P2Y1介导μ阿片受体调控结肠感觉传导环路的作用与机制研究

Opioids relieves pain while inducing constipation. The study showed a decreased sensitivity to the defecation reflex afterμopioid receptors (MORs) were activated contribute to opioid-induced constipation(OIC), but the mechanism is unclear. According to the results of our previous studies, MOR, together with P2Y1 and ATP transferase decreasingd, is increasing in colon with OIC, and the electrophysiological characteristics of MOR and P2Y1 in the colon are similar. According to effect that P2Y1 can regulate the sensory conduction of the colon, we speculate that MORs were abnormally activated and the disorder of colon sensory conduction was induced by P2Y1 after taking opioid analgesics for a long time, and the plasticity of the defecation reflex was changed. It resulted in the intractable constipation. Now our research group try to study the mechanism that P2Y1 modulates the colonic sensory information transmission using the conditional gene knockout mouse models by the technologies of the pharmacology and electrophysiology, research the mechanism that MOR selectively regulates the releases of ATP from the intestinal nerve cells by methods of the patch clamp and Bradford method for measurement of protein, explore that MOR and P2Y1 receptor form the neural pathway in colonic enteric nervous system (ENS) by the methods of the multiple labeling immunofluorescence, the neural tracer and the immune electron microscopy, and evaluate the relationship of P2Y1 receptor to the colonic the disorder of the colonic sensory information transmission of mouse with OIC by the approaches of the molecular biology and the intestinal function. The intrinsic relationship and the pathogenesis of P2Y1 receptor-mediated OIC are clarified through the above studies, which contributes to the effective treatment of OIC in clinic.

阿片类在镇痛同时诱发便秘，主要是激活结肠μ阿片受体（MOR），降低排便反射的感觉敏感性，但机制不明。预实验发现，阿片类诱发便秘（OIC）模型结肠内MOR表达明显升高，但P2Y1和ATP转移酶表达下降，在结肠MOR与P2Y1的电生理特点有相似性。结合P2Y1调控结肠感觉传导，推测长期服用阿片类镇痛剂，异常激活MOR，诱发P2Y1调控的结肠感觉传导紊乱，排便反射通路的可塑性改变，导致顽固性便秘。本项目拟利用OIC模型在体神经干预，通过观察结肠功能，分子生物学方法检测MOR和P2Y1等的表达，分析P2Y1与OIC感觉异常的相关性；免疫荧光多重标记、神经示踪和免疫电镜等技术勾勒MOR和P2Y1等在结肠构成的神经通路；利用条件性敲除P2Y1基因小鼠和培养结肠初级传入神经元，在体外运用药理学、膜片钳和Bradford法蛋白测量技术，探索MOR调控肠细胞释放嘌呤类递质，经P2Y1调节结肠感觉传递机制。

阿片类制剂激活外周μ阿片受体（MOR）导致结肠功能紊乱和便秘，目前机制不明。P2Y1是嘌呤类受体的一种，在胃肠道内广泛表达，与胃肠道平滑肌舒张功能密切相关。根据本实验前期研究推测，MOR对结肠功能活动的调控可能经P2Y1受体介导。 本实验利用阿片类诱发的大鼠便秘模型，探索P2Y1等在阿片类诱发便秘（opioid induced constipation；OIC）大鼠远端结肠黏膜下层内的表达特点及其与OIC发生的相关性。通过观察模型大鼠粪便的性状及含水量的变化评价模型；免疫荧光组织化学方法观察P2Y1等在大鼠远端结肠黏膜下丛内的分布特性；RT-PCR、Western Blot等方法检测P2Y1、MOR等在OIC大鼠远端结肠黏膜下层内的表达变化，评价P2Y1与OIC发生的相关性；运用药理学和电生理学方法，探讨P2Y1和MOR介导OIC的机制。 实验结果显示，造模7d后，OIC大鼠粪便呈腊肠状小碎块且含水量降低。免疫荧光组织化学染色显示，在远端结肠黏膜下丛神经细胞内P2Y1与 NeuN有共存。RT-PCR、Western Blot结果显示，在OIC大鼠远端结肠黏膜下层内，P2Y1的mRNA和蛋白表达水平明显降低，而MOR的mRNA和蛋白表达水平增加。体外张力实验结果显示，P2Y1受体激动剂腺苷5′-[β-硫基]二磷酸三锂盐（ADPβS）抑制了远端结肠段自发性收缩的幅度，而拮抗剂MRS2500增强了自发性收缩幅度；继续施加内吗啡-2（EM2）抑制了ADPβS的作用，但对MRS2500的作用无显著影响；施加MOR拮抗剂β-FNA对 ADPβS的作用效果无明显影响，但可抑制MRS2500的作用。在体外，给予5Hz频率0.4ms的电场刺激（EFS）抑制了远端结肠组织片段的自发性收缩，进一步给予ADPβS可增强EFS的作用，而给予MRS2500的作用效果则相反；继续施加EM2降低了ADPβS对EFS的影响，但对MRS2500的作用效果无显著改变；而施加β-FNA促进了ADPβS的作用，同时也显著抑制了MRS2500的作用。 综上所述推测，MOR对结肠运动的调节可能与调控结肠神经系统中嘌呤类神经递质如ATP等的释放，进而激活平滑肌细胞表面的P2Y1，调节结肠的运动功能有关。