KA 诱导的兴奋毒性通过激活铁蛋白自噬诱导神经元铁死亡。

OBJECTIVES: This study aims to elucidate the role of Fe overload in kainic acid (KA)-induced excitotoxicity, investigate the involvement of ferritinophagy selective cargo receptor NCOA4 in the pathogenesis of excitotoxicity. METHODS: Western blotting was used to detect the expression of FTH1, NCOA4, Lamp2, TfR, FPN, and DMT1 after KA stereotaxic injection into the unilateral striatum of mice. Colocalization of Fe with lysosomes in KA-treated primary cortical neurons was observed by using confocal microscopy. Desferrioxamine (DFO) was added to chelate free iron, a CCK8 kit was used to measure cell viability, and the Fe levels were detected by FerroOrange. BODIPY C11 was used to determine intracellular lipid reactive oxygen species (ROS) levels, and the mRNA levels of PTGS2, a biomarker of ferroptosis, were measured by fluorescent quantitative PCR. 3-Methyladenine (3-MA) was employed to inhibit KA-induced activation of autophagy, and changes in ferritinophagy-related protein expression and the indicated biomarkers of ferroptosis were detected. Endogenous NCOA4 was knocked down by lentivirus transfection, and cell viability and intracellular Fe levels were observed after KA treatment. RESULTS: Western blot results showed that the expression of NCOA4, DMT1, and Lamp2 was significantly upregulated, while FTH1 was downregulated, but there were no significant changes in TfR and FPN. The fluorescence results indicated that KA enhanced the colocalization of free Fe with lysosomes in neurons. DFO intervention could effectively rescue cell damage, reduce intracellular lipid peroxidation, and decrease the increased transcript levels of PTGS2 caused by KA. Pretreatment with 3-MA effectively reversed KA-induced ferritinophagy and ferroptosis. Endogenous interference with NCOA4 significantly improved cell viability and reduced intracellular free Fe levels in KA-treated cells. CONCLUSION: KA-induced excitotoxicity activates ferritinophagy, and targeting ferritinophagy effectively inhibits downstream ferroptosis. Interference with NCOA4 effectively attenuates KA-induced neuronal damage. This study provides a potential therapeutic target for excitotoxicity related disease conditions.

目的：本研究旨在阐明铁过载在海人酸（KA）诱导的兴奋性毒性中的作用，探讨铁蛋白自噬选择性货物受体 NCOA4 在兴奋性毒性发病机制中的作用。

方法：Western blot 法检测 KA 立体定向注射到小鼠单侧纹状体后 FTH1、NCOA4、Lamp2、TfR、FPN 和 DMT1 的表达。通过共聚焦显微镜观察 KA 处理的原代皮质神经元中铁与溶酶体的共定位。用去铁胺（DFO）螯合游离铁，用 CCK8 试剂盒测量细胞活力，用 FerroOrange 检测铁水平。用 BODIPY C11 测定细胞内脂质活性氧（ROS）水平，用荧光定量 PCR 测定铁死亡生物标志物 PTGS2 的 mRNA 水平。用 3-甲基腺嘌呤（3-MA）抑制 KA 诱导的自噬激活，检测铁蛋白自噬相关蛋白表达和铁死亡相关生物标志物的变化。用慢病毒转染敲低内源性 NCOA4，观察 KA 处理后细胞活力和细胞内铁水平的变化。

结果：Western blot 结果显示，NCOA4、DMT1 和 Lamp2 的表达明显上调，而 FTH1 下调，但 TfR 和 FPN 没有明显变化。荧光结果表明，KA 增强了神经元中游离铁与溶酶体的共定位。DFO 干预可有效挽救细胞损伤，减少细胞内脂质过氧化，降低 KA 引起的 PTGS2 转录水平升高。3-MA 预处理可有效逆转 KA 诱导的铁蛋白自噬和铁死亡。内源性干扰 NCOA4 可显著提高 KA 处理细胞的活力并降低细胞内游离铁水平。

结论：KA 诱导的兴奋性毒性激活铁蛋白自噬，靶向铁蛋白自噬可有效抑制下游铁死亡。干扰 NCOA4 可有效减轻 KA 诱导的神经元损伤。本研究为兴奋性毒性相关疾病提供了潜在的治疗靶点。