新生期再编程介导慢性内脏痛的表观调控机制研究

Irritable bowel syndrome (IBS) is a common gastrointestinal disorder characterized by chronic visceral pain in association with altered bowel habits. However, its precise mechanisms of visceral hypersensitivity remain elusive and treatment options are very limited and often disappointing in efficacy, with some being managed with opiates. Recently, we have developed and validated a rat model of visceral hyperalgesia that mimics some of the features of human IBS. This model is thus eminently suitable for a mechanistic approach to the pathogenesis of chronic visceral pain. Inspired by our previous data, we now propose to determine the potential role of neonatal reprogramming and ionic and molecular basis of peripheral sensitization in colonic visceral pain model. In this regard, we hypothesize that the pathogenesis of visceral pain is a "Developmental Origin of Adult Disease" and involves peripheral sensitization and plasticity of hypothalamic-pituitary-adrenal (HPA) axis function by neonatal reprogramming. The epigenetic regulations of key nociceptive genes by neonatal reprogramming contribute to visceral hypersensitivity induced by neonatal colonic inflammation (NCI). To test this hypothesis, we propose the following three specific aims: Specific Aim 1: To determine whether NCI induces neonatal reprogramming and alterations in HPA functions, thus contributing to visceral hypersensitivity in adult rats; Specific Aim 2: To identify the ionic and molecular basis for increased excitability of colon specific DRG neurons in adult rats after NCI. Specific Aim 3: To determine epigenetic mechanisms by which key nociceptive genes were upregulated by neonatal reprogramming in a rat model of IBS induced by NCI. Behavior experiments will be employed on normal and neonatal acetic acid-treated rats. Patch clamp recordings and calcium imaging will be performed in vitro on single colon-specific DRG neurons labeled with DiI acutely isolated from these rats. Expression of key nociceptive gene (i.e., CBS, voltage-gated sodium and potassium channels, DNMTs, HATs and HDACs) will be examined with immunocytochemistry and western blotting analysis. DNA methylation status will be determined by methylation specific PCR (MSP) and Bisulfite sequencing. Histone modification will be examined by chromatin immunoprecipitation (ChIP) assay and electrophoretic mobility shift assays EMSA. We believe that our studies may open new era towards "developmental origins of adult disease" and epigenetic mechanisms of chronic pain in functional gastrointestinal disorders. This added knowledge would provide new strategies for treatment of chronic visceral pain in patients with functional bowel diseases such as IBS.

慢性内脏痛是肠易激综合症(IBS) 的重要临床症状之一,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：新生期肠道炎症刺激导致血浆激素水平紊乱、硫化氢合酶表达上调、去甲基化功能增强以及肠特异性神经元兴奋性增加，由此推测：新生发育易感期肠道炎性刺激诱发"新生期再编程"，导致成年个体产生外周敏感化和内脏痛过敏；"新生期再编程"对下丘脑-垂体-肾上腺轴的调控和对关键性痛基因的表观调控介导了上述变化。我们的具体研究目标是:阐明新生发育易感期结肠炎性刺激诱发"新生期再编程"参与成年个体内脏痛过敏的形成和对HPA-轴功能的调控特征;研究"新生期再编程"诱导肠道特异性传入神经元兴奋性增加的离子通道及分子基础；探讨"新生期再编程"诱导的表观调控机制在成年个体内脏痛过敏的作用特征。本课题将在整体动物，离子通道和分子水平揭示慢性内脏痛的病理机制,也望在表观调控水平为临床治疗内脏痛提供新的更有效举措。

慢性内脏痛是肠易激综合症(IBS)的重要临床症状之一，因发病机制不清导致临床治疗非常困难。本课题的研究假设是：新生发育易感期肠道炎性刺激诱发"新生期再编程"，导致成年个体产生外周敏感化和内脏痛过敏；"新生期再编程"对下丘脑-垂体-肾上腺轴的调控和对关键性痛基因的表观调控介导了上述变化。具体研究目标包括：阐明新生发育易感期结肠炎性刺激诱发"新生期再编程"参与成年个体内脏痛过敏的形成和对HPA-轴功能的调控特征；研究"新生期再编程"诱导肠道特异性传入神经元兴奋性增加的离子通道及分子基础；探讨"新生期再编程"诱导的表观调控机制在成年个体内脏痛过敏的作用特征。本项目已按原计划执行，运用多种实验方法和技术在整体动物、离子通道和分子水平对慢性痛特别是功能性内脏痛的病理机制进行了全面细致地探讨。取得的主要研究成果包括：(1) 采用新生鼠易感期不良刺激诱导成年大鼠内脏痛模型，揭示了背根神经节中电压门控性钠通道的表达调控机制、神经元兴奋性的调节及其在慢性内脏痛中的作用；(2) 阐明了去甲基化和甲基化相关酶 (DNMT3b/TET3) 及下游靶基因参与慢性内脏痛和糖尿病性外周神经病理性疼痛的机制；(3) 明确了背根神经节中H2S合成酶CBS和ATP受体P2X3基因参与内脏痛的调节，并受到NFkB信号的调控；明确了Toll样受体4在内脏痛中的重要作用；(4) 初步阐明HPA轴中去甲肾上腺素信号系统参与新生发育期不良刺激诱发的慢性内脏痛的调控；(5) 筛选出参与内脏痛新生期再编程的microRNA-19，及其表达调控的上下游机制。另外，还初步研究了内脏牵涉痛的机制，为后续研究奠定了基础。以上研究结果已在JCR分区I区II区杂志发表论文19篇。在该项目资助下，培养博士研究生7名，硕士研究生21名，博士后2名，申报发明专利2项。项目负责人多次主办国内外高水平学术会议，并在多个学术团体担任主任委员或理事，多本杂志的副主编或编委等职。资助经费按规定使用。结题汇报考核为优。