肢体缺血预适应调控miR-21/PDCD4通路对脓毒症急性肾损伤的作用及其机制研究

Acute kidney injury (AKI) is a common and life-threatening complication in patients with sepsis. Inflammation and cell apoptosis are important pathophysiological mechanisms of septic AKI. It has been demonstrated that miR-21 is an important gene in regulating apoptosis and inflammation, involving in the development and repair of AKI. Our previous studies showed that renal ischemic preconditioning upregulated miR-21 expression in kidneys in time-dependent manner, inhibited programmed cell death 4 (PDCD4) expression, and protected against renal ischemia-reperfusion injury via inhibiting apoptosis. Our preliminary experiment exhibited that limb ischemic preconditioning (LIPC) upregulated miR-21 in gastrocnemius muscle, as well as in kidneys, and reduced endotoximia-induced renal injury in mice, but its mechanisms are not clear. We hypothesize that limb ischemic preconditioning increased expression of miR-21 in local tissue, and the miR-21 is released into circulation, transferred to remote organs as a potent endocrine factor, exerts anti-apoptotic and anti-inflammatory effects. In this study, with the septic AKI model in miR-21 knock out mice and wild mice, we further explore the protective effects of LIPC on apoptosis and inflammation, and its underlying mechanisms, and elucidate the potential mechanisms that limb ischemia regulates miR-21/PDCD4 pathway in local tissue and remote organ (kidney). This study will provides a new strategy and target for the prevention of septic AKI.

脓毒症患者急性肾损伤（AKI）的发生率高、预后差。炎症反应和细胞凋亡是脓毒症性AKI的重要病理生理机制。已证实miR-21参与细胞凋亡和免疫炎症的调控，在AKI的发生和修复中起重要作用。我们发现，肾脏缺血预处理可上调miR-21表达，抑制程序性细胞死亡因子4（PDCD4），抑制细胞凋亡，减轻肾脏缺血性损伤。预实验亦初步证实小鼠后肢缺血预处理可上调局部组织和肾脏miR-21的表达，且能减轻内毒素血症诱发的肾损伤，但具体机制尚不清楚。我们推测肢体缺血预适应上调局部组织miR-21表达，miR-21再通过内分泌机制，到达远隔器官，发挥抗凋亡和抗炎作用。本项目在前期研究基础上，拟通过miR-21基因敲除小鼠和野生型鼠的脓毒症AKI模型，深入探讨肢体缺血预适应对脓毒症性肾损伤的防治作用及其机制，阐明肢体缺血调控局部和远隔器官miR-21/PDCD4通路的分子机制，从而为AKI的防治提供新策略和靶点。

脓毒症诱发的急性肾损伤（acute kidney injury, AKI）是脓毒症常见的急性并发症，发生率高、预后差，是脓毒症患者的重要死因。目前仍缺乏安全、有效的防治措施。研究证实肢体远程缺血预适应（remote ischemic preconditioning, rIPC）作为一种内源性保护机制，具有远隔器官的保护作用。本项目旨在研究rIPC对脓毒症AKI的干预作用，并探讨可能的机制。首先利用脂多糖和盲肠结扎穿孔(CLP)建立两种脓毒症小鼠模型，证实rIPC可显著抑制系统性和脏器局部炎症反应、减少实质细胞凋亡，从而减轻CLP和脂多糖诱发的小鼠肾脏、肝脏、肺脏损伤。继之，探索rIPC的肾保护机制，重点探讨miR-21在介导脓毒症AKI肾保护中的作用机制。发现rIPC可诱导骨骼肌低氧诱导因子1α（hypoxia-inducible factor 1 ahpha, HIF-1α）激活，在转录水平上调miR-21表达；通过构建miR-21基因敲除小鼠和抑制骨骼肌HIF-1α基因表达，证实rIPC通过激活骨骼肌HIF-1α/miR-21通路促进远隔肾脏miR-21表达，进而通过调控其下游靶蛋白 PTEN和PDCD4的表达，发挥抗炎、抗凋亡作用，抑制肾损伤。最后，探讨肢体缺血预适应诱导的骨骼肌miR-21通过何种途径保护远隔肾脏。我们通过体外、体内实验，发现缺血预处理后小鼠外周血外泌体miR-21表达上调；缺氧预处理后细胞上清中外泌体miR-21表达上调。富含miR-21的外泌体输注，可以明显减轻脓毒症小鼠的炎症反应和急性肾损伤；另外，缺氧预处理的小管上皮细胞上清分离的外泌体，体外干预肾小管上皮细胞，对脂多糖处理的上皮细胞具有保护作用。最终证实rIPC诱导骨骼肌miR-21上调，通过外泌体途径将miR-21运送至远隔肾脏，进而发挥抗炎、抗凋亡作用，抑制脓毒症AKI。该研究不仅拓展了肢体缺血预适应在防治急性肾损伤中的研究，也为以HIF-1α/miR-21/PDCD4 为靶点干预脓毒症性急性肾损伤提供理论依据，从而为AKI 的防治提供新的靶点和策略。