Apelin-13 suppressed pro-inflammatory responses by inhibiting oxidative stress in LPS-treated macrophages. (A) BMDMs were treated with apelin-13 (1 umol/L), NAC (2 mmol/L), DPI (10 umol/L), or F13A (1 umol/L) for 1 h before stimulation with LPS (1 ug/mL) for 6 h. The mRNA levels of TNF-α, IL-1β, and IL-6 were determined by real-time PCR. (B) TNF-α, IL-1β, and IL-6 levels in culture supernatants were measured by ELISA. (C) Protein levels of NOX4 were measured by Western blot analysis. (D) BMDMs were transfected with NOX4 siRNA before stimulation with LPS (1 ug/mL) for 6 h. The mRNA levels of TNF-α, IL-1β, and IL-6 were determined by real-time PCR. (E) Concentrations of pro-inflammatory cytokines were detected in culture supernatants. (F) Protein levels of NOX4 were measured by Western blot analysis. (G) H2O2 concentrations in BMDMs were measured. (H and I) Intracellular ROS was detected by the probe DCF-DA. Data were expressed as mean ± SD. *P< 0.05 vs control group, #P< 0.05 vs the LPS group, &P< 0.05 vs the LPS + Apelin, $P< 0.05 vs NT siRNA group, ^P< 0.05 vs the LPS+NT siRNA (n=3).
Abbreviations: LPS, lipopolysaccharide; BMDMs, bone marrow-derived macrophages; NAC, N-Acetyl Cysteine; DPI, diphenylene iodonium; TNF-α, tumor necrosis factor α; IL-1β, interleukin-1β; IL-6, interleukin-6; ELISA, enzyme linked immunosorbent assay; NOX4, NADPH oxidase (NOX) 4; siRNA, small-interfering RNA; H2O2, hydrogen peroxide; ROS, reactive oxygen species.