多巴胺通过D5受体调节十二指肠黏膜屏障的机制研究

Mucosal barrier damage with high permeability plays an important role in the peptic ulcer formation, and tight junction is the structural foundation of the mucosal barrier. Depletion of catecholamine or destruction of endogenous dopaminergic system can weaken the duodenal mucosal resistance, enhance mucosal permeability and then produce duodenal ulcers, suggesting that DA is involved in the regulation of duodenal mucosal barrier, but the mechanism is not clear. It has been reported that cAMP analogues is able to decrease the permeability of the epithelial paracellular pathway by altering the structure of tight junction. In our preliminary study, DA binding with D1-like receptor family (D1 and D5) can elevate intracellular cAMP and increase duodenal mucosal resistance. In the transgenic mice with D5 up-regulation, DA significantly increased intracellular cAMP and mucosal resistance, and decreased FITC-dx4 permeability in duodenal mucosa, suggesting an enhanced barrier function in duodenal mucosa. Our hypothesis is that DA, when binding with D5 receptors, might regulate the structure, distribution and expression of tight junction complex and permeability of the paracellular pathway, and then improve duodenal mucosal barrier function though intracellular cAMP signaling pathway. Several animal models, laser microdissection and short circuit current will be adopted in this project to investigate the underlying mechanism and provide theoretical and experimental evidences for the clinical treatment and prevention of duodenal ulcer.

黏膜屏障损伤、通透性增加是消化性溃疡发生的重要环节，而紧密连接是黏膜屏障的结构基础。耗竭体内儿茶酚胺或损毁多巴胺（DA）能系统可以降低十二指肠黏膜电阻、增大其通透性，并诱发溃疡，提示DA参与调控十二指肠黏膜屏障，但机制尚不清楚。文献报道cAMP类似物可以通过改变紧密连接结构降低上皮细胞的通透性。我们前期发现DA可以激活D1受体家族（D1和D5）升高细胞内cAMP水平并增大十二指肠黏膜电阻；在D5受体上调的转基因小鼠中，DA可以引起十二指肠黏膜电阻增大而荧光标记葡聚糖（FITC-dx4）通透性降低，提示黏膜屏障功能增强。我们推测：DA与D5受体结合经cAMP通路调控紧密连接复合物的结构、分布和表达等，改变上皮的通透性，进而影响十二指肠黏膜屏障功能。本研究将借助动物模型、镭射显微切割和短路电流等技术深入探讨DA调节十二指肠黏膜屏障功能的机制，为临床十二指肠溃疡的诊治提供可靠的理论与实验依据。

消化性溃疡尤其是十二指肠溃疡为临床常见病，多发病。抗多巴胺(dopamine，DA)药物诱发十二指肠溃疡，而D2受体激动剂和促进DA释放均有效降低溃疡的发生，提示DA及其受体参与十二指肠粘膜屏障的保护作用。肠粘膜屏障主要由上皮细胞和细胞间紧密连接两部分构成，调控肠黏膜离子转运和通透性。我们前期已报道DA可以通过广泛表达于十二指肠黏膜的D1类受体（包括D1和D5）介导十二指肠黏膜上皮离子转运。那么D1类受体是否参与十二指肠上皮黏膜通透性的调节？我们借助人源性D5受体转基因小鼠和外周DA系统功能上调6-OHDA大鼠模型，采用免疫组织化学、酶联免疫、短路电流和葡聚糖通透实验等进行研究。结果发现DA引起十二指肠黏膜上皮短路电流降低的同时升高黏膜的通透性，而预处理D1家族受体拮抗剂SCH-23390可显著抑制上述作用并逆转十二指肠黏膜电阻降低。免疫荧光结果显示D5受体而不是D1受体广泛表达于十二指肠黏膜，提示D5受体参与调节十二指肠黏膜屏障功能。为了验证上述推测，我们借助人源性D5受体转基因小鼠，通过检测D5受体荧光强度和DA引起的短路电流和细胞内cAMP的改变量确定模型成功。实验结果发现人源性D5受体上调的D5S390G转基因小鼠十二指肠黏膜D5受体荧光强度显著增强、DA引起的短路电流和细胞内cAMP改变量明显增大，而其十二指肠黏膜的基础电流和黏膜通透性均显著升高，黏膜电阻和紧密连接蛋白ZO-1和occludin表达明显降低，说明D5S390G转基因小鼠十二指肠黏膜屏障功能损伤；与之相反，人源性D5受体下调的D5F173L转基因小鼠十二指肠黏膜基础电流和黏膜通透性明显降低。进一步发现双侧黑质DA能神经元损毁的6-OHDA大鼠模型，纹状体DA含量减少，血浆中促炎因子以及消化道通透性显著升高，而抗炎因子降低，胃肠排空和转运速度减慢。此外，十二指肠黏膜的D5受体上调，十二指肠黏膜通透性升高而黏膜电阻和紧密连接蛋白ZO-1和occludin表达明显降低。因此，D5受体在DA调节十二指肠黏膜通透性中发挥重要作用，为DA参与黏膜屏障功能调节和胃肠道黏膜保护作用机制提供新的实验依据。