miR-31参与去甲肾上腺素介导慢性内脏痛敏的表观调控机制研究

The pathogenesis of irritable bowel syndrome（IBS）remains largely unknown and treatment options are very limited. Recent studies in our laboratory indicate that Neonatal Maternal Deprivation (NMD) results in visceral hypersensitivity in adult rats, and further studies show that adverse environmental stress at adult age imposed on NMD rats significantly exacerbated visceral pain responses. We also detected a higher norepinephrine (NE) level in blood plasma in NMD+AS rats than that in CON+AS rats. NE inhibitor treatment significantly reversed visceral hypersensitivity of NMD+AS rats; P2X3R expression and ATP-evoked currents in colon specific dorsal root ganglion (DRG) were significantly enhanced after NMD+AS treatment. More interestingly, we also found that after NMD+AS treatment, the expression of microRNA-31 (miR-31) was downregulated in colon DRGs; incubation of NE on colon specific DRG neurons decreased the expression of miR-31. Based on these exciting results, we propose the following experiment hypothesis: NMD+AS activates the NE signaling pathways to downregulate miR-31 expression, thus sensitizing P2X3R, and eventually produces chronic visceral hyperalgesia. Inhibitors of NE receptor and miR-31 Lentiviral over expression strategies will be employed to investigate the following specific aims: (1) To define the function of NE in visceral pain model; (2) To determine the roles of NE in sensitization of P2X3R; (3) To explore the mechanisms of miR-31 involved in NE-P2X3R signaling pathway in visceral pain model. We hope that our study will further explain pathological mechanisms of stress-induced chronic visceral pain and that this signaling pathway would be a novel potential target for treatment of chronic visceral pain in patients with IBS.

IBS的病理机制尚不清楚，治疗手段也非常短缺。我们前期的研究结果显示新生期母爱剥夺(NMD)导致成年大鼠内脏痛高敏，近期研究发现NMD大鼠成年期进行成年应激（AS）会加剧内脏痛反应、升高血浆NE水平、增强肠DRG神经元ATP电流及P2X3R的表达，并下调miR-31的表达；另外给予NE受体拮抗剂可翻转内脏高敏反应，NE孵育肠DRG神经元可下调miR-31的表达。由此我们拟提出以下研究假设:复合应激（NMD+AS）通过激活肾上腺素能信号系统下调miR-31的表达，介导P2X3R表达上调且功能增强，诱导慢性内脏痛。从而确立了以下研究内容：1）初步阐明NE在IBS慢性内脏痛敏中的作用；2）探讨NE对P2X3R的功能和表达的调控作用；3) 探讨miR-31参与内脏痛模型中NE对P2X3R的调控作用。预期通过本研究将进一步阐明IBS慢性内脏痛的病因和病理机制，并有望为临床治疗IBS提供新的治疗靶点。

肠易激综合征（Irritable Bowel Syndrome，IBS）是一种常见的胃肠功能疾病，由于IBS没有明显的病理指标异常，因此病理机制不详，研究进展缓慢。我们前期的研究结果显示新生期母爱剥夺(neonatal maternal deprivation, NMD)导致成年大鼠内脏痛高敏，但是在成年鼠6周的时候没有明显的痛觉高敏，为了更好的研究IBS的病理机制，我们对6周NMD大鼠增加了成年应激（Adult Stress, AS），看是否可以诱导内脏高敏，并研究其介导机制。近期研究发现NMD大鼠成年期进行成年应激（AS）会加剧内脏痛反应、升高血浆NE水平、全细胞膜片钳实验发现NMD+AS大鼠肠DRG神经元兴奋性增加，肠DRG中β2蛋白表达显著增加，腹腔注射β2受体阻断剂可以显著降低NMD+AS大鼠的内脏疼痛反应，降低肠特异DRG神经元的兴奋性，以上数据说明NMD大鼠增加成年应激可以诱导内脏高敏，而这种高敏反应可能是由去甲肾上腺素的β2受体参与介导的。为了深入研究其病理机制，我们又给与了P2X3受体阻断剂，发现其可以显著增加NMD+AS大鼠疼痛阈值，缓解疼痛反应，Western Blot实验检测发现NMD+AS也显著升高肠DRG的P2X3的蛋白表达，增加了肠特异性DRG神经元ATP电流密度；进一步研究发现，NMD+AS大鼠给与β2受体阻断剂可以显著降低肠DRG中P2X3的蛋白表达，降低ATP的电流密度，由此说明β2受体通过增加P2X3受体的功能与表达介导NMD+AS诱导的内脏高敏。生物信息学预测发现，调控P2X3受体基因的miRNA是miR-31,PCR实验检测发现NMD+AS大鼠肠DRG的miR-31表达显著降低，结合负反馈调控机制，说明P2X3和miR-31之间可能存在调控作用，给与NMD+AS大鼠鞘内注射miR-31的agomir，可以显著升高疼痛阈值，缓解内脏疼痛，并且给与miR-31的过表达慢病毒也可以显著缓解NMD+AS大鼠的内脏痛敏，另外我们还发现在离体培养的肠DRG神经元上孵育NE，可以显著降低miR-31的表达。综上所述，NMD诱导的内脏高敏可能是通过激活肾上腺素能信号系统下调miR-31的表达,进而介导P2X3受体表达上调且功能增强来完成的。因此，本研究进一步阐明了IBS慢性内脏痛的病因和病理机制，并为临床治疗IBS提供新的治疗靶点。