HIF-2α/A2AR调控Treg细胞参与小肠缺血再灌注损伤机制

Intestinal ischemia/reperfusion (IR) injury is one of the major obstacles in clinic. Hypoxia-inducible factor (HIF), A2A adenosine receptors (A2AR) and regulatory T lymphocytes (Treg) Cells are involved in the injury, but the interrelation among them is still unknown. Our previous investigation has led to the finding that HIF-2α had a closely relation with intestinal IR injury and palyed an important role in intestinal protection which may be mediated by A2AR which may be dependent on Treg Cells. But the exact mechanism of HIF-2α regulating the A2AR protection to IR injury and how A2AR stimulation affects Treg cells are not well understood．After preliminary experiment and literature review, we hypothesize that the protective mechanism would be the Treg Cells influenced by the A2AR pathway directed by HIF-2α. To explore the mechanism, we utilized the Cre/Loxp recombination system to generate HIF-2α knockout/overexpress mice. Afterwards, mice were subjected to intestinal IR and the cells were subjected to hypoxia/reoxygenation (HR) injury and mixed lymphocyte reaction, to testify the A2AR of Treg cells on the protection effects of HIF-2α activation. Using antibody chips, we will investigate how HIF-2α regulates A2AR in the changes of inflammatory cytokines and chemokines. Finally, we utilized the technique of EMSA and Luciferase Reporter Assay to verify whether the upstream of the A2AR has some region with HIF-2α gene. Then we will clarify the exact pathway that after the HIF-2α-mediated A2AR, Treg acquired stronger intestinal IR protective activity. So it might be a viable therapeutic option that exogenous influences could be developed to mimic both processes and creat a synergistic effect in attenuating IR injury.

小肠缺血再灌注（IR）损伤是临床的重要问题。缺氧诱导因子（HIF）、腺苷A2A受体（A2AR）、调节性T（Treg）细胞分别在其中发挥作用，但相互关系尚不清楚。本课题组前期的研究结果提示HIF-2α与IR损伤有密切关系，并通过A2AR发挥保护作用，预实验结果显示机制中涉及Treg细胞的参与。推测HIF-2α可能通过上调Treg细胞表面的A2AR促使Treg细胞增殖来实现小肠IR保护作用。拟采用Cre/Loxp技术获得HIF基因敲除及过表达小鼠，应用“小鼠小肠IR损伤模型”、“缺氧再复氧损伤细胞模型”、“离体共培养抑制”等阐明Treg细胞上A2AR在不同HIF基因背景下所发挥的作用。通过EMSA技术和荧光素酶报告实验验证Treg细胞中A2AR基因上游存在与HIF-2α结合的启动子。进一步阐明HIF-2α调节A2AR进而通过Treg细胞发挥小肠IR损伤作用的机制，为IR损伤防治提供新干预靶点。

小肠缺血再灌注(IR)损伤是临床的重要问题。缺氧诱导因子(HIF)及腺苷与器官IR损伤的关系密切，但二者之间在小肠 IR 中具体联系尚不明确。IR在细胞层面表现为缺氧再复氧损伤。HIF-1a是诱导低氧反应基因和修复细胞氧内环境稳定的核心调节因子，对缺血缺氧条件下保护细胞免于细胞凋亡，保持正常功能状态有着至关重要的作用。我们通过缺氧培养模拟IR过程，以CACO2细胞为研究对象，以常氧培养下的细胞为对照组，1% O2 缺氧环境下，检测发现HIF-1a mRNA及蛋白表达水平均增高。既往以缺血损伤为模型的实验中发现细胞外的腺苷作为免疫调节因子，能够保护细胞免受过度的免疫介导的损害。ATP或ADP可被CD39转换为5-AMP，进一步被CD73转换为腺苷，在缺血损伤中发挥免疫调节作用。我们的研究发现缺氧能够提高CD39及CD73的mRNA表达，促使单个细胞分泌腺苷水平升高；而在缺氧环境中予以HIF-1a抑制剂glyceryl trinitrate，除能抑制缺氧诱导的HIF-1a表达，CD39、CD73 mRNA及蛋白表达也被抑制，单个细胞分泌的腺苷水平也下降。在体内实验中，通过小鼠小肠IR损伤模型，我们发现IR损伤后，小肠组织中的HIF-1a、CD39、CD73 以及腺癌受体A2AR mRNA表达水平升高，提示IR损伤后，HIF-1a、CD39及CD73表达升高。腹腔注射HIF-1a抑制剂glyceryl trinitrat能够抑制IR导致HIF-1a表达升高，同时CD39、CD73及A2AR表达也被抑制。因此此项研究的开展可能提供一种HIF-1α/A2AR的信号通路对IR损伤中炎症反应的保护性治疗方案。