【目的】 体外观察中医经典方剂小续命汤中主要成分槲皮素、木犀草素、山奈酚对缺血性脑卒中神经元损伤的修复作 用及机制。【方法】 将PC12细胞分为5组：对照组、氧糖剥夺（OGD）组、槲皮素组、木犀草素组、山奈酚组。氧糖剥夺组及 3个药物处理组细胞通过氧糖剥夺模拟缺血性脑卒中神经元损伤。膜联蛋白V（Annexin V）-异硫氰酸荧光素（FITC）/碘化丙啶 （PI ）双荧光染色法检测缺氧不同时间后细胞的凋亡水平，5-乙炔基-2’-脱氧尿嘧啶核苷（EdU）法检测缺氧不同时间后细胞 增殖水平，确定合适缺氧时间点。通过细胞计数试剂盒 8（CCK-8）法检测不同浓度的槲皮素、木犀草素、山奈酚对 PC12细 胞的毒性，选择合适作用浓度。采用比色法检测细胞中 Fe2+ 水平。采用 Western Blot 法检测细胞中谷胱甘肽过氧化物酶 4 （GPX4）、长链酰基辅酶 A 合成酶 4（ACSL4）、磷酸化蛋白激酶 B（p-AKT）、白细胞介素 6（IL-6）、丝裂原活化蛋白激酶 3 （MAPK3）、肿瘤蛋白 53（TP53）、血管内皮生长因子 A（VEGFA）、肿瘤坏死因子 α（TNF-α）蛋白表达水平。采用实时荧光定 量聚合酶链反应（PCR）法检测 ACSL4 mRNA 表达水平。【结果】 PC12细胞进行缺氧处理后，细胞凋亡率升高、增殖率降低， 且随着缺氧实验时间延长，差异越大。缺氧处理使细胞发生铁死亡，而中药成分槲皮素、木犀草素、山奈酚联合缺氧处理 可抑制缺氧诱导的神经元铁死亡及炎症损伤，具体表现在：槲皮素、木犀草素均能够显著上调缺氧处理后的 PC12 细胞中 GPX4、p-AKT 蛋白表达水平，槲皮素可抑制 MAPK3、ACSL4 蛋白表达，木犀草素可下调 ACSL4 mRNA 表达水平、抑制 MAPK3蛋白表达及Fe2+ 水平；此外，二者还能够显著抑制缺氧诱导的神经元炎症，表现为IL-6和TNF-α蛋白表达水平的下 降。山奈酚能够上调缺氧处理后的 PC12细胞中 GPX4蛋白表达水平，并下调 ACSL4蛋白和 mRNA表达水平，此外，山奈酚 还能够显著降低缺氧处理后的PC12细胞中Fe2+ 水平。【结论】 小续命汤中主要成分槲皮素、木犀草素、山奈酚可通过抑制缺 氧诱导神经元铁死亡及炎症反应发挥保护作用。

Objective To investigate in vitro the reparative effects and underlying mechanisms of the main components （quercetin， luteolin， and kaempferol） of the classic traditional Chinese medicine formula Xiao Xuming Decoction on neuronal injury in ischemic stroke. Methods PC12 cells were divided into five groups： control group， oxygen-glucose deprivation （OGD） group， quercetin group， luteolin group， and kaempferol group. Neuronal injury in ischemic stroke was simulated via OGD in the OGD group and the three drug treatment groups. Annexin V-FITC/PI double fluorescence staining was used to detect apoptosis levels at different time points post-hypoxia. The 5-Ethynyl-2’-deoxyuridine （EdU） assay was employed to assess cell proliferation levels at different time points post-hypoxia，to determine the optimal hypoxia time point. The Cell Counting Kit-8（CCK-8） assay was used to evaluate the cytotoxicity of different concentrations of quercetin，luteolin，and kaempferol on PC12 cells to select appropriate working concentrations. A colorimetric method was applied to detect intracellular Fe2+ levels. Western Blot was performed to detect the protein expression levels of glutathione peroxidase 4（GPX4）， long-chain acyl-CoA synthetase 4（ACSL4），phosphorylated protein kinase B （p-AKT），interleukin-6（IL-6）， mitogen-activated protein kinase 3（MAPK3），tumor protein p53（TP53），vascular endothelial growth factor A （VEGFA）， and tumor necrosis factor-alpha （TNF- α） in the cells. Real-time quantitative polymerase chain reaction （RT-qPCR） was used to measure ACSL4 mRNA expression levels. Results Following hypoxia， PC12 cells exhibited increased apoptosis rates and decreased proliferation rates， with the differences becoming more pronounced with prolonged hypoxia. Hypoxia treatment induced ferroptosis in the cells，whereas co-treatment with the herbal components quercetin，luteolin，and kaempferol inhibited hypoxia-induced neuronal ferroptosis and inflammatory injury. Specifically： both quercetin and luteolin significantly upregulated the protein expression levels of GPX4 and p-AKT in PC12 cells after hypoxia treatment. Quercetin inhibited the protein expression of MAPK3 and ACSL4 as well as the level of Fe2+ . Luteolin downregulated ACSL4 mRNA expression levels and inhibited MAPK3 protein expression and Fe2+ level. Furthermore， both components significantly suppressed hypoxia-induced neuronal inflammation，manifested as decreased protein expression levels of IL-6 and TNF-α. Kaempferol upregulated both the protein expression level of GPX4 and downregulated both the protein and mRNA expression levels of ACSL4 in PC12 cells after hypoxia treatment. Additionally，kaempferol significantly reduced Fe2+ levels in hypoxic PC12 cells. Conclusion The main components of Xiao Xuming Decoction—quercetin， luteolin， and kaempferol—exert protective effects against hypoxia-induced neuronal injury by inhibiting ferroptosis and the inflammatory response.

R285.5

广东省基础与应用基础研究基金企业联合基金项目（面上项目）（编号：2022A1515220183）