Granulatoside A, a starfish steroid glycoside, enhances PC12 cell neuritogenesis induced by nerve growth factor through an activation of MAP kinase
Granulatoside A, a starfish steroid glycoside, enhances PC12 cell neuritogenesis induced by nerve growth factor through an activation of MAP kinase
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DOI：
10.1002/cdmc.200600190
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发表时间：
2006-12-01
期刊：
影响因子：
3.4
通讯作者：
Ojika, Makoto
中科院分区：
文献类型：
--
作者：
Qi, Jianhua;Han, Chunguang;Ojika, Makoto
神经生长因子(NGF)是神经营养因子家族中第一个也是最具特征的成员，是神经元生长、分化、存活和功能维持的重要因子。[2]由于来自大鼠嗜铬细胞瘤细胞的PC12细胞系表达对NGF的神经元分化，这不仅对于筛选研究，而且对于研究PC12细胞神经发生的信号转导途径都是有用的模型。[3]我们先前使用PC12细胞系系统寻找模拟NGF神经生成活性的化合物，结果分离了一系列名为linck oside的类固醇苷类化合物(例如，从冲绳蓝星Linckia laevigata中分离得到一种已知的代谢物，颗粒皂苷A(1)，它最初是从海星绒毛膜中分离出来的。[5]虽然颗粒皂苷A(1)不能诱导PC12细胞中的任何突起生长，但它有效地增强了NGF的神经生长活性。在这里，我们报道了颗粒苷A(1)的新的神经生长因子增强活性，简要描述了其构效关系，以及涉及丝裂原活化蛋白(MAP)激酶的细胞机制。该代谢物最初是从另一种海星中分离出来的，并通过与报道的数据进行比较来鉴定。[5]颗粒皂苷A(1)在浓度为40μM(图1，2a)的情况下不能诱导PC12细胞中神经生长因子样神经元的分化(图1，2a)，尽管它的结构与一些活性的林肯皂苷相似。[4]然而，在微量的神经生长因子(1.5ngmL±1)的存在下，几乎不能诱导轴突生长(图1，2a)。2B)，颗粒皂苷A(1)以剂量依赖的方式显著诱导轴突生长(图1)。当1在40μM存在时，神经再生达到最大值95%，相当于神经生长因子40 ngmL±1的活性(图1，2c)。未加药剂培养的细胞未见突起生长(图2d)。当浓度高于80μM时，该化合物表现出轻微的细胞毒性。1对PC12细胞的IC50值大于200μM(数据未显示)，表明1是一种具有有趣生物活性的低毒药物。通过改变颗粒苷A(1)和神经生长因子的浓度，观察两者对神经发生的协同作用(图3)。随着1浓度的升高和NGF浓度的降低，作用增强，说明1能有效地促进NGF的生成，但不能促进NGF的生成。例如，使用1ngmL1ngmL1的神经生长因子和40μM的组合可获得峰值活性，与单独使用40ngmL1的神经生长因子的活性相当，这意味着神经生长因子的活性大约增加了40倍。
Nerve growth factor (NGF) is the first and best characterized member of the neurotrophin family and an important factor for growth, differentiation, survival, and function maintenance of neurons.[1] Although NGF is expected to have therapeutic potential, it is also unstable and cannot readily pass through the blood-brain barrier. Thus, attempts have been made to identify the exogenous substances that mimic and/or enhance its physiological action.[2] Since the PC12 cell line derived from rat pheochromocytoma cells expresses neuronal differentiation in response to NGF, this is a useful model not only for screening studies but also for investigating the signal transduction pathways of PC12 cell neuritogenesis.[3] Our previous search for compounds that mimic the neuritogenic activity of NGF using the PC12 cell line system resulted in the isolation of a series of steroid glycosides named linckosides (for example, linckoside B (4)) from the Okinawan blue starfish Linckia laevigata.[4] A further examination of the related compounds led to identification of a known metabolite, granulatosideA (1), which was originally isolated from the starfish Choriaster granulatus.[5] Although granulatoside A (1) did not induce any neurite outgrowth in PC12 cells, it potently enhanced the neuritogenic activity of NGF. Here we report the novel NGF-enhancing activity of granulatoside A (1), a brief description of the structure–activity relationship, and the cellular mechanism involving mitogen-activated protein (MAP) kinase. Granulatoside A (1) was obtained in 0.002% yield (based on the wet weight of the starfish L. laevigata) using the HPLC process that previously yielded linckoside F.[4c] This metabolite was originally isolated from a different starfish and identified by comparing its spectral properties with the reported data.[5] Granulatoside A (1) did not induce the NGF-like neuronal differentiation in PC12 cells up to a concentration of 40 μM (Figure 1, 2a), despite its structural similarity to some active linckosides.[4] However, in the presence of a trace amount of NGF (1.5 ngmLÀ1) that scarcely induced neurite outgrowth (Figure 1, 2b), granulatoside A (1) significantly induced neurite outgrowth in a dose-dependent manner (Figure 1). In the presence of 1 at 40 μM, the neuritogenesis reached the maximum of 95%, which was comparable to the activity of 40 ngmLÀ1 of NGF (Figure 1, 2c). The cells cultured without the agents did not exhibit any neurite outgrowth (Figure 2d). At a concentration higher than 80 μM, the compound showed a little cytotoxicity. The IC50 value of 1 against PC12 cells was estimated to be higher than 200 μM by MTT assay (data not shown), suggesting that 1 is a low-toxicity agent with interesting biological activity.The synergistic effect exerted on the PC12 cell neuritogenesis by granulatoside A (1) and NGF was examined by varying their concentrations (Figure3). The effect significantly increased as the concentration of 1 became higher and the concentration of NGF became lower, indicating that 1 potently enhanced NGF but not vice versa. For example, the peak activity was achieved using the combination of 1 ngmLÀ1 of NGF and 40 μM of 1, and was comparable to the activity by 40 ngmLÀ1 of NGF alone, meaning an approximately 40-fold enhancement of the activity of NGF.