Calycosin-7-O-β-D-Glucoside Ameliorates Palmitate-Induced Lipid Accumulation in HT22 Cells

Abstract

Background: The pathogenesis of Alzheimer's disease (AD) is complex. Recent research suggests that AD patients have early disorders in brain cholesterol metabolism. Cholesterol and its derivatives accumulate in neurons, leading to p-Tau overproduction and synaptic dysfunction, initiating AD progression. Calycosin-7-O-β-D-glucoside (CG), a distinctive constituent of Astragali Radix, holds a representative position. Many clinical trials have demonstrated that CG can attenuate cerebral ischemia/reperfusion injury and preserve the structural integrity of the blood-brain barrier. However, whether CG alleviates tau-mediated neurodegeneration by increasing cholesterol efflux after lipid accumulation remains unexplored.

Methods: Ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS/MS) and multivariate data analysis were employed to investigate metabolic changes in HT22 cells induced by sodium palmitate following 24 hours of CG treatment. The potential therapeutic mechanisms of CG on AD were further examined through Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis.

Results: Metabolomic analysis characterized 24 potential biomarkers, revealing that CG could ameliorate cholesterol metabolic pathways. The results of cell experiments revealed that CG can increase the expression of enzyme cholesterol 24-hydroxylase (CYP46A1) (p < 0.05) and the level of 24 hydroxycholesterol (24-OHC) (p < 0.05), reduce the expression of p-Tau (Thr231)/Tau (p < 0.01), inhibit the formation of lipid droplets.

Conclusion: CG may inhibit the accumulation of cholesterol and its derivatives in neurons by affecting the CYP46A1-CE-Tau axis, offering a potential therapeutic strategy for AD.

Fig. 1.

The viability of HT22 cells exhibits dose-dependent responses to CG. (A) Total cholesterol (T-CHO) levels in HT22 cells after exposure to varying concentrations of PA. (B) Chemical structure of CG. (C) Viability of HT22 cells treated with different concentrations of CG for 24 or 48 hours assessed by the CCK-8 assay. (D) T-CHO content in HT22 cells following treatment with various concentrations of PA and CG. Data are presented as the mean $\pm$ standard error of the mean (SEM) from three independent experiments. Statistical significance is indicated as *p 0.05 and **p 0.01 compared to the control group; ^#p 0.05 and ^#⁢#p 0.01 compared to the PA group, not significant is indicated as ns. PA, palmitic acid; CG, calycosin-7-O-beta-D-glucoside; CCK-8, cell counting kit-8.

Fig. 2.

Multifactorial analysis of metabolomic data in CG-treated HT22 cells. (A) OPLS-DA score plots comparing metabolites between the control, model, and CG-treated groups. (B) Volcano plot illustrating significant differences in metabolites between the model and control groups, and between the CG and model groups. (C) Heat map on the relative levels of 24 key metabolites across the control (green), model (blue), and CG (red). The color scale ranges from dark blue (lowest) to dark red (highest) metabolite levels. Abbreviations: C, control group; M, model group; CG, CG group; CG, calycosin-7-O-beta-D-glucoside; PA, palmitic acid; CE, cholesterol ester; PGF1, prostaglandin F1; LTE4, leukotriene E4.

Fig. 3.

Metabolite pathway analysis in CG-treated HT22 cells. (A) KEGG pathway enrichment analysis of metabolites in HT22 cells treated with CG. (B) Schematic representation of selected metabolic pathways influenced by PA and CG treatment. Abbreviations: KEGG, Kyoto Encyclopedia of Genes and Genomes; CG, calycosin-7-O-beta-D-glucoside; PA, palmitic acid; VAP, viral attachment protein; ORP5, oxysterol-binding protein-related protein 5; SOAT, sterol O-acyltransferase; FC, cholesterol; SORT1, neurotensin receptor 3; CIDEB, cell death-inducing DFFA-like effector B; VLDL, very low density lipoprotein; CYP7A1, cytochrome P450 7A1; ER, endoplasmic reticulum; TG, triacylglycerol.

Fig. 4.

Effects of CG on cholesterol metabolism in HT22 cells. (A) Western blot analysis was utilized to determine the protein expression levels of p-Tau (Thr231), Tau and CYP46A1 in HT22 cells following PA induction and CG treatment. (B,C) The signal intensity was measured quantitatively through densitometric analysis and expressed as the fold difference in comparison to the control group, both for cells treated with CG and Efavirenz. (D) ELISA was employed to assess the concentrations of 24-OHC in DMEM, following PA induction and CG treatment. (E) Staining with Oil red O. Data are presented as the mean $\pm$ SD from three independent experiments. Statistical significance is indicated as *p 0.05 compared to the control group; ^#p 0.05, ^#⁢#p 0.01 compared to the model group. CG, calycosin-7-O-beta-D-glucoside; PA, palmitic acid; CYP46A1, cholesterol 24-hydroxylase; DMEM, Dulbecco’s modified eagle medium; SD, standard deviation; ELISA, enzyme-linked immunosorbent assay.