【作者】 张磊；

【导师】 侯晓华；

【作者基本信息】 华中科技大学 ， 消化内科， 2017， 博士

【摘要】 目的:内脏高敏感是肠易激综合征(irritable bowelsyndrome,IBS)最重要的生物标志,与反复发作的腹痛有关,其具体的分子机制尚未完全明确。前期研究发现,感染后IBS模型鼠肠系膜传入神经自发性放电活动增强,且对机械和化学刺激更为敏感,表明肠神经系统(enteric nervous system,ENS)处于高敏状态,而ENS高敏状态是持续内脏高敏感的关键。中枢海马神经元突触可塑性是中枢记忆形成的标志,与疼痛的中枢致敏有关,是否ENS也有类似形成“记忆”的基础导致ENS持续高敏呢?EphrinB2/EphB2受体酪氨酸激酶分布于突触前后膜,尤其在兴奋性突触中表达,对突触形成和功能维持有重要作用,参与中枢突触可塑性调控。有“肠脑”之称的ENS与中枢存在共性,其突触可塑性的调控是否可能存在相同的通路呢?因此,我们推测EphrinB2/EphB2可能参与调控ENS突触可塑性,使ENS处于高敏状态,产生外周“记忆”,进而参与内脏高敏感的形成。方法:(1)收集正常人及IBS-D、IBS-C患者结肠活检黏膜组织,通过免疫荧光和免疫印迹检测ephrinB2/ephB2在人结肠ENS中的分布和表达,突触可塑性相关即刻早期基因c-fos和arc表达,突触相关蛋白PSD95和Synaptophysin表达,兴奋性谷氨酸转运体VGluT及受体NMDAR表达,及其与腹痛的相关性。(2)采用旋毛虫感染诱导的PI-IBS内脏高敏感模型,分为正常组(NC)、PI-IBS组(VH)和PI-IBS干预组(VH+ EphB2Fc)。模型评价采用HE染色炎症评分评估肠道炎症水平,AWR评分及肠系膜传入神经放电记录评估内脏高敏感状态。通过免疫荧光和免疫印迹/RT-PCR检测ephrinB2/ephB2在ENS中的分布和表达及其与内脏高敏感的相关性,突触可塑性相关即刻早期基因c-fos、arc、bdnf、ptgs2、creb、creme和icer表达。通过透射电镜检测ENS突触囊泡密度,免疫荧光检测ENS突触密度,免疫印迹检测突触前后相关蛋白PSD95和Synaptophysin、Synaptobrevin表达,评估突触结构可塑性。通过免疫荧光和免疫印迹/RT-PCR检测兴奋性谷氨酸转运体VGluT及受体NMDAR表达和磷酸化,评估突触功能可塑性。(3)通过体外LMMP组织培养,并给与EphrinB2-Fc(eB2Fc)活化ephB2信号通路,并分别加入Erk/MAPK通路抑制剂PD98059和U0126、PI3K/Akt通路抑制剂LY294002 和 PLCγ 抑制剂 U73122、PKC 抑制剂 Chelerytrine、CaMKII抑制剂 KN93,观察ENS突触结构可塑性改变。通过原代ENS神经元培养,观察eB2Fc对ENS神经元纤维新生(初级神经突、次级神经突和神经突分支节点数)、神经元分化成熟和突触萌芽改变。(4)通过体外LMMP组织培养,并给与EphrinB2-Fc(eB2Fc)活化ephB2信号通路,并分别加入Src激酶抑制剂PP2、非特异性NR抑制剂MK801及选择性NR2B阻断剂ifenprodil,观察ENS突触功能可塑性改变。通过原代ENS神经元培养,观察eB2Fc对ENS突触可塑性因子c-fos及谷氨酸诱导的[Ca2+]i的影响。结果:(1)EphrinB2/ephB2相对特异性表达于人体结肠黏膜ENS,而且在IBS-D、IBS-C患者中呈高表达水平。IBS患者结肠黏膜ENS存在明显突触前后可塑性改变。EphrinB2/ephB2表达与IBS患者腹痛症状程度呈明显正相关,且与c-fos水平呈显著正相关。(2)EphrinB2/ephFB2表达于大鼠结肠肌间、黏膜下神经丛及其神经纤维。VH鼠AWR评分增加、疼痛阈值降低,肠系膜传入神经自发放电显著增加且对刺激的敏感性增加;EphrinB2表达、ephB2磷酸化水平显著增加,与疼痛呈相关性负相关。突触可塑性相关即刻早期基因c-fos、arc表达增加。VH鼠存在明显突触结构可塑性,ENS突触密度增加,突触囊泡密度增加,突触蛋白PSD95和Synaptophysin、Synaptobrevin表达增加。VH鼠存在明显突触功能可塑性,NR1、NR2B表达及NR2B磷酸化(Y1472)增加,VGluTl、VGluT2表达增加。EphB2Fc明显阻断VH鼠ENS突触结构和功能可塑性,并改善PI-IBS鼠内脏敏感性。(3)EphrinB2Fc促进LMMP组织ENS突触结构可塑性改变,并且可被PI3K/Akt抑制剂LY294002显著阻断,被Erk/MAPK通路抑制剂PD98059和U0126部分阻断,PLCγ/PKC、CaMKII阻断无明显作用。EphrinB2Fc促进ENS神经元纤维新生(初级神经突、次级神经突和神经突分支节点数增加)、神经元分化成熟(神经突生长的神经元比例增加)和突触萌芽增加。(4)EphrinB2Fc促进LMMP组织ENS突触功能可塑性改变,增加NR2B(Y1472)磷酸化水平,及CREB(S133)磷酸化和c-fos、arc表达水平,并且可被PP2显著抑制。MK801、ifenprodil 显著抑制 CREB(S133)磷酸化和 c-fos、arc 表达水平。EphrinB2Fc促进ENS活化c-fos mRNA表达,增加谷氨酸诱导的[Ca2+]i。结论:EphrinB2/ephB2表达于肠神经元,且与IBS患者腹痛症状程度及IBS模型内脏高敏感明显相关,在ENS突触可塑性调控具有关键作用。EphrinB2/ephB2通过活化下游PI3K/Akt、Erk/MAPK通过参与ENS突触结构可塑性形成。EphrinB2/ephB2通过Src激酶诱导的NR2B磷酸化,介导[Ca2+]i增加并Ca2+依赖的即刻早期基因表达,参与ENS突触功能可塑性形成。更多还原

【Abstract】 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(PLCγ 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 PLCγ/PKC and CaMKⅡ 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.更多还原