Stress and Visceral Hyperalgesia: Epigenetic Mechanisms

压力和内脏痛觉过敏：表观遗传机制

• 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
• 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

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，慢性应激是引起腹痛的诱因，这是临床中常见但知之甚少的表现。我们将研究慢性应激激活表观遗传调控途径导致腹痛（内脏痛觉过敏）的新假设。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（甲基化增加会降低基因表达）和组蛋白乙酰化水平（组蛋白乙酰化增加会增强基因表达）来调节基因表达。应激激活下丘脑-垂体-肾上腺轴，导致人类和啮齿动物血液中皮质醇和皮质酮（CORT）水平升高，皮质醇和CORT通过细胞内皮质激素受体GR和mr调节基因表达。急性应激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。慢性应激可下调CB1表达，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，组蛋白乙酰化位点将使用ChIP进行解剖。我们提出慢性应激会选择性地调节这些通路，这些通路是在支配结肠的感觉背根神经节（DRG）神经元中传递疼痛的。研究将在慢性避水应激大鼠的DRG中进行，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。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双重标记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起作用。我们假设内脏痛觉过敏可能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。该亚群将使用免疫组织化学标记和激光捕获显微镜来鉴定不同的DRG神经元亚群，并结合相关目标的定量PCR。特异性受体（GR/MR）和信号转导通路（甲基化/乙酰化）对观察到的CB和TRP通路变化的作用将通过靶向递送特异性激动剂/拮抗剂药物和基因沉默（si-RNA）试剂原位证实，以及这些干预措施与行为的相关性，例如对结肠直肠膨胀的内脏运动反应。我们相信，提出的研究将对我们对压力如何影响疼痛感知的理解产生重大影响。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。