Role of superoxide production by NOS in skeletal muscles in the development of shock after ischemia/reperfusion.

骨骼肌中 NOS 产生的超氧化物在缺血/再灌注后休克发展中的作用。

• 批准号: 15590582
• 负责人: KURISAKI Emiko
• 金额: $ 2.3万
• 依托单位: Fukushima Medical University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15590582/
• 关键词: ischemia; repeefusion; shock; nitric oxide synthase (NOS); superoxide; cholesterol peroxides; histochemistry; HPLC; HPLC; 緊縛性ショック; 過酸化ストレス; 遠隔臓器; 免疫組織化学; 一酸化窒素代謝物濃度; Nitric Oxide Synthase(NOS); NOS阻害薬

To clarify the role of superoxide (O_2-) produced by NOS after ischemia/reperfusion (I/R), a hind limb tourniquet ischemia animal model was used. A rubber band was applied to the inguinal region of the hind limb for 3 hours under isoflurane anesthesia followed by reperfusion. O_2- production in skeletal muscle cells was observed in mice using an in situ detection technique with hydroethidine. Five minutes after the start of reperfusion, burst production of O_2- was detected in the cytoplasm as well as on and/or near the membrane of muscle cells subjected to I/R. Remarkably increased NADPH diaphorase activiry, and immunoreactivities using an anti-NOS antibody and anti-4-hydroxy-nonenal (HNE) antibody were also observed in I/R muscle, whereas there was no significant increase of immunoreadrivity using anti-nitrotyrosine (NT). These findings suggested that O_2- production by NOS as well as xanthine oxides was involved in the mechanism of O_2- production after reperfusion. Positive imunoreactivity using anti-HNE was observed in the liver and the kidney and immunoreactivity using anti-NT was positive in the heart after reperfusion. Cholesterol peroxides and NO metabolites were determined by HPLC. Levels of cholesterol peroxides increased in the muscle subjected to I/R and in the kidney after reperfusion. Levels of NO metabolites in plasma also increased between 15 and 60 min after reperfuiion. HPLC results were consistent with histochemical findings. These results suggested that O2-and/or NO produced by NUS was responsible for not only local injury but also remote organ injury after I/R. In the present study, we showed that O_2- production by NOS plays a role as an initiator of the development of shock after I/R.

为探讨一氧化氮合酶（NOS）产生的超氧化物（O_2-）在缺血/再灌注（I/R）中的作用，本研究采用后肢止血带缺血动物模型。在异氟烷麻醉下，将橡皮筋应用于后肢的腹股沟区3小时，然后再灌注。用氢乙啶原位检测技术观察了小鼠骨骼肌细胞内O_2-的产生。再灌注后5分钟，在缺血/再灌注肌细胞的胞浆内及细胞膜上和（或）膜附近检测到O_2-的爆发性产生。在I/R肌中还观察到显著增加的NADPH黄递酶活性，以及使用抗NOS抗体和抗4-羟基壬烯醛（HNE）抗体的免疫反应性，而使用抗硝基酪氨酸（NT）的免疫反应性没有显著增加。提示NOS和黄嘌呤氧化物产生O_2 ~-参与了再灌注后O_2 ~-的产生机制。再灌注后，抗HNE在肝脏和肾脏呈阳性反应，抗NT在心脏呈阳性反应。用HPLC法测定胆固醇过氧化物和NO代谢产物。胆固醇过氧化物水平增加的肌肉进行I/R和再灌注后的肾脏。再灌注后15 ~ 60 min血浆NO代谢产物水平也明显升高。HPLC结果与组织化学结果一致。提示NUS产生的O_2 ~-和/或NO不仅对缺血再灌注后的局部损伤起作用，而且对远隔器官的损伤也起作用。本研究表明，NOS产生的O_2-在I/R后休克的发生发展中起着启动作用。