刺芒柄花素通过下调P53/SAT1/ACSL4介导的铁死亡改善新生小鼠缺氧缺血性脑损伤

摘要：

目的 探讨刺芒柄花素（FMN）对新生小鼠缺氧缺血性脑损伤（HIBD）的神经保护作用，并阐明其机制是否与调控P53/SAT1/ACSL4信号通路以抑制神经元铁死亡有关。 方法 选取24只出生9~11 d的C57BL/6J新生小鼠，通过结扎左侧颈总动脉并置于低氧环境（92% N2，8% O2）中40 min以构建HIBD模型。造模成功后，将小鼠随机分为4组（n=6）：假手术组（Sham）、模型组（HIBD）、FMN低剂量治疗组（HIBD+FMN-L，50 mg/kg）和高剂量治疗组（HIBD+FMN-H， 100 mg/kg）。腹腔注射给药，3 d后取脑皮层缺血半暗带组织进行检测。采用Western blotting和双重免疫荧光技术分析铁死亡通路关键蛋白P53、SAT1及ACSL4的表达水平；同时测定组织内亚铁离子（Fe²+）、超氧化物（DHE）、丙二醛（MDA）和谷胱甘肽（GSH）的含量，以评估铁死亡及相关氧化应激指标。体外选用HT22神经元，建立氧糖剥夺（OGD）模型并分组为：对照组（Control）、模型组（OGD）、FMN干预组（OGD+FMN，100 μmol/L）、P53激动剂Nutlin-3干预组（OGD+Nutlin-3，10 μmol/L）以及FMN与Nutlin-3联合处理组（OGD+FMN+Nutlin-3）。在药物预处理1 h及OGD处理6 h后，检测上述铁死亡相关蛋白，并利用特异性探针或生化方法评估细胞内Fe²+（FerroOrange）、活性氧（DCFH-DA）、脂质过氧化（BODIPY-C11、MDA）、GSH水平、线粒体膜电位（JC-1）及细胞活性（CCK-8）。 结果 体内实验结果表明，与HIBD组相比，FMN治疗能够显著抑制P53、SAT1和ACSL4的蛋白表达（P<0.05），并有效改善铁死亡相关生化指标，具体为Fe²+、ROS和MDA含量显著降低，同时抗氧化物质GSH水平得以恢复（P<0.05）。体外实验结果与体内发现相互印证，FMN处理缓解了OGD诱导的HT22细胞铁死亡，表现为铁死亡关键蛋白表达下调（P<0.05），Fe²+积聚、脂质过氧化和ROS水平受到抑制，并且GSH含量、线粒体膜电位及细胞活力得到显著改善（P<0.05）。机制验证方面，当使用Nutlin-3特异性激活P53信号后，FMN所产生的上述保护效应均被明显逆转或削弱（P<0.05），P53信号在FMN抑制铁死亡过程中发挥重要的上游调节作用。 结论 刺芒柄花素通过下调P53/SAT1/ACSL4通路介导的神经元铁死亡的途径，对新生小鼠HIBD产生神经保护效应。

关键词: 刺芒柄花素, 缺氧缺血性脑损伤, 铁死亡, P53, SAT1, ACSL4

Abstract:

Objective To investigate the neuroprotective effects of formononetin (FMN) against hypoxic-ischemic brain damage (HIBD) in neonatal mice and the underlying mechanism. Methods Twenty-four neonatal C57BL/6J mice were randomly divided (n=6) into sham-operated group, HIBD model group, HIBD+FMN-L (50 mg/kg) group, and HIBD+FMN-H (100 mg/kg) group. Mouse models of HIBD were established by left common carotid artery ligation followed by hypoxia (92% N₂, 8% O₂) for 40 min. FMN at the two doses was administered by intraperitoneal injection, and 3 days later, brain tissues from the cortical ischemic penumbra were collected for assessing expressions of ferroptosis-related proteins (P53, SAT1, and ACSL4) using Western blotting and immunofluorescence staining and for detecting the levels of Fe²⁺, superoxide, malondialdehyde (MDA), and glutathione (GSH). In cultured HT22 neurons with oxygen-glucose deprivation (OGD), the effects of 100 μmol/L FMN, 10 μmol/L Nutlin-3 (a P53 agonist), or their combination on expressions of ferroptosis proteins, intracellular Fe²⁺, reactive oxygen species (ROS), lipid peroxidation, GSH, mitochondrial membrane potential, and cell viability were evaluated. Results In the neonatal mouse models of HIBD, FMN treatment significantly suppressed the protein expression of P53, SAT1, and ACSL4, reduced Fe²⁺, ROS, and MDA levels and increased GSH content in the cortical ischemic penumbra. In HT22 neurons with OGD, FMN obviously alleviated OGD-induced ferroptosis as shown by lowered expressions of the key ferroptosis proteins, reduced Fe²⁺ accumulation and lipid peroxidation, and significant increases of GSH levels, mitochondrial membrane potential, and cell viability. Mechanistic experiments showed that activation of P53 signaling by Nutlin-3 markedly reversed the protective effects of FMN. Conclusion FMN produces neuro-protective effects against HIBD in neonatal mice by mitigating neuronal ferroptosis, primarily through downregulation of the P53/SAT1/ACSL4 signaling pathway.

Key words: formononetin, hypoxic-ischemic brain damage, ferroptosis, P53, SAT1, ACSL4

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