(1) Hybridization Experiments to Obtain Mouse Macrophages Lacking S100A4
As shown in the schematic diagram, F1-generation heterozygous mice were obtained by crossing S100A4flox/flox mice and Lyz-Cre mice. The F1 generation of heterozygous mice was self-crossed. Among the F2 generation mice obtained, the target mice containing S100A4flox/floxCre + lacked S100A4 in their macrophages (Fig. 1A). F2-generation mice were identified by mouse tail PCR, and three mice (5131, 5133, and 5134) were identified as S100A4 positive (producing only a 223 bp band) (Fig. 1B). According to the Lyz analysis, both primer pairs, P1 and P2, produced positive results for Lyz (Fig. 1C), suggesting that the three mice (5131,
5133, and 5134) were target mice.
(2) Metabolomic Analysis Identifies Differentially Abundant Metabolites
To determine the biological repeatability between the same group of samples, Spearman rank correlation was used as the evaluation index of biological repeat correlation. The closer r is to 1, the better the repeatability. The results showed that the r index between the two groups of samples was close to 1 (Fig. 2A), indicating that macrophages lacking S100A4 had greater differences than macrophages in the control group did. Among the differentially abundant metabolites, 161 common metabolites exhibited changes in abundance, with 84 showing differences in abundance in the control group/S100A4 knockout group. Among those metabolites, 77 were enriched, and 77 were depleted (Fig. 2B-C). These differentially abundant metabolites were further classified, and the enriched metabolites were mainly concentrated in the UDP-N-acetylmuramoyl-L-ala-D-glutamate, arachidonate, DG, ganoderic acid G, methyl 15-cyanopentadecanoate, 1,25-dihydroxy-19-novitamin D3, forsythoside B, N-(2-cyaoethyl)valine, fenitrooxone, and epoxyeicosatrienoic acid metabolic pathways. The relatively depleted metabolites were mainly associated with the 4-(methylsulfanyl)-2-oxbutanoylcarnitine, furanodienone, 2',2'-difluorodexyuridine, phendimetrazine, N-methylvaline, histidylphenylalanine, oxytocin 1–8, methylthiouracil, 4(1H)-pyridinone, 2-ethy-3-hydroxy-1-(2-hydroxyethyl), and L-methionine 10 metabolic pathways (Fig. 2D).
(3) Synergistic Relationships Between Differentially Abundant Metabolites
There is a synergistic or mutually exclusive relationship between differentially abundant metabolites. For example, if the trend of changes in the abundance of a metabolite is the same, there is a positive correlation; if the trend of changes in the abundance of a metabolite is opposite, there is a negative correlation. The highest correlation is 1, which is a complete positive correlation (red), and the lowest correlation is -1, which is a complete negative correlation (blue). The results showed that there was both synergy and antagonism between different metabolites. For example, thiothixene and histidylphenylalanine have an antagonistic relationship, but thiothixene and bacilysin have a synergistic relationship (Fig. 3).
(4) Major Metabolites whose Abundance Was Altered
Among the major metabolites, lysophosphatidylserine (LysoPS) (Fig. 4A), ceramide (Cer) (Fig. 4B), FMN (Fig. 4C), thiothixene (Fig. 4D), 5-(3Sa,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrotieno[3,4-d]imidazol-4yl]-N’-propylpentanehydrazide (Fig. 4E), (5S)-5,6-dihydroxy-2-oxohexanal (Fig. 4F), 4(1H)-pyridinone,2-ethyl-3-hydroxy-1-(2-hydroxyethyl) (Fig. 4G), histidylphenylalanine (Fig. 4H), PE(P-18:0/18:1{12Z}-2OH(9, 10) (Fig. 4I), and L-glutamate (Fig. 4J) were the 10 with largest variations in abundance.
(5) KEGG Analysis
Cluster analysis was performed on metabolites whose contents changed, and the most enriched metabolites were benzenoids; lipids and lipid-like molecules; nucleosides, nucleotides, and analogs; organic acids and analogs; organic acids and derivatives; organic nitrogen compounds; organic oxygen compounds; organoheterocyclic compounds; phenylpropanoids; and polyketides (Fig. 5A). To understand the role of these metabolites, we subjected these differentially abundant metabolites to KEGG cluster analysis and found enrichment in amino acid metabolism, biosynthesis of other secondary metabolites, cancer: overview, digestive system, lipid metabolism, membrane transport, metabolism of cofactors and vitamins, metabolism of other amino acids, nervous system, translation, xenobiotic biodegradation and metabolism (Fig. 5B).