Pathologicl significance and its inhibition of apoptosis on the development and its progression of liver injury.

细胞凋亡对肝损伤发生发展的病理意义及其抑制作用。

基本信息

批准号：
10470142
负责人：
KAKUMU Shinichi
金额：
$ 8.45万
依托单位：
Aichi Medical University School 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：
1998
资助国家：
日本
起止时间：
1998 至 2000
项目状态：
已结题

项目摘要

Toxic effects of chloramphenicol, an antibiotic inhibitor of mitochondrial protein synthesis, on rat liver derived RL-34 cell line were completely blocked by a combined treatment with substances endowed with direct or indirect antioxidant properties. A stable, nitroxide free radical scavenger, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl, and a protein synthesis inhibitor, cycloheximide, suppressed in a similar manner the following manifestations of the chloramphenicol cytotoxicity : (1) Oxidative stress state as evidenced by FACS analysis of cells loaded with carboxy-dichlorodihydrofluorescein diacetate and Mito Tracker CMTH_2MRos ; (2) megamitochondoria formation detected by staining of mitochondria with Mito Tracker CMXRos under a laser confocal microscopy and electron microscopy ; (3) apoptotic changes of the cell detected by the phase contrast microscopy, DNA laddering analysis and cell cycle analysis. Since increases of ROS generation in chloramphenicol-treated cells were the first sign of the chloramphenicol toxicity, we assume that oxidative stress state is a mediator of above described alternations of RL-34 cells including MG formation. Pretreatment of cells with cycloheximide or 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl, which is known to be localized into mitochondria, inhibited the megamitochondria formation and succeeding apoptotic changes of the cell. Protective effects of cycloheximide, which enhances the expression of Bcl-2 protein, may further confirm our hypothesis that the megamitochondria formation is a cellular response to an increased ROS generation and raise a possibility that antiapoptotic action of the drug is exerted via the protection of the mitochondria functions.

氯霉素（线粒体蛋白合成的抗生素抑制剂）对大鼠肝脏衍生的RL-34细胞系的毒性作用，通过与直接或间接抗氧化剂特性的物质的联合治疗完全阻断了大鼠肝脏衍生的RL-34细胞系。一种稳定的，氮氧化物自由基清除剂，4-羟基-2,6,6,6-四甲基哌啶-1-氧基和蛋白质合成抑制剂，环己酰亚胺，以类似方式抑制，以下方式抑制了以下表现。氧化应激状态可以通过FACS分析对带有羧基二氯二氢氟乙酸酯和mito跟踪器CMTH_2MROS的细胞的FACS分析所证明。（2）在激光共聚焦显微镜和电子显微镜下用线粒体CMXros染色的巨米核形成；（3）通过相对比显微镜检测到的细胞的凋亡变化，DNA梯子分析和细胞周期分析。由于氯霉素处理的细胞中ROS的产生增加是氯霉素毒性的第一个迹象，因此我们假设氧化应激状态是上述RL-34细胞（包括MG形成）的上述交替的介体。用环己酰亚胺或4-羟基-2,6,6,6-四甲基哌啶-1-氧基对细胞进行预处理，已知将其定位于线粒体中，抑制了细胞的巨金蛋白方针的形成和细胞的成功变化。可增强Bcl-2蛋白表达的环己酰亚胺的保护作用，可以进一步证实我们的假设，即巨蛋白酶结构形成是对增加的ROS产生的细胞反应，并提高了该药物的抗凋亡作用的可能性，该作用是通过保护药物的抗凋亡作用。线粒体功能。