自组装肽-MSC靶向给药系统治疗缺血再灌注急性肾损伤的作用及机制研究

Inflammation and fibrosis are key factors that affect the progression of acute kidney injury (AKI) and its transit chronic kidney disease (CKD). Mesenchymal stem cells (MSCs) have the ability of migration and homing to the injury sites as well as the anti-inflammatory effect. Combined with functionalized self-assembling peptide (SAP) and MSC, we design a therapeutic carrier that can target delivery and release of anti-inflammatory and anti-fibrotic proteins. This therapeutic carrier will synergistically improve the therapeutic efficacy of MSC transplantation for AKI..The project intends to: 1) Design and synthesis of functionalized SAPs that consist of MSC surface binding, MMP-sensitive degradation and protein linked sequence, and analyzed the morphology, physical/chemical and degradation properties. 2) Conjunction of anti-inflammatory/anti-fibrotic proteins to SAP nanofibers by biotin-avidin method, evaluating the protective effects of SAP-engineered MSC on the hypoxia/reoxygenation (H/R)-induced renal tubular epithelial cell injury. 3) SAP-engineered MSCs transplantation for the treatment of ischemic/reperfusion injury (IRI) -AKI rats, and analyzed the in vivo distribution and protein release conditions, and its effects on the renal function, histopathology, pro-inflammatory and pro-fibrotic factor expression, evaluating the therapeutic efficacy of SAP-engineered MSC on AKI. 4) Combined with metabolomics and molecular biology method to analyze the changes of signaling molecules related to apoptosis, inflammation and fibrosis in renal cells, and explore the potential mechanism. This study will help to improve the therapeutic efficacy of AKI, and reduce the risk of AKI transit to CKD, provide insight into the treatment of AKI in further clinical studies.

炎症反应和纤维化是急性肾损伤（AKI）及其向慢性肾病（CKD）发展的关键因素。利用MSC向损伤肾脏迁移及抗炎特性，基于自组装肽构建能同时靶向运载并释放抗炎/抗纤维化蛋白的智能给药系统，协同MSC作用治疗AKI。.本项目拟：1）设计合成具有MSC结合-MMP降解-蛋白偶联三个功能的自组装肽，检测其形态和理化性质；2）通过多肽-MSC运载HGF/BMP-7等，体外研究其对肾小管上皮细胞H/R损伤的作用，筛选具有显著保护作用的组合；3）多肽-MSC系统移植治疗AKI大鼠，研究其体内定植和蛋白释放情况，及其对肾功能、组织病理、炎症/纤维化因子表达等影响，综合评价其治疗AKI的疗效；4）采用代谢组学和分子生物学方法研究多肽-MSC能否阻断肾脏细胞凋亡、炎症和纤维化等相关信号通路，阐明可能的效应机制。本研究有助于提高AKI治愈率，降低其向CKD转归风险，为临床MSC移植治疗AKI研究提供新的思路。

急性肾损伤（AKI）是威胁人类健康的重要疾病之一，目前临床仍缺乏有效的促进AKI后肾脏再生的方法。本项目研究针对AKI后氧化应激、线粒体损伤和炎症反应等关键病因，提出结合自组装肽（SAP）纳米材料及间充质干细胞（MSC）治疗的优势，构建能递送治疗药物或MSC活性成分的功能性纳米材料，降低AKI后急性期损伤及炎症反应，同时促进肾脏再生，从而协同改善肾脏长期预后。在这一研究思路指导下，我们主要开展了以下几个方面的工作：（1）设计合成新型SAP水凝胶局部递送药物治疗肾损伤。我们新合成的KLDD水凝胶能在体外延长线粒体抗氧化剂（MT）的释放时间，在AKI小鼠体内降低肾脏线粒体氧化应激，恢复线粒体能量代谢与线粒体生物合成，从而减轻肾脏细胞凋亡、坏死及慢性炎症反应，促进受损肾脏恢复。KLD2R/Hep复合水凝胶能序贯缓释两种不同药物（anti-TNF-ɑ/HGF）并保持其释放后的生物活性；快速释放的anti-TNF-ɑ降低AKI后炎性反应，而持续释放的HGF可促进肾小管再生，发挥协同效应减轻慢性肾脏纤维化发生。（2）采用SAP水凝胶局部递送MSC来源的胞外囊泡（MSC-EV）治疗肾损伤。KMP2水凝胶能够有效的在体内外缓慢释放MSC-EV，并保持其原有生物活性。AKI小鼠体内给予载EV的水凝胶治疗，通过增强MSC-EV的抗炎/抗凋亡及促进血管再生潜能，改善AKI后慢性肾脏纤维化。（3）具肾保护作用药物的鉴定及其作用机制研究。PGA可通过改善AKI后肾脏炎症反应、线粒体损伤和脂代谢紊乱等机制恢复肾脏代谢稳态而降低肾损伤。RSV可通过激活Sirt1抑制Smad3磷酸化及Smad3/Smad4乙酰化，阻断促纤维化的关键信号通路TGF-β/Smad而减轻肾脏纤维化。Ploretin可通过抑制NLRP3炎症小体信号和调节UA代谢而减轻肾脏炎性/纤维化损伤，为后续采用SAP水凝胶或EV递送肾保护药物治疗AKI建立实验基础。这些研究结果提示，基于SAP纳米水凝胶的递送系统能有效保护药物/EV的生物活性，延长其体内外释放及作用时间，从而降低肾脏氧化应激、线粒体和炎性损伤，有可能成为促进受损肾脏再生的新型疗法。