Bruton酪氨酸激酶致糖尿病肾脏巨噬细胞激活的作用及分子机制

Bruton's tyrosine kinase (Btk), a member of the Tec family of non-receptor tyrosine kinases, has been demonstrated to be an important molecular of Toll-like receptor (TLR) signal pathway. In provious study, we found out that activation of the downstream proinflammatory pathways of TLR in renal macrophages could lead to the enhancement of inflammatory response in the kidney and the development and progress of diabetic nephropathy (DN). Our preliminary experiments showed that p-Btk expression was increased in renal tissue from diabetic mice and macrophages cultured in high glucose, and Btk inhibitors could inhibit the activation of macrophages. Therefore, we hypothesize that Btk can activate proinflammatory pathways in renal macrophages, resulting in inflammatory response in the kidney, and involves the development and progress of DN. In this study, diabetic model of mice and macrophages cultured in high glucose are utilized. The main object of study is the renal macrophages and Btk knock out and RNA interference are also used. We aim to investigate the impact of renal macrophage Btk on the development and progress of DN, and study the relation of Btk to the activation of downstreams of including MAPKs and NF-kapa B as Well as other signal pathway such as JAK/STAT1 and PI3K/Akt. Based on the above experiments, the molecular mechanism of Btk to regulate renal macrophage activation will be deeply studied. The results of this study will provide new ideas and targets in the prevention and management of DN.

Bruton 酪氨酸激酶(Btk)是非受体类酪氨酸激酶Tec家族成员之一，研究发现它是TLR信号通路的一个关键分子。我们前期发现巨噬细胞TLR下游信号通路的活化可增强肾脏炎症反应与糖尿病肾病(DN)发生、发展，预实验发现糖尿病小鼠肾组织及高糖培养的巨噬细胞p-Btk表达增加，Btk抑制剂可抑制高糖环境下巨噬细胞激活。因此，我们假设Btk可能通过激活促炎症信号通路活化肾脏巨噬细胞，促发炎症反应，参与DN的发生、发展。本研究拟采用小鼠糖尿病模型、体外高糖培养的巨噬细胞为主要研究对象，应用Btk 基因敲除小鼠、RNA干扰等手段，观察肾脏巨噬细胞Btk与DN的相关性，并明确其与TLR下游的MAPK和NF-κB信号通路及JAK/STAT1和PI3K/Akt信号通路活化的关系，深入探讨Btk调控巨噬细胞活化的分子机制。本项目的实施有望为DN的防治提供新的思路和线索。

近年来，研究者认为糖尿病肾病是一种慢性炎症性疾病，肾脏的微炎症状态加速了肾脏功能下降，巨噬细胞作为固有免疫的主要细胞，在糖尿病肾脏炎症中起着重要的作用.，因此在控制血糖、血压的基础上控制肾脏的炎症水平及降低巨噬细胞的浸润及激活则为一种新的治疗手段。Btk仅表达于髓系细胞，减低其表达进而降低炎症水平状态，从而保护肾脏。该研究以链脲佐菌素诱导的1型糖尿病小鼠及骨髓来源的巨噬细胞为研究对象，探讨Bruton酪氨酸激酶（Btk）致糖尿病肾脏巨噬细胞激活及可能分子机制。其主要成果包括：Btk的敲除可降低糖尿病小鼠肾脏损伤、24小时尿蛋白排泄率、肾脏的炎症水平，此现象的发生可能与MAPK及NF-κB信号通路的抑制的有关；随之体外实验发现，在高浓度葡萄糖的刺激下，骨髓来源的巨噬细胞易被激活转化为M1型巨噬细胞，并且巨噬细胞的趋化、分泌炎症因子的功能明显增强，Btk的抑制剂PCI-32765可明显抑制高糖对巨噬细胞的诱导刺激作用，并且PCI-32765抑制了信号通路ERK的激活及NF-κB p65的核转移。由此可见，Btk在巨噬细胞的激活中占据了重要的作用，降低Btk糖尿病小鼠肾脏的表达可改善小鼠肾脏的损伤。