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首页> 外文期刊>American Journal of Physiology >Glutathione depletion is associated with decreased Bcl-2 expression and increased apoptosis in cholangiocytes.
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Glutathione depletion is associated with decreased Bcl-2 expression and increased apoptosis in cholangiocytes.

机译：谷胱甘肽耗竭与胆管细胞Bcl-2表达减少和细胞凋亡增加有关。

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Cholangiocytes are the target of a group of liver diseases termed the cholangiopathies that include conditions characterized by periductal inflammation and cholangiocyte apoptosis. Because inflammation is associated with oxidative stress, we developed the hypothesis that cholangiocytes exposed to oxidative stress will be depleted of endogenous cytoprotective molecules, leading to cholangiocyte apoptosis. To begin to test this hypothesis, we explored the relationships among glutathione (GSH) depletion, expression of Bcl-2 (a protooncogene that inhibits apoptosis), and apoptosis in a nonmalignant human cholangiocyte cell line. Monolayers of human bile duct epithelial cells, derived from normal liver and immortalized by SV40 transformation, were depleted of GSH using buthionine sulfoximine (BSO). Bcl-2 expression was assessed by quantitative immunoblot analysis, and apoptosis quantified by fluorescence microscopy using the DNA binding dye 4', 6'-diamidino-2-phenylindole. Bcl-2 message was assessed by RNase protection assay, and Bcl-2 protein synthesis and half-life by pulse-chase analysis. Exposure of human cholangiocytes in culture to BSO reduced GSH levels by 93 +/- 3% (P < 0.01). In addition, treatment of cholangiocytes with BSO reduced Bcl-2 levels by 87 +/- 2% (P < 0.01) and was associated with a time-dependent increase in the number of cells undergoing apoptosis; approximately 11 +/- 1% of cultured cells demonstrated morphological changes of apoptosis by 72 h compared with 1.5 +/- 0.1% in untreated cholangiocytes (P < 0. 01). Maintenance of GSH levels by addition of glutathione ethyl ester in the presence of BSO blocked the BSO-associated increase in apoptosis in BSO-treated cholangiocytes and also prevented the decrease in Bcl-2 protein. BSO treatment of cholangiocytes did not change steady-state levels of bcl-2 mRNA or Bcl-2 protein synthesis. However, Bcl-2 protein half-life decreased 57% in BSO-treated vs. untreated cells. Our results using a human cholangiocyte cell line demonstrate that reduction in the cellular levels of an antioxidant such as GSH results in increased degradation of Bcl-2 protein and an increase in apoptosis. These data provide a mechanistic link between the consequences of oxidative stress and cholangiocyte apoptosis, an observation that may be important in the pathogenesis of the inflammatory cholangiopathies.
机译：胆管细胞是称为胆管病的一组肝脏疾病的靶标，其包括以导管周围炎症和胆管细胞凋亡为特征的疾病。因为炎症与氧化应激有关，所以我们提出了一个假设，即暴露于氧化应激的胆管细胞将耗尽内源性细胞保护分子，从而导致胆管细胞凋亡。为了验证这一假设，我们探讨了谷胱甘肽（GSH）耗竭，Bcl-2（抑制凋亡的原癌基因）表达和非恶性人类胆管细胞系细胞凋亡之间的关系。使用丁硫氨酸亚砜亚胺（BSO）去除了源自正常肝脏并通过SV40转化而永生的人胆管上皮细胞单层。通过定量免疫印迹分析评估Bcl-2表达，并使用DNA结合染料4'，6'-二mid基-2-苯基吲哚通过荧光显微镜法定量凋亡。 Bcl-2信息通过RNase保护分析评估，Bcl-2蛋白质合成和半衰期通过脉冲追踪分析评估。将人胆管细胞暴露于BSO可将GSH水平降低93 +/- 3％（P <0.01）。此外，用BSO处理胆管细胞可使Bcl-2水平降低87 +/- 2％（P <0.01），并且与凋亡相关的细胞数呈时间依赖性增加。与未处理的胆管细胞中的1.5 +/- 0.1％相比，到72 h时约11 +/- 1％的培养细胞表现出凋亡的形态学变化（P <0. 01）。通过在BSO存在下添加谷胱甘肽乙酯来维持GSH水平，可以阻止BSO相关的胆管细胞凋亡与BSO相关的凋亡增加，并且还可以阻止Bcl-2蛋白的减少。 BSO处理胆管细胞不会改变bcl-2 mRNA或Bcl-2蛋白合成的稳态水平。但是，与未经处理的细胞相比，经BSO处理的细胞的Bcl-2蛋白半衰期降低了57％。我们使用人胆管细胞细胞系的结果表明，抗氧化剂（例如GSH）的细胞水平降低导致Bcl-2蛋白降解增加和凋亡增加。这些数据提供了氧化应激与胆管细胞凋亡之间的机械联系，这一发现可能对炎症性胆管病的发病机制很重要。

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