背景与目的 肠易激综合征(IBS)是一种以腹痛伴有排便频率和/或粪便性状改变的功能性胃肠病，目前发病机制仍待阐明。研究发现IBS患者存在多层面免疫系统激活，尤其是部分IBS患者血清中存在多种抗神经元抗体(aNA)，可引起针对肠神经系统(ENS)的免疫反应，导致ENS内神经元损伤。慢性假性肠梗阻(CIPO)患者aNA阳性血清能促使神经细胞自噬，而IBS患者抗肠神经元抗体(AENA)阳性血清能导致肠神经元凋亡。本研究目的：明确IBS患者抗HuD抗体阳性血清或商品化抗HuD抗体能否导致神经细胞自噬，并阐释其损伤机制，探究抗HuD抗体在IBS发病中的病理生理作用。 材料与方法 采用符合罗马Ⅲ诊断标准的IBS患者和健康受试者血清，参考前期应用间接免疫荧光法检测AENA、HuProt™芯片检测血清中差异性抗体的结果。分别选择IBS患者AENA阴性且差异性抗体阴性血清(AENA-/PR0-组)、AENA阳性且抗HuD抗体阴性血清(AENA+/HuD-组)、AENA阴性且抗HuD抗体阳性血清(AENA-/HuD+组)、商品化单克隆鼠抗人HuD抗体(anti-HuD组)、健康受试者血清(HCs组)孵育培养SH_Sy5Y细胞24小时，并用胎牛血清(FBS组)孵育作为阴性对照，用雷帕霉素(rapamycin组)诱导自噬作为阳性对照。倒置显微镜下观察细胞形态，CCK-8检测细胞活性，间接免疫荧光法(抗LC3-Ⅱ/抗PGP9.5/DAPI染色)检测神经细胞自噬，western blot检测自噬相关蛋白LC3-Ⅱ、P62、p-mTOR、Beclin-1的表达；透射电镜观察细胞超微结构的改变，明确有无自噬体的形成。 结果 倒置显微镜下见anti-HuD组细胞生长密度减小，胞体缩小，细胞形态不规则，突起减少，折光性减弱；FBS组、HCs组、AENA-/PRO-组、AENA+/HuD-组和AENA-/HuD+组细胞形态正常，生长良好。CCK-8检测细胞活性显示：anti-HuD组细胞活性显著低于FBS组、HCs组、AENA-/PR0-组、AENA+/HuD-组和AENA-/HuD+组(P＜0.05)。间接免疫荧光显示：除rapamycin组外，其余各组均未见胞浆内抗LC3-Ⅱ抗体绿色荧光浓聚，提示无自噬体形成。Western blot检测自噬相关蛋白表达量，结果显示FBS组、HCs组、AENA-/PRO-组、AENA+/HuD-组、AENA-/HuD+组和anti-HuD组间LC3-Ⅱ蛋白表达量无显著差异(均P＞0.05)，且均显著低于rapamycin组(P＜0.05)。HCs 组、AENA-/PRO-组、AENA+/HuD-组和 AENA-/HuD+组P62蛋白表达量显著高于FBS组、anti-HuD组和rapamycin组(P＜0.05)， raparaycin组P62蛋白表达量低于FBS组(P＜0.05)；AENA+/HuD-组、AENA-/HuD+组和anti-HuD组p-mTOR蛋白表达量显著高于FBS组(P＜0.05)，AENA-/HuD+组和anti-HuD组p-mTOR蛋白表达量显著高于HCs组，而rapamycin组p-mTOR蛋白表达量较其余各组均显著减少(P＜0.05)；AENA-/HuD+组、anti-HuD组和rapamycin组Beclin-1蛋白相对表达量显著高于FBS组(P＜0.05)。电镜观察见HCs组、 AENA-/PRO-组、AENA+/HuD-组和AENA-/HuD+组神经细胞胞质疏松，形成空洞，且可见脂滴和糖原堆积；Anti-HuD组同样可见部分细胞存在胞质疏松、脂滴增多和糖原堆积现象，且可见死亡细胞；除rapamycin组外，其余各组内均未见自噬体形成。 结论 商品化单克隆鼠抗人HuD抗体可显著引起SH-Sy5Y细胞损伤和死亡，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。抗HuD抗体所致神经元损伤可能与部分IBS患者的发病相关。 ①本研究为国家国际科技合作项目(2014DFA31850)和国家自然科学基金课题(81870379)研究内容 关键词 肠易激综合征，抗HuD抗体，自噬

 Background and Objective Irritable bowel syndrome (IBS), as a functional gastrointestinal disorder, is characterized by abdominal pain/discomfort with altered defecation and bowel habits. The pathophysiological mechanisms of IBS are complicated, and some studies have demonstrated activation of immune system in multiple levels with IBS patients. Several anti-neuronal antibodies (aNA) were found in the sera of IBS patients which indicate that the specific effects on the enteric nervous system (ENS) through immunoreaction and damage of enteric neurons. aNA positive sera in patients with CIPO could evoke neuronal autophagy and anti-enteric neuronal antibodies (AENA) positive sera in patients with IBS could induce neuronal apoptosis. The aims of this study were to detect the effects of anti-HuD antibodies positive sera from IBS patients and purified mouse-anti-human-HuD monoclonal antibodies to the neuronal cells via activation autophagy, and to identify the significance of anti-HuD antibodies on the pathogenesis of IBS. Material and Methods IBS patients met the Rome Ⅲ criteria and healthy controls (HCs) were enrolled, and the sera AENA was tested by indirect immunofluorescence and differential antibodies were identified by HuProt™ microarray in the previous study. SH-Sy5Y cells were cultured and incubated with different types of sera from IBS patients, including sera with AENA and differential antibodies negative (AENA-/PRO-), AENA positive and anti-HuD antibodies negative (AENA+/HuD-), AENA negative and anti-HuD antibodies positive (AENA-/HuD+),commercial anti-HuD monoclonal antibodies (anti-HuD), and sera of HCs with negative AENA for about 24 hours. Fetal bovine serum (FBS) was used to incubate cells as negative control,and rapamycin was used to induce autophagy as positive control. The cell morphological changes were observed under the inverted microscope. The viability of cells was detected by CCK-8. Indirect immunefluorescence (anti-PGP9.5/anti-LC3-Ⅱ/DAPI) was used to monitor neurons autophagy. Western blot was used to detect the expression of autophagy associated proteins LC3-Ⅱ,P62,p-mTOR,and Beclin-1. The changes of cell ultrastructure as well as autophagosome were observed by the transmission electron microscope. Results The neuronal cells in anti-HuD group grew with lower density, shrunken cell bodies, fewer processes, and reduced refraction after incubation. In FBS, HCs, AENA-/PRO-,AENA+/HuD-,AENA-/HuD+ groups, the shapes of cells were normal and the growth were in good conditions. The cell viability of anti-HuD groups was significantly lower than FBS, HCs, AENA-/PRO-,AENA+/HuD-,AENA-/HuD+ groups (all P<0.05).Indirect immunofluorescence showed there were no anti-LC3-Ⅱ antibodies stainings of green fluorescences in cytoplasm, which indicated no autophagosome formation except rapamycin group. There were no significant differences of LC3-Ⅱ expression among FBS,HCs,AENA-/PRO-,AENA+/HuD-,AENA-/HuD+, and anti-HuD groups, but those were significant lower than rapamycin group (all P<0.05). The expressions of P62 in HCs, AENA-/PRO-,AENA+/HuD-,AENA-/HuD+ groups were significant higher than FBS, anti-HuD and rapamycin groups (all P<0.05). The expressions of p-mTOR in AENA-/HuD+,AENA-/HuD+ and anti-HuD groups were significant higher than FBS groups (all P<0.05). The expressions of p-mTOR in AENA-/HuD+ and anti-HuD groups were significant higher than HCs groups (P<0.05). The expressions of Beclin-1 in AENA-/HuD+,anti-HuD, and rapamycin groups was significant higher than FBS groups (P<0.05). In the transmission electron microscope, we observed loosy cytoplasms and vacuolization as well as accumulation of lipid droplets and glycogen in HCs,AENA-/PRO-,AENA+/HuD-,AENA-/HuD+ groups; and cytoplasm rarefaction, accumulation of lipid droplets and glycogen,as well as dead cells in anti-HuD group; and the autophagosome were only observed in rapamycin group. Conclusions The commercial anti-HuD monoclonal antibodies might induce damage and death of SH-Sy5Y cells and these effects mediate by inhibition of autophagy processing, activation of apoptosis or necrosis signaling pathways. Sera of positive anti-HuD antibodies from IBS patients might induce slight injury on SH-Sy5Y cells and alter the expression of autophagic proteins on signaling pathways. We speculate some substances in human sera, especially in sera of IBS patients, might alter the glucose and lipid metabolism of SH-Sy5Y cells, resulting in damage to the cells. The neuronal injury induced by anti-HuD antibody may be linked to the pathogenesis in a subset of IBS patients. Key words irritable bowel syndrome, anti-HuD antibodies, autophagy