Mechanisms of Altered Gastrointestinal Dysfunction

描述（由申请人提供）：肠易激综合征（IBS）患者患有慢性腹痛和相关的胃肠道症状-腹泻，便秘，腹胀和尿急。那些有严重症状的人也可能对伤害性刺激有内脏超敏反应，提示异常的神经疼痛处理机制。事实上，我们发现有相当一部分（约35%）的IBS患者对有害的实验性刺激（如结肠膨胀）有内脏过敏；但其机制尚不清楚。我们在之前美国国立卫生研究院支持的研究中发现了这种机制的线索（R01NS053090）；我们在这个亚群的结肠组织中发现了独特的microRNAs （miRNAs）。在这里，我们将通过测试以下假设来跟进这一线索：关键miRNA的异常表达失调下游miRNA靶点（阿片受体；NMDA受体）的表达，这有助于肠易激综合征患者的内脏过敏。为了确定独特的mirna在下游受体靶基因的表观遗传调控中的细胞内和细胞间作用，我们的实验室开发了创新的方法。这些方法将锁定核酸（LNA）修饰的寡核苷酸探针独特的miRNA识别特性与使用酰胺信号扩增（TSA）技术的荧光原位杂交（FISH）相结合。我们将在体内鞘内注射miRNA抑制剂（LNAs）或miRNA模拟物（前体），在我们验证的大鼠内脏超敏反应模型中，通过实验调节靶向阿片（Aim 1）和谷氨酸（Aim 2）受体的特定miRNA的表达（Zhou等人，Pain, 2008）。因此，我们将使用多种策略的组合来开发阿片样物质和谷氨酰胺受体的miRNA靶向网络的综合高置信度模型。在Aim 1中，我们将确定miR-29a/b簇和miR-29b-1-5p表达的增加是否通过下调阿片信号通路来增强内脏超敏反应。在Aim 2中，我们将确定miR-23a/b簇和125b-1-3p表达的降低是否通过上调谷氨酸信号传导机制促进内脏超敏反应。我们提出的研究应该确定在肠易激综合征（IBS）患者肠道组织中针对下游阿片和谷氨酸受体的异常表达的mirna的关键作用，这将弥补我们对导致慢性疼痛和内脏伤害感受改变的伤害感受通路失调的潜在机制的认识上的重要空白。更重要的是，这一结果将导致创新治疗策略的发展，通过合成寡核苷酸来抑制或模拟特定的mirna，这些mirna调节下游靶基因（如阿片或谷氨酸受体）的表观遗传调控，驱动内脏伤害感受。这些转化研究的最终目标是开发治疗肠易激综合征和其他功能性GI疾病患者的新疗法。

DESCRIPTION (provided by applicant): Patients with irritable bowel syndrome (IBS) suffer from chronic abdominal pain and associated GI symptoms - diarrhea, constipation, bloating & urgency. Those with severe symptoms may also have visceral hypersensitivity to nociceptive stimuli, suggesting aberrant neural pain processing mechanisms. Indeed, we found a significant subset (~35%) of IBS patients with visceral hypersensitivity to noxious experimental stimuli (e.g., colonic distension); but the mechanism was unclear. We found a clue to that mechanism during our previous NIH-supported study (R01NS053090); we identified unique microRNAs (miRNAs) in colon tissues of this subset. Here we will follow up on that clue by testing the following hypothesis: Aberrant expression of key miRNAs dysregulates expression of downstream miRNA targets (opioid receptors; NMDA receptors) which contributes to visceral hypersensitivity in IBS patients. To determine the intra- and inter-cellular roles of unique miRNAs in the epigenetic regulation of down- stream receptor target genes, our lab has developed innovative methods. These methods combine unique miRNA recognition properties of locked nucleic acid (LNA) modified oligonucleotide probes with fluorescence in situ hybridization (FISH) using tyramide signal amplification (TSA) technology. We will use in vivo intrathecal injection of miRNA inhibitors (LNAs) or miRNA mimics (precursors) to experimentally regulate expression of specific miRNAs that target opioid (Aim 1) and glutamate (Aim 2) receptors in our validated rat model of visceral hypersensitivity (Zhou et al., Pain, 2008). Thus, we will use a combination of strategies to develop a comprehensive high-confidence model of miRNA targeting networks for opioid and glutamine receptors. In Aim 1 we will determine whether increased miR-29a/b cluster and miR-29b-1-5p expression enhances visceral hypersensitivity via downregulation of opioid signaling pathways. In Aim 2, we will determine whether decreased miR-23a/b cluster and 125b-1-3p expression facilitates visceral hypersensitivity via upregulation of glutamate signaling mechanisms. Our proposed studies should identify critical contributions of aberrantly expressed miRNAs that target downstream opioid and glutamate receptors in intestinal tissues of IBS patients with visceral hypersensitivity This will bridge an important gap in our knowledge of the underlying mechanisms of dysregulation of nociceptive pathways that lead to chronic pain and altered visceral nociception in patients who suffer from common functional GI disorders. More importantly, the results should lead to the development of innovative therapeutic strategies via development of synthesized oligonucleotides that inhibit or mimic specific miRNAs that modulate epigenetic regulation of down-stream target genes (e.g. opioid or glutamate receptors) that drive visceral nociception. The ultimate goal of the proposed translational studies is to develop new therapies for treating our patients with IBS and other functional GI disorders.