Satoh Takumi, Izumi Masanori, Inukai Yuki, Tsutsumi Yasutaka, Nakayama Naoto, Kosaka Kunio, Shimojo Yosuke, Kitajima Chieko, Itoh Ken, Yokoi Toshio, Shirasawa Takuji
Department of Welfare Engineering, Faculty of Engineering, Iwate University, Ueda 4-3-5, Morioka, Iwate 020-8551, Japan.
Neurosci Lett. 2008 Apr 4;434(3):260-5. doi: 10.1016/j.neulet.2008.01.079. Epub 2008 Feb 13.
In a previous study, we found that carnosic acid (CA) protected cortical neurons by activating the Keap1/Nrf2 pathway, which activation was initiated by S-alkylation of the critical cysteine thiol of the Keap1 protein by the "electrophilic"quinone-type of CA [T. Satoh, K. Kosaka, K. Itoh, A. Kobayashi, M. Yamamoto, Y. Shimojo, C. Kitajima, J. Cui, J. Kamins, S. Okamoto, T. Shirasawa, S.A. Lipton, Carnosic acid, a catechol-type electrophilic compound, protects neurons both in vitro and in vivo through activation of the Keap1/Nrf2 pathway via S-alkylation of targeted cysteines on Keap1. J Neurochem., in press]. In the present study, we used HT22 cells, a neuronal cell line, to test CA derivatives that might be more suitable for in vivo use, as an electrophile like CA might react with other molecules prior to reaching its intended target. CA and carnosol protected the HT22 cells against oxidative glutamate toxicity. CA activated the transcriptional antioxidant-responsive element of phase-2 genes including hemeoxygenase-1, NADPH-dependent quinone oxidoreductase, and gamma-glutamyl cysteine ligase, all of which provide neuroprotection by regulating cellular redox. This finding was confirmed by the result that CA significantly increased the level of glutathione. We synthesized a series of its analogues in which CA was esterified at its catechol hydroxyl moieties to prevent the oxidation from the catechol to quinone form or esterified at those moieties and its carbonic acid to stop the conversion from CA to carnosol. In both cases, the conversion and oxidation cannot occur until the alkyl groups are removed by an intracellular esterase. Thus, the most potent active form as the activator of the Keap1/Nrf2 pathway, the quinone-type CA, will be produced inside the cells. However, neither chemical modulation potentiated the neuroprotective effects, possibly because of increased lipophilicity. These results suggest that the neuroprotective effects of CA critically require both free carboxylic acid and catechol hydroxyl moieties. Thus, the hydrophilicity of CA might be a critical feature for its neuroprotective effects.
在之前的一项研究中，我们发现迷迭香酸（CA）通过激活Keap1/Nrf2途径来保护皮质神经元，该途径的激活是由“亲电”醌型CA对Keap1蛋白关键半胱氨酸硫醇进行S-烷基化引发的[T. Satoh, K. Kosaka, K. Itoh, A. Kobayashi, M. Yamamoto, Y. Shimojo, C. Kitajima, J. Cui, J. Kamins, S. Okamoto, T. Shirasawa, S.A. Lipton, 迷迭香酸，一种儿茶酚型亲电化合物，通过对Keap1上靶向半胱氨酸进行S-烷基化激活Keap1/Nrf2途径，在体外和体内均能保护神经元。《神经化学杂志》，即将发表]。在本研究中，我们使用神经元细胞系HT22细胞来测试可能更适合体内使用的CA衍生物，因为像CA这样的亲电试剂在到达其预期靶点之前可能会与其他分子发生反应。CA和鼠尾草酸保护HT22细胞免受氧化型谷氨酸毒性的影响。CA激活了包括血红素加氧酶-1、NADPH依赖性醌氧化还原酶和γ-谷氨酰半胱氨酸连接酶在内的二期基因的转录抗氧化反应元件，所有这些基因都通过调节细胞氧化还原反应来提供神经保护作用。CA显著提高谷胱甘肽水平的结果证实了这一发现。我们合成了一系列其类似物，其中CA在其儿茶酚羟基部分进行酯化以防止从儿茶酚氧化为醌形式，或者在这些部分及其碳酸部分进行酯化以阻止从CA转化为鼠尾草酸。在这两种情况下，直到烷基被细胞内酯酶去除，转化和氧化才会发生。因此，作为Keap1/Nrf2途径激活剂的最有效的活性形式，醌型CA将在细胞内产生。然而，两种化学修饰都没有增强神经保护作用，可能是因为亲脂性增加。这些结果表明，CA的神经保护作用严重依赖游离羧酸和儿茶酚羟基部分。因此，CA的亲水性可能是其神经保护作用的关键特征。
