Numerous studies suggest that glutamate toxicity is a major contributor to neuronal dysfunction and death in several neurodegenerative diseases. In our previous study, isoliquiritigenin (ISL) isolated from Glycyrrhiza uralensis showed neuroprotective effects against neuronal cell death mediated by intracellular reactive oxygen species (ROS) generation and loss of mitochondrial membrane potential. However, the mechanisms by which ISL protects against glutamate-induced oxidative stress are unknown. In the present study, we focused on the cellular and molecular mechanisms underlying the inhibition of ROS production and induction of mitochondrial dysfunction by ISL in glutamate-stimulated HT22 mouse hippocampal neuron cells. The results revealed that ISL inhibited glutamate-induced mitochondrial ROS production and decline of glutathione levels and ATP generation in HT22 cells. Interestingly, we discovered that ISL prevents glutamate-induced mitochondrial fission by inhibiting the dephosphorylation of Drp1 at the serine 637 residue, which is a regulatory factor of mitochondrial dynamics, and both a S637D mutation of Drp1, which resulted in a phosphorylation-mimetic form of Drp1 at Ser637, and mitochondria-targeted antioxidant Mito-TEMPO inhibited glutamate-induced mitochondrial fission. Furthermore, ISL also prevented the increase of intracellular calcium accompanied by activation of calcineurin, which is a key regulator of dephosphorylation of Drp1 (Ser637), in glutamate-treated HT22 cells. Taken together, our results demonstrated that ISL protects against glutamate-induced mitochondrial fission by inhibiting the increase of mitochondrial ROS and intracellular calcium, which are accompanied by dephosphorylation of Drp1 (Ser637), and consequently attenuates glutamate-induced neuronal cell death. Therefore, these findings suggest that ISL exhibits the potential for protection against glutamate toxicity. These results may contribute to the development of new drugs and novel strategies for the treatment of neurodegenerative disorders related to glutamate toxicity.

大量研究表明，谷氨酸毒性是几种神经退行性疾病中神经元功能障碍和死亡的主要诱因。在我们以前的研究中，从甘草中分离出异黄体生成素（ISL）提示对细胞内活性氧（ROS）产生和线粒体膜电位丧失介导的神经元细胞死亡具有神经保护作用。但是，ISL防御谷氨酸诱导的氧化应激的机制尚不清楚。在本研究中，我们集中于谷氨酸刺激的HT22小鼠海马神经元细胞中ISL抑制ROS产生和诱导线粒体功能障碍的细胞和分子机制。结果显示，ISL抑制了HT22细胞中谷氨酸诱导的线粒体ROS的产生以及谷胱甘肽水平和ATP生成的下降。有趣的是，我们发现ISL通过抑制丝氨酸637残基上的Drp1的去磷酸化来防止谷氨酸诱导的线粒体裂变，它是线粒体动力学的调节因子，Drp1的S637D突变（导致Ser637处的Drp1磷酸化）和线粒体靶向的抗氧化剂Mito-TEMPO抑制了谷氨酸诱导的线粒体裂变。此外，ISL还阻止了谷氨酸处理的HT22细胞中钙调神经磷酸酶的活化，钙调神经磷酸酶是Drp1（Ser637）脱磷酸的关键调节剂，因此可以防止细胞内钙的增加。两者合计，我们的结果表明，ISL通过抑制线粒体ROS和细胞内钙的增加来保护谷氨酸诱导的线粒体裂变，这些增加伴随着Drp1（Ser637）的去磷酸化，从而减轻了谷氨酸诱导的神经元细胞死亡。所以，这些发现表明，ISL具有防止谷氨酸毒性的潜力。这些结果可能有助于开发新药和治疗与谷氨酸毒性有关的神经退行性疾病的新策略。