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Fig. 6 | Cellular & Molecular Biology Letters

Fig. 6

From: Formyl peptide receptor 2 activation by mitochondrial formyl peptides stimulates the neutrophil proinflammatory response via the ERK pathway and exacerbates ischemia–reperfusion injury

Fig. 6

The ERK1/2 pathway mediates mtFP–FPR2 axis-activated neutrophil proinflammatory functions. 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 cocultured with mtFP, mtFP + WRW4, or mtFP + SCH772984. After 2 h of coculture, the expression levels of p-ERK1/2, t-ERK1/2, and GAPDH in neutrophils were determined by western blot analysis. B Neutrophils were plated in the top chamber (pore size, 3 μm) of a transwell system. Synthesized mitochondrial peptide mtFP were added to the bottom chambers, and the FPR2 inhibitor WRW4 or the ERK1/2 pathway inhibitor SCH772984 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 mtFP, mtFP + WRW4, or mtFP + SCH772984. After 2 h of coculture, the H2O2 concentration in the neutrophil culture medium supernatant was measured by the Fenton reaction method. C Meanwhile, the levels of IL-1β and IL-8 in the supernatant were determined by enzyme-linked immunosorbent assays. D, E Serum from sham surgery, IRI, and SCH772984-treated rats was sampled at 24 h after surgery. Serum creatinine and blood urea nitrogen levels were measured. Kidney tissues were sectioned for histological examination. Scale bar, 100 μm. *P < 0.05, **P < 0.01, ***P < 0.001. ns, P > 0.05

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