Figure 3.

Apelin-13 inhibited the release of pro-inflammatory cytokines in LPS-treated macrophages through the APJ receptor. (A) RAW264.7 cells and BMDMs were treated with 1ug/mL LPS and the indicated concentrations of apelin-13 for 6 h. The mRNA levels of TNF-α, IL-1β, and IL-6 were measured by real-time PCR. (B) TNF-α, IL-1β, and IL-6 levels in culture supernatants were measured by ELISA. (C) BMDMs were treated with LPS (1ug/mL) or apelin-13 (1 umol/L) at different times. Pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 concentrations were measured in culture supernatants. (D) BMDMs were pre-incubated with apelin-13 (1umol/L) for 1 h before being exposing to 1ug/mL LPS for 6 h. APJ mRNA levels were measured by real-time PCR. (E) Protein levels of APJ were measured by Western blot analysis. (F) BMDMs were treated with apelin-13 (1 umol/L) or F13A (1 umol/L) for 1h before being stimulated 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. (G) TNF-α, IL-1β, and IL-6 levels in culture supernatants were measured by ELISA. (H) Colocalization of APJ and F4/80 in lung sections was examined by immunofluorescence. Scale bar, 100 μm. Data were expressed as mean ± SD. *P<0.05 versus control group, ^#P<0.05 versus the LPS treatment group, ^&P< 0.05 vs the LPS + Apelin (n=3).

Abbreviations: LPS, lipopolysaccharide; BMDMs, bone marrow-derived macrophages; ELISA, enzyme linked immunosorbent assay; TNF-α, tumor necrosis factor α; IL-1β, interleukin-1β; IL-6, interleukin-6.