Protection against ischemia-reperfusion injury using a short interfering RNA that inhibits the activity of myeloperoxidase

使用抑制髓过氧化物酶活性的短干扰 RNA 预防缺血再灌注损伤

• 批准号: 18390428
• 负责人: ARAI Toshiyuki
• 金额: $ 10.65万
• 依托单位: Kyoto University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2006
• 资助国家: 日本
• 起止时间: 2006 至 2007
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18390428/
• 关键词: Neutreopils; Myeronperoxidaes; RNA interference; Short interfering RNA; Real time PCR; Reactive oxygen species; Human umbilical endothelial cells; Interleukin-8; ヒト臍帯静脈内皮細胞; インターロイキン-8; RNA干渉; siRNA; フローサイトメーター; 蛍光試薬DCFH; ミエロペルオキシダーゼ; 一重項酸素

In order to investigate the feasibility of protection against ischemia-reperfusion injury (IRI) using a short interfering RNA (siRNA) that inhibits the activity of myeloperoxidase (MPO) of human neutrophils, the following studies were Performed.The siRNA to diminish the MPO activity was designed and synthesized. Both effective (positive) and ineffective (negative) siRNAs were prepared. Then, the siRNA was electrically transferred into neutrophils using NucleofectorR system. The Suction of the expression of mRNA of MPO by a positive siRNA was successfully performed, which was confirmed using a real-time PCR. However, when the neutrophils were activated by opsonized zymosan, the generation of reactive oxygen species (ROS) was not diminished in the neutrophils incorporated with a positive siRNA compared to that in the neutrophils incorporated with a negative siRNA. That is, the RNA interference was not effective to inhibit the MPO activity of neutrophils.As an alternative capture, we tried to inhibit the ROS generation in endothelial cells using human umbilical vein endothelial cells (HUVECs). First, it was revealed that some stimulants, such as lipopolysaccaride (LPS), induced the expression of interleukin-8 (IL-8) and that IL-8, in turn, induced ROS generation in HUVECs. The siRNA to diminish the expression of IL-8 was designed and synthesized. Both positive and negative siRNAs were prepared. Then, the siRNA was electrically transferred into HUVECs using NucleofectorR system. The Suction of the expression of mRNA of IL-8 by a positive siRNA was successfully performed, which was confirmed using a real-time PCR. And, when the HUVECs were stimulated by LPS, the generation of ROS was diminished in the HUVECs incorporated with a positive siRNA compared to that in the HUVECs incorporated with a negative siRNA. It was known that the ROS generated by endothelial cells hurt themselves during IRI. So, we can say that protection against WI using a siRNA was achieved.

为了探讨抑制人中性粒细胞髓过氧化物酶（MPO）活性的小干扰RNA（siRNA）对缺血再灌注损伤（IRI）保护作用的可行性，设计并合成了抑制MPO活性的siRNA。制备有效（阳性）和无效（阴性）siRNA。然后，使用NucleofectorR系统将siRNA电转移到中性粒细胞中。通过实时荧光定量PCR证实了siRNA对MPO mRNA表达的抑制作用。然而，当中性粒细胞被调理酵母聚糖激活时，与掺入阴性siRNA的中性粒细胞相比，掺入阳性siRNA的中性粒细胞中活性氧（ROS）的产生并没有减少。作为另一种捕获方法，我们尝试用人脐静脉内皮细胞（HUVECs）抑制内皮细胞中ROS的产生。首先，研究表明，脂多糖（LPS）等一些刺激剂可诱导白细胞介素-8（IL-8）的表达，而IL-8反过来又诱导HUVEC中活性氧的产生。设计并合成了抑制IL-8表达的siRNA。制备阳性和阴性siRNA。然后，使用NucleofectorR系统将siRNA电转移到HUVECs中。利用实时荧光定量PCR技术，成功地进行了IL-8 mRNA表达的siRNA抽吸。并且，当HUVECs被LPS刺激时，与掺入阴性siRNA的HUVECs相比，掺入阳性siRNA的HUVECs中ROS的产生减少。众所周知，在IRI过程中，内皮细胞产生的ROS会损伤自身。因此，我们可以说实现了使用siRNA针对WI的保护。

项目成果

Inhibitory effects of 6-formylpterin on HIF-la protein accumulation.

6-甲酰蝶呤对 HIF-1α 蛋白积累的抑制作用。

• 发表时间: 2007
• 期刊: Biol Pharm Bull 30
• 作者: Sommari P;Yamashita K;Miyoshi T;et. al.
• 通讯作者: et. al.

The radical scavenger edaravone(3-methyl-1-phenyl-2-pyrazolin-5-one)reacts with a pterin derivative and produces a cytotoxic substance that induces intracellular ROS generation and cell death.

自由基清除剂依达拉奉（3-甲基-1-苯基-2-吡唑啉-5-酮）与蝶呤衍生物反应，产生细胞毒性物质，诱导细胞内ROS生成和细胞死亡。

• 发表时间: 2008
• 期刊: J Pharmacol Exp Ther 324
• 作者: Arai T;Nonogawa M;Makino K;et. al.
• 通讯作者: et. al.

Gene expression in enhanced apoptosis of human lymphoma U937 cells treated with the combination of different free radical generators and hyperthermia

不同自由基发生器和热疗联合处理人淋巴瘤U937细胞凋亡增强中的基因表达

• 发表时间: 2007
• 期刊: Free Radical Res 41
• 作者: Wada;S.;Tabuchi;Y.;Kondo;T.;Cui;Z-G.;Zhao;Q-L.;Takasaki;I.;Salunga;TL.;Ogawa;R.;Arai;T.;Makino;K.;Furuta;I
• 通讯作者: I

Inhibitory effects of 6-formylpterin on HIF-la protein accumulation

6-甲酰蝶呤对 HIF-1α 蛋白积累的抑制作用

• 发表时间: 2007
• 期刊: Biol Pharm Bull 30
• 作者: Sommani;P.;Yamashita;K.;Miyoshi;T.;Tsunemine;H.;Kodaki;T.;Mori;H.;Hirota;K.;Arai;T.;Sasada;M.;Makino;K
• 通讯作者: K

Effects of intravenous anesthesia with propofol on regional cortical blood flow and intracranial pressure in surgery for moyamoya disease

• DOI: 10.1016/j.surneu.2006.11.064
• 发表时间: 2007-10-01
• 期刊: SURGICAL NEUROLOGY
• 作者: Kikuta, Ken-ichiro;Takagi, Yasushi;Hashimoto, Nobuo
• 通讯作者: Hashimoto, Nobuo