Chronic Stress and Abdominal Pain: Novel Mechanisms

慢性压力和腹痛：新机制

基本信息

批准号：
8478935
负责人：
JOHN W WILEY
金额：
$ 33.82万
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：
2013
资助国家：
美国
起止时间：
2013-04-01 至 2018-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8478935
关键词：
Abdominal Pain Acetylation Adrenal Cortex Hormones Adult Affect Animal Model Animals Area Behavior Binding CNR1 gene Cell Line Cells Chronic Chronic stress Clinical Clinical Medicine Co-Immunoprecipitations Colon Complex Control Animal Corticosterone CpG Islands DNA Methylation DNA Methyltransferase DNA Modification Methylases DNA Sequence DNA-Protein Interaction Data Development Endocannabinoids Epigenetic Process Esthesia Exposure to Feedback Fluorescence Resonance Energy Transfer Gastrointestinal tract structure Gene Expression Gene Silencing Genes Genetic Transcription Glucocorticoid Receptor Harvest Health Histone Acetylation Human Cell Line Hyperalgesia In Situ In Vitro Intervention Label Lasers Lead Link Lower Extremity Measures Mediating Memory Methods Methylation Microscopy Moods Motor Mus Neuraxis Neurons Nociception Organ Pain Pain Disorder Pathway interactions Pattern Pelvis Perception Peripheral Play Polymerase Chain Reaction Population Process Promoter Regions Proteins Psychological Stress RNA Rattus Reagent Receptor Down-Regulation Receptor Signaling Regulation Regulatory Pathway Reporting Rodent Role Seminal Signal Transduction Pathway Site Small Interfering RNA Spinal Ganglia Stress TRPV1 gene Total Internal Reflection Fluorescent Up-Regulation Visceral Visceral pain Water base biological adaptation to stress chromatin immunoprecipitation chromatin remodeling colorectal distension gene function genome-wide histone acetyltransferase histone modification hypothalamic-pituitary-adrenal axis inhibitor/antagonist laser capture microdissection novel promoter receptor receptor expression receptor function response sciatic nerve somatosensory targeted delivery transmission process

项目摘要

DESCRIPTION (provided by applicant): Chronic Stress and Abdominal Pain: Novel Mechanisms Chronic stress activates the Hypothalamic-Pituitary-Adrenal (HPA) axis and is a known inducer of abdominal pain. Recent reports indicate that chronic psychological stress causes changes in epigenetic regulation of gene function in CNS regions associated with memory and mood. Epigenetics refers to stable and/or heritable changes in gene function without changes in the DNA sequence via DNA methylation, histone modification and chromatin remodeling. The field of epigenetics has emerged rapidly in the past decade based on seminal studies demonstrating genome-wide distribution of methylation and acetylation sites in primary cells and human cell lines. It is unknown whether chronic stress regulates peripheral pain pathways via epigenetic mechanisms. The endovanilloid transient receptor potential (TRP) pathway plays a pivotal role in pain transmission and the TRPV1 receptor is known to be regulated by endocannabinoids (CB) acting on CB1 receptors which inhibit TRPV1 function. Chronic stress down-regulates the function of the CB pathway resulting in up-regulation of TRPV1 receptor function and abdominal pain. We will examine the hypothesis that chronic stress induces visceral pain via epigenetic regulation of CB1 and TRPV1 receptors in a region- and cel- specific manner in dorsal root ganglion (DRG) neurons innervating pelvic organs but not the somatosensory distribution to the lower extremities. Specifically, our preliminary data support two highly novel hypotheses: 1. Chronic intermittent stress promotes DNMT1-mediated methylation of glucocorticoid receptor (GR) promoter sites resulting in decreased GR expression that is linked to reduced levels and function of the anti-nociceptive endocannabinoid CB1 receptor, and enhances histone acetylation linked to increased expression and function of the pro-nociceptive endovanilloid TRPV1 receptor in dorsal root ganglion (DRG) neurons; and 2. Chronic stress-induced, corticosterone (CORT)-mediated epigenetic changes are region- and cell-specific, and "hardwired" to nociceptive DRGs innervating the GI tract (colon) vs. somatosensory (sciatic nerve) distribution. The hardwired expression pattern predisposes nociceptive neurons innervating the GI tract to hyperalgesia in response to colorectal distension in the setting of chronic intermittent stress. These studies will include the application of cuttin-edge methods to identify putative regulatory CpG methylation sites at the promoters of stress response genes and chromatin immunoprecipitation (ChIP) analysis of relevant histone modification targets. The subpopulation of nociceptive neurons will be identified using both immunohistochemical markers with retrograde labeling and laser capture microscopy to harvest distinct populations of DRG neurons in conjunction with quantitative single-cell PCR of relevant targets. Confirmation of the role of specific receptors and signal transduction pathways to the changes observed in CB and TRP pathways will be confirmed using targeted delivery of gene silencing (si-RNA) reagents in situ and correlation of these interventions with behavior, e.g. visceral motor response to colorectal distension. We will also examine whether the formation of CB1-TRPV1 receptor complexes plays a role in stress-associated visceral hyperalgesia in DRG neurons and transfected cells using FRET/TIRF microscopy and electrophysiological recordings. Clarifying the mechanisms underlying epigenetic regulation of chronic stress-induced visceral pain will have a significant impact on our understanding of how pain pathways are regulated and, likely, the management of functional pain disorders affecting the GI tract.

描述（由申请人提供）：慢性压力和腹痛：新机制慢性压力会激活下丘脑-垂体-肾上腺（HPA）轴，是已知的腹痛诱发因素。最近的报告表明，慢性心理压力会导致中枢神经系统区域与记忆和情绪相关的基因功能的表观遗传调节发生变化。表观遗传学是指基因功能的稳定和/或可遗传的变化，而不通过DNA甲基化、组蛋白修饰和染色质重塑而改变DNA序列。表观遗传学领域在过去十年中迅速兴起，其基础是开创性的研究，证明了原代细胞和人类细胞系中甲基化和乙酰化位点的全基因组分布。目前尚不清楚慢性压力是否通过表观遗传机制调节外周疼痛通路。内香草素瞬时受体电位 (TRP) 通路在疼痛传递中发挥着关键作用，已知 TRPV1 受体受内源性大麻素 (CB) 的调节，内源性大麻素 (CB) 作用于 CB1 受体，从而抑制 TRPV1 功能。慢性压力会下调 CB 通路的功能，导致 TRPV1 受体功能上调和腹痛。我们将检验以下假设：慢性应激通过对支配盆腔器官的背根神经节（DRG）神经元中的 CB1 和 TRPV1 受体以区域和细胞特异性方式进行表观遗传调控，从而诱发内脏疼痛，但不会影响下肢的体感分布。具体来说，我们的初步数据支持两个非常新颖的假设：1.慢性间歇性应激促进 DNMT1 介导的糖皮质激素受体 (GR) 启动子位点甲基化，导致 GR 表达减少，这与抗伤害性内源性大麻素 CB1 受体的水平和功能降低有关，并增强与背根神经节 (DRG) 中促伤害性内香草素 TRPV1 受体表达和功能增加相关的组蛋白乙酰化神经元； 2. 慢性应激诱导的皮质酮 (CORT) 介导的表观遗传变化具有区域和细胞特异性，并且与支配胃肠道（结肠）与躯体感觉（坐骨神经）分布的伤害性 DRG 具有“硬连线”。在慢性间歇性应激的情况下，这种固有的表达模式使支配胃肠道的伤害性神经元容易出现痛觉过敏，以响应结直肠扩张。这些研究将包括应用尖端方法来识别应激反应基因启动子处的假定调节性 CpG 甲基化位点，以及相关组蛋白修饰靶点的染色质免疫沉淀 (ChIP) 分析。将使用逆行标记的免疫组织化学标记物和激光捕获显微镜来识别伤害性神经元的亚群，以收获不同的 DRG 神经元群，并结合相关靶标的定量单细胞 PCR。特定受体和信号转导途径对 CB 和 TRP 途径中观察到的变化的作用的确认将通过原位基因沉默 (si-RNA) 试剂的靶向递送以及这些干预措施与行为的相关性来确认，例如行为。内脏运动对结直肠扩张的反应。我们还将使用 FRET/TIRF 显微镜和电生理记录检查 CB1-TRPV1 受体复合物的形成是否在 DRG 神经元和转染细胞中与应激相关的内脏痛觉过敏中发挥作用。阐明慢性应激引起的内脏疼痛的表观遗传调控机制将对我们理解疼痛通路的调节方式以及影响胃肠道的功能性疼痛疾病的治疗产生重大影响。