肠易激综合征自身免疫性肠神经元损伤机制的研究

Sera with anti-enteric neuronal antibodies(AENA) in patients with irritable bowel syndrome (IBS) promote the myenteric neuronal apoptosis in vitro, which indicate AENA might target enteric neurons and induce autoimmune inflammatory neuropathy in enteric nervous system(ENS). The current project aims ①to establish a rat model of autoimmune neuropathy by intra-peritoneal injection of passive immunization and to validate AENA positive sera of IBS patients (including anti-HuD antibody positive sera) could cause morphological changes of enteric neurons and thus induce the changes of intestinal secretion which reflex the abnormality of regulation by submucosal plexus(SMP). ②to incubate the primary cultured myenteric neurons of guinea pigs and sub-cultured of human neuroblastoma cell line (SH-Sy5Y) with AENA positive sera of IBS patients and of different control groups, to detect the neuronal apoptosis mediated by different apoptotic pathways using the indirect immunofluorescence triple staining, and to semi-quantitatively analyze the expression of apoptosis-related proteins which involved in the different pathways of neuronal cell apoptosis by employing the western blot analysis. The above experiments are intended to reveal the mechanism of AENA induced neuronal apoptosis. ③to observe colonic submucosal neuronal morphology of IBS patients with sera positive AENA and positive anti-HuD antibody (including autoimmune inflammatory changes and ultrastructures of apoptotic neurons), to detect patients′ visceral sensitivity by barostat and to capture the characteristics of their clinical manifestations. The results from current project could clarify that autoimmune inflammatory neuropathy mediated by sera AENA do exist in a subset of IBS patients, which provide evidences to explore a new therapy targeted on autoimmune inflammatory neuropathy in ENS of IBS patients.

肠易激综合征（IBS）患者血清中存在的抗肠神经元抗体（AENA）可在体外促进肠神经元的凋亡，推测AENA可针对性引起自身免疫性肠神经元损伤。本项目①采用腹腔注射被动免疫途径构建免疫性肠神经损伤大鼠模型，验证IBS患者AENA阳性血清（包括抗HuD抗体阳性）可引起大鼠肠神经元形态和肠神经调控的肠道分泌功能改变；②用IBS患者AENA阳性和对照血清孵育豚鼠肌间神经元、SH-Sy5Y细胞株，间接免疫荧光三重染色检测不同凋亡机制介导的神经元凋亡，western bolt检测神经细胞表达的不同凋亡途径相关蛋白，揭示AENA诱导凋亡机制；③观察血清AENA阳性IBS患者结肠黏膜下肠神经形态（包括免疫性炎症改变和凋亡神经元超微结构）、恒压器检测内脏敏感性、分析其临床表现特征。证明：在部分IBS患者存在主要由AENA介导自身免疫性肠神经病，为从自身免疫性肠损伤的机制着手研究新的治疗提供证据。

肠易激综合征（IBS）患者血清存在的抗神经元抗体（AENA）可在体外促进肠神经元的凋亡，推测AENA可针对性引起自身免疫性肠神经元损伤。本项目通过腹腔注射IBS患者抗AENA阳性血清（包括抗HuD抗体阳性）被动免疫途径构建免疫性肠神经损伤大鼠模型，验证IBS患者AENA阳性血清可引起大鼠肠神经元形态和肠神经调控的肠道分泌功能改变；在体外实验，用IBS患者AENA阳性和对照血清孵育豚鼠肌间神经元、SH-Sy5Y细胞株，检测不同自噬/凋亡机制介导的神经元凋亡；观察血清AENA阳性IBS患者结肠黏膜下肠神经形态、恒压器检测内脏敏感性、分析其临床表现特征。主要研究结果：单次腹腔注射0.5ml抗HuD抗体阳性血清进行被动转移造模可导致大鼠肠道动力减慢和内脏高敏感，可模拟便秘型IBS（IBS-C）部分临床特点，与被动转移商品化小鼠抗人HuC/D抗体产生的改变类似；IBS患者AENA阳性血清被动转移试验对肠道传输功能和内脏痛觉功能均无明显影响。在体外实验中，商品化单克隆鼠抗人HuD抗体可显著引起SH-Sy5Y细胞损伤/凋亡和死亡，该作用可能是通过抑制细胞自噬，激活细胞凋亡或坏死等其他途径实现的。IBS患者抗HuD抗体阳性血清可致SH-Sy5Y细胞损伤并影响自噬通路相关蛋白的表达。人血清，特别是IBS患者血清中的部分物质可影响SH-Sy5Y细胞的糖脂代谢过程，对细胞造成损伤。在肠神经元培养实验中，IBS患者抗HuD抗体阳性血清和商品化抗HuC/D抗体有抑制豚鼠小肠肌间神经元自噬的倾向，人血清中的部分物质可对豚鼠小肠肌间神经元造成明显损伤。此外，IBS患者血清AENA与抗脑神经抗体比较发现：AENA抗体阳性率明细高于抗脑神经抗体阳性率（76.8% 比14%）,AENA抗体与肠道症状相关，而抗脑神经抗体主要与合并的头痛、失眠有关，进一步提示AENA抗体主要靶向损伤和影响肠神经，仅有小部分患者影响CNS。以上结果表明，在部分IBS患者存在主要由AENA主要是抗HuD抗体介导自身免疫性肠神经损伤，为从自身免疫性肠损伤的机制着手研究新的治疗提供证据。