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目的以细胞谷氨酸兴奋性损伤为疾病模型,探讨AKAPs对PKA的锚定及PKA活性介导姜黄素的神经保护作用及其作用机制。方法用谷氨酸处理SHSY5Y细胞,建立谷氨酸兴奋性损伤的细胞实验模型;用Western blotting,CO-IP,Confocal的方法,研究PKA的细胞内的定位及活性参与姜黄素的神经保护作用的机制。结果谷氨酸可以剂量依赖的方式损伤SHSY5Y细胞,能够明显降低细胞活力、引起核固缩和核碎裂。预给予姜黄素则能有效地抵抗谷氨酸兴奋性损伤,能够明显地提高细胞活力、减轻核固缩和核碎裂,并且其神经保护作用存在剂量依赖性和时间依赖性。MTT及CO-IP实验显示:用H89抑制PKA活性以及用Ht31干扰PKA的空间定位能明显抑制姜黄素的神经保护作用并抑制MAPK通路各关键蛋白的序贯激活,这表明姜黄素的神经保护作用与PKA的活性和定位密切相关;此外,姜黄素还能调节AKAP79锚定的PKA的活性及空间定位,这可能与姜黄素调节NMDA-R的活性减少钙内流,从而减轻下游钙相关蛋白的损伤作用。结论姜黄素可以抵抗谷氨酸兴奋性损伤,且其作用机制依赖于AKAP对PKA的锚定和解锚定作用,与姜黄素增强细胞活力、抑制钙超载和钙相关蛋白的迁移活化、激活细胞存活相关的信号通路密切相关。
OBJECTIVE: To investigate the neuroprotective effect of AKAPs on the PKA and the neuroprotective effect of PKA mediated by glutamate excitotoxicity. Methods SHSY5Y cells were treated with glutamate to establish a cell model of glutamate excitotoxicity injury. Western blotting, CO-IP and Confocal were used to study the intracellular localization and activity of PKA involved in the neuroprotective effect of curcumin mechanism. Results Glutamate could injure SHSY5Y cells in a dose-dependent manner, significantly reducing cell viability and causing nuclear pyknosis and nuclear fragmentation. Curcumin pretreatment can effectively resist the glutamate excitotoxic injury, can significantly improve cell viability, reduce nuclear condensation and nuclear fragmentation, and its neuroprotective effect in a dose-dependent and time-dependent manner. MTT and CO-IP experiments showed that inhibition of PKA activity by H89 and spatial localization of PKA by Ht31 significantly inhibited the neuroprotection of curcumin and inhibited the sequential activation of key proteins of MAPK pathway, indicating that the neuroprotective effect of curcumin Which is closely related to the activity and localization of PKA. In addition, curcumin can also modulate the activity and spatial location of AKAP79-anchored PKA. This may be related to the fact that curcumin regulates the activity of NMDA-R to reduce the influx of calcium and thus reduce the expression of downstream calcium-related proteins Damage effect. CONCLUSION: Curcumin can resist the excitotoxicity of glutamate, and its mechanism of action depends on the anchoring and anchoring effect of AKAP on PKA. Curcumin can enhance cell viability, inhibit calcium overload and calcium-related protein migration and activation, and activate cell survival Related signal pathways are closely related.