D2R-A2aR-mGluR5拮抗/协同机制在肠胶质细胞参与肠缺血再灌注损伤机制中的作用

The mechanism underlying intestinal ischemia and reperfusion(IR) -induced damage remains unclear. The previous studies have revealed that enteric glial cells(EGC) display the dual roles under IR stimulation: protecting intestinal mucosal function as well as participating in the inflammation process. Clarifying the seemingly paradoxical role of EGC under IR is the main goal of the present study. Several works demonstrated that G protein-coupled receptor(GPCR) adenosine 2a receptor(A2aR) has antagonistic receptor-receptor interaction with another GPCR dopamine D2 receptor(D2R), while has synergistically interaction with metabotropic glutamate receptor 5(mGluR5).Via regulating the excellular glutamate(Glu)concentration, the receptor-receptor interaction conversion between A2aR-D2R and A2aR-mGluR5 could be the potential molecular mechanism to switch the role of EGC between "intestinal protector" and "inflammation promoter" through activating PKA or PKC signaling pathway. Accordingly, co-immunoprecipitation as well as high performance liquid chromatography techniques will be used to verify the antagonistic or synergistically interaction among A2aR,D2R and mGluR5 and Glu overflow of EGC. The selective antagonists as well as agonists for these receptors will be applied to reveal the precise mechanism underlying GPCR interaction. Furthermore, a variety of EGC-derived cytokines involved in the intestinal protection or proinflammatory will be analyzed to confirm the important role of the interaction between different GPCRs in the modulation of EGC's functions. In addition, IR mouse model will be established to investigate the influence of the abovementioned GPCRs in IR injury. Intestinal luminal perfusion of Glu at vary concentrations will be used to clarify the key role of Glu overflow in the intestine in mediating IR-induced inflammation. The selective antagonists and agonists for A2aR,D2R and mGluR5 will be used to screen the potential targets which may has protection effect against IR injury. In general, the present study is designed to try to improve the outcome of the critical care patients suffering from IR insults.

严重创伤、大型手术所诱发的小肠缺血再灌注(IR)损伤是影响患者预后的重要因素。前期研究表明，肠胶质细胞(EGC)除在肠粘膜保护发挥着重要作用，同时参与了小肠IR损伤进程，但具体功能转换机制仍不清楚。研究显示，在高浓度谷氨酸(Glu)刺激下，G蛋白耦联受体腺苷酸A2a受体(A2aR)与代谢性Glu受体5(mGluR5)协同作用参与促炎症反应；而多巴胺D2受体(D2R)可能通过拮抗A2aR发挥保护作用。我们推测A2aR与D2R、mGluR5的拮抗/协同机制可能为EGC功能转换的重要机制，Glu浓度是介导此机制的关键介质。本研究拟利用免疫共沉淀及高效液相色谱技术验证EGC A2aR与mGluR5、D2R之间的拮抗/协同机制及对Glu外流的作用，观察上述互动机制对EGC肠粘膜保护及促炎症反应的影响，建立小肠IR模型，研究上述拮抗/协同机制及Glu在小肠IR损伤机制中的作用，筛选小肠IR保护新靶点。

严重创伤、大型手术所诱发的小肠缺血再灌注(IR)损伤是影响患者预后的重要因素。前期研究表明，肠胶质细胞(EGC)除了在肠粘膜保护中发挥着重要作用，同时还参与了小肠IR损伤进程，但具体功能转换机制仍不清楚。.本研究设想，G蛋白耦联受体腺苷酸A2a受体(A2aR)与代谢性Glu受体5(mGluR5)协同作用参与促炎症反应；而多巴胺D2受体(D2R)可能通过拮抗A2aR发挥保护作用。因此，我们推测A2aR与D2R、mGluR5的拮抗/协同机制可能是EGC功能转换的重要机制。.本研究利用小鼠IR模型，发现在肠道缺血缺氧损伤条件下，EGC 中A2aR的表达明显增强。为了进一步研究A2aR在IR条件下的作用，我们建立了A2aR敲除小鼠的IR模型，发现A2aR 基因的缺失明显加剧了急性IR 刺激诱导的肠黏膜结构损伤，同时伴随着肠上皮间紧密连接蛋白ZO-1的表达显著下调；同时，体外细胞实验同样发现，A2aR拮抗剂ZM241385 加重了缺氧刺激下肠上皮屏障的破坏，而A2aR激动剂CGS21680 则缓解了肠上皮屏障的损伤。进一步研究发现，正常情况下 EGC 细胞内.A2aR 蛋白与 D2R 蛋白是结合的；而当细胞受到缺氧再复氧及高浓度谷氨酸盐（600μ M）刺激时， A2aR 则与 mGluR5 结合，启动促炎效应。通过本项目，不但能够进一步完善EGC参与肠道IR损伤及保护的复杂机制，还能筛选小肠IR损伤保护的干预新靶点。

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