Stress and Visceral Hyperalgesia: Epigenetic Mechanisms

压力和内脏痛觉过敏：表观遗传机制

基本信息

批准号：
8541353
负责人：
JOHN W WILEY
金额：
$ 33.43万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-09-20 至 2013-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8541353
关键词：
Abdominal Pain Acetylation Adrenal Cortex Hormones Affect Agonist Animals Area Behavior Binding Biological Assay Biomedical Research Blood CNR1 gene COS Cells Cells Chronic Chronic stress Clinical Colon Complex Corticosterone DNA Methylation DNA-Protein Interaction Data Endocannabinoids Epigenetic Process Esthesia Experimental Designs Feedback Fluorescence Resonance Energy Transfer Future Gastrointestinal tract structure Gene Expression Gene Silencing Genes Genetic Transcription Glucocorticoid Receptor Glucocorticoids Goals Harvest Histone Acetylation Hormones Human Hydrocortisone Hyperalgesia In Situ In Vitro Intervention Label Lasers Link Mediating Medicine Methods Methylation Microscopy Mineralocorticoids Monitor Motor Mutate Neurons Nociception Pain Pain Disorder Pathway interactions Pattern Perception Peripheral Pharmaceutical Preparations Play Promoter Regions Proteins Psychological Stress RNA Rattus Reagent Receptor Gene Regulation Regulatory Pathway Rodent Role Sensory Signal Transduction Pathway Site Spinal Ganglia Stress TRPV1 gene Testing Therapeutic Intervention Total Internal Reflection Fluorescent Visceral Visceral pain Water acute stress biological adaptation to stress colorectal distension experience histone acetyltransferase histone modification hypothalamic-pituitary-adrenal axis inhibitor/antagonist novel promoter receptor receptor expression response targeted delivery transmission process

项目摘要

DESCRIPTION (provided by applicant): Chronic stress is a well-known trigger for abdominal pain, a common yet poorly understood presentation in the clinical setting. We will examine the novel hypothesis that chronic stress activates epigenetic regulatory pathways resulting in enhanced abdominal pain (visceral hyperalgesia). Epigenetic pathways regulate gene expression by modifying the level of methylation of upstream promoter regions of genes (increased methylation decreases gene expression) and histone acetylation (increased histone acetylation enhances gene expression). Stress activates the hypothalamic- pituitary-adrenal axis resulting in increased blood levels of the hormone cortisol in humans and corticosterone (CORT) in rodents which regulate gene expression via the intracellular corticoid receptors GR and MR. Acute stress activates the anti-nociceptive endocannabinoid CB1 receptor gene expression which, in turn, inhibits the function of pro-nociceptive endovanilloid TRPV1 receptors. Chronic stress down-regulates CB1 expression and up-regulates TRPV1 expression. We propose that chronic stress will increase methylation of GR promoter sites resulting in decreased CB1 expression, and augment histone acetylation resulting in increased TRPV1 expression, culminating in visceral hyperalgesia. DNA methylation sites will be characterized using methylation-specific PCR analysis and pyrosequencing, and histone acetylation sites dissected using ChIP. We propose that chronic stress will modulate these pathways selectively in primary sensory dorsal root ganglion (DRG) neurons innervating the colon that transmit pain. Studies will be performed in DRGs obtained from chronic water-avoidance stressed rats, control rats treated with CORT in situ and control DRG explants treated with CORT in vitro. We will examine whether the formation of GR-MR and CB1-TRPV1 receptor complexes plays a role in stress-related visceral hyperalgesia in DRG neurons transfected with either dual labeled GR and MR, or CB1 and TRPV1 using FRET/TIRF microscopy. We hypothesize that visceral hyperalgesia will be linked to epigenetic mechanisms that selectively affect promoter sites for the GR, CB1 and TRPV1 receptors in nociceptive DRG neurons innervating the colon. This subpopulation will be identified using immunohistochemical markers and laser capture microscopy to harvest different subpopulations of DRG neurons in conjunction with quantitative PCR of relevant targets. Confirmation of the role of specific receptors (GR/MR) and signal transduction pathways (methylation/acetylation) to the changes observed in CB and TRP pathways will be confirmed using targeted delivery of specific agonist/antagonist drugs and gene silencing (si-RNA) reagents in situ, and correlation of these interventions with behavior, e.g. visceral motor response to colorectal distension. We believe that the proposed studies will have high impact regarding our understanding of how stress affects pain perception. Therapeutic interventions that target epigenetic mechanisms are touted as a pathway to personalized medicine in the future.

描述（由申请人提供）：众所周知，慢性压力是腹痛的诱因，腹痛是临床上常见但知之甚少的表现。我们将研究一个新的假设，即慢性压力激活表观遗传调控途径，导致腹痛加剧（内脏痛觉过敏）。表观遗传途径通过改变基因上游启动子区域的甲基化水平（甲基化增加降低基因表达）和组蛋白乙酰化水平（组蛋白乙酰化增加增强基因表达）来调节基因表达。压力激活下丘脑-垂体-肾上腺轴，导致人类皮质醇激素和啮齿类动物皮质酮 (CORT) 血液水平升高，通过细胞内皮质激素受体 GR 和 MR 调节基因表达。急性应激会激活抗伤害性内源性大麻素 CB1 受体基因表达，进而抑制促伤害性内源性大麻素 TRPV1 受体的功能。慢性应激会下调 CB1 表达并上调 TRPV1 表达。我们认为，慢性应激会增加 GR 启动子位点的甲基化，导致 CB1 表达减少，并增加组蛋白乙酰化，导致 TRPV1 表达增加，最终导致内脏痛觉过敏。 DNA 甲基化位点将使用甲基化特异性 PCR 分析和焦磷酸测序进行表征，并使用 ChIP 解剖组蛋白乙酰化位点。我们认为，慢性压力将选择性地调节支配结肠传递疼痛的初级感觉背根神经节（DRG）神经元中的这些通路。研究将在从慢性避水应激大鼠、用 CORT 原位处理的对照大鼠和用 CORT 体外处理的对照 DRG 外植体获得的 DRG 中进行。我们将使用 FRET/TIRF 显微镜检查 GR-MR 和 CB1-TRPV1 受体复合物的形成是否在用双标记 GR 和 MR 或 CB1 和 TRPV1 转染的 DRG 神经元中与应激相关的内脏痛觉过敏中发挥作用。我们假设内脏痛觉过敏与表观遗传机制有关，该机制选择性地影响支配结肠的伤害性 DRG 神经元中 GR、CB1 和 TRPV1 受体的启动子位点。将使用免疫组织化学标记物和激光捕获显微镜来识别该亚群，以收获 DRG 神经元的不同亚群，并结合相关靶标的定量 PCR。将使用特定激动剂/拮抗剂药物的靶向递送和基因沉默（si-RNA）来确认特定受体（GR/MR）和信号转导途径（甲基化/乙酰化）对CB和TRP途径中观察到的变化的作用原位试剂，以及这些干预措施与行为的相关性，例如内脏运动对结直肠扩张的反应。我们相信，拟议的研究将对我们理解压力如何影响疼痛感知产生重大影响。针对表观遗传机制的治疗干预措施被吹捧为未来个性化医疗的途径。