胆红素与5-羟色胺3受体的氢键结合改变阻塞性黄疸的痛阈

There is a special phenomenon in obstructive jaundice. That is less sensitive to nociceptive stimulation, which is also considered as up-regulation of pain threshold. It makes patients pay less attention to the illness due to less pain, which delays the treatment. It also causes relative over-dosages of opioid analgesics, which puts the patients into risk of lives.However, its mechanism remains unclear. In this case, bilirubin acting on 5-HT3 receptors and facilitating GABA signal pathway will be investigated in the present study to clarify mechanism of the changing of pain threshold in jaundice. Firstly, basic pain threshold and postoperative morphine requirements in jaundice patients will be observed after blocking of 5-HT3 receptors. Secondly, after blocking of 5-HT3 receptors both in gene and protein level in jaundice mice, pain threshold and spinal GABA release will be detected. The patch-clamp technique will also be used to exam the changes of inhibiting postsynaptic currents. These will validate whether activation of 5-HT3 receptors will facilitate GABA pathway and cause the changing of pain threshold. Finally, the bilirubin effect on currencts of different 5-HT3 subtypes expressed in xenopus oocytes will be observed and molecular docking studies will be utilized to predict the binding affinity of bilirubin and different 5-HT3 receptor subtypes, which will clear the interaction of bilirubin and 5-HT3 receptors in changing pain threshold of jaundice. This study will provide a new interpretation of the elevating pain threshold in obstructive jaundice. It will also prevent covering the severity the illness or excessive usage of analgesics in patients with jaundice due to less sensitive to nociceptive stimulation.

阻塞性黄疸存在特殊痛觉现象：痛阈增高。痛阈增高易掩盖病情，延误治疗；并造成镇痛药的相对过量使用，威胁生命。但其机制仍然不明。我们认为胆红素与5-HT3受体结合，将易化GABA通路，增高黄疸的痛阈。本研究首先比较阻断5-HT3受体后，黄疸患者基础痛阈和术后吗啡需求量的不同。然后，在基因及蛋白水平阻断5-HT3受体，观察黄疸小鼠痛阈及脊髓GABA释放的变化；并采用膜片钳技术检测脊髓片GABA介导突触后电流的改变，以验证痛阈增高是否与5-HT3受体激活并易化GABA通路有关。最后，检测胆红素对爪蟾卵母细胞中表达的5-HT3受体各个亚型介导的内向电流的影响，并采用同源建模和分子对接分析胆红素和5-HT3受体不同亚型的结合模式，以明确痛阈增高是胆红素与5-HT3受体氢键结合的结果。本项目将为阻塞性黄疸痛阈增高的原因提供新的解释，避免因痛阈增高掩盖病情或过量使用镇痛药物，减少不良事件的发生。

背景 胆汁淤积患者表现为痛觉过敏，对伤害刺激不敏感，容易掩盖病情并造成镇痛药物使用相对过量。本研究旨在探索胆汁淤积患者体内升高的胆红素介导痛觉过敏的中枢机制。 方法 通过胆总管结扎（bile duct ligation, BDL）建立胆汁淤积模型，检测血清和脑脊液中的肝功能指标变化，并测试BDL与胆红素鞘内注射后大鼠痛阈的改变。Western blot和免疫荧光检测5-羟色胺3（5-HT3）受体和γ-氨基丁酸（GABA）受体各亚基表达变化。ELISA和电生理测试胆红素对GABA神经元功能的影响。最后，通过放射性配体结合、同源建模和分子对接分析胆红素同5-HT3受体的亲和力，并构建两者的结合位点和结合方式。 结果 BDL后血清和脑脊液中游离胆红素明显升高，且BDL后和鞘内注射胆红素均能增高大鼠痛阈，伴随5-HT3A受体和GABAA受体表达增加。 5-HT3受体和GABA受体拮抗剂能够不同程度逆转BDL大鼠痛阈增高，同时胆红素增加脑脊液GABA浓度和脊髓背角抑制性突触后电流，并可激活5-HT3A受体诱发内向电流，这种效应可被昂丹司琼抑制。最后，胆红素进入5-HT3A受体形成的疏水间隙，两者具有较高的互补性，并有氢键形成。 结论 胆汁淤积中枢增高的胆红素通过与脊髓背角5-HT3受体的氢键结合增高胆汁淤积的痛阈。本研究为胆汁淤积患者痛阈升高的机制研究提供新的思路，避免黄疸患者因痛阈升高掩盖病情，也为阻塞性黄疸患者合理使用阿片类镇痛药物，减少围术期并发症和死亡率提供理论依据。