Cultured porcine trabecular meshwork cells display altered lysosomal function when subjected to chronic oxidative stress
Cultured porcine trabecular meshwork cells display altered lysosomal function when subjected to chronic oxidative stress
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DOI：
10.1167/iovs.08-1915
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发表时间：
2008-09-01
影响因子：
4.4
通讯作者：
Epstein, David L.
中科院分区：
文献类型：
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作者：
Liton, Paloma B.;Lin, Yizhi;Epstein, David L.
目的.探讨慢性氧化应激对小梁网细胞溶酶体功能的影响。在存在或不存在蛋白酶抑制剂亮抑酶肽（10 μ M）的情况下，将猪TM细胞的汇合培养物在生理（5%O-2）或高氧条件（40%O-2）下生长2周。使用括号内指示的荧光探针定量以下参数：自体荧光、细胞内活性氧物质（ROS; H2 DCFDA）、线粒体膜电位（JC-1）、线粒体含量（Mitotracker Red; Invitrogen-Molecular Probes，尤金，OR），溶酶体含量（吖啶橙子和Lysotracker Red [Invitrogen-Molecular Probes]）、自噬泡含量（MDC）、SA-β-半乳糖苷酶（FDG）和组织蛋白酶活性（z-FR-AMC）。通过qPCR和Western印迹分析定量组织蛋白酶水平。透射电子显微镜进行超微结构分析。猪TM细胞长时间暴露于高氧环境导致ROS产生和氧化物质增加。电子显微镜照片显示脂褐素加载的溶酶体的细胞质积累。增加的溶酶体和自噬空泡内容物被证实与特定的荧光团。几种组织蛋白酶的mRNA和蛋白水平上调与氧化应激。这种表达上调与溶酶体活性增加无关。结果表明，TM细胞长期暴露于氧化应激导致溶酶体隔室内不可降解物质的积累，导致溶酶体活性降低。由于溶酶体系统负责细胞器的持续更新，溶酶体活性受损可能导致细胞TM功能随年龄的进行性失效。
PURPOSE. To investigate the effects of chronic oxidative stress on lysosomal function in trabecular meshwork (TM) cells.METHODS. Confluent cultures of porcine TM cells were grown for 2 weeks in physiological (5% O-2) or hyperoxic conditions (40% O2) in the presence or absence of the protease inhibitor leupeptin (10 mu M). The following parameters were quantified using the fluorogenic probes indicated within parentheses: autofluorescence, intracellular reactive oxygen species (ROS; H 2 DCFDA), mitochondrial membrane potential (JC-1), mitochondrial content (Mitotracker Red; Invitrogen-Molecular Probes, Eugene, OR), lysosomal content (acridine orange and Lysotracker Red [Invitrogen-Molecular Probes]), autophagic vacuole content (MDC), SA-beta-galactosidase (FDG), and cathepsin activities (z-FR-AMC). Cathepsin levels were quantified by qPCR and Western blot analysis. Ultrastructural analysis was performed by transmission electron microscopy.RESULTS. Prolonged exposure of porcine TM cells to a hyperoxic environment led to an increase in ROS production and oxidized material. Electron micrographs revealed the cytoplasmic accumulation of lipofuscin-loaded lysosomes. Augmented lysosomal and autophagic vacuole content was confirmed with specific fluorophores. The mRNA and protein levels of several cathepsins were upregulated with oxidative stress. This upregulated expression did not correlate with increased lysosomal activity.CONCLUSIONS. The results indicate that chronic exposure of TM cells to oxidative stress causes the accumulation of nondegradable material within the lysosomal compartment, leading to diminished lysosomal activity. Since the lysosomal system is responsible for the continuous turnover of cellular organelles, impaired lysosomal activity may lead to progressive failure of cellular TM function with age.