靶向调控ITAM/ITIM-FcγRs及SYK信号级联在糖尿病肾病早期炎症机制中的作用

Diabetic nephropathy(DN) is a low grade inflammatory disease trigged by metabolic disorder. Inflammation has an important role in the early stage of DN pathogenesis. Inflammatory cytokines, such as C-reactive protein(CRP),have effects on cell through binding immunoglobulin G Fc receptors (FcγRs) in the surface of cell. We previously found that the expression of FcγRs with an immunoreceptor tyrosine-based activation motif(ITAM-FcγRs) increased in the kidney of diabetic CRP-Tg mouse induced by STZ. ITAM-FcγRs increased, FcγRs with an immunoreceptor tyrosine-based inhition motif(ITIM-FcγR) decreased and spleen tyrosine kinase(SYK) activated in cultured rat glomerular mesangial cells(GMC) ambient high glucose. A role for activated SYK signal cascade leading to the cell responses and pro-inflammatory cytokines production has been established in antibody-dependent kidney disease. Based on previous findings, we hypothesized that the unbalance of ITAM /ITIM-FcγRs and activation of SYK play a central role of inflammatory mechanism in the early stage of DN. Therefore, we plan to construct the GMC, tubular epithelial cells and SD rats models of ITAM- FcγRs , SYK gene inhibition and ITIM-FcγR overpxression(wild-type)by transfection in vivo and vitro in the current study. Then, the cells will be stimulated by high glucose and the rats treated with streptozotocin. The expression and activity of ITAM-FcγRs, ITIM-FcγR and SYK gene influences on inflammatory signaling pathways and molecular markers related with fibrosis will be tested at different time points. We aim to investigate the role of ITAM/ITIM-FcγRs unbalance and their effects on SYK cascade in the early stage of DN and to identify new target for treatment and prevention of the disease.

炎症机制在早期糖尿病肾病（DN）中起重要作用。以CRP为代表的因子通过与免疫球蛋白G Fc受体（FcγRs）相结合而促进炎症。我们前期研究发现：糖尿病CRP转基因小鼠肾脏表达含免疫受体酪氨酸激活基序的FcγRs(ITAM-FcγRs)上调；高糖刺激系膜细胞ITAM-FcγRs增多，含免疫受体酪氨酸抑制基序的FcγR(ITIM-FcγR)减少，脾酪氨酸激酶（SYK）活化。由此我们假设：ITAM/ITIM-FcγRs表达失衡影响SYK信号级联是DN早期炎症机制的核心。因此,本课题采用体内外转染方法,构建ITAM-FcγRs、SYK基因抑制、ITIM-FcγR过表达的细胞和SD大鼠，高糖刺激细胞和STZ诱导动物模型，在不同时间点检测ITAM/ITIM-FcγRs表达及对SYK信号级联的影响，从分子-细胞-整体水平探讨调控ITAM/ITIM-FcγRs影响SYK信号级联在早期DN的作用机制。

糖尿病肾病（diabetic nephropathy, DN）是糖尿病最主要的微血管并发症，在世界范围内都是终末期肾病（end stage renal disease, ESRD）的首位病因。其发病机制至今尚未完全阐明，缺乏有效的干预措施。探讨DN的发病机制和早期防治具有重大的社会意义和经济价值。既往研究发现炎症机制在早期糖尿病肾病（DN）中起重要作用。我们前期研究发现：糖尿病CRP转基因小鼠肾脏表达含免疫受体酪氨酸激活基序的FcγRs(ITAM-FcγRs)上调；高糖刺激系膜细胞ITAM-FcγRs增多，含免疫受体酪氨酸抑制基序的FcγR(ITIM-FcγR)减少，脾酪氨酸激酶（SYK）活化。本课题在前期研究基础上采用基因重组技术，构建SYKshRNA的质粒，分别进行体外转染人肾小管上皮细胞株HK-2和体内转染C57BL6糖尿病小鼠模型，在不同时间点，检测ITAM/ITIM-FcγRs表达及对SYK及其信号级联-核转录因子-κB （NF-κB）、下游信号通路关键炎症因子白介素-1β(IL-1β)、肿瘤坏死因子-α（TNF-α）表达的影响，分析肾组织病理变化。我们的研究发现DN成模小鼠肾组织炎症细胞浸润明显，尿蛋白增加，ITAM-FcγRs表达上调，SYK表达及磷酸化增加，NF-κB信号通路激活，炎症因子IL-1β、TNF-α表达上调。予以SYKshRNA干预后，DN小鼠肾组织炎症细胞浸润明显减轻，尿蛋白减少，部分抑制激活的NF-κB信号通路，降低炎症因子IL-1β、TNF-α表达，证实ITAM/ITIM-FcγRs表达失衡激活SYK信号级联是DN早期炎症机制的核心，为靶向调控SYK这一“信号开关”分子防治DN早期炎症损伤，延缓DN肾小球硬化及肾小管间质纤维化奠定理论基础。