Stress and Visceral Hyperalgesia: Epigenetic Mechanisms

描述(由申请人提供)：慢性压力是众所周知的腹痛的诱因，在临床环境中是一种常见但知之甚少的表现。我们将检验这一新的假设，即慢性应激激活表观遗传调节通路，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的甲基化水平(甲基化增加降低基因表达)和组蛋白乙酰化(增加组蛋白乙酰化增强基因表达)来调节基因表达。应激激活下丘脑-垂体-肾上腺轴，导致人类血液中皮质醇和啮齿动物皮质酮(Corticosterone，CORT)水平升高，后者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，进而抑制伤害性内源性TRPV1受体的功能。慢性应激下调CB1的表达，上调TRPV1的表达。我们认为慢性应激会增加GR启动子位置的甲基化，导致CB1表达降低，并增强组蛋白乙酰化，导致TRPV1表达增加，最终导致内脏痛觉过敏。DNA甲基化位点将通过甲基化特异的聚合酶链式反应分析和焦磷酸测序来表征，组蛋白乙酰化位点将使用芯片进行解剖。我们认为慢性应激将选择性地调节支配结肠的初级感觉背根神经节(DRG)神经元中的这些通路，这些神经元负责传递疼痛。研究将在慢性避水应激大鼠、原位皮质醇处理的对照大鼠和体外皮质醇处理的对照背根神经节外植体中进行。我们将利用FRET/TIRF显微镜研究GR-MR和CB1-TRPV1受体复合体的形成是否在双标记GR和MR或CB1和TRPV1双重标记的DRG神经元的应激相关内脏痛敏中起作用。我们假设内脏痛敏与表观遗传机制有关，表观遗传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受体的启动子位置。这一亚群将使用免疫组织化学标记和激光捕获显微镜采集不同亚群的DRG神经元，并结合相关靶点的定量聚合酶链式反应进行鉴定。特异性受体(GR/MR)和信号转导通路(甲基化/乙酰化)对CB和Trp通路中观察到的变化的作用将通过靶向递送特定激动剂/拮抗剂药物和原位基因沉默(si-RNA)试剂以及这些干预与行为的相关性(例如对结直肠扩张的内脏运动反应)来确认。我们相信，拟议的研究将对我们理解应激如何影响疼痛感知产生很大影响。针对表观遗传机制的治疗干预被吹捧为未来个性化医学的一条途径。

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.