Modulation of Visceral Hypersensitivity in IBS by Lipid Mediators

内脏痛觉过敏(VH)常见于肠易激综合征(IBS)患者。负责任的人 机制尚不清楚。我们的初步数据显示，易腹泻的IBS患者的结肠活检组织中含有亲- 炎症分子(S)，可改变背根神经节神经元的电生理特性，并诱导 内脏高敏感性(VH)。体内和体外研究表明，促伤害性作用可以被抑制 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。我们还显示，一种新的内源性抗炎脂质介体--分解素，是 在结肠粘膜合成，可能在治疗上有助于逆转PGE2对DRG的作用 兴奋性。我们推测PGE2是IBS结肠粘膜MC释放的关键促炎分子 PTS，可以激活G蛋白偶联的EP2受体，刺激腺苷环化酶(AC)，导致 在cAMP中，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激活的溶血素减少cAMP的形成，降低感觉神经元的兴奋性。 从而降低VH。目的1.证明IBS结肠活检组织中产生的PGE2水平升高 通过提高IBS-D PTS大鼠结肠粘膜蛋白水解酶活性。蛋白酶、前列腺素E_2和其他多不饱和 IBS患者和健康对照的结肠活检和活检上清液中的脂肪酸将被量化。在……里面 活体疼痛行为研究将检验预先使用EP2拮抗剂或沉默EP2受体是否可以 预防大鼠结肠内注入IBS活检上清液引起的VH。全细胞膜片钳研究将 结肠投射背根神经节神经元基础膜特性和兴奋性变化的特征 暴露于IBS活检上清液，表明这些变化是由PGE2介导的。目的2.免疫印迹、RT- 聚合酶链式反应和全细胞膜片钳研究将检测细胞内信号通路和膜 IBS活检上清液中介导PGE2作用的通道。使用siRNA技术和 显示IBS结肠上清液中PGE2的药理抑制剂通过EP2-AC-cAMP-PKA途径发挥作用 调制IH、IA和IC电流。通过沉默表达来证明HCN2、Kv4.2和TRPV1的参与 使用荧光素标记的siRNA对这些分子进行检测。目的3.证明解毒剂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 溶血素对IBS-D上清液或前列腺素E_2处理的背根节神经元的影响信号转导途径 将通过沉默甲酰肽受体(FPR2)的表达和百日咳的治疗来研究 毒素可灭活胃肠道α。鞘内注射解毒剂或慢性给药后疼痛行为的体内研究 二十二碳六烯酸(DHA)(解析素的前体)喂养将展示这一治疗意义 一类新的化合物。我们的建议将确定对IBS PTS中观察到的VH负责的新机制 并探索将其作为一类新型治疗剂用于VH的治疗。

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.