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Oxidative Stress of Asbestos or its Substitute to the Lung

石棉或其替代品对肺的氧化应激

基本信息

批准号：
12470103
负责人：
IGUCHI Hiroshi
金额：
$ 6.53万
依托单位：
Hyogo College of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2002
项目状态：
已结题

项目摘要

1. Alveolar macrophages of rats, RAW264.7 or J774 cells which is macrophage cell line established from mouse, induced nitric oxide(NO) Synthase(NOS), and increased the prduction of NO in the culture with asbestos or man-made mineral fiber(MMMF), such as glass fiber, ceramic fiber, rock wool or slug wool by way of substitute for asbestos.2. Increase in NO Synthesis was confirmed in the culture of AM from rats instilled asbestos into trachea.3. Generation of superoxide anion, O_2^- as well as NO was significantly enhanced in the culture of AM, RAW264.7 or J774 with asbestos or MMMF.4. When cultured with asbestos or MMMF, RAW264.7, AM or J774 decreased significantly intracellular glutathione levels in both reduced-and ozxdized-form.5. Taken altogether, these findings imply that oxidative stress in AM, RAW264.7 or J774 was strongly euhanced on exposure to asbestos or MMMF.6. It is well known that NO or peroxynitrite ONOO which is product of NO and O_2^- causes a nitration reaction with free thiol group in protein or wih reduced-form glutathione(GSH). These reaction products are RS-NO or GS-NO. We succeeded in developing a simple and new method for measuremert of RS-NO or GS-NO by using fluorescent dye. Using this method, we found an increased amount of GS-NO or RS-NO in conditioned media of AM, RAW264.7 or J774 cells cultured with asbestos or MMMF. This increase reflects at least in part a decrease in glutathione level in the cultured cells, suggesting the enhancement of oxidative stress or change of protein functions.7. Moreover, when cultured with asbestos or MMMF, AM, RAW264.7 or J774 responded to increase the production of proin- flammatory cytokines such as TNF-α, IL-1β, IL-6. The increases were found in BALF (Bronchoalveolar lavage fluid) from rats inntilled asbestos into trachea. These findings suggeet that collagen synthesis increases and will progress to the development of pneumoconiosis, that is, lung fibrosis soon or later.

1. 2.用石棉或人造矿物纤维（如玻璃纤维、陶瓷纤维、岩棉或海绒棉等）代替石棉培养大鼠肺泡巨噬细胞RAW264.7或小鼠巨噬细胞系J774细胞诱导一氧化氮合成酶（NOS），增加NO的产生。在气管内灌注石棉的大鼠AM培养中证实了NO合成的增加。石棉或mmmf对AM、RAW264.7和J774的超氧阴离子、O_2^-和NO的生成均有显著的促进作用。当用石棉或MMMF培养时，RAW264.7， AM或J774在还原和氧化形式下显著降低细胞内谷胱甘肽水平。综上所述，这些发现表明AM、RAW264.7或J774中的氧化应激在暴露于石棉或mmmf时被强烈增强。众所周知，NO和O_2^-的产物NO或过氧亚硝酸盐ONOO与蛋白质中的游离巯基或还原型谷胱甘肽（GSH）发生硝化反应。这些反应产物是RS-NO或GS-NO。我们成功地开发了一种简单的荧光染料测定RS-NO或GS-NO的新方法。用这种方法，我们发现在石棉或MMMF培养的AM、RAW264.7或J774细胞的条件培养基中，GS-NO或RS-NO的数量增加。这种增加至少部分反映了培养细胞中谷胱甘肽水平的下降，表明氧化应激的增强或蛋白质功能的改变。此外，当与石棉或MMMF一起培养时，AM、RAW264.7或J774反应增加了炎性因子如TNF-α、IL-1β、IL-6的产生。支气管肺泡灌洗液（支气管肺泡灌洗液）升高。这些发现提示胶原蛋白合成增加，迟早会发展为尘肺病，即肺纤维化。