Abstract: Objective To investigate the effects of Mcart1 gene knockout on macrophage metabolism and phenotype. Methods Two types of bone marrow-derived macrophages (BMDMs) were obtained from Mcart1^flox/flox and Mcart1^Lyz2-cre mice. Basal respiration of both cell types was measured using a Seahorse analyzer. The activity of α-ketoglutarate dehydrogenase (OGDH) was assessed in Mcart1 stably knockout RAW264.7 cells (Mcart1^-/--RAW264.7) and wild-type RAW264.7 cells (RAW264.7). The two types of BMDMs were induced into M1 macrophages using LPS, and whole-cell reactive oxygen species (ROS) levels were measured by flow cytometry, while dynamic expression of inflammatory mediators was monitored by qPCR. The two types of BMDMs were also induced into M2 macrophages using IL-4, and expression of M2 anti-inflammatory and tissue repair mediators was measured by qPCR, while protein levels of classical M2 activation markers were detected using Western blot. Results Compared with Mcart1^flox/flox BMDMs, Mcart1 knockout significantly reduced basal respiration in BMDMs, and Mcart1 stable knockout decreased OGDH activity in macrophages(P＜0.01). Mcart1 knockout significantly enhanced ROS production in M1 macrophages and upregulated early inflammatory mediators Il1b, Tnf, and Nos2(P＜0.05), while having no significant effect on the expression of M2 anti-inflammatory and tissue repair mediators Arg1, Mrc1, Retnla, Cd163, Fn1, or classical activation proteins p-Stat6 and p-Stat3 in M2 macrophages. Conclusions Mcart1 knockout significantly suppresses basal respiration in macrophages, inhibits OGDH enzyme activity, increases oxidative stress and inflammatory mediator levels in M1 macrophages, and does not significantly affect functional mediator levels in M2 macrophages.

Key words: Mcart1, NAD⁺, reactive oxygen species, α-ketoglutarate dehydrogenase;macrophage polarization