Th17/IL-17途径在小鼠糖尿病肾病进展中的作用及其分子机制研究

Until recently, a third subset of IL-17-producing effector T helper cells, called Th17 cells, has now been discovered and characterized. Since IL-17 induces cytokine and growth factor production in various cell types, there has been increasing attention to the role of IL-17 in the pathogenesis of various inflammatory diseases. Although significant association between Th17/IL-17 pathway and type 2 diabetes mellitus as well as microangiopathy has been reported , the role of the IL-17 pathway in the pathogenesis of diabetic nephropathy (DN) has not been well studied previously. Therefore，in the current study, experiments in vivo will be conducted, including observing correlation between the differentiation of Th17 cells, secretion of IL-17 and the stage of DN progression, and then comparing the change of DN progression on condition whether inhibiting IL-17 or not，to demonstrate the role of the Th17/IL-17 pathway in the pathogenesis of DN in T2DM mice. Subsequently, diabetic and physiological environment will be simulated by experiments in vitro to demonstrate, on transcription and translation level, the molecular mechanism of inflammatory reaction regulated by IL-17 in circumstance of high glucose in murine mesangial cells. After stimulating murine mesangial cells with IL-17, we examine whether chemotatic factors will be induced by IL-17 and seek the possible signal pathway on which the induced chemotatic factors might be dependent. In conclusion, the project will further clarify the mechanism of type 2 diabetes mellitus as well as micro vascular complications. Moreover, it also contribute to offering the evidence that targeting the Th17/IL-17 pathway may be a promising therapeutic strategy for the treatment of type 2 diabetes mellitus.

Th17细胞是近年新发现的一类T细胞亚群，分泌特征性细胞因子IL-17。Th17/IL-17途径在炎性疾病中的作用逐渐成为研究热点。Th17/IL-17途径与2型糖尿病（T2DM）及糖尿病微血管病变关系密切，但其在糖尿病肾病（DN）进展中的作用及分子机制还不清楚。本项目通过体内实验，考察Th17细胞分化、IL-17表达与DN分期的关系，比较正常和抑制IL-17条件下T2DM小鼠DN的进展变化，阐释Th17/IL-17途径在DN进展中的作用；在体外实验中，模拟糖尿病及正常生理环境，IL-17干预鼠肾小球系膜细胞后，观察IL-17对炎性趋化因子表达的影响及其所依赖的信号通路，着力揭示高糖环境下IL-17在转录和翻译水平上调控肾小球系膜细胞炎性反应的分子机制。通过本研究，有助于阐明T2DM及其微血管并发症的发病机制，并为选择T2DM抗炎治疗的新靶点提供理论和实验依据。

项目研究了Th17/IL-17途径在小鼠糖尿病肾病进展中的作用及其分子机制。在体内实验中，我们考察了不同周龄db/db小鼠肾功能和肾组织病理学变化，检测了不同周龄db/db小鼠的Th17细胞分化状况及肾组织中IL-17及Th17相关转录因子的mRNA和蛋白表达的变化，发现（1）随周龄增加，db/db小鼠的脾脏Th17/CD4+T细胞比例、血清IL-17水平逐渐升高，均高于同周龄的db/m小鼠；（2）db/db小鼠肾皮质IL-17及其相关转录因子RORγt的mRNA及蛋白表达均较同周龄db/m小鼠升高。（3）IL-17抗体治疗后，db/db小鼠肾功能及肾组织中IL-17 mRNA水平明显降低；Th17细胞相关转录因子RORγt、RORα的mRNA表达也被抑制。上述结果表明：IL-17参与了糖尿病肾病的发生发展，抑制IL-17能够延缓糖尿病肾病的发生发展。在体外实验，我们观察了IL-17干预高糖环境下肾小球系膜细胞炎性反应的分子机制和相关信号通路，发现（1）高糖作用24hr、48hr和72hr均能够促进系膜细胞IL-17 mRNA表达，其中以48h上调效果最为显著，约为对照组的2.5倍。western结果表明高糖环境下系膜细胞中IL-17的蛋白水平较正常糖环境相比也明显升高。（2）50,100和200ng/ml的mrIL-17分别干预高糖培养的系膜细胞2、4、6、12、24hr，均能提高高糖环境下MCP-1和MIP-2的表达，其中以100 ng/ml作用效果最显著。其作用在12h达峰值。结果提示IL-17在体外能够促进肾小球系膜细胞表达炎性趋化因子。（3）mrIL-17干预后高糖环境培养肾小球系膜细胞后，磷酸化的ERK1/2、JNK蛋白以及NF-κB蛋白表达升高；而MAPK途径抑制剂U0126和NF-κB途径抑制剂PDTCS干预系膜细胞后，mrIL-17诱导的MCP-1和MIP-2的蛋白表达水平降低。上述结果表明，MAPK和NF-κB途径介导了IL-17对高糖环境下系膜细胞中炎性趋化因子表达的调节。. 本研究明确了Th17/IL-17途径在小鼠DN发生发展中的作用，揭示出高糖环境下IL-17通过调控炎性趋化因子MCP-1和MIP-2的表达介导了肾小球系膜细胞的炎性反应及参与调控的信号通路，为阐明T2DM及DN的发病机制具有重要意义。