Metabolomic Analysis

The samples were extracted using a 400 µL methanol: acetonitrile (1:1, v/v) solution. The mixture then sonicated at 40 kHz for 30 min at 5°C. The samples were placed at -20°C for 30min to precipitate proteins. After centrifugation at 13000g at 4°C for 15min, the supernatant was carefully transferred to new microtubes and evaporated to dryness under a gentle stream of nitrogen. For UHPLC-MS/MS analysis, the samples were reconstituted in 100 µL loading solution of acetonitrile: water (1:1, v/v) by brief sonication in a 5°C water bath. Extracted metabolites were spun for 15 min at 13000g at 4°C on a bench-top centrifuge and cleared supernatant were transferred to sample vials for LC-MS/MS analysis. The instrument platform for LC-MS analysis is UHPLC-Q-Exactive system of Thermo Fisher Scientific. The selection of significantly different metabolites was determined based on the Variable importance in the projection (VIP) obtained by the OPLS-DA model and the p-value of student’s t test, and the metabolites with VIP>1, p<0.05. The analysis was performed using the free online platform of majorbio cloud platform (cloud.majorbio.com).