Saitoh Tomohiro, Tsuchiya Yuichi, Kinoshita Toshihiko, Itoh Motohiro, Yamashita Shigeru
Department of Obstetrics and Gynecology Toho University Medical Center Sakura Hospital 564-1 Shimoshizu Sakura-shi 285-8741 Chiba Japan.
Department of Biochemistry Toho University School of Medicine 5-21-16 Omori-nishi Ota-ku 143-8540 Tokyo Japan.
Reprod Med Biol. 2008 Dec 13;8(1):3-9. doi: 10.1007/s12522-008-0001-x. eCollection 2009 Mar.
The viability of mammalian eggs after ovulation is reported to be improved by the presence of ascorbic acid in the culture medium. However, the pro-survival mechanisms of ascorbic acid are poorly understood. The molecular pathways of apoptosis are evolutionarily conserved among animal species, and eggs are technically and ethically more suitable for biochemical analyses than mammalian eggs. We used egg cytoplasmic extracts to examine the direct intracellular effects of ascorbic acid.
Incubation of egg extracts for more than 4 h induces the spontaneous release of cytochrome from mitochondria. This event triggers the activation of caspases, cleavage of substrate proteins, and execution of apoptosis. Multiple signal transduction pathways including proteolysis and protein phosphorylation are also involved in this process. We examined whether any of these events might be inhibited by the addition of ascorbic acid.
Ascorbic acid showed no effect against cytochrome release, but prevented caspase activation and substrate cleavage. Ascorbic acid also blocked the proteolysis of apoptosis inhibitor proteins and the dephosphorylation of p42 MAP kinase. However, dehydroascorbic acid (oxidized form of ascorbic acid) and acetate (unrelated acid) were equally effective, indicating that these effects were primarily due to their acidity. In addition, dehydroascorbic acid inhibited caspase activities directly in vitro.
The anti-apoptotic effect of ascorbic acid in egg extracts is mainly due to cytoplasmic acidification rather than its intracellular antioxidant activity. Instead, oxidative conversion of ascorbic acid into dehydroascorbic acid may inhibit apoptosis through the inhibition of caspases.
据报道，培养基中存在抗坏血酸可提高排卵后哺乳动物卵子的存活率。然而，抗坏血酸的促存活机制尚不清楚。凋亡的分子途径在动物物种中具有进化保守性，并且从技术和伦理角度来看，卵子比哺乳动物卵子更适合进行生化分析。我们使用卵细胞质提取物来研究抗坏血酸的直接细胞内作用。
卵提取物孵育超过4小时会诱导线粒体中细胞色素的自发释放。这一事件会触发半胱天冬酶的激活、底物蛋白的切割以及凋亡的执行。包括蛋白水解和蛋白质磷酸化在内的多种信号转导途径也参与了这一过程。我们研究了添加抗坏血酸是否会抑制这些事件中的任何一个。
抗坏血酸对细胞色素释放没有影响，但可防止半胱天冬酶激活和底物切割。抗坏血酸还阻断了凋亡抑制蛋白的蛋白水解和p42丝裂原活化蛋白激酶的去磷酸化。然而，脱氢抗坏血酸（抗坏血酸的氧化形式）和乙酸盐（无关酸）同样有效，表明这些作用主要归因于它们的酸性。此外，脱氢抗坏血酸在体外直接抑制半胱天冬酶活性。
抗坏血酸在卵提取物中的抗凋亡作用主要是由于细胞质酸化，而非其细胞内抗氧化活性。相反，抗坏血酸氧化转化为脱氢抗坏血酸可能通过抑制半胱天冬酶来抑制凋亡。
