肠胶质细胞源性ATP激活肠神经元P2X3受体促进IBS内脏痛的机制研究

Visceral pain is a hallmark symptom in irritable bowel syndrome (IBS) patients that profoundly affects their daily work and quality of life. Aberrant activation of the peripheral enteric neurons in the enteric nervous system (ENS) plays essential roles in generation of visceral pain in IBS patients. However, the underlying mechanisms of visceral pain remain largely unclear. Recent studies have suggested that enteric glial cells (EGCs) not only provide structural and nutritional support for ENS, but also release abundant ATP to modulate enteric neuroplasticity via activating synaptic ionotropic P2 receptors on adjacent neurons. We have recently demonstrated that activation of P2X3 receptor mediates visceral pain in stress-induced IBS and that its expression level is increased in submucous plexus. We therefore hypothesize that ATP released by enteric glial cells could be activating synaptic P2X3 receptor on neighboring enteric neurons, thus promoting enteric functional neuroplasticity to exacerbate visceral pain in IBS patients. We intend to establish a single-prolonged stress animal model for visceral hyperalgesia. By using this model, we will examine visceromotor responses and abdominal withdrawal reflexes of the animals and use patch-clamp to study enteric neuroplasticity. We will also apply various molecular techniques such as immunofluorescence staining, western blotting and luciferase assays to critically study the molecular mechanisms underlying visceral pain in IBS. Together, our proposed studies may identify potential molecular targets for treating stress-related visceral pain in IBS patients.

肠易激综合征（IBS）内脏痛严重影响人们工作和生活质量，肠神经系统中肠神经元异常激活对IBS内脏痛-外周致敏有非常重要的作用，但相关分子机制不详。新近研究表明，肠胶质细胞（EGCs）不仅营养、支持肠神经系统，更可释放ATP，激活P2受体下游信号通路，调节神经元可塑性。我们前期研究发现P2受体亚型P2X3参与了应激诱导的IBS内脏痛，同时在结肠粘膜下神经丛表达明显增多，因此我们推测EGCs源性ATP可能激活突触间隙肠神经元P2X3受体，重塑其功能活性，促进IBS内脏痛。本课题拟采用内脏运动反射、腹壁撤退反射、Western blot、免疫荧光标记、荧光素酶法和全细胞膜片钳等技术，从整体-分子-细胞三个层次，观察IBS内脏痛情况下ATP/P2X3信号轴在EGCs激活肠神经元的作用及机制，揭示肠神经元功能可塑性和IBS内脏痛外周敏化的分子机制和潜在的治疗新靶标。

PTSD与内脏痛觉敏化的形成有密切的关系, 但相关机制不详。本项目成功建立了PTSD应激相关的内脏痛敏动物模型，较真实的模仿了患者经历的各种创伤应激事件；采用不同梯度结直肠扩张（CRD） 的方法检测内脏运动反射(VMR) 及腹壁撤退反射（AWR）的变化，结果显示在建模7、14天时，内脏运动反射(VMR) 及腹壁撤退反射（AWR），内脏敏感性增加，腹腔内注射EGCs阻断剂后，建模7、14天后内脏运动反射(VMR) 及腹壁撤退反射（AWR）恢复正常；通过免疫荧光染色技术及Western blot方法检测研究在应激状态下IBS内脏痛敏的动物模型中，在ENS上建模7天时P2X3受体与GFAP的共表达，NEUN受体与GFAP的共表达，表达水平高低与内脏敏感性评分呈正相关；通过免疫荧光染色技术及Western blot方法检测研究脊髓上在建模7天时P2X3受体与GFAP的共表达P2X3受体与GFAP的共表达均明显升高；通过免疫荧光染色技术及Western blot方法检测研究在应激状态下IBS内脏痛敏的动物模型中，DRG上GFAP表达明显升高，而DRG上NEUN表达变化不明显；用免疫荧光染色技术检测研究在应激状态下IBS内脏痛敏的动物模型中，观察大鼠结肠中在建模7天时核心生物钟基因per2的表达下调，运用其调节剂后per2的表达恢复，说明per2对应激所致的内脏痛敏具有保护性作用。 在该课题资助下，进行了较广泛的学术交流，发表以第一作者发表SCI会议论文1篇；以第一作者发表SCI个案报道1篇；以第一作者发表CSCD综述1篇；以第一作者发表会议论文1篇；以通讯作者发表CSCD个案1篇。