Fig. 4

mtFP activates neutrophil proinflammatory responses through FPR2 in vitro. Primary neutrophils were purified from rat femur or tibia bone marrow using gradient centrifugation and cultured in pure RPMI 1640 medium. All in vitro experiments were conducted within 24 h after rat sacrifice. A Neutrophils were plated in the top chamber (pore size, 3 μm) of a transwell system. MMK-1 or mtFP was added to the bottom chambers, and the FPR2 inhibitor WRW₄ was added to the top chambers. Two hours after top chamber insertion, migrated cells in the bottom chambers were counted. To measure H₂O₂ levels in cell culture medium, neutrophils were cocultured with MMK-1, MMK-1 + WRW₄, mtFP, or mtFP + WRW₄. After 2 h of coculture, the H₂O₂ concentration in the neutrophil culture medium supernatant was measured. B Neutrophils were cocultured with MMK-1 or MMK-1 + WRW₄. After 2 h of coculture, neutrophils were extracted, and the levels of FPR2 and GAPDH were determined by western blot analysis (quantified in D). C Neutrophils were cocultured with mtFP or mtFP + WRW₄. After 2 h of coculture, neutrophils were extracted, and the levels of MMP-9 and GAPDH were determined by western blot analysis (quantified in D). E Meanwhile, MMP-9 secretion and beta-hexosaminidase assays in supernatant were tested. F IL-1β, IL-1Ra, and IL-8 concentrations in supernatant were determined using enzyme-linked immunosorbent assays, with their intracellular mRNA expression levels determined by qPCR. G Neutrophils were cocultured with mtFP or mtFP + WRW₄. After 2 h of coculture, neutrophils were extracted. Cell viability was measured using an Annexin V/PI assay and flow cytometry. H Neutrophils were cocultured with mtFP or mtFP + WRW₄. After 2 h of coculture, neutrophils were extracted, and the expression of CD62L was measured via flow cytometry. The results are representative of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. ns, P > 0.05.