结肠腔内多巴胺在结肠上皮细胞内的代谢以及对结肠黏膜屏障功能的调节

Accumulating evidence has shown that a substantial amount of free dopamine (DA) exists in colonic lumen. The level of DA in colonic lumen is much higher than that in colonic tissue or plasma. It is far from clear that whether the luminal DA could regulate the colonic mucosa barrier function or be metabolized in the colonic epithelium cells. Our preliminary data showed abundant expression of the metabolic enzymes of DA (MAO-A, MAO-B and COMT) in the colonic epithelial cells, and wide distribution of DA receptors and dopamine transporter (DAT) on the epithelial cell membrane. Administration of exogenous DA at the luminal side of the isolated colonic mucosa markedly elevated the levels of DA, DOPAC and HVA in the colonic tissue, which was inhibited by pretreatment with DAT inhibitor GBR12909. Besides, we also found the distribution of D5 receptors on the colonic goblet cells, while DA promoted colonic mucus secretion via D5 receptor. We presumed that the colonic luminal DA could regulate the colonic mucosa barrier function through combining with DA receptor. It also could be transported into colonic epithelium cells via DAT, where it would be metabolized by MAO and COMT. The present study aims to explore the process of the luminal DA metabolized in the colonic epithelium cells, and the role of the luminal DA on the regulation of colonic mucosa barrier function by DA receptor knockout mice, isolated colonic mucosa and primary cultured colonic epithelial cells, by means of HPLC/isotopic liquid scintillation counts/mucosal permeability measurement etc., which may provide a rationale for the function and metabolism of gut luminal DA and shed light on diagnosis and treatment of the colonic mucosa barrier disturbance associated diseases.

研究报道结肠腔内存在大量多巴胺（DA），其含量远高于结肠组织和血液。这些DA是否调节结肠黏膜屏障功能、是否在结肠上皮细胞内代谢尚不清楚。我们预实验发现：结肠上皮内含有大量代谢DA的酶；DA受体和转运体（DAT）广泛分布在结肠上皮细胞；离体结肠腔面膜侧给予DA显著升高黏膜中DA和DA代谢产物的含量，抑制DAT则明显减弱上述过程。我们还发现杯状细胞表达D5受体且调节结肠黏液分泌。我们推测结肠腔内DA可能作用于结肠黏膜上的DA受体调节黏膜屏障功能，并经DAT转入结肠上皮细胞内被其酶代谢分解。本研究拟采用DA受体基因敲除小鼠，结合在体灌肠/离体结肠黏膜/原代上皮细胞培养等，运用HPLC、同位素液闪计数和黏膜通透性检测等方法深入研究结肠腔内DA在结肠上皮细胞内的代谢及其对结肠黏膜屏障功能的影响，以期为结肠腔内DA的作用及其代谢过程提供理论和实验依据，为与黏膜屏障功能紊乱相关的疾病提供诊疗思路与靶点。

研究发现结肠腔内存在大量多巴胺（DA），其含量远高于结肠组织和血液。这些DA是否调节结肠黏膜屏障功能、是否在结肠上皮细胞内代谢尚不清楚。本研究采用免疫荧光结合激光共聚焦技术发现多巴胺转运体（DAT）位于大鼠结肠上皮细胞的腔面膜侧，多巴胺代谢酶单胺氧化酶（MAO）和儿茶酚氧位甲基转移酶（COMT）在大鼠和人的结肠黏膜上皮细胞均有广泛表达。通过在体结肠腔侧灌流DA及离体结肠组织孵育腔侧给予DA结合HPLC测定等方法，发现结肠腔DA可以被DAT转运到黏膜上皮组织，结肠上皮细胞的MAO和COMT可以代谢转运到上皮细胞的DA。通过D5受体转基因小鼠、内源性结肠DA含量上调的大鼠模型和内源性结肠DA含量下降的小鼠模型结合离体结肠组织孵育的方法发现DA通过杯状细胞的D5受体促进黏液释放。通过D1R敲除小鼠和腺相关病毒干扰D2R小鼠以及原代肠胶质细胞培养的方法，发现低剂量的DA通过作用于胆碱能神经元的D1R促进ACh的释放，从而促进结肠胶质细胞释放胶质源性神经营养因子（GDNF）的释放，然而高剂量的DA可以通过作用于EGC上的D2R和胆碱能神经元上的D2R抑制结肠GDNF释放。应用6-OHDA破坏中枢黑质多巴胺能神经元制备的帕金森大鼠模型，发现其结肠DA升高，而GDNF水平降低，结肠黏膜通透性升高。本研究发现结肠腔DA可以转运到结肠上皮细胞并被代谢；DA可以促进结肠黏液的释放，还可以与胆碱能神经元互作调节结肠GDNF的释放，为DA参与调节结肠黏膜屏障功能提供新的理论与实验依据。