目的 研究绞股蓝总皂苷(GPs)对tau蛋白过表达细胞代谢的影响,探讨GPs治疗阿尔茨海默病(AD)的机制。方法 通过采用CRISPR/Cas9技术构建微管相关蛋白tau (MAPT)过表达N2a细胞系(MAPT细胞);用CCK-8法检测GPs对N2a、MAPT细胞存活率的影响;体外培养N2a (对照组)、MAPT细胞(模型组),向MAPT细胞中加入GPs (50、100 μg·mL^-1)进行干预,共同孵育24 h后收集细胞,采用Western blotting技术检测总tau蛋白量水平;利用代谢组学技术检测GPs对MAPT细胞内源性代谢产物的影响,明确差异代谢产物及通路。结果 50、100、200 μg·mL^-1 GPs对N2a细胞活力无明显影响;5、50、100、200 μg·mL^-1 GPs对MAPT细胞活力无显著性影响;与对照组相比,MAPT细胞内tau蛋白水平明显增加(P<0.001);与模型组比较,50 μg·mL^-1 GPs可明显减少MAPT细胞内总tau蛋白含量(P<0.05);代谢组学结果显示,按照差异代谢产物数量占比从高到底依次为脂质和类脂分子,有机酸及其衍生物,核苷、核苷酸和类似物等。通过京都基因与基因组百科全书(KEGG)通路分析,差异代谢产物主要富集在核苷酸代谢、氨基酸合成、甘油磷脂代谢等通路。与模型组比较,给药后参与甘油磷脂代谢通路的胞磷胆碱和磷脂酰胆碱水平显著增加(P<0.001),甘油磷酰胆碱、甘油-3-磷乙醇胺水平显著降低(P<0.001)。结论 GPs可以减少细胞内tau蛋白的异常过表达,通过增加胞磷胆碱和磷脂酰胆碱水平、减少甘油磷酰胆碱和甘油-3-磷乙醇胺水平调控甘油磷脂代谢通路。

Objective To investigate the effect of gypenosides (GPs) on improving the abnormal cell metabolism induced by abnormal tau protein overexpression, and to explore the mechanism of GPs in the treatment of Alzheimer's disease. Methods Microtubule-associated protein tau (MAPT) overexpressing N2a cell were constructed by CRISPR/Cas9 technique. The effects of GPs on the survival ratio of N2a and MAPT cells were detected by Cell Counting Kit-8 (CCK-8) to determine the safe dose range of GPs. N2a and MAPT cells were cultured in vitro, and different concentrations of GPs (50,100 μg·mL^-1) were added to MAPT cells for intervention. After co-incubation for 24 h, the cells were collected and the total tau protein levels were detected by western blotting. Metabolomics technology was used to detect the effects of GPs on metabolites of MAPT cells and identify the different metabolites and pathways. Results GPs (50, 100, 200 μg·mL^-1) had no significant effect on N2a cell viability. GPs (5, 50, 100, 200 μg·mL^-1) had no significant effect on MAPT cell viability. Compared with N2a control group, the tau protein level in MAPT cells was significantly increased (P < 0.001), and the total tau protein content in MAPT cells was significantly decreased by 50 μg· mL^-1 GPs. The metabolomics results showed that the different metabolites enriched in lipid and lipid molecules, organic acids, nucleosides, nucleotides and analogues, etc. KEGG pathway analysis demonstrated differential metabolites were mainly concentrated in nucleotide metabolism, amino acid synthesis, glycerol phospholipid metabolism and other pathways. After GPs treatment, the level of CDP-Choline and phosphocholine (PC) were increased (P < 0.001), while the contents of glycerophosphocholine (GPC) and glycerin-3-phosphoethanolamine were significantly decreased (P < 0.001). Conclusion GPs can attenuate the abnormal overexpression of tau protein, and regulate the metabolism pathway of glycerophospholipid by increasing CDP-Choline and PC, reducing GPC and sn-Glycerol-3-phosphoethanolamine.

R965

北京市医院管理中心“青苗”计划(QML20210806);北京市第三批中药骨干人才