Widely targeted metabolomics analysis
Widely targeted metabolomics was performed as previously described.27,28 Briefly, 50 µL of plasma was transferred to an extraction solution (acetonitrile:methanol = 1:4, v/v) containing internal standards and then centrifuged at 12,000 rpm for 10 min (4 °C). The supernatant (180 µL) was collected for further analysis. Metabolites were separated and identified using ultra performance liquid chromatography (ExionLC AD, https://sciex.com.cn/), quadrupole-time of flight (TripleTOF 6600, AB SCIEX), and triple quadrupole-linear ion trap mass spectrometry (QTRAP®, https://sciex.com/ ). The orthogonal projections to latent structures discriminant analysis (OPLS-DA) model and variable importance in projection (VIP) values were generated using the R package MetaboAnalystR. Metabolites with p < 0.05, VIP ≥ 1, and fold change ≥ 1.2 or ≤ 0.83 were considered significantly differential metabolites. Pathway enrichment analyses for these metabolites were performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database (http://www.genome.jp/kegg ).
Animals and carbon tetrachloride ( CCl4)-induced liver injury
Eight-week-old C57BL/6 mice were purchased from SPF Biotechnology Co., Ltd. (Beijing, China) and bred in a specific pathogen-free facility at the Experimental Animal Center of Nanfang Hospital. Food and water were provided ad libitum under a 12/12 h light/dark cycle.
To induce acute liver injury, mice were administered one intraperitoneal injection of 10 mL/kg of 5% CCl4 (Rhawn Co., Ltd., Shanghai, China) dissolved in corn oil, and the control group was administered one intraperitoneal injection of just corn oil (Macklin, Shanghai, China). Mice were treated with metabolites or phosphate buffered saline (PBS) at 2 h after injection of CCl4. Several available differential metabolites were divided into water-soluble and non-water-soluble metabolites. For the non-water-soluble metabolites, mice with liver injury were administered 100 mg/kg of the corresponding metabolites by oral gavage. For water-soluble metabolites, mice with liver injury were intraperitoneally injected with 100 mg/kg of the corresponding metabolites. Blood was collected 24 and 48 h after CCl4 injection. All mice were sacrificed 72 h after injection, and blood and liver samples were collected. The protocols for animal experiments were approved by the Institutional Animal Ethics Committee of the Experimental Animal Center of Nanfang Hospital.