Center for Vascular Medicine, Section Angiology, University of Munich Medical Center, City Campus, Munich, Germany.
J Ethnopharmacol. 2013 Jan 9;145(1):162-7. doi: 10.1016/j.jep.2012.10.045. Epub 2012 Nov 2.
Supplementation with aged garlic extract (AGE) has been shown to restore impaired endothelium-dependent vasodilator response in subjects with acutely elevated plasma homocysteine (Hcy) levels after an oral methionine load and in patients with chronic coronary artery disease. Moreover, AGE has been shown to inhibit the progression of coronary calcifications in patients with coronary artery disease. The molecular mechanisms, by which AGE preserves endothelial function is unknown. Our objective was to explore whether AGE preserves endothelial nitric oxide (NO) output even under conditions of elevated Hcy levels by preventing oxidative inactivation of the NO synthase cofactor tetrahydrobiopterin.
Endothelial (EA.hy 926) cells were incubated with hypoxanthine, aminopterin, thymidine and methionine (HAT/MET) to increase cellular Hcy levels, and with and without AGE. Agonist stimulated NO output was measured using the fluorescent probe DAF-2, and cellular thiol levels (Hcy, cysteine, reduced and oxidized glutathione) and cellular tetrahydrobiopterin levels were measured by high performance liquid chromatography.
HAT/MET incubation resulted in significantly increased cellular Hcy levels, unaffected by coincubation with AGE. Elevated Hcy went along with significantly decreased NO output (to 34.4 ± 4.4% of control) and levels of tetrahydrobiopterin (from 4.67 ± 2.17 to 2.17 ± 0.97 pmol/mg). Incubation with AGE (5mg/mL) in HAT/MET-treated cells prevented the declines in NO output and tetrahydrobiopterin levels. AGE increased cellular levels of cysteine and total glutathione, and prevented glutathione and tetrahydrobiopterin oxidation induced by elevated Hcy.
Incubation with AGE preserved normal NO output from endothelial cells even under conditions of elevated Hcy levels by increasing cellular thiol antioxidant and prevention of tetrahydrobiopterin oxidation. This suggests that AGE might be useful in the prevention of endothelial dysfunction.
ETHNOPHARMACOLOGICAL 相关性：补充 aged garlic extract (AGE) 已被证明可以恢复因口服蛋氨酸负荷而导致血浆同型半胱氨酸 (Hcy) 水平升高的受试者和慢性冠状动脉疾病患者受损的内皮依赖性血管舒张反应。此外，AGE 已被证明可抑制冠状动脉疾病患者冠状动脉钙化的进展。AGE 保存内皮功能的分子机制尚不清楚。我们的目的是探索 AGE 是否通过防止一氧化氮合酶辅因子四氢生物蝶呤的氧化失活来保留内皮一氧化氮 (NO) 的输出，即使在 Hcy 水平升高的情况下也是如此。
将内皮 (EA.hy 926) 细胞用次黄嘌呤、氨甲蝶呤、胸苷和蛋氨酸 (HAT/MET) 孵育以增加细胞内 Hcy 水平，并与 AGE 一起孵育或不孵育。使用荧光探针 DAF-2 测量激动剂刺激的 NO 输出，并用高效液相色谱法测量细胞硫醇水平（Hcy、半胱氨酸、还原型和氧化型谷胱甘肽）和细胞四氢生物蝶呤水平。
HAT/MET 孵育导致细胞内 Hcy 水平显著升高，与同时孵育 AGE 无关。Hcy 升高伴随着 NO 输出显著降低（降至对照的 34.4±4.4%）和四氢生物蝶呤水平降低（从 4.67±2.17 降至 2.17±0.97 pmol/mg）。在 HAT/MET 处理的细胞中孵育 AGE（5mg/mL）可防止 NO 输出和四氢生物蝶呤水平下降。AGE 增加了细胞内半胱氨酸和总谷胱甘肽水平，并防止了由 Hcy 升高引起的谷胱甘肽和四氢生物蝶呤氧化。
即使在 Hcy 水平升高的情况下，AGE 孵育也可通过增加细胞硫醇抗氧化剂并防止四氢生物蝶呤氧化来维持内皮细胞的正常 NO 输出。这表明 AGE 可能有助于预防内皮功能障碍。
