目的 探讨银杏二萜内酯（ginkgo diterpene lactone，GDL）对D-半乳糖诱导的衰老小鼠的保护作用及潜在作用机制。方法 将小鼠随机分为对照组、模型组及GDL低、高剂量（5、10 mg/kg）组和二甲双胍（200 mg/kg）组，每组6只。小鼠连续8周颈部sc D-半乳糖（500 mg/kg），从第3周开始，给予相应药物干预。通过水迷宫实验评价小鼠的记忆和认知功能；β-半乳糖苷酶染色评价小鼠海马神经元的衰老情况；Western blotting检测海马组织细胞周期相关蛋白的表达。利用网络药理学和分子对接探索GDL抗衰老的机制，并采用Western blotting和免疫荧光法对分子对接结果进行验证。结果 与对照组比较，模型组小鼠在目标象限停留时间减少（P＜0.05），海马神经元β-半乳糖苷酶活性显著升高（P＜0.001），海马组织衰老标志蛋白p53、p21、p16的表达明显升高（P＜0.001）。与模型组比较，GDL组小鼠在目标象限停留时间增加（P＜0.05），β-半乳糖苷酶活性降低（P＜0.001），p53、p21和p16蛋白表达水平显著降低（P＜0.05、0.01、0.001）。网络药理学和分子对接结果提示GDL预防衰老可能与磷脂酰肌醇3-激酶（phosphatidylinositol 3-kinase，PI3K）/蛋白激酶B（protein kinase B，Akt）通路和细胞凋亡有关。动物实验验证表明GDL能够上调PI3K/Akt信号通路蛋白的表达（P＜0.05、0.01、0.001），并抑制其下游凋亡蛋白剪切型半胱氨酸天冬氨酸蛋白酶-3（cleaved cystein-asparate protease-3，cleaved Caspase-3）、B细胞淋巴瘤-2相关X蛋白（B-cell lymphoma-2 associated X protein，Bax）的表达（P＜0.01、0.001）。结论 GDL可改善D-半乳糖诱导的衰老小鼠的认知功能，抑制衰老诱导的细胞凋亡，其机制可能与调控PI3K/Akt信号通路有关。

Objective To investigate the protective effect and potential mechanisms of ginkgo diterpene lactone (GDL) on D-galactose-induced aging in mice. Methods Mice were randomly divided into control group, model group, GDL low-, high-dose (5, 10 mg/kg) groups and metformin (200 mg/kg) group, with six mice in each group. Mice were given D-galactose (500 mg/kg) in the neck for eight consecutive weeks, and corresponding drug intervention was administered starting from the third week. The memory and cognitive functions of mice were evaluated through water maze experiments; Aging status of hippocampal neurons was evaluated by β-galactosidase staining; Western blotting was used to detect the expressions of cell cycle related proteins in hippocampal tissue. The anti-aging mechanism of GDL was explored using network pharmacology and molecular docking, and molecular docking results was validated by Western blotting and immunofluorescence. Results Compared with control group, the time spent in the target quadrant of mice in model group was reduced (P < 0.05), β-galactosidase activity in hippocampal neurons was significantly increased (P < 0.001), the expressions of aging marker proteins p53, p21 and p16 in hippocampal tissue were significantly increased (P < 0.001). Compared with model group, the time spent in the target quadrant of mice in GDL group was increased (P < 0.05), β-galactosidase activity in hippocampal neurons was significantly decreased (P < 0.001), the expressions of p53, p21 and p16 proteins in hippocampal tissue were significantly decreased (P < 0.05, 0.01, 0.001). The results of network pharmacology and molecular docking suggested that the prevention of aging by GDL may be related to phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway and cell apoptosis. Animal experiments showed that GDL could up-regulate the expressions of PI3K/Akt signaling pathway proteins (P < 0.05, 0.01, 0.001) and inhibit the expressions of downstream apoptotic proteins such as cleaved cysteine aspartate protease-3 (cleaved Caspase-3) and B-cell lymphoma associated X protein (Bax) (P < 0.01, 0.001). Conclusion GDL could improve the cognitive function of D-galactose-induced aging mice, inhibit aging induced cell apoptosis, and its mechanism may be related to the regulation of PI3K/Akt signaling pathway.

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）