蛋白激酶 CK2介导SphK-1/S1P通路调节糖尿病肾脏纤维化的研究

It has been paid great attention that the pathology of renal fibrosis in diabetes is close associated withchronic renal inflammation. Our previous studies have demonstrated that SphK-1/S1P signaling pathway is a crucial regulator in diabetic renal fibrosis. However, the molecular mechanism by which SphK-1/S1P signaling pathway influences inflammation activation remains further elucidation. Recently, mounting evidence has shown that protein kinase CK2 activation closely related to glucose metabolism disorder and inflammatory reaction. Whether CK2 involves in the pathogenesis of diabetic renal fibrosis through SphK-1/S1P signaling pathway arise our researching interest. Preliminary experiments by us indicated that CK2 might play a role in diabetic renal fibrosis which connected with SphK-1/S1P signaling pathway. Based on these foundations, the current project was performed to confirm the regulatory effects of CK2 on diabetic renal fibrosis with the exploition of in vivo animal models, in vitro cell models, and protein-protein interaction methods. Then, further efforts will be made to explore the potential mechanisms of CK2 in diabetic renal through mediating the regulation of SphK-1/S1P signaling pathway on inflammatory nuclear factors, NF-κB and AP-1. Taken together, this work provide objective experimental evidence for revealing new mechanisms of inflammation activation in diabetic renal fibrosis and developing new therapeutic target to delay diabetic nephropathy, which has great study significance.

糖尿病肾脏纤维化病变是肾脏慢性炎性反应的观点受到高度重视。我们已证实SphK-1/S1P信号通路对糖尿病肾脏纤维化成分起关健调节作用，然其影响炎性活化的机制尚需进一步阐明。近年来有充分证据表明蛋白激酶CK2的活化与机体糖代谢紊乱、炎性反应病变关系密切。CK2能否影响糖尿病肾脏纤维化的病理进程？该作用是否与SphK-1/S1P信号通路密切相关？引起我们特别关注。预实验结果显示了CK2可能具有该作用及其与SphK-1/S1P通路相关联的重要信息。本项目在此基础上，拟通过体外细胞模型、体内动物模型、蛋白质相互作用等途径系统研究确证CK2影响糖尿病肾脏纤维化的作用；探讨CK2介导SphK-1/S1P通路影响NF-κB、AP-1等炎症相关核因子的活化，参与调节糖尿病肾脏纤维化的可能作用机制。为阐明糖尿病肾脏纤维化炎性反应活化的新机制、探索抗DN新的潜在作用靶点提供客观的实验依据，具有重要的研究意义。

项目背景与主要研究内容：糖尿病肾脏纤维化病变是肾脏慢性炎性反应的观点受到高度重视。我们已证实SphK-1/S1P信号通路对糖尿病肾脏纤维化成分起关健调节作用，然其影响炎性活化的机制尚需进一步阐明。近年来有充分证据表明蛋白激酶CK2的活化与机体糖代谢紊乱、炎性反应病变关系密切。CK2能否影响糖尿病肾脏纤维化的病理进程？该作用是否与SphK-1/S1P信号通路密切相关？引起我们特别关注。预实验结果显示了CK2可能具有该作用及其与SphK-1/S1P通路相关联的重要信息。本项目在此基础上，拟通过体外细胞模型、体内动物模型、蛋白质相互作用等途径系统研究确证CK2影响糖尿病肾脏纤维化的作用；探讨CK2介导SphK-1/S1P通路影响NF-κB1等炎症相关核因子的活化，参与调节糖尿病肾脏纤维化的可能作用机制。. 重要研究结果与意义：. 1. 体内、外研究结果显示：抑制CK2活性或小分子干扰CK2α明显抑制高糖处理GMCs的FN、ICAM-1表达，明显抑制高糖处理GMCs的IκBα的降解及p65的核聚积，明显抑制高糖处理GMCs的NFκB的DNA结合活性及转录活性；抑制CK2活性或基因沉默CK2腺病毒处理明显改善糖尿病动物的肾脏纤维化，明显减少糖尿病动物肾脏中的IκB降解及p65的核聚积；TBB治疗或基因沉默CK2腺病毒治疗明显改善糖尿病动物肾纤维化，TBB治疗或基因沉默CK2腺病毒治疗可抑制糖尿病动物肾小球IκB降解和NF-κB核积聚。证实：CK2在调节糖尿病肾脏炎性纤维化病变过程中发挥重要作用。 . 2. 进一步研究结果显示：敲低SphK1下调GMCs中FN和ICAM-1的蛋白水平，并抑制NF-κB的核转位；敲低CK2α抑制过表达外源性SphK1引起的FN、ICAM-1升高，同时降低NF-κB的核转位及DNA结合活性；过表达外源性SphK1增加正常糖或高糖培养GMCs的pTyr255 CK2α蛋白水平，而敲低SphK1则抑制高糖刺激引起的pTyr255 CK2α蛋白水平升高； SphK1与CK2α在GMCs中存在相互作用，而高糖刺激则能增强两者之间的相互作用。证实：SphK1/S1P通路通过调节蛋白激酶CK2α的活化、影响NF-κB通路，参与调节了糖尿病肾脏纤维化的病理进程。