Chronic Stress and Abdominal Pain: Novel Mechanisms

慢性压力和腹痛：新机制

基本信息

批准号：
8815954
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 2016-02-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8815954
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 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 Play Polymerase Chain Reaction Population Process Promoter Regions Proteins Psychological Stress RNA Rattus Reagent Receptor Down-Regulation Receptor Signaling 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 epigenetic regulation gene function genome-wide histone acetyltransferase histone modification hypothalamic-pituitary-adrenal axis inhibitor/antagonist laser capture microdissection novel peripheral pain 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）：慢性应激和腹痛：新型机制慢性应激激活下丘脑 - 垂体 - 肾上腺（HPA）轴，并且是腹痛的已知诱导剂。最近的报道表明，慢性心理压力会导致与记忆和情绪相关的中枢神经系统区域中基因功能的表观遗传调节的变化。表观遗传学是指基因功能的稳定和/或可遗传的变化，而无需通过DNA甲基化，组蛋白修饰和染色质重塑，而无需改变DNA序列。在过去的十年中，基于开创性研究，表观遗传学领域迅速出现，该研究表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。尚不清楚慢性应激是否通过表观遗传机制调节外周痛途径。内托氨基类瞬态受体电位（TRP）途径在疼痛传播中起关键作用，并且已知TRPV1受体受到作用于抑制TRPV1功能的CB1受体的内源性大麻素（CB）调节。慢性应力下调CB途径的功能，导致TRPV1受体功能和腹痛的上调。我们将研究以下假设：慢性应激通过在背根神经节（DRG）神经元中以区域和cel的表观遗传调节来诱导内脏疼痛，从而导致骨盆神经元（DRG）神经元，而不是骨盆神经元，而不是躯体感觉分布到下极差。具体而言，我们的初步数据支持两个高度新颖的假设：1。慢性间歇性应力促进DNMT1介导的糖皮质激素受体（GR）启动子位点的甲基化甲基化，导致GR表达降低，从而与降低的水平和功能相关，抗性摄影性内核酸内细胞运动员和增强的抗性CB1型氧化氢和功能的功能，并将其链接起来。背根神经节（DRG）神经元中的内托氏菌TRPV1受体；和2。慢性应激诱导的皮质酮（CORT）介导的表观遗传变化是区域和细胞特异性的，并且“硬有线”以使GI道（结肠）与体感（Sciatic Never）分布的DRG减小。在慢性间歇性压力的情况下，硬有线表达模式使胃肠道的伤害性神经元因结直肠延伸而响应于痛觉过敏。这些研究将包括使用cuttin-边缘方法在应力反应基因的启动子和染色质免疫沉淀（CHIP）分析相关组蛋白修饰靶标的启动子上鉴定推定的调节性CPG甲基化位点。使用逆行标记和激光捕获显微镜的免疫组织化学标记将鉴定出伤害性神经元的亚群，以收集与相关靶标的定量单细胞PCR一起收集不同DRG神经元的种群。确认特定受体的作用和信号转导途径对CB和TRP途径中观察到的变化的作用将通过靶向递送基因沉默（SI-RNA）试剂原位确认，并将这些干预与行为的干预相关，例如。对结直肠延伸的内脏运动响应。我们还将检查使用FRET/TIRF显微镜和电生理记录中，CB1-TRPV1受体复合物的形成是否在DRG神经元和转染的细胞中与应激相关的内脏性痛觉过敏起着作用。阐明慢性应激引起的内脏疼痛的表观遗传调节的机制将对我们对疼痛途径的调节方式的理解产生重大影响，并且很可能管理影响GI道的功能性疼痛障碍。