MECHANISMS OF VISCERAL PAIN DRIVEN BY SMALL INTESTINAL MICROBIOTA

项目总结/摘要 肠易激综合征是一种全球流行的疾病（约11%），其特征是粪便改变 形式/频率和腹痛每周一天或多天。IBS中的腹痛，像其他形式的 内脏痛通常是弥漫性的，并且局部化不好，使得难以描绘病理部位，并且作为一种 结果，治疗选择很少。肠道微生物产物已被证明是重要的管腔信号 但这些研究主要集中在结肠上。我们最近发现小肠 微生物组成与胃肠道（GI）症状如腹痛有关，但其机制 小肠微生物群/微生物产物在腹痛病理生理学中的潜在作用 仍然是一个关键的知识差距。为了解决这一差距，我们将阐明近端的感觉神经支配， 小肠，并确定细胞和分子途径，小肠检测和 传递导致内脏高敏感性的管腔微生物信号。在我们广泛的初步 研究中，我们建立了一个专门的模型来研究人类小肠微生物组的生理作用， 在无菌（GF）小鼠中，代谢物对分离的DRG和上皮细胞的影响，以及一种新的离体脊髓- 小肠制备，用于研究管腔信号通过管腔代谢物向脊髓的传递 通过（1）EC细胞进行信号传导，EC细胞是通过神经递质血清素向神经元发出信号的上皮细胞 （2）背根神经节（DRG）中的感觉神经元，一级神经元， 疼痛通路的传入神经元。使用这些模型，我们确定了不同的细菌和细菌代谢物 激活EC细胞和胸背根神经节神经元。根据我们的初步发现，我们假设， 肠道细菌产物通过激活小肠感觉传入而导致内脏高敏感性 直接和通过激活EC细胞的神经上皮连接。我们将分两部分来讨论这个假设 使用尖端刺激/采集方法的特定目的，包括在 类器官，EC细胞和DRG神经元的电生理学，来自新型转基因小鼠的离体制剂， 和腺相关病毒（AAV）表征来自小肠的感觉输入，以及光遗传学 来研究神经上皮信号在具体目标1中，我们将确定EC激活的潜在机制。 细胞和DRG神经元，并在具体目标2，我们将确定涉及的感觉转导通路 对不同微生物产物的反应。这些研究将是第一个提供功能和分子 表征DRG中支配小肠的感觉神经元，确定 亚群被管腔中的微生物产物激活和/或致敏，并测试EC细胞是否 参与感觉传导通路。我们的发现将使新的微生物的发展 针对小肠中不同微生物途径的腹痛疗法。

PROJECT SUMMARY/ABSTRACT Irritable bowel syndrome is a globally prevalent disorder (~11%) characterized by an alteration in stool form/frequency and abdominal pain one or more days per week. Abdominal pain in IBS, like other forms of visceral pain, is often diffuse and poorly localized, making it difficult to delineate the site of pathology, and as a result there are few therapeutic options. Gut microbial products have been shown to be important luminal signals for abdominal pain, but these studies have largely focused on the colon. We recently found that small intestinal microbial composition is associated with gastrointestinal (GI) symptoms like abdominal pain, but the mechanisms underlying the role of small intestinal microbiota/microbial products in the pathophysiology of abdominal pain remains a critical knowledge gap. To address this gap, we will elucidate the sensory innervation of the proximal small intestine and identify the cellular and molecular pathways by which the small intestine detects and transduces luminal microbial signals that contribute to visceral hypersensitivity. In our extensive preliminary studies, we established a dedicated model to study the physiologic effects of human small intestinal microbiome in germ free (GF) mice, metabolite effects on isolated DRGs and epithelial cells, and a novel ex vivo spinal cord- small intestine preparation to study the transmission of luminal signals to the spinal cord via luminal metabolite signaling through (1) EC cells, that are epithelial cells which signal to neurons via serotonin, a neurotransmitter in the gut that modulates visceral pain, and (2) sensory neurons in dorsal root ganglia (DRG), the first-order afferent neurons of pain pathways. Using these models, we identified distinct bacteria and bacterial metabolites that activate EC cells and thoracic DRG neurons. Based on our preliminary findings, we hypothesize that small intestinal bacterial products contribute to visceral hypersensitivity by activating small intestine sensory afferents directly and through neuro-epithelial connections by activating EC cells. We will address the hypothesis in two Specific Aims using cutting-edge stimulation/acquisition approaches, including combination of Ca2+ imaging in organoids, electrophysiology of EC cells and DRG neurons, ex vivo preparations from novel transgenic mice, and adeno-associated viruses (AAVs) characterizing the sensory input from the small intestine, and optogenetics to study neuro-epithelial signaling. In Specific Aim 1, we will determine mechanisms underlying activation of EC cells and DRG neurons, and in Specific Aim 2, we will determine the sensory transduction pathways involved in responding to distinct microbial products. These studies will be the first to provide a functional and molecular characterization of sensory neurons in the DRG that innervate the small intestine, determine which subpopulations are activated and/or sensitized by microbial products in the lumen, and test whether EC cells are involved in the sensory transduction pathway. Our findings will allow the development of novel microbial therapies for abdominal pain that target distinct microbial pathways in the small intestine.