创新单抗融合蛋白调控代谢和炎症及其下游细胞焦亡治疗糖尿病肾病的作用及机制

Diabetic nephropathy (DN), one of the leading causes of uremia, is related to abnormal metabolism, chronic inflammation, and pyroptosis. It was reported that targeting vascular endothelial growth factor B (VEGF-B) was likely to ameliorate DN through modulating the glucose and lipid metabolism. Therefore, A novel anti-VEGF-B antibody has been generated, and its therapeutic effect on DN has been studied in our preliminary research. Our data showed that the anti-VEGF-B antibody could significantly attenuate the albuminuria but had limited effect on the nephritic inflammation and pyroptosis. We have demonstrated that interleukin-22 (IL-22) can suppress the diabetic nephropathy via attenuating the expression of inflammatory cytokine IL-1β and pyrotosis-related protein NLRP3, so the IL-22 will be fused to anti-VEGF-B antibody to endow the antibody with anti-inflammation and -pyroptosis functions. Then, the therapeutic effects of the fusion protein against DN will be investigated in vitro and in vivo, as well as the role of the fusion protein in modulating the lipid metabolism, glycometabolism, inflammation and pyroptosis. Through the evaluations on the related signaling pathways of lipid metabolism, glycometabolism, inflammation and pyroptosis, such as NLRP3/Caspase 1, p42/44 MAPK and NRP-1/FATPs, our research will elucidate how the anti-VEGF-B and IL-22 fusion protein ameliorates the process of DN, which will highlight a novel approach for diabetic nephropathy therapy.

糖尿病肾病是尿毒症的主要病因，发病机制与代谢、炎症及其下游的细胞焦亡相关。有报道称靶向VEGF-B有望通过调控糖脂代谢治疗糖尿病肾病，但其对炎症和细胞焦亡的调控能力较弱。我们前期研究显示IL-22能抑制IL-1β等炎性因子及细胞焦亡相关蛋白NLRP3的表达并减轻糖尿病肾病小鼠的肾脏损伤。因此我们拟首次制备抗VEGF-B单抗，研究其对糖尿病肾病的治疗作用与机制，并进一步将VEGF-B单抗与IL-22融合表达制备一种创新的单抗融合蛋白，探讨该融合蛋白对糖尿病肾病的治疗作用。随后将研究融合蛋白对糖脂代谢、炎症及下游细胞焦亡的调控，并进一步检测其相关信号通路分子如Caspase 1、IRS、IL-1β、NRP-1、FATPs等的表达以研究融合蛋白治疗糖尿病肾病分子机制。最后通过研究代谢、炎症及其下游细胞焦亡之间的相互作用，深入研究融合蛋白治疗糖尿病肾病的机制，为治疗糖尿病肾病药物的研发提供新思路。

背景.糖尿病肾病（DN）现已成为导致终末期肾脏病最常见的原因。但DN发病机制复杂且尚不明确，临床上尚无针对DN有效的治疗方法。有研究表明拮抗血管内皮生长因子B（VEGF-B）后可以通过减轻肾脏脂毒性来改善糖尿病肾病，但尚无证据表明单独VEGF-B可以减轻糖尿病肾病其他致病因素的作用。此外有研究显示白介素22（IL-22）可通过减轻肾脏炎症反应来改善糖尿病肾病。本研究旨在构建具有抗-VEGFB抗体以及IL-22生物学效应的融合蛋白，并进一步研究该融合蛋白对DN的治疗作用及机制。.内容及结果.1.聚丙烯酰胺凝胶电泳与排阻色谱法检测anti-VEGFB/IL22融合蛋白与抗-VEGFB单克隆抗体的分子量及纯度；表面等离子共振技术发现anti-VEGFB/IL22融合蛋白、抗-VEGFB单克隆抗体与VEGF-B抗原具有较高亲和力；anti-VEGFB/IL22融合蛋白不仅激活IL-22信号通路蛋白p-STAT3、p-AKT表达，也可减轻高糖诱导的氧化应激和线粒体功能障碍；anti-VEGFB/IL22融合蛋白可以阻断VEGF-B诱导的FATP4的高表达。.2.高脂饮食喂养db/db小鼠后成功构建糖尿病肾病小鼠；给予anti-VEGFB/IL22融合蛋白治疗8周后，糖尿病肾病小鼠ACR、血肌酐、血尿素氮、血糖、血甘油三酯、血胆固醇水平显著降低；anti-VEGFB/IL22融合蛋白减轻糖尿病肾病小鼠肾脏组织病理损伤，改善糖尿病肾病小鼠足突脱落、足突融合、基底膜增厚。.3.anti-VEGFB/IL22融合蛋白降低糖尿病肾病小鼠肾脏组织炎症反应相关蛋白：NLRP3、Cleaved Caspase-1、Mature IL-1β表达水平，减轻炎症因子（TNF-α、NF-κB）表达，减轻氧化应激水平及线粒体功能障碍；油红O染色及免疫荧光染色分析发现anti-VEGFB/IL22融合蛋白可以减轻糖尿病肾病小鼠肾脏组织中中性脂聚集程度，降低ADFP及FATP4表达水平；anti-VEGFB/IL22也可以减轻糖尿病肾病小鼠肾脏组织中的糖原聚集，增加糖尿病肾病小鼠肝脏及肾脏组织中胰岛素信号通路相关蛋白p-IRS-1、p-ERK/2、p-AKT表达水平。.意义.成功构建并纯化一种新的anti-VEGF-B/IL-22融合蛋白，同时有抗VEGF-B抗体和IL-22的生物活性，证实anti-VE