孕期应激诱发成年子代功能性消化不良胃部痛觉过敏的分子神经机制研究

Functional dyspepsia (FD) is one of major functional gastrointestinal disorders that affects about 20~30% of the population in the world. The primary symptoms of FD include delayed gastric emptying and chronic epigastric visceral pain/discomfort that usually worsened by ingestion of a meal. This disorder has baffled clinical gastroenterologist and basic scientists alike because its symptoms occur in the absence of overall inflammation and structural or biochemical changes. However, its precise mechanisms of gastric hypersensitivity remain elusive and treatment options are very limited and often disappointing in efficacy. Recently, we have developed and validated a rat model of gastric hyperalgesia that mimics some of the features of human FD. This model is thus eminently suitable for a mechanistic approach to the pathogenesis of chronic visceral pain. Inspired by our preliminary data, we now propose to determine the potential role of prenatal maternal stress (PMS) and ionic and molecular basis of peripheral sensitization in epigastric visceral pain model. In this regard, we hypothesize that the pathogenesis of gastric hypersensitivity is a "Fetal Origin of Adult Disease" and involves peripheral sensitization and plasticity of hypothalamic-pituitary-adrenal (HPA) axis function in adult offspring. The epigenetic regulations of key nociceptive genes such as acid-sensing ion channels by stress hormones contribute to gastric hypersensitivity in adult offspring induced by PMS. To test this hypothesis, we propose the following three specific aims: Specific Aim 1: To determine whether PMS induces gastric hypersensitivity and alterations in HPA functions in adult offspring; Specific Aim 2: To identify the ionic and molecular basis for increased excitability of gastric specific DRG/NG neurons in adult offspring. Specific Aim 3: To determine mechanisms by which key nociceptive genes were upregulated by neonatal reprogramming in a rat model of FD induced by PMS. Behavior experiments will be employed on adult offspring from normal and PMS rats. Patch clamp recordings and calcium imaging will be performed in vitro on single gastric innervating DRG/NG neurons labeled with DiI acutely isolated from these rats. Expression of key nociceptive gene (i.e., ASICs, voltage-gated sodium and potassium channels, glucocorcoid receptors) will be examined with immunocytochemistry and western blotting analysis. We believe that our studies may would provide gastrointestinal new strategies for treatment of chronic visceral pain in patients with functional bowel diseases such as FD.

功能性消化不良是消化科最常见的疾病之一，临床主要表现进食后的上腹疼痛和胃排空延缓，因发病机制不清导致临床治疗非常困难。我们前期的研究发现：孕期应激(Prenatal Maternal Stress, PMS)可导致成年子代产生胃部痛觉过敏，并伴有血浆皮质酮水平升高和支配胃的初级感觉神经元兴奋性增加，由此推测：孕期应激激活了下丘脑－垂体－肾上腺轴导致终端应激激素水平升高，敏化了酸敏感离子通道，最终导致成年个体产生胃部痛觉过敏。为了验证以上假设，我们提出了以下研究目标：重点研究酸敏感离子通道(Acid-sensing ion channels, ASICs)在"胎源性"应激诱发成年子代胃部痛觉过敏中的作用特征；初步阐明皮质酮活化ASICs的调控机制。本课题将在整体动物，离子通道和分子水平揭示FD胃部痛觉过敏的病理机制，也望在基因水平为临床治疗慢性内脏痛提供新的更有效举措。

功能性消化不良(FD)是消化科最常见的疾病之一，因发病机制不清导致临床治疗非常困难。生命早期的不良刺激，如心理应激和炎症等，可能导致成年出现FD。孕期应激（PMS）可以影响子代的组织结构和功能，导致胃肠道疾病的发生，而在胚胎发育过程中表观遗传学修饰对细胞内的微环境变化非常敏感。本项目的研究目的是探讨酸敏感性离子通道（ASICs）在PMS诱导的胃部痛觉高敏模型中的作用及转录因子核因子κB（NFκB）通过与asic1基因启动子区的结合上调ASIC1的表达参与PMS成年子代大鼠胃部痛觉高敏的表观调控机制。研究运用胃部球囊扩张（GD）及肌电图（EMG）分析技术评测PMS大鼠的胃部痛觉高敏行为反应；运用全细胞膜片钳技术检测神经元的兴奋性和ASICs电流的变化；运用甲基化特异性PCR（MSP）方法检测大鼠胃相关DRG内asic1基因启动子区的甲基化状态；运用染色质免疫沉淀（ChIP）技术检测胃相关DRG内NFκB与asic1基因启动子区的结合情况。我们研究发现PMS成年子代大鼠不但出现明显的胃部痛觉高敏，还出现肠道痛觉过敏。在研究胃部痛觉过敏的机制时，我们发现胃特异性DRG神经元的兴奋性增加，ASICs的表达和电流密度增大；鞘内注射ASICs抑制剂Amiloride能够减轻PMS成年子代大鼠的胃部痛觉过敏，减少ASICs的电流密度；进一步研究发现PMS成年子代大鼠胃相关DRG内NFκB的表达上调，且asic1基因启动子区CpG岛去甲基化明显增加；NF-κB与asic1基因CpG岛内两个位点的结合均显著增强。鞘内注射NF-κBp65 shRNA 慢病毒能显著抑制PMS成年子代大鼠胃相关DRG内NFκB和ASIC1的表达，并降低胃特异性神经元的兴奋性；NF-κBp65 shRNA慢病毒还能缓解PMS诱导的成年子代大鼠胃部痛觉高敏。实验结果提示PMS能促进转录因子NFκB通过与启动子区DNA去甲基化的asic1基因结合，ASIC1表达上调，参与了PMS成年子代大鼠胃部痛觉高敏，同时发现应激激素很可能是内脏痛的重要的诱导因素。本课题将在整体动物，离子通道和分子水平揭示FD胃部痛觉过敏的病理机制，也望在基因水平为临床治疗功能性胃肠疾病的慢性内脏痛提供新的更有效举措。