Chronic Stress and Abdominal Pain: Novel Mechanisms

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-垂体-肾上腺（HPA）轴，是已知的腹痛诱因。最近的报道表明，慢性心理应激导致中枢神经系统中与记忆和情绪相关的区域基因功能的表观遗传调控发生变化。表观遗传学是指通过DNA甲基化、组蛋白修饰和染色质重塑，在不改变DNA序列的情况下，基因功能发生稳定和/或可遗传的变化。表观遗传学领域在过去十年中迅速兴起，其基础是对原代细胞和人类细胞系中甲基化和乙酰化位点全基因组分布的开创性研究。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。内源性大麻素（endocannabinoids， CB）作用于CB1受体，抑制TRPV1的功能，从而调节TRPV1受体。慢性应激下调CB通路功能，导致TRPV1受体功能上调，引起腹痛。我们将研究慢性应激通过CB1和TRPV1受体的表观遗传调控，以区域和细胞特异性的方式诱导内脏疼痛，而不是通过支配盆腔器官的背根神经节（DRG）神经元，而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，并增强组蛋白乙酰化，与背根神经节（DRG）神经元中促伤害性内源性大麻素TRPV1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）和躯体感觉（坐骨神经）分布的伤害性DRGs“硬连接”。这种固有的表达模式使支配胃肠道的伤害性神经元在慢性间歇性应激环境下对结肠直肠膨胀的反应中容易产生痛觉过敏。这些研究将包括应用前沿方法识别应激反应基因启动子中可能的CpG甲基化调控位点，以及染色质免疫沉淀（ChIP）分析相关组蛋白修饰靶点。伤害性神经元亚群将通过免疫组织化学标记与逆行标记和激光捕获显微镜来鉴定，以收集不同的DRG神经元群，并结合相关目标的定量单细胞PCR。通过原位靶向递送基因沉默（si-RNA）试剂，以及这些干预措施与行为（如结肠直肠膨胀的内脏运动反应）的相关性，将证实特异性受体和信号转导途径在CB和TRP途径中观察到的变化中的作用。我们还将使用FRET/TIRF显微镜和电生理记录来研究CB1-TRPV1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起作用。阐明慢性应激性内脏疼痛的表观遗传调控机制将对我们理解疼痛通路是如何被调节的，并可能对影响胃肠道的功能性疼痛障碍的管理产生重大影响。

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.