Regulation Of Macrophage M1 Polarization & Mitochondrial Homeostasis By Urolithin-A

To study the effects of Urolithin A (UroA) on M1 polarization and underlying mechanisms related to metabolic shift in macrophages. M1 polarization was induced by treating RAW 264.7 cells with LPS+IFN-γ before different concentrations of UroA (2.5, 5, 10μmol/L) were added. Then flow cytometry, qRT-PCR and ELISA were conducted to verify M1 polarization markers and the levels of ROS&mtROS were also measured. Proteome sequencing was further performed to get insights about potential mechanism. Mitochondrial injuries were evaluated by TEM, Mito-Tracker staining and JC-1 fluorescent probe. Additionally, energy productions were assessed using ATP assay kits, with the glycolysis levels quantified by a Seahorse analyzer. M1 polarization was inhibited by UroA as assessed by flow cytometry, PCR and ELISA and the concentrations of ROS&mtROS were reduced in UroA groups(P<0.05). As Proteome sequencing identified several downregulated proteins related to glycolysis (such as GLUT1, HK1) and enrichment analyses revealed alterations related to mitochondrial functions, subsequent experiments further confirmed UroA ameliorated mitochondrial impairments in M1 macrophages evidenced by improved mitochondrial ultra-structure, elevated MMP level and mitochondrial mass indicated by JC-1 and Mito-Tracker staining. Extracellular acidification rates (ECAR) also decreased significantly in UroA group, indicating a metabolic pattern away from glycolysis, although no significant difference in total ATP production was observed. UroA could alleviate mitochondrial injuries while inhibiting M1 polarization and glycolytic capacity of macrophages in vitro, suggesting UroA may regulate macrophage functions via metabolic reprogramming.