目的 探讨银杏二萜内酯（ginkgo diterpene lactone，GDL）对血小板活化因子（platelet-activating factor，PAF）诱导神经元损伤的保护作用及相关机制。方法 构建PAF诱导神经元损伤的细胞模型与动物模型，给予GDL或PERK激动剂CCT020312进行处理。分别使用CCK-8和Annexin V-FITC细胞凋亡检测试剂盒测定小鼠海马神经元细胞系HT22细胞活力和凋亡；使用乳酸脱氢酶（lactate dehydrogenase，LDH）检测试剂盒检测HT22细胞培养上清液中LDH活性；采用Fluo-4钙离子检测试剂盒测定HT22细胞钙离子水平；采用分子对接探究相关分子机制；使用Western blotting检测HT22细胞与小鼠脑组织的细胞凋亡相关蛋白[半胱氨酸天冬氨酸蛋白酶-3（cystein-asparate protease-3，Caspase-3）、cleaved Caspase-3、B细胞淋巴瘤-2（B-cell lymphoma-2，Bcl-2）、Bcl-2相关X蛋白（Bcl-2 associated X protein，Bax）]和内质网应激相关蛋白[蛋白激酶R样内质网激酶（protein kinase R-like ER kinase，PERK）、p-PERK、真核翻译起始因子-2α（eukaryotic initiation factor-2α，eIF-2α）、p-eIF2α、转录激活因子4（activating transcription factor 4，ATF4）、C/EBP同源蛋白（C/EBP-homologous protein，CHOP）]的表达。结果 体外细胞实验结果显示，与对照组比较，模型组细胞活性显著降低（P＜0.001），LDH释放率升高（P＜0.001），钙离子水平升高（P＜0.001），凋亡细胞显著增加（P＜0.001），cleaved Caspase-3、Bax蛋白表达水平显著升高（P＜0.001），Bcl-2蛋白表达水平显著下降（P＜0.001），内质网应激相关蛋白表达水平显著升高（P＜0.05、0.001）；与模型组比较，GDL显著升高了细胞活力（P＜0.05、0.001），显著上调Bcl-2的表达（P＜0.05、0.001），显著下调cleaved Caspase-3、Bax、p-PERK、p-eIF2α、ATF4、CHOP的表达（P＜0.05、0.01、0.001）；CCT020312处理后GDL的效果均被逆转（P＜0.001）。体内动物实验结果显示，与对照组比较，模型组小鼠脑组织Bax蛋白表达水平显著升高（P＜0.001），Bcl-2蛋白表达水平显著下降（P＜0.001），内质网应激相关蛋白表达水平显著升高（P＜0.001）；与模型组比较，GDL显著上调Bcl-2的表达（P＜0.01、0.001），显著下调p-PERK、p-eIF2α、ATF4、CHOP、Bax的表达（P＜0.001）；CCT020312处理后GDL的效果均被逆转（P＜0.001）。结论 PAF可以激活神经元内质网应激的PERK/ATF4/CHOP通路来诱导钙超载，从而使神经元凋亡；而GDL可以通过抑制PERK/ATF4/CHOP通路来抑制内质网应激、钙超载和凋亡，以减轻PAF诱导的神经元损伤。

Objective To investigate the protective effect and related mechanism of ginkgo diterpene lactone (GDL) on platelet-activating factor (PAF)-induced neuronal injury. Methods A cellular model and an animal model of PAF-induced neuronal injury were constructed, GDL or PERK agonist CCT020312 were administered. The cell viability and apoptosis of HT22 cells were determined using CCK-8 and Annexin V-FITC apoptosis assay kits, respectively; Activity of lactate dehydrogenase (LDH) in HT22 cell culture supernatant was measured using a LDH assay kit, and calcium level in HT22 cells was determined using a Fluo-4 calcium assay kit; Molecular docking was used to explore the relevant molecular mechanisms; Western blotting was used to detect the expressions of apoptosis-related proteins [cystein-asparate protease-3 (Caspase-3), cleaved Caspase-3, B-cell lymphoma-2 (Bcl-2), Bcl-2 associated X protein (Bax)] and endoplasmic reticulum stress-related proteins [protein kinase R-like ER kinase (PERK), p-PERK, eukaryotic initiation factor-2α (eIF-2α), p-eIF2α, activating transcription factor 4 (ATF4), C/EBP-homologous protein (CHOP)] in HT22 cells and brain tissues of mice. Results The results of in vitro cell experiments showed that, compared with control group, cell activity was significantly reduced in model group (P < 0.001), release rate of LDH was increased (P < 0.001), level of calcium ions was increased (P < 0.001), number of apoptotic cells was significantly increased (P < 0.001), the expression levels of cleaved Caspase-3 and Bax proteins were significantly increased (P < 0.001), the expression level of Bcl-2 protein was significantly decreased (P < 0.001), and the expression levels of endoplasmic reticulum stress-related proteins were significantly increased (P < 0.05, 0.001). Compared with model group, GDL significantly increased cell viability (P < 0.05, 0.001), significantly up-regulated Bcl-2 expression (P < 0.05, 0.001), and significantly down-regulated the expression of cleaved Caspase-3, Bax, p-PERK, p-eIF2 α, ATF4 and CHOP (P < 0.05, 0.01, 0.001); The effect of GDL after CCT020312 treatment was reversed (P < 0.001). The results of in vivo animal experiments showed that compared with control group, the expression level of Bax protein in brain tissue of mice in model group was significantly increased (P < 0.001), the expression level of Bcl-2 protein was significantly decreased (P < 0.001), and the expression levels of endoplasmic reticulum stress-related proteins were significantly increased (P < 0.001); Compared with model group, GDL significantly up-regulated the expression of Bcl-2 (P < 0.01, 0.001), and significantly down-regulated the expressions of p-PERK, p-eIF2α, ATF4, CHOP and Bax (P < 0.001); The effect of GDL after CCT020312 treatment was reversed (P < 0.001). Conclusion PAF can active PERK/ATF4/CHOP pathway of neuronal endoplasmic reticulum stress to induce calcium overload and thus neuronal apoptosis, whereas GDL can inhibit endoplasmic reticulum stress, calcium overload and apoptosis by inhibiting PERK/ATF4/CHOP pathway to attenuate PAF-induced neuronal injury.

R285.5

江苏省基础研究计划自然科学基金—前沿引领技术基础研究专项（BK20232014）；连云港市“521”人才工程支持项目（LYG06521202221）