Metabolomics study on the effect of linezolid on the proliferation of MEG-01 megakaryocytes and the generation of platelet precursors
OBJECTIVE To investigate the metabolic changes in MEG-01 megakaryocytes after treatment with linezolid (LZD) from metabolomic perspective and its impact on the the proliferation of cells and generation of platelet precursors. METHODS MEG-01 cells were seeded in proliferation medium and divided into blank control group (untreated),solvent control group (4‰DMSO),and 100,200,400,800 μg/mL LZD groups. The proliferation status of cells in each group was observed under the microscope;cell proliferation and viability were assessed. Cells were also seeded in differentiation medium and divided into blank control group (untreated),solvent control group (4‰DMSO),and 400 μg/mL LZD group;after 14 days of culture,platelet precursor generation was observed under the microscope;immunofluorescence staining was performed to count the proportion of cells producing pseudopodia,the relative length of pseudopodia was measured,and the expression levels of CD41 and CD42b mRNA were assessed. Cells from the solvent control group and the 400 μg/mL LZD group,cultured in differentiation medium for 14 days,were extracted and subjected to non-targeted metabolomics and targeted energy metabolomics analysis using liquid chromatography-tandem mass spectrometry. The relative content of pyruvate in cells,after being cultured for 14 days with the addition of pyruvate (10 mmol/L) or LZD (400 μg/mL)+pyruvate (10 mmol/L),was measured and observed,as well as its effects on cell proliferation and platelet precursor generation. RESULTS 400 μg/mL LZD could significantly inhibit the proliferation of MEG-01 cells and the generation of platelet precursors (P<0.01). Non-targeted metabolomic analysis of MEG-01 cells after 400 μg/mL LZD treatment revealed significant changes in energy metabolism-related pathways such as mTOR signaling pathway,alanine,aspartate and glutamate metabolism,and central carbon metabolism in cancer. Targeted energy metabolomic analysis further showed that the relative contents of adenosine triphosphate,guanosine triphosphate,and pyruvate in MEG-01 cells were significantly reduced (P<0.01),while the relative contents of lactate were significantly increased (P<0.01). Compared with the LZD group,the relative content of pyruvate,cell count,the proportion of cells producing pseudopodia and the relative length of pseudopodia were significantly increased in the LZD+pyruvate group (P<0.01). CONCLUSIONS LZD may reduce pyruvate production by inhibiting mitochondrial energy metabolism,thereby suppressing megakaryocyte proliferation and platelet precursor production,ultimately leading to the occurrence of thrombocytopenia.
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维普
