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.