miR-29b靶向TET3参与慢性内脏痛敏的脊髓胶质细胞机制研究

Irritable bowel syndrome (IBS) is a common gastrointestinal disorder characterized by chronic visceral hypersensitivity in association with altered bowel habits. However, its precise mechanisms of visceral pain remain unclear, leading to limited and disappointed treatment options. Some evidence has shown that increased sensitivity of peripheral and central neurons is a critical mechanism for chronic visceral pain. We have developed and validated neonatal colonic inflammation (NCI) model previously, a rat model of adult visceral hyperalgesia that mimics some features of human IBS. And Our preliminary studies showed that the spinal synaptic transmission was significantly enhanced in NCI rats compared with age-matched control rats. But the mechanism was not very clear. Besides, we also found the expression of P2X7R, TET3 and GATA1 was obviously increased, but miR-29b was remarkedly down-regulated in spinal dorsal horn of NCI rats. There are eight miR-29b binding sites in TET3 mRNA 3’UTR predicted by bioinformatics. In addition, P2X7R and TET3 were both specifically expressed in astrocytes of spinal dorsal horn in NCI rats. Based on these results, we proposed the following research hypothesis: NCI leads to a significant down-regulation of miR-29b, negatively regulates the translation of TET3, then resulting in the increased expression of P2X7R due to DNA demethylation in its CpG island and enhanced binding with GATA1, then enhanced spinal synaptic transmission by astrocytes activation, eventually produces chronic visceral hypersensitivity. In order to confirm this hypothesis, we propose the following three specific aims: Specific Aim 1: To determine the role of TET3 regulated by miR-29b in visceral hypersensitivity induce by NCI; Specific Aim 2: To identify the DNA demethylation of p2x7r gene promoter catalyzed by TET3 upregulation in spinal dorsal horn of NCI rats; Specific Aim 3: To explore the contribution of transcript factor GATA1 in regulating P2X7R expression. We believe that our studies may explain the theory of DNA methylation and demethylation homeostasis in chronic pain of functional gastrointestinal disorders. The new mechanism would provide new strategies for treatment of chronic visceral pain in IBS patients.

肠易激综合征的重要症状之一是慢性内脏痛敏，目前其发病机制不清。已有的证据显示中枢敏化机制参与了慢性内脏痛敏。羟甲基化酶TET可以通过DNA去甲基化调节靶基因表达，但自身也受到上游机制调控。我们前期工作显示，新生期结肠炎性刺激（NCI）诱导的慢性内脏痛敏大鼠的脊髓突触传递增强，且TET3、P2X7R和GATA1表达均显著上调，但miR-29b表达显著下调；P2X7R和TET3特异性表达在星形胶质细胞；miR-29b与TET3 mRNA 3’UTR有结合位点。由此，我们提出本研究假设：NCI大鼠脊髓背角中miR-29b表达下调，靶向调控TET3表达上调，促使p2x7r基因去甲基化，转录因子GATA1与之结合增强，P2X7R表达增加，导致突触传递增强，产生内脏痛敏。本项目旨在阐明脊髓胶质细胞中miR-29b-TET3-P2X7R调控通路在慢性内脏痛敏中的作用特征，有望为临床提供新的潜在治疗靶点。

背景：肠易激综合征是一种常见的病因不明的功能性肠道疾病，其特征是广泛的慢性腹痛并伴有排便改变。越来越多的证据表明，新生期出现的损伤和炎症可能会对成年期的组织结构和功能产生长期影响，包括易患胃肠道疾病。本研究旨在探讨脊髓星形胶质细胞中转录因子GATA结合蛋白1（GATA1）引导的p2x7r基因发生DNA去甲基化的表观调控机制对新生期结肠炎性刺激（NCI）诱导的成年大鼠慢性内脏痛觉过敏的影响。方法：在NCI诱导的成年期内脏痛模型大鼠中，我们研究了脊髓中GATA1靶向p2x7r基因的DNA去甲基化。我们通过荧光素酶分析、染色质免疫沉淀、膜片钳和体内外敲降GATA1表达的手段来探究GATA1在NCI大鼠慢性内脏痛敏中的作用。此外，我们设计并应用诱饵寡核苷酸（decoy oligodeoxynucleotide, Decoy ODN）来探索GATA1对p2x7r CpG岛DNA甲基化的影响。结果：我们发现NCI导致肠相关节段脊髓背角星形胶质细胞中GATA1、TET3和P2X7R的表达增加，并且抑制这些分子的表达或功能可显著增加NCI模型大鼠的CRD阈值、抑制星形胶质细胞的激活、降低脊髓自发性兴奋性突触后电流（spontaneous excitatory postsynaptic current, sEPSC）的频率。NCI导致肠相关节段脊髓背角中p2x7r位点发生去甲基化，这种作用依赖于GATA1-TET3相互作用以及GATA1与p2x7r启动子区的结合增强作用。重要的是，我们证明了在敲除GATA1或TET3表达后，p2x7r基因的去甲基化（以及伴随而来的p2x7r表达的增加）被逆转，并且鞘内注射选择性阻断GATA1结合位点的Decoy ODN可以明显增加p2x7r启动子区CpG岛的甲基化水平。结论：以上结果表明，GATA1和TET3协同介导了NCI大鼠肠相关脊髓背角角星形胶质细胞中p2x7r基因的DNA去甲基化，最终导致NCI大鼠慢性内脏痛觉过敏。因此，通过靶向GATA1和p2x7r位点结合能力有望成为临床干预IBS慢性内脏痛敏潜在的治疗策略。