炎症相关miRNA负反馈调节NF-κB轴在肾脏缺血再灌注损伤中的作用与机制研究

Acute kidney injury is one of the main complications among critically illed patients, and ischemia reperfusion injury (IRI) is the predominant cause. In our previous study, it was demonstrated that oxidized stress induced inflammatory response is an important pathophysiological event during kidney IRI. Inflammation induced microvascular endothelial cell injury is the key process of IRI, and NF-κB is one of the most important molecules during inflammation, which induced the expression of both pro-inflammatory and anti-inflammatory factors, including miRNAs. Our preliminary study showed that miR-98 and miR-374 were up-regulated in IRI kidney. Literature and bioinformatics search, as well as reporter gene tests all demonstrated that the targets of the 2 miRNAs were all molecules in the up- or down-stream of NF-κB . Thus we speculated that inflammatory miRNAs might be protective against renal IRI via a negative feedback regulatory effect against NF-κB. Therefore, the present study was disigned to clarify this effect and mechanism in renal IRI induced endothelial injury in vitro, and assure this mechanism in vivo using miRNA agomir/antagomir or adenovirus transfection. The purpose of this study is clarify the new negative feedback regulatory mechanism of endothelial injury during renal IRI.

急性肾损伤是危重病患者最常见的并发症之一，缺血再灌注损伤(IRI)是其主要病因。我们前期已证实氧化应激诱导的炎症反应是肾脏IRI的病理生理过程。炎症诱导的内皮损伤是IRI的关键环节，NF-κB是其中的关键分子，可诱导促炎与抗炎分子转录，包括miRNA。预实验利用芯片发现IRI肾脏高表达miR-98和miR-374，并通过文献与生物信息学检索、报告基因检测证实它们的靶基因均为NF-κB上游或下游分子。据此推测上述炎症miRNA可能通过负反馈调节NF-κB轴对肾脏IRI发挥保护作用。因此本课题拟利用基因转染、小分子阻断等技术证实NF-κB轴与炎症miRNA的关系及其在肾脏内皮细胞损伤中的作用，再用miRNA小分子试剂或腺病毒转染进行在体验证，力图阐明IRI时肾脏内皮损伤的新型负反馈调控机制。

本研究旨在探讨mir-98和mir-374在肾微血管内皮细胞缺血再灌注损伤后的作用和机制。通过生物信息学检索查找了miR-98和miR-374的潜在靶基因，在人脐静脉内皮细胞（HUVEC）的缺氧复氧模型中，检测了miR-98和miR-374及其潜在靶基因的表达变化，发现miR-98在缺氧复氧损伤后4h即出现上升；24h后，约升至NC组5倍（p<0.05）；基因转染显示miR-98 mimics 组caspase-3 mRNA表达水平仅为对照组的1/5；Anti miR-98组caspase-3 mRNA表达水平为Anti miR-98 NC组近2倍（P<0.05），说明miR-98与caspase-3存在负反馈调控作用。同时，双荧光素酶报告基因检测发现：mmu-miR-98对带有目的基因CASP3 3’UTR的luciferase的表达具有调控作用（p<0.0001）；而mmu-miR-98对带有突变的CASP3 3’UTR的luciferase的表达不具有调控作用，验证了miR-98与caspase-3之间的靶向关系。实验也进一步确认了miR-98表达升高可以减少缺氧复氧损伤中HUVEC的凋亡。因此，认为miR-98可以通过caspase-3参与调控HUVEC的凋亡。实验还观察到HUVEC缺氧复氧损伤后，转染HIF-1α siRNA，miR-98的表达率较对照组下降约1倍，提示HIF-1α可能影响miR-98表达。在体研究也证实了miR-98升高对小鼠肾脏缺血再灌注损伤的肾功能有保护作用。所以，实验认为在小鼠肾脏缺血再灌注损伤时，miR-98升高可能通过抑制caspase-3的表达保护肾微血管内皮细胞，从而保护肾功能。