ATP和P2X3受体参与雌激素调节子宫内膜异位症疼痛信号转导通路的作用及机制研究

Endometriosis is a common gynecological disease with its incidence increasing yearly. Pain is the characteristic symptom of endometriosis, and the key to endometriosis treatment is pain. So to clarify the mechanism of endometriosis-associated pain is becoming hotspot in this field. Our previous studies found that adenosine triphosphate (ATP) content and P2X3 receptor expression levels in endometriotic lesions are increased in women with endometriosis, which are associated with pain and menstrual cycle, but the exact mechanism is still unclear. ATP and P2X3 receptor may play an important role in the process of formation and maintenance of endometriosis pains adjusted by estrogen. In this project, molecular biology, behavioral science and whole-cell patch clamp will be performed to measure ATP content and P2X3 receptor expression levels in the different lesions of endometriosis patients after GnRHa intervention; we will use the SD rat model of endometriosis as the in vivo model, and the culture of dorsal root ganglion (DRG) and endometrial cells as the in vitro model, measuring the changes of ATP content and P2X3 receptor expression levels in the lesions and DRG and spinal cord neurons as well as biological behavioral changes before and after different interventions by estrogen and the related factors, so as to investigate the key role of ATP and P2X3 receptor in signal transduction pathway of endometriosis-associated pain, find key molecules involving in endometriosis pain transduction pathway, and even provide more effective treatments for endometriosis-associated pain by using a molecular targeted therapy.

子宫内膜异位症（内异症）是妇科的常见病与多发病，发病率逐年上升。疼痛是内异症治疗的关键，因此，阐明内异症疼痛的发生机制，是内异症目前研究的热点与难点。前期研究发现，内异症病灶可检测到与患者疼痛和月经周期相关的ATP高浓度和P2X3受体高表达，但机制不清。ATP和P2X3受体可能在雌激素调控内异症疼痛的形成和维持中发挥重要作用。本项目拟应用分子生物学、行为学以及全细胞膜片钳等技术检测内异症患者GnRHa干预前后病灶ATP含量与P2X3受体表达水平；构建内异症SD大鼠模型，建立离体子宫内膜细胞和DRG细胞培养模型，测定模型大鼠和模型细胞雌激素及相关因子干预前后病灶、DRG、脊髓背角和离体细胞ATP含量与P2X3受体表达变化以及疼痛生物学行为改变。探讨ATP和P2X3受体参与雌激素调节内异症疼痛信号转导中的关键作用，寻获内异症疼痛信号转导中的关键分子，为将来研制临床上能治疗内异症疼痛的靶向药物。

子宫内膜异位症（内异症）是妇产科常见的疾病，有文献报道其在育龄妇女中的发病率可达10%以上。疼痛作为内异症的主要临床症状之一，其发生机制仍未阐明，是目前内异症研究的热点与难点，临床上也缺乏有效的治疗方法。P2X3是一种ATP的膜受体，作为一种疼痛性受体，受体限制性的分布于背根神经节、三叉神经节以及结状神经节的小和／或中等直径的辣椒素敏感的神经元，已经被证实参与许多神经源性疼痛的疾病。本课题首先证明了异位内膜组织同异位症患者在位内膜和正常内膜相比，异位组织ATP的含量以及P2X3的表达水平更高，且与患者的疼痛程度相关。当内异症患者接受GnRH-a治疗后，患者的临床疼痛症状明显改善，同时异位病灶中的ATP含量，P2X3 mRNA和蛋白的表达量也显著降低。在异位内膜细胞中，IL-1β和ATP的刺激可以激活MAPK-ERK-CREB通路从而在转录水平上提高P2X3的表达水平。在大鼠内异症动物模型上，异位内膜组织和DRG组织也表现出ATP和P2X3的高表达，并且与动物表现出来的疼痛行为学过敏呈正相关。在DRG细胞水平，E2和ADP的刺激均能引起P2X3转录水平的上升，且运用ATF3/AP-1通路抑制剂SP600125后能有效抑制ADP刺激所导致的ATF3/AP-1通路的激活以及P2X3的上调。此外，我们设计了脂质纳米材料CSOSA/NLC和CSOSA/LPs来分别包装P2X3特异性抑制剂A-317491和ATF3/AP-1通路抑制剂SP600125，在离体细胞实验和在体内异症大鼠模型实验均取得了理想的治疗结果。综上，本课题详细阐述了ATP和P2X3参与内异症疼痛及其可能的调控方式，并运用P2X3特异性抑制剂A-317491和ATF3/AP-1通路抑制剂SP600125在动物模型上有效抑制内异症所导致的P2X3的受体敏感性以及P2X3受体的表达上调，治疗了内异症所致的疼痛行为学过敏。上述结果说明P2X3在内异症疼痛的产生和维持中有重要作用，为进一步了解内异症的疼痛机制，以及今后寻找内异症疼痛的靶向治疗药物提供新的角度和思路。