氙气预适应通过miR-21调控机制预防脓毒症急性肾损伤的实验研究

As a common critical disease, acute kidney injury (AKI) is a serious threat to human health, and an important field of critical care medicine. AKI induced by sepsis (septic AKI) accounts for 50% of the overall incidence of AKI, with high mortality and poor prognosis, and is still lack of effective prevention and treatments. Cell apoptosis and inflammation are important pathophysiological mechanisms of septic AKI. MiR-21 is an important gene in regulating apoptosis and inflammation. In our previous studies, we found that xenon preconditioning protected from ischemic renal injury and nephrotoxicity by upregulating miR-21 in the kidney and inhibitting apoptosis. Thus, we speculate that xenon preconditioning may also prevent septic AKI. Preliminary experiment showed that xenon reduced renal damage induced by lipopolysaccharide (LPS) in mice, but its mechanism is still not clear. In this study, with the acute kidney injury model induced by LPS in miR-21 transgenic mice and wild mice, we further explore the protective effects of xenon on apoptosis and inflammation, and elucidate the potential mechanisms underlying xenon preconditioning with bioinformatics, lock nucleotide modification and RNA interference technology. This study not only extends the research of xenon as a potential organ protective agent in the prevention and treatment of AKI, but also provides a new target for the prevention of septic AKI.

急性肾损伤（AKI）是严重威胁人类健康的常见危重病，是危重病救治的重要领域。其中脓毒症引起者占50%，且死亡率极高，是AKI防治的难点。细胞凋亡、炎症反应是脓毒症性AKI的重要病理生理机制。miR-21是调控凋亡和免疫炎症的重要基因。申请人前期研究发现，氙气预适应通过上调肾脏miR-21表达，抑制细胞凋亡，减轻药物性、缺血性肾损伤。因而推测氙气预适应也可能通过miR-21干预脓毒症AKI的发生发展。预实验亦初步证实氙气能够减轻脂多糖诱发的小鼠肾损伤，但具体作用机制尚不清楚。本项目在前期研究基础上，拟采用脂多糖诱导miR-21转基因小鼠和野生鼠AKI模型，从抗凋亡、抗炎等方面深入探讨氙气预适应对脓毒症AKI的防治作用；并利用生物信息学、锁核苷酸修饰及RNA干扰等技术深入阐明miR-21及其下游信号通路在氙气预适应机制中的作用，从而为脓毒症AKI的防治提供新的靶点和思路。

脓毒症是急性肾损伤(AKI)常见病因，脓毒症性AKI患者死亡率高，预后差，目前尚缺乏有效防治措施。研究发现，氙气作为一种吸入性麻醉剂，具有独立于麻醉作用之外的器官保护作用。本项目首先通过脂多糖 (LPS) 诱导的脓毒症小鼠模型，观察了氙气预适应对脓毒症性AKI的干预作用。发现氙气预适应明显减轻LPS引起的肾功能损伤，降低血和肾组织中的炎症因子（TNF-α、IL-6）表达，减少肾脏细胞凋亡和炎细胞浸润。进一步机制研究发现，氙气呈时间依赖性上调肾组织HIF-1α和miR-21表达，下调miR-21的靶基因磷酸酯酶-张力蛋白同源物(PTEN)和程序化细胞死亡蛋白4(PDCD4)的表达，抑制核因子κB (NF-κB)的活性，并提高Akt磷酸化水平，抑制凋亡相关基因Cleaved Caspase 3表达。HIF-1α在转录水平上调控miR-21的表达。当体内敲减miR-21表达后，PTEN和PDCD4表达上调，肾脏细胞凋亡和炎症反应加重，氙气预适应的肾保护作用明显减低。表明氙气预适应可通过激活miR-21及其下游信号通路，发挥抗炎、抗凋亡作用，减轻脓毒症性急性肾损伤。在此基础上，项目组又进一步探讨了肾脏预缺血诱导miR-21高表达对脓毒症多脏器损伤（肾脏、心脏、肝脏和肺）的干预作用及其机制。发现肾脏缺血预适应能明显减轻脓毒症引起的急性多脏器损伤（包括：肾、心、肝、肺），表现为血肌酐和转氨酶浓度降低，心脏收缩功能改善；降低血和各脏器组织中的炎症因子表达；减少各脏器组织的细胞凋亡。当体内敲减或基因敲除miR-21后，肾脏缺血预适应的多脏器保护作用明显减弱。进一步机制研究发现，肾脏缺血预适应是通过上调HIF-1α，进而在转录水平上调miR-21表达，经外泌体途径，将肾脏高表达的miR-21转运至远隔器官，激活其下游靶信号通路（PDCD4/ NF-κB和PTEN/Akt）发挥抗炎、抗凋亡作用。.本项目的顺利完成具有一定的科学意义。证实了氙气预适应的器官保护作用，为氙气预适应将来应用于临床、发挥其独立于麻醉作用之外的脏器保护作用提供了理论和实验基础，为脓毒症性AKI的防治提供了新的手段和新的靶点；同时也为远程缺血预适应预防急性肾损伤提供了依据。