目的 研究银杏二萜内酯K（ginkgolide K，GK）对D-半乳糖诱导的小鼠原代星形胶质细胞抗衰老作用及可能机制。方法 以小鼠原代星形胶质细胞为研究对象，建立D-半乳糖诱导的衰老模型，设置对照组、模型组和GK组，GK（50 μg/mL）预处理3 h后，以150 mmol/L D-半乳糖造模6 d，采用β-半乳糖苷酶染色法检测衰老星形胶质细胞；Western blotting法测定衰老标志蛋白（p21、p53和Lamin B1）、沉默信息调节因子1（silent information regulator 1，SIRT1）、脑源性神经营养因子（brain-derived neurotrophic factor，BDNF、自噬标志蛋白微管相关蛋白1轻链3-Ⅱ/I（microtubule associated protein 1 light chain 3-Ⅱ/I，LC3-Ⅱ/I）和自噬选择性底物p62表达；ELISA检测衰老相关分泌表型[白细胞介素-6（interleukin-6，IL-6）、IL-1β、肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）]；免疫荧光检测增殖标志分子Ki67表达。结果 与对照组比较，D-半乳糖显著诱导原代星形胶质细胞β-半乳糖苷酶阳性表达（P＜0.001），促进衰老标志蛋白p21、p53的上调和Lamin B1的下调（P＜0.05、0.01），减少增殖标志分子Ki67的表达（P＜0.01），表明成功构建了公认的细胞衰老模型。与模型组比较，GK显著减少β-半乳糖苷酶阳性表达（P＜0.001），下调p21、p53和上调Lamin B1蛋白的表达（P＜0.05、0.01），增加Ki67的表达（P＜0.01），表明GK具有抗衰老作用。此外，GK显著促进BDNF蛋白表达（P＜0.05），减少TNF-α的分泌（P＜0.05），上调SIRT1和自噬标志蛋白LC3-Ⅱ/I表达（P＜0.05、0.01），减少p62蛋白表达（P＜0.01）。结论 GK可有效抑制原代星形胶质细胞的衰老，其作用机制可能与上调SIRT1、BDNF表达和促进细胞自噬有关，从而多维度协同达到抗细胞衰老的效应。

Objective To study the anti-aging effect and possible mechanism of ginkgolide K (GK) on murine primary astrocytes induced by D-galactose. Methods The aging model induced by D-galactose was established in murine primary astrocytes, and the aging astrocytes were set up in control group, model group and GK group. After pretreatment with GK (50 μg/mL) for 3 h, the aging astrocytes were modeled with 150 mmol/L D-galactose for 6 d, and β-galactosidase staining was used to detect the aging astrocytes. Western blotting was used to detect the aging marker proteins (p21, p53 and Lamin B1), silent information regulator 1 (SIRT1), brain-derived neurotrophic factor (BDNF), autophagy marker protein microtubule associated protein 1 light chain 3-II/I (LC3-II/I) and autophagy selective substrate p62 expressions. ELISA was used to detect aging-related secretory phenotype [interleukin-6 (IL-6), IL-1β, tumor necrosis factor-α (TNF-α)]; The expression of Ki67 was detected by immunofluorescence. Results Compared with control group, D-galactose significantly induced the positive expression of β-galactosidase in primary astrocytes (P < 0.001), promoted the up-regulation of aging marker proteins p21 and p53 and the down-regulation of Lamin B1 (P < 0.05, 0.01), and reduced the expression of proliferation marker molecule Ki67 (P < 0.01), which indicated that a recognized cell aging model was successfully established. Compared with model group, GK significantly reduced the positive expression of β-galactosidase (P < 0.001), down-regulated the expressions of p21, p53 and up-regulated Lamin B1 protein expression (P < 0.05, 0.01), and increased the expression of Ki67 (P < 0.01), indicating that GK had anti-aging effect. In addition, GK significantly promoted the expression of BDNF protein (P < 0.05), decreased the secretion of TNF-α (P < 0.05), increased the expressions of SIRT1 and autophagy marker protein LC3-Ⅱ/I/I (P < 0.05, 0.01), and decreased the expression of p62 protein (P < 0.01). Conclusion GK can effectively inhibit the aging of primary astrocytes, and its mechanism may be related to up-regulating the expressions of SIRT1 and BDNF and promoting autophagy, so as to achieve the anti-aging effect in multiple dimensions.

2021年国家中医药管理局岐黄学者项目