Abstract: 【Abstract】 Objective To analyze changes in energy metabolism and oxylipin metabolism in macrophages after stimulation by Mycobacterium marinum （M. marinum） using targeted metabolomics, and to provide insights into the mechanisms underlying the immune defense by macrophages against M. marinum infections. Methods Mouse bone marrow-derived macrophages were obtained from the bilateral femurs of mice, and cultured cells were divided into two groups: the active M. marinum group and the inactivated M. marinum group. Bacterial suspensions were prepared using M. marinum clinical isolates; the active M. marinum group was treated with live M. marinum suspensions for 12 hours, while the inactivated M. marinum group with inactivated M. marinum suspensions for 12 hours. Cell morphology was observed through microscopy, and cell length was measured. Cell lysates collected from both groups were subjected to liquid chromatography-tandem mass spectrometry analysis to detect energy and oxylipin metabolites. A t-test was utilized to compare the lengths of macrophages between the two groups, while principal component analysis and orthogonal partial least squares-discriminant analysis were conducted to identify differential metabolites. Results Under the microscope, macrophages in the active M. marinum group formed more granuloma-like cell aggregates compared with those in the inactivated M. marinum group; the macrophages were significantly thinner and longer in the inactivated M. marinum group （439.52 ± 91.67 μm） than in the active M. marinum group （289.96 ± 70.11 μm, P < 0.001）. Principal component analysis and orthogonal partial least squares-discriminant analysis of energy metabolism and oxylipin metabolism in macrophages demonstrated good separation between the two groups. As for the energy metabolism, a total of 12 differential metabolites were identified, with the amino acid metabolism showing the most significant changes. Specifically, there was a significant increase in the content of L-citrulline, while the content of L-leucine and serine decreased. As for the oxylipin metabolism, 20 differential metabolites were identified, with the arachidonic acid metabolism showing the most significant changes. Conclusions Macrophages stimulated by live M. marinum exhibited altered amino acid metabolism and arachidonic acid metabolism compared with those stimulated by inactivated M. marinum, characterized by an increase in L-citrulline content, a decrease in L-leucine and serine levels, and alterations in arachidonic acid content.

Key words: Mycobacterium marinum, Macrophages, Metabolomics, Energy metabolism, Citrulline, Leucine, Serine, Arachidonic acid