EETs通过自噬/凋亡途径在肾缺血再灌注损伤中的作用及机制研究

Renal ischemia-reperfusion (I/R) injury is a leading cause of acute kidney injury (AKI). The complex mechanisms and lack of effective therapy greatly influence the outcome of AKI, which dramatically increases the financial burden on individuals and society. Our previous results showed that, EETs, the metabolites of arachidonic acid, protected against AKI through ameliorating apoptosis. And the levels of EETs changed during renal I/R injury. Based on recent study, instead of being independent from each other, apoptosis and autophagy are two closely-related, genetically-regulated and evolutionarily-conserved processes, which can convert into each other and form a cross-impact relationship. Moreover, they can regulate cell fate and play important roles in the occurrence and development of many diseases. Whether EETs influence the crosstalk between autophagy and apoptosis in AKI process? And what are the mechanisms of this phenomenon? In the current study, pharmacology administration or gene intervention in mice will be used to establish higher or lower systemic levels of EETs prior to experimental I/R-induced AKI. Based on the crosstalk between autophagy/apoptosis and its potential signaling pathway, approaches such as immunofluorescence, RT-qPCR, Western blot and LC-MS/MS will be used to evaluate the changes of renal function and structure, genes, and metabolites at different time points during the study. Our results can potentially pave the way for a targeted pharmacological intervention in disease progression or induction.

肾缺血/再灌注（I/R）是急性肾损伤（AKI）的首要原因，其机制复杂，缺乏有效干预措施, 严重影响AKI的预后并加重个人及社会的经济负担。我们前期发现，表氧化二十碳三烯酸（EETs）的水平在肾脏I/R损伤中发生着动态变化，且EETs可通过减轻细胞凋亡在AKI中起到保护作用。最近研究发现，自噬和凋亡并不是独立存在的两个过程，二者可相互转化，相互影响，共同调控细胞的生存和死亡，并在多种疾病的发生发展过程中发挥重要作用。EETs是否影响AKI进程中自噬/凋亡的交互作用？其病理生理机制如何？本课题拟从基因和药物两个方面干预内、外源性EETs水平，建立肾I/R小鼠模型，并以自噬/凋亡的相关蛋白及信号通路为切入点，应用IF、RT-qPCR、Western blot及LC-MS/MS等技术，以期阐明EETs在肾脏I/R损伤不同时期的动态变化对自噬/凋亡的作用和机制，为AKI的药物防治提供新思路。

缺血再灌注（Ischemia-reperfusion, I/R）是急性肾损伤的主要原因之一。 缺血性急性肾损伤(acute kidney injury ,AKI)时磷脂酶 A2(phospholipases A2 , PLA2)的激活是起始阶段各种早期事件的主要因素。PLA2激活在心脑 I/R损伤中起关键作用，但其对急性损伤的作用尚不清楚，环氧化二十碳三烯酸(epoxyeicosatrienoic acids, EETs)和20- 羟基乙酸(20–hydroxyArachidonic Acid，20-HETE)分别是由游离花生四烯酸（arachidonic acid ，AA)经CYP环氧化酶和羟基化酶合成的，且二者作用相反。 本研究旨在探讨EET对肾I/R损伤的作用及其机制。 首先，在测量肾脏中游离和酯化的环二十烷类化合物的水平时，我们发现EETs，不同于20-HETE，其在缺血期间没有积累，这种不平衡可能导致AKI的形成。 其次，我们对肾I/R诱导的AKI动物模型使用小型动物MRI扫描，发现EET类似物可以在血流恢复的早期即改善肾脏血氧含量。 第三，EET类似物可激活关键的细胞存活途径，从而可能在I/R早期抑制细胞凋亡。 第四，我们的数据表明，EET通过激活SIRT1-Foxo3a 信号通路改善I/R引起的肾损伤，从而保护肾脏免受I/R损伤。 最后，EET可减轻肾I/R损伤，其机制可能与通过Toll样受体通路调节炎症小体NLRP3的产生和细胞焦亡有关。 因此，我们认为目前我们正在开发合成的EET类似物可能为治疗心血管和肾脏疾病提供新的治疗选择。