Koriyama Yoshiki, Ohno Mamoru, Kimura Takahiro, Kato Satoru
Department of Molecular Neurobiology, Graduate School of Medicine, Kanazawa University, 13-1 Takaramachi, Kanazawa 920-8640, Japan.
Brain Res. 2009 Nov 3;1296:187-95. doi: 10.1016/j.brainres.2009.08.016. Epub 2009 Aug 15.
N-beta-Alanyl-5-S-glutathionyl-3,4-dihydroxyphenylalanine (5-S-GAD), an antibacterial substance isolated from the flesh fly, has been described as having multipotential biological activities toward various tissues. In a previous paper, we reported a novel neuroprotective action of 5-S-GAD on rat retinal ganglion cell apoptosis induced by optic nerve injury and intraocular N-methyl-D-aspartate treatment in vivo. In the present study, we further investigated the protective mechanism of this small peptide against other types of apoptosis in cultured cells of the established rat retinal ganglion cell line RGC-5. Hydrogen peroxide and serum deprivation treatments induced intracellular reactive oxygen species levels and lipid peroxidation, revealed by 4-hydroxy-2-nonenal production, in RGC-5 cells within 9-12h. The treatments also induced cell death accompanied by nuclear condensation, DNA laddering and increases in apoptotic Bax and caspase-3 proteins in RGC-5 cells within 12-24h. 5-S-GAD at 25-50 microM clearly suppressed the cell death and apoptotic features induced by these treatments. 5-S-GAD restored the nuclear condensation, DNA laddering and increases in apoptotic proteins. Furthermore, 5-S-GAD directly activated anti-apoptotic phospho-Akt and Bcl-2 proteins in RGC-5 cells. 5-S-GAD also quenched the reactive oxygen species production and inhibited the lipid peroxidation induced by oxidative stress. Therefore, 5-S-GAD may complementarily protect RGC-5 cells against apoptosis through dual actions as a radical scavenger and an inducer of anti-apoptotic phospho-Akt and Bcl-2. Taken together, 5-S-GAD is a high-potential tool for rescuing the retinal ganglion cell apoptosis induced by a variety of glaucomatous conditions.
N-β-丙氨酰基-5-S-谷胱甘肽基-3,4-二羟基苯丙氨酸（5-S-GAD）是一种从麻蝇中分离出的抗菌物质，已被描述为对多种组织具有多种生物学活性。在之前的一篇论文中，我们报道了5-S-GAD对体内视神经损伤和眼内N-甲基-D-天冬氨酸处理诱导的大鼠视网膜神经节细胞凋亡具有新的神经保护作用。在本研究中，我们进一步研究了这种小肽对已建立的大鼠视网膜神经节细胞系RGC-5培养细胞中其他类型凋亡的保护机制。过氧化氢和血清剥夺处理在9-12小时内诱导RGC-5细胞内活性氧水平和脂质过氧化，通过4-羟基-2-壬烯醛的产生得以揭示。这些处理还在12-24小时内诱导RGC-5细胞死亡，伴有核浓缩、DNA梯状条带形成以及凋亡相关的Bax和半胱天冬酶-3蛋白增加。25-50微摩尔的5-S-GAD明显抑制了这些处理诱导的细胞死亡和凋亡特征。5-S-GAD恢复了核浓缩、DNA梯状条带形成以及凋亡蛋白的增加。此外，5-S-GAD直接激活RGC-5细胞中的抗凋亡磷酸化Akt和Bcl-2蛋白。5-S-GAD还消除了活性氧的产生并抑制了氧化应激诱导的脂质过氧化。因此，5-S-GAD可能通过作为自由基清除剂以及抗凋亡磷酸化Akt和Bcl-2诱导剂的双重作用，互补性地保护RGC-5细胞免受凋亡。综上所述，5-S-GAD是挽救由多种青光眼性疾病诱导的视网膜神经节细胞凋亡的一种极具潜力的工具。
