Chronic Stress and Abdominal Pain: Novel Mechanisms

慢性压力和腹痛：新机制

• 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
• 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

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：新机制慢性应激激活下丘脑-肾上腺-肾上腺（HPA）轴，是已知的腹痛诱导剂。最近的报告表明，慢性心理应激导致与记忆和情绪相关的CNS区域的基因功能的表观遗传调节的变化。表观遗传学是指通过DNA甲基化、组蛋白修饰和染色质重塑而不改变DNA序列的基因功能的稳定和/或可遗传的变化。表观遗传学领域在过去十年中迅速兴起，其基础是精液研究，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。慢性应激是否通过表观遗传机制调节外周疼痛通路尚不清楚。内源性香草素瞬时受体电位（endovaniloid transient receptor potential，TRP）通路在疼痛传递中起着关键作用，并且已知TRPV 1受体受到作用于CB 1受体的内源性大麻素（endocannabinoids，CB）的调节，CB 1受体抑制TRPV 1功能。慢性应激下调CB通路的功能，导致TRPV 1受体功能上调和腹痛。我们将研究慢性应激通过CB 1和TRPV 1受体以区域和细胞特异性方式在支配盆腔器官的背根神经节（DRG）神经元中的表观遗传调节而诱导内脏痛的假设，但不是下肢的体感分布。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DNMT 1介导的糖皮质激素受体（GR）启动子位点的甲基化，导致GR表达降低，这与抗伤害性内源性大麻素CB 1受体的水平和功能降低有关，并增强组蛋白乙酰化，这与背根神经节（DRG）神经元中促伤害性内源性香草素TRPV 1受体的表达和功能增加有关;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，并且与支配胃肠道（结肠）与躯体感觉（坐骨神经）分布的伤害性DRG“硬连线”。在慢性间歇性应激的情况下，神经支配胃肠道的伤害性神经元对结直肠扩张的痛觉过敏反应的固有表达模式是易感的。这些研究将包括应用尖端方法来确定应激反应基因启动子处的假定调控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。伤害感受神经元的亚群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结合相关靶标的定量单细胞PCR来鉴定，以收获DRG神经元的不同群体。将使用原位靶向递送基因沉默（si-RNA）试剂以及这些干预与行为（例如，对结肠直肠扩张的内脏运动反应）的相关性来确认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。我们还将研究CB 1-TRPV 1受体复合物的形成是否在DRG神经元和转染细胞的应激相关内脏痛觉过敏中发挥作用，使用FRET/TIRF显微镜和电生理记录。阐明慢性应激诱导内脏痛的表观遗传调控机制将对我们理解疼痛通路如何调控以及影响胃肠道的功能性疼痛疾病的管理产生重大影响。