[關(guān)鍵詞]
[摘要]
目的 研究絞股藍(lán)總皂苷(GPs)對(duì)tau蛋白過表達(dá)細(xì)胞代謝的影響,探討GPs治療阿爾茨海默病(AD)的機(jī)制。方法 通過采用CRISPR/Cas9技術(shù)構(gòu)建微管相關(guān)蛋白tau (MAPT)過表達(dá)N2a細(xì)胞系(MAPT細(xì)胞);用CCK-8法檢測(cè)GPs對(duì)N2a、MAPT細(xì)胞存活率的影響;體外培養(yǎng)N2a (對(duì)照組)、MAPT細(xì)胞(模型組),向MAPT細(xì)胞中加入GPs (50、100 μg·mL-1)進(jìn)行干預(yù),共同孵育24 h后收集細(xì)胞,采用Western blotting技術(shù)檢測(cè)總tau蛋白量水平;利用代謝組學(xué)技術(shù)檢測(cè)GPs對(duì)MAPT細(xì)胞內(nèi)源性代謝產(chǎn)物的影響,明確差異代謝產(chǎn)物及通路。結(jié)果 50、100、200 μg·mL-1 GPs對(duì)N2a細(xì)胞活力無明顯影響;5、50、100、200 μg·mL-1 GPs對(duì)MAPT細(xì)胞活力無顯著性影響;與對(duì)照組相比,MAPT細(xì)胞內(nèi)tau蛋白水平明顯增加(P<0.001);與模型組比較,50 μg·mL-1 GPs可明顯減少M(fèi)APT細(xì)胞內(nèi)總tau蛋白含量(P<0.05);代謝組學(xué)結(jié)果顯示,按照差異代謝產(chǎn)物數(shù)量占比從高到底依次為脂質(zhì)和類脂分子,有機(jī)酸及其衍生物,核苷、核苷酸和類似物等。通過京都基因與基因組百科全書(KEGG)通路分析,差異代謝產(chǎn)物主要富集在核苷酸代謝、氨基酸合成、甘油磷脂代謝等通路。與模型組比較,給藥后參與甘油磷脂代謝通路的胞磷膽堿和磷脂酰膽堿水平顯著增加(P<0.001),甘油磷酰膽堿、甘油-3-磷乙醇胺水平顯著降低(P<0.001)。結(jié)論 GPs可以減少細(xì)胞內(nèi)tau蛋白的異常過表達(dá),通過增加胞磷膽堿和磷脂酰膽堿水平、減少甘油磷酰膽堿和甘油-3-磷乙醇胺水平調(diào)控甘油磷脂代謝通路。
[Key word]
[Abstract]
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.
[中圖分類號(hào)]
R965
[基金項(xiàng)目]
北京市醫(yī)院管理中心“青苗”計(jì)劃(QML20210806);北京市第三批中藥骨干人才