多巴胺对肠胶质细胞的调节机制及其在结肠黏膜屏障中的作用

Mucosal barrier maintains intestinal homeostasis and steady state of the internal environment. Recently, enteric glial cells (EGC) have been reported to play a key role in keeping the function of intestinal mucosal barrier. The intermediate filament glial fibrillary acidic protein (GFAP) and the calcium-binding protein S100β are the markers of EGC. Gastrointestinal (GI) disorders have been reported to show high prevalence in Parkinson’s disease patients due to progressive degeneration of dopamine (DA) system. Moreover, the enhanced expression of GFAP and S100β protein are observed in the colon of Parkinson’s disease patients with obvious GI dysfunction, which suggests that DA may regulate the expression of GFAP and S100β protein in EGC. However, the underlying mechanism is not clear. In our previous studies, rat with increased GI DA content showed that the levels of GFAP protein, glial cell line-derived neurotrophic factor (GDNF) and the transepithelial resistance of the colon were dramatically decreased. The immunoreactivity(IR) of DA receptor including D2 and D5 was distributed in EGC of the colon. The mRNA level of GFAP was increased in the colon of D2 receptor deficient mice. We hypothesize that DA might regulate the expression of GFAP and S100β protein and the release of biological active substance in EGC by acting directly on DA receptor and then influence the colonic mucosal barrier function. In this study, EGC and epithelial cell line co-culture, animal models (removal of endogenous DA) and dopamine receptor deficient mice will be utilized to investigate the mechanism underlying the potential effect(s) of dopamine on the EGC and its role on colonic mucosal barrier. This current project is intended to provide a new research perspective for the regulation of DA on colon mucosal barrier function, and provide a platform for the treatment of diseases associated with intestinal mucosal barrier dysfunction.

肠黏膜屏障维持肠道稳态与内环境稳定。近年来研究发现肠胶质细胞（EGC）在维持肠黏膜屏障起关键作用。多巴胺（DA）系统退行性变的帕金森病人胃肠道功能障碍发病率高，其结肠EGC标记蛋白GFAP和S100β显著升高，提示DA可能调节EGC标记性蛋白的表达，但机制不清。前期发现胃肠道DA含量升高的大鼠，结肠GFAP蛋白表达、胶质源性神经营养因子含量和黏膜电阻均显著降低；结肠EGC有DA受体（D2和D5）的表达；D2受体敲除小鼠结肠GFAP升高。我们推测DA可能通过直接作用于EGC的DA受体，调控EGC标记性蛋白的表达和生物学活性物质的释放，进而调节结肠黏膜屏障功能。本研究将借助EGC与上皮细胞共培养和基因敲除动物等探讨DA对EGC标记性蛋白表达和生物活性物质释放的调节机制及其在结肠黏膜屏障中的作用。该项目将为DA调节结肠黏膜屏障功能提供新的视角，也为肠黏膜屏障损伤相关疾病的治疗提供新的思路。

肠胶质细胞对肠黏膜屏障功能发挥重要的作用。研究发现多巴胺（dopamine, DA）系统发生退行性变的帕金森病人，结肠EGC标记物胶质纤维酸性蛋白（glial fibrillary acidic protein，GFAP）和钙结合蛋白S100β显著升高，其结肠黏膜通透性升高，提示DA可能调节结肠EGC标记性蛋白的表达，进而影响黏膜屏障功能，但是具体机制尚不清楚。本研究采用免疫荧光双标方法发现多巴胺受体D2和D5在大鼠、小鼠及人的结肠EGC上均有表达。通过离体孵育结肠组织的方法，发现DA可以剂量依赖性地促进小鼠结肠胶质细胞释放GDNF和GSNO。并且DA可以促进原代EGC释放GDNF，D1受体拮抗剂SCH23390可以阻断DA的作用，但是D2受体拮抗剂Sulpride无明显作用。同时D1受体激动剂可以模拟DA的作用，而D2受体激动剂Quinpirole无明显作用。结肠内源性DA含量降低的MPTP小鼠，其GDNF水平均明显降低。结肠内源性DA升高的6-OHDA大鼠，其结肠D5受体蛋白水平下降，同时GDNF含量降低，黏膜电阻降低，而黏膜炎症因子和FITC标记的荧光葡聚糖通透性均升高。我们还发现6-OHDA大鼠肠道微生物紊乱。以上结果说明DA通过肠胶质细胞上的D5受体促进其GDNF的释放。结肠内源性DA降低或者D5受体下调，可以引起GDNF释放减少，降低黏膜电阻，增加黏膜通透性。该研究为DA调节结肠黏膜屏障功能提供新的视角与实验依据。