Modulation of Visceral Hypersensitivity in IBS by Lipid Mediators

脂质介质对 IBS 内脏过敏的调节

• 批准号: 9767798
• 负责人: Gintautas Grabauskas
• 金额: $ 42.99万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-01 至 2020-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9767798
• 关键词: Abdominal Pain; Action Potentials; Adenylate Cyclase; Afferent Neurons; Analgesics; Anti-inflammatory; Antidotes; Ascending colon; Biopsy; Biopsy Specimen; Capsaicin; Cells; Chemicals; Chronic; Colon; Complex; Coupled; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Data; Descending colon; Diarrhea; Dinoprostone; Disease; Docosahexaenoic Acids; Electrophysiology (science); Enzyme-Linked Immunosorbent Assay; Exposure to; FPR2 gene; Fluorescein; Frequencies; GTP-Binding Proteins; Gut Mucosa; Hyperalgesia; Hypersensitivity; Immune system; In Vitro; Inflammation; Inflammation Mediators; Inflammatory; Intrathecal Injections; Ion Channel; Irritable Bowel Syndrome; Label; Measurement; Measures; Mechanical Stimulation; Mediating; Membrane; Membrane Potentials; Mucous Membrane; Nervous system structure; Neurons; Nociception; Pain; Pain intensity; Pathway interactions; Patients; Peptide Hydrolases; Pertussis Toxin; Pharmacology; Play; Polyunsaturated Fatty Acids; Potassium; Property; Rattus; Resistance; Resolution; Rest; Reverse Transcriptase Polymerase Chain Reaction; Role; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Small Interfering RNA; TRPV1 gene; Technology; Testing; Therapeutic; Therapeutic Agents; Visceral; Western Blotting; behavioral study; experience; experimental study; fMet-Leu-Phe receptor; feeding; gastrointestinal system; in vivo; indium arsenide; inhibitor/antagonist; lipid mediator; mast cell; neuronal excitability; novel; novel therapeutics; pain behavior; pain reduction; patch clamp; prevent; receptor; therapeutic target

Visceral hyperalgesia (VH) is frequently observed in irritable bowel syndrome (IBS) patients. The responsible mechanism is unclear. Our preliminary data show colonic biopsies from diarrhea-prone IBS pts contain pro- inflammatory molecule(s) which can modify the electrophysiological properties of the DRG neurons and induce visceral hypersensitivity (VH). In vivo and in vitro studies show the pro-nociceptive effects can be inhibited by either blocking mast cell (MC) dependent PGE2 synthesis pathway or by injecting EP2 receptor antagonists intrathecally. We also show resolvins, a novel class of endogenous anti-inflammatory lipid mediators, are synthesized in the colonic mucosa and may be therapeutically useful to reverse the actions of PGE2 on DRG excitability. We hypothesize PGE2 is a key pro-inflammatory molecule released by colonic mucosa MC of IBS pts, can activate G protein coupled EP2 receptors and stimulates adenylyl cyclase (AC) resulting in an increase in cAMP, which modulates IH, IA and IC currents via different signal transduction pathways. On the other hand, resolvins activating via FPR2/Giα reduce cAMP formation and decrease excitability of sensory neurons and consequently reduce VH. Aim 1. Demonstrate IBS colonic biopsies contain elevated levels of PGE2 generated by increased proteolytic activities in colonic mucosa of IBS-D pts. Proteases, PGE2 and other polyunsaturated fatty acids will be quantified in colonic biopsies and biopsy supernatants from IBS pts and healthy controls. In vivo pain behavior studies will examine if pretreatment with an EP2 antagonist or silencing EP2 receptor can prevent VH in rats exposed to colonic instillation of IBS biopsy supernatant. Whole-cell patch-clamp studies will characterize the changes in basic membrane properties and excitability of colon projecting DRG neurons exposed to IBS biopsy supernatant and show these changes are mediated by PGE2. Aim 2. Western blot, RT- PCR and whole-cell patch-clamp studies will examine the intracellular signaling pathways and membrane channels that mediate the actions of PGE2 in IBS biopsy supernatant. Use siRNA technologies and pharmacological inhibitors to show PGE2 in IBS colonic supernatants acts via the EP2-AC-cAMP-PKA pathway to modulate IH, IA and IC currents. Demonstrate HCN2, Kv4.2 and TRPV1 participation by silencing expression of these molecules using fluorescein labeled siRNAs. Aim 3. To demonstrate resolvins is an effective antidote for the excitatory actions of PGE2 in the IBS-D supernatant, whole-cell patch-clamp recordings will examine the effects of resolvins on DRG neurons treated with IBS-D supernatant or PGE2. Signal transduction pathways will be studied by silencing the expressions of formyl peptide receptor (FPR2) and treatment with pertussis toxin to inactivate Giα. In vivo pain behavior studies following intrathecal injections of resolvins or chronic docosahexaenoic acid (DHA) (precursor of resolvin) feeding will demonstrate therapeutic significance of this novel class of compounds. Our proposal will identify a new mechanism responsible for VH observed in IBS pts and explore the use of resolvins as a class of novel therapeutic agent to treat VH.

内脏痛觉过敏（VH）常见于肠易激综合征（IBS）患者。负责人 机制尚不清楚。我们的初步数据显示，易腹泻 IBS 患者的结肠活检含有促 炎症分子可以改变 DRG 神经元的电生理特性并诱导 内脏超敏反应（VH）。体内和体外研究表明，促伤害感受作用可以被抑制 阻断肥大细胞 (MC) 依赖的 PGE2 合成途径或注射 EP2 受体拮抗剂 鞘内注射。我们还表明，消解素是一类新型的内源性抗炎脂质介质， 在结肠粘膜中合成，可能在治疗上有助于逆转 PGE2 对 DRG 的作用 兴奋性。我们假设 PGE2 是 IBS 结肠粘膜 MC 释放的关键促炎分子 pts，可以激活 G 蛋白偶联 EP2 受体并刺激腺苷酸环化酶 (AC)，导致腺苷酸环化酶 (AC) 增加 cAMP 中的 cAMP，通过不同的信号转导途径调节 IH、IA 和 IC 电流。另一方面， 通过 FPR2/Giα 激活的分解素可减少 cAMP 形成并降低感觉神经元的兴奋性 从而降低VH。目标 1. 证明 IBS 结肠活检中产生的 PGE2 水平升高 IBS-D 患者结肠粘膜蛋白水解活性增加。蛋白酶、PGE2 和其他多不饱和酶 对 IBS 患者和健康对照的结肠活检和活检上清液中的脂肪酸进行定量。在 体内疼痛行为研究将检查用 EP2 拮抗剂或沉默 EP2 受体进行预处理是否可以 预防暴露于结肠灌注 IBS 活检上清液的大鼠的 VH。全细胞膜片钳研究将 表征结肠投射 DRG 神经元的基本膜特性和兴奋性的变化 暴露于 IBS 活检上清液并显示这些变化是由 PGE2 介导的。目标 2. 蛋白质印迹，RT- PCR 和全细胞膜片钳研究将检查细胞内信号通路和膜 介导 IBS 活检上清液中 PGE2 作用的通道。使用 siRNA 技术和 药物抑制剂显示 IBS 结肠上清液中的 PGE2 通过 EP2-AC-cAMP-PKA 途径发挥作用 调制 IH、IA 和 IC 电流。通过沉默表达证明 HCN2、Kv4.2 和 TRPV1 的参与 使用荧光素标记的 siRNA 对这些分子进行分析。目标 3. 证明消解素是一种有效的解毒剂 对于 IBS-D 上清液中 PGE2 的兴奋作用，全细胞膜片钳记录将检查 resolvins 对 IBS-D 上清液或 PGE2 处理的 DRG 神经元的影响。信号转导途径 将通过沉默甲酰肽受体（FPR2）的表达和百日咳治疗来研究 毒素使 Giα 失活。鞘内注射消退素或慢性疼痛后的体内疼痛行为研究 二十二碳六烯酸 (DHA)（分解素前体）喂养将证明其治疗意义 新型化合物。我们的提案将确定一种新机制，负责在 IBS 患者中观察到的 VH 并探索使用消解素作为一类新型治疗剂来治疗 VH。