GLCCI1效应弱化在TNF-α诱导GRIP1解离所致哮喘个体激素敏感性降低过程中的作用机制

Studies have shown that TNF-α can induce steroid resistant asthma by dissociating GRIP1 from the GR transcriptional regulatory complexes, and another fact is the mutation of GLCCI1 rs37973 can result in inhibition response to GC by decreasing the expression of GLCCI1, which will down-regulate GR function. We recently discovered that, TNF-α is capable of inhibiting the expression of GLCCI1. Therefore, it is speculated that TNF-α might induce the dissociation of GRIP1 from the GR transcriptional regulatory complexes by inhibiting the biological effects of GLCCI1, which reduces the responsiveness to GC in patients with asthma. Based on the above, the project intends to carry out the following works: (1) To investigate the regulatory relationship between TNF-α and the variant of GLCCI1 rs37973 in the patients with steroid resistant asthma. (2) To explore the negative effects of GLCCI1 on “GRIP1 dissociation induced by TNF-α” in the patients with steroid resistant asthma. (3) The GLCCI1 KO mice will be used to build asthmatic models, and the aim is to evaluate the weights of GLCCI1 in the pathogenesis of steroid resistant asthma induced by TNF-α.

研究表明，TNF-α能够诱使GRIP1从GR转录调控复合体中解离导致哮喘患者对激素敏感性降低，而GLCCI1 rs37973位点突变所致GLCCI1表达下调亦能通过影响GR功能进而降低激素效应。我们发现，TNF-α能够诱导GLCCI1表达下调，因此推测GLCCI1可能通过弱化自身效应对TNF-α进行应答，诱导GRIP1从GR转录调控复合体中解离最终降低哮喘患者激素敏感性。基于上述，本项目拟开展以下工作：（1）探讨哮喘患者激素敏感性变化过程中，rs37973多态性所致GLCCI1表达差异与TNF-α的对应调控关系。（2）利用rs37973多态性进行分组，探讨哮喘患者激素敏感性变化过程中GLCCI1对“TNF-α诱导GRIP1解离”的负向应答机制。（3）利用GLCCI1基因敲除小鼠建立哮喘模型，进一步探讨GLCCI1对激素效应的影响及其在TNF-α诱导哮喘个体激素敏感性降低过程中的作用权重。

GLCCI1表达下调可能与哮喘患者激素敏感性降低存在关联，结合前期研究基础，本项目拟探讨 GLCCI1表达弱化对糖皮质激素受体作用元件的调控作用借以明确哮喘激素敏感性降低的发生机制。.纳入轻中度哮喘患者，给予糖皮质激素吸入治疗，记录治疗前后GLCCI1表达水平增幅与及第一秒用力呼气量（FEV1）变化幅度之间的对应关系。用野生型和GLCCI1基因敲除（KO）小鼠构建OVA哮喘模型，并用GLCCI1 siRNA干预 Beas2B细胞和A549细胞，分别用地塞米松进行处理。检测各组小鼠气道阻力及肺泡灌洗液总细胞数，RT-PCR检测各组小鼠肺组织CD38、FKBP5、GILZ、CCL2、CCL3、CCL4、CCL7、IFN-γ、TNF-α、IL-4、IL-6和IL-13的表达水平，RT-PCR和WB方法检测各组小鼠肺组织和各组细胞中GLCCI1、GR、GRIP1、IRF1和IRF3的表达情况，CO-IP检测地塞米松干预小鼠肺组织和上皮细胞中GR和GRIP1、IRF1和GRIP1以及IRF3和GRIP1之间的结合情况。. 结果发现，在激素吸入治疗前后哮喘患者GLCCI1 mRNA表达变化水平及FEV1变化幅度呈正相关关系。与野生型小鼠相比，地塞米松治疗GLCCI1 KO哮喘小鼠肺组织炎症、气道阻力、肺泡灌洗液总细胞数降低不明显，CD38表达水平增高且GILZ和FKBP5表达水平未见明显增高。同时，GLCCI1 表达降低可上调地塞米松治疗组中CCL4、CCL7、IFN-γ、TNF-α、IL-6的表达水平。与野生型哮喘小鼠相比，地塞米松干预导致GLCCI1 KO哮喘小鼠肺组织中GR和GRIP1表达水平降低但IRF1和IRF3表达水平增高，GR和GRIP1的结合水平亦明显降低，而IRF1和GRIP1以及IRF3和GRIP1结合反而增强。针对Beas2B细胞和A549细胞的体外实验结果亦与上述在体研究相一致。. 综上，GLCCI1表达下调能够导致哮喘患者及哮喘小鼠对糖皮质激素敏感性降低，其机制可能与GLCCI1表达下调通过增强IRF1和GRIP1、IRF3和GRIP1的结合从而竞争性抑制GR和GRIP1的结合存在密切关联。