微RNA-21在肾缺血预处理中的保护作用及其机制

Mechanism of miR-21 in delayed ischemia preconditioning and its protection against subsequent ischemia reperfusion injury in kidney

目的探讨微RNA-21（miR-21）对脯氨酸羟化酶2（proline hydroxylase domain2，PHD2）的调控作用，及其对肾脏晚期缺血预适应（ischemic preconditioning，IPC）小鼠的保护作用。方法建立肾脏晚期缺血预适应小鼠模型，分为两组：（1）缺血再灌注组（IR组）：假手术后第4天双侧肾蒂夹闭35min后恢复灌注；（2）缺血预适应组（IPC组）：第1天双侧肾蒂夹闭15min，手术第4天处理同IR组。于术后第5天处死所有小鼠，留取血液及肾脏标本。HE染色观察肾组织病理变化；常规生化检测血肌酐（Scr）变化；实时荧光定量PCR法检测。肾脏miR-21表达；Western印迹法检测肾组织PHD2和低氧诱导因子1α（HIF—1α）蛋白表达变化。体外给予人肾小管上皮细胞（HK．2）1％O2处理6h，采用Lipofectamine2000法转染锁核酸（LNA）修饰的anti-miR-21寡核苷酸抑制miR-21表达，实时荧光定量PCR法检测细胞miR-21、PHD2以及HIF—1α mRNA表达；Western印迹法检测细胞PHD2与HIF—1α蛋白表达。结果体内实验结果发现，与IR组相比，IPC组小鼠血肌酐和肾组织病理损伤有明显改善（均P〈0．01）；与IR组相比，IPC组小鼠肾组织miR，21表达上调（P〈0．01）；PHD2蛋白表达下调（P〈0．05），HIF—1α蛋白表达上调（P〈0．05）。体外HK-2细胞低氧模型实验结果提示，抑制细胞miR-21表达可致PHD2蛋白水平明显升高（P〈0．05）。结论miR-21对PHD2具有负向调控作用。晚期IPC可能通过诱导miR-21表达，减少PHD2表达，间接上调HIF-1α，对肾脏缺血再灌注损伤起到保护作用。

Objective To investigate the molecular mechanism of protection of ischemia preconditioning on renal isehemia reperfusion injury. Methods Male C57/BL6N mice were randomly divided into two groups： in IR group, 35 min ischemia was induced by occlusion of both renal pedieles followed by 24 h perfusion （I/R）. 15 min isehemia was induced 4 days before I/R in IPC group. Blood sample and kidney were collected in IR and IPC group after 24 h perfusion. Serum creatinine （Scr） and histological changes were used to evaluate the renal injury. PHD2 and HIF- 1α were evaluated by Western blotting, miR- 21 expression was confirmed by real-time PCR. In vitro, hypoxic model was established by 1% O2 in HK-2 cells. Knockdown of miR-21 in hypoxic model was perfermed by locked nucleic acid modified-anti-miR-21 transfection. The levels of miR-21, HIF-letand PHD2 mRNA were confirmed by real-time PCR. The levels of HIF-1α and PHD2 proteins were tested by Western blotting. Results In vivo, Compared with IR group, the renal function and histological changes were improved in IPC group （P 〈 0.01）. Compared with IR group, the expression of miR-21（P 〈 0.01） and HIF-lct （P 〈 0.05） were increased in IPC group, while PHD2 was reduced （P 〈 0.01）. In vitro, hypoxia reduced miR-21. The inhibition of miR-21 could increased the expression of PHD2 （P〈 0.05）. Conclusions Ischemia preconditioning may exert protection against renal ischemia reperfusion injury by inhibiting PHD2.