENS neurons,enhanced the[Ca2+]i induced by glutamate.Conclusions:EphrinB2/ephB2 expressed in the ENS,and was significantly correlated with abdominal pain and visceral hypersensitivity in IBS,which played a key role in the regulation of synaptic plasticity in ENS.EphrinB2/ephB2 participates in the formation of synaptic structural plasticity in ENS by activating downstream PI3K/Akt and Erk/MAPK signalling.EphrinB2/ephB2 promoted NR2B phosphorylation by Src kinase,leading to the eIqr ession of Ca2+-dependent immediate early gene mediated by[Ca2+]i increasing,and participates in the formation of synaptic functional plasticity of ENS.