探讨银杏二萜内酯（ginkgo diterpene lactone，GDL）对氧化应激诱导的细胞衰老的保护作用及机制。方法建立H₂O₂诱导PC12神经元衰老细胞模型，分别给予血小板活化因子拮抗剂WEB-2086或GDL预处理，采用CCK-8试剂盒检测细胞活性； β-半乳糖甘酶染色法检测衰老细胞率； DAPI染色法观察细胞平均核体积； Western blotting检测p21、p53、核纤层蛋白B1（Lamin B1）、血小板活化因子受体（platelet activating factor receptor，PAFR）、沉默调节蛋白1（Sirtuin 1，SIRT1）、磷酸化信号转导和转录激活蛋白3（phosphorylated signal transducer and activator of transcription 3，p-STAT3）、脑源性神经营养因子（brain-derived neurotrophic factor，BDNF）、神经营养因子3（neurotrophin 3，NT3）和神经生长因子（nerve growth factor，NGF）蛋白表达； TUNEL染色法观察细胞凋亡。结果与对照组比较，模型组显著降低了细胞的活性（P＜ 0.05），衰老细胞数显著增加（P＜0.01），PAFR表达显著升高（P＜0.001）。与模型组比较，WEB-2086显著抑制PAFR表达（P＜0.001），显著升高SIRT1蛋白表达（P＜0.05），显著减少β-半乳糖甘酶衰老细胞率（P＜0.05）； GDL显著升高了细胞活力（P＜0.05），显著上调Lamin B1、SIRT1、BDNF、NT3的表达（P＜0.05、0.01），显著下调PAFR、p-STAT3、p21、p53蛋白表达（P＜0.05、0.01）。结论GDL可以保护神经元并抑制细胞凋亡，延缓H2O2代表的氧化应激诱导PC12神经元的衰老，其机制可能与拮抗PAFR、调节SIRT1/STAT3信号通路有关。

Objective To explore the protective effect and mechanism of ginkgo diterpene lactone (GDL) on cell senescence induced by oxidative stress. Methods The cell model of PC12 neuron aging induced by H₂O₂ was established, and platelet activating factor antagonist WEB-2086 or GDL was pretreated respectively, cell activity was detected by CCK-8 kit; β-Galactosidase staining was used to detect the aging cell rate; The average nuclear volume of cells was observed by DAPI staining; p21, p53, Lamin B1, platelet activating factor receptor (PAFR), Sirtuin 1 (SIRT1), phosphorylated signal transducer and activator of transcription 3 (p-STAT3), brain-derived neurotrophic factor (BDNF) and neurotrophic factor 3 (NT3) and nerve growth factor (NGF) protein expressions were detected by Western blotting; Apoptosis was observed by TUNEL staining. ResultsCompared with control group, the activity of cells in model group was significantly decreased (P ＜ 0.05), the number of aging cells was significantly increased (P ＜ 0.01) and the expression of PAFR was significantly increased (P ＜ 0.001). Compared with model group, WEB-2086 significantly inhibited the expression of PAFR (P ＜ 0.001), significantly increased the expression of SIRT1 protein (P ＜ 0.05), and significantly reduced the aging cell rate of β-galactosidase (P ＜ 0.05). GDL significantly increased the cell viability (P ＜ 0.05), significantly increased the expressions of Lamin B1, SIRT1, BDNF and NT3 (P ＜ 0.05, 0.01), and significantly decreased the expressions of PAFR, p-STAT3, p21 and p53 (P ＜ 0.05, 0.01). Conclusion GDL can protect neurons, inhibit apoptosis and delay the aging of PC12 neurons induced by oxidative stress represented by H₂O₂, and its mechanism may be related to antagonizing PAFR and regulating SIRT1/STAT3 signaling pathway.

2021年国家中医药管理局青年岐黄学者项目