Quercetin Modulates Liver Metabolic Profile in the Chronic Unpredictable Mild Stress Rat Model Based on Metabolomics Technology

GraphPad Prism 5 was used to generate all charts. A heatmap of metabolite was performed by using the software R version 4.0.2.The UPLC BEH C18 column (100×2.1mm, 1.7μm) was used for chromatographic separation. The mobile phase consists of 0.1% formic acid (solvent A) and acetonitrile (solvent B).

Liquid phase gradient setting was as follows: 0 to 0.5min, 98% A; 0.5 to 1.5min, 80% A; 1.5 to 6min, 30% A; 6 to 12min, 2% A; 12 to 14min, 30% A; 14 to 16min, 98% A. The flow rate was set to 0.45mL/min and the injection volume was 2μL. The temperature of the autosampler and the column were set to 4℃ and 35℃. The QC sample was injected after every 10 measurements to monitor the stability of the instrument and evaluate the reproducibility of the UPLC-MS systems.Date feature alignment, nontargeted signal detection, and signal integration were performed by Progenesis QI software.

Subsequently, EZinfo software (version 2.0) was used for the statistical analysis of metabolite features. Principal component analysis (PCA) was used to assess the repeatability of technical and methodological. Then, partial least squares discriminant analysis (PLS-DA) and orthogonal partial least squares discriminant analysis (OPLS-DA) was performed to examine the best separation between the groups.

Furthermore, the model was validated by a cross‐validation tool (SIMCA‐P software; version 14.0) to avoid the risk of overfitting. The S-plot and the value of variable importance in the projection (VIP) scores >1.0 were used to select differential metabolites. Finally, differential metabolites with fold change ≥ 2, and P＜0.05 were filtered as significantly changed metabolites.Preliminary identification of metabolites was performed using the Human Metabolome Database (HMDB).

Finally, metabolites were identified based on m/z and MS/MS spectra compared with public databases. Metabolites pathway analysis was performed with Metaboanalyst (mataboanalyst.ca) using the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database (genome.jp/kegg/kegg2.html) and the Small Molecule Pathway Database (SMPDB, smpdb.ca).