Department of Medical Biophysics and Immunology, The Campbell Family Institute for Breast Cancer Research at Princess Margaret Hospital, Ontario Cancer Institute, University Health Network, Toronto, Ontario, Canada.
Cell Death Differ. 2013 Oct;20(10):1350-8. doi: 10.1038/cdd.2013.70. Epub 2013 Jun 21.
Erythrocytes endure constant exposure to oxidative stress. The major oxidative stress scavenger in erythrocytes is glutathione. The rate-limiting enzyme for glutathione synthesis is glutamate-cysteine ligase, which consists of a catalytic subunit (GCLC) and a modifier subunit (GCLM). Here, we examined erythrocyte survival in GCLM-deficient (gclm(-/-)) mice. Erythrocytes from gclm(-/-) mice showed greatly reduced intracellular glutathione. Prolonged incubation resulted in complete lysis of gclm(-/-) erythrocytes, which could be reversed by exogenous delivery of the antioxidant Trolox. To test the importance of GCLM in vivo, mice were treated with phenylhydrazine (PHZ; 0.07 mg/g b.w.) to induce oxidative stress. Gclm(-/-) mice showed dramatically increased hemolysis compared with gclm(+/+) controls. In addition, PHZ-treated gclm(-/-) mice displayed markedly larger accumulations of injured erythrocytes in the spleen than gclm(+/+) mice within 24 h of treatment. Iron staining indicated precipitations of the erythrocyte-derived pigment hemosiderin in kidney tubules of gclm(-/-) mice and none in gclm(+/+) controls. In fact, 24 h after treatment, kidney function began to diminish in gclm(-/-) mice as evident from increased serum creatinine and urea. Consequently, while all PHZ-treated gclm(+/+) mice survived, 90% of PHZ-treated gclm(-/-) mice died within 5 days of treatment. In vitro, upon incubation in the absence or presence of additional oxidative stress, gclm(-/-) erythrocytes exposed significantly more phosphatidylserine, a cell death marker, than gclm(+/+) erythrocytes, an effect at least partially due to increased cytosolic Ca(2+) concentration. Under resting conditions, gclm(-/-) mice exhibited reticulocytosis, indicating that the enhanced erythrocyte death was offset by accelerated erythrocyte generation. GCLM is thus indispensable for erythrocyte survival, in vitro and in vivo, during oxidative stress.
红细胞不断受到氧化应激的影响。红细胞中主要的氧化应激清除剂是谷胱甘肽。谷胱甘肽合成的限速酶是谷氨酸-半胱氨酸连接酶，它由一个催化亚基（GCLC）和一个修饰亚基（GCLM）组成。在这里，我们研究了 GCLM 缺陷（gclm(-/-)）小鼠的红细胞存活情况。gclm(-/-)小鼠的红细胞内谷胱甘肽含量显著降低。延长孵育时间会导致 gclm(-/-)红细胞完全溶解，这可以通过外源性抗氧化剂 Trolox 的输送来逆转。为了测试 GCLM 在体内的重要性，用苯肼（PHZ；0.07mg/g b.w.）处理小鼠以诱导氧化应激。与 gclm(+/+)对照组相比，gclm(-/-)小鼠的溶血明显增加。此外，在 PHZ 处理后 24 小时内，gclm(-/-)小鼠的脾脏中受伤红细胞的积累明显大于 gclm(+/+)小鼠。铁染色表明 gclm(-/-)小鼠的肾小管中有红细胞来源的色素含铁血黄素沉淀，而 gclm(+/+)对照组则没有。事实上，在处理后 24 小时，gclm(-/-)小鼠的肾功能开始下降，血清肌酐和尿素明显升高。因此，虽然所有接受 PHZ 处理的 gclm(+/+)小鼠都存活下来，但 90%接受 PHZ 处理的 gclm(-/-)小鼠在处理后 5 天内死亡。在体外，在没有或存在额外氧化应激的情况下孵育时，gclm(-/-)红细胞暴露的磷脂酰丝氨酸（一种细胞死亡标志物）明显多于 gclm(+/+)红细胞，这种效应至少部分是由于细胞浆 Ca(2+)浓度增加所致。在静息状态下，gclm(-/-)小鼠表现出网织红细胞增多，表明增强的红细胞死亡被加速的红细胞生成所抵消。因此，在氧化应激期间，GCLM 对于红细胞的体外和体内存活都是必不可少的。
