Figure 2

Ghrelin inhibits LPS-induced IL-6 release in a GHS-R dependent manner. To determine whether ghrelin modulates the secretion of inflammatory cytokines in mid-brain neurones, we induced innate-immune activation via LPS (1 μg/ml). Neurones (3 to 5 x 10⁵ per ml) were treated with ghrelin (1, 10, 100 nM) for 4 h prior to LPS (1 μg/ml) challenge; subsequently, supernatants were collected 24 h later for detection of IL-6 via ELISA. A) Pre-treatment with ghrelin at 1, 10, and 100 nM significantly reduced LPS-induced IL-6 secretion. Data are normalized to cell number and expressed as a percentage of the control value (mean ± s.e.m), which was 774.8 ± 306.7 pg/ml, and represent four independent experiments performed in-triplicate for each concentration point. B) Pre-incubation of ghrelin with the ghrelin-receptor antagonist, [D-Lys-3]-GHRP-6, blocked ghrelin’s inhibitory effect on LPS-induced IL-6 secretion. The data (expressed as in A) with a control value of 815.8 ± 319.8 pg/ml represent the mean ± s.e.m of five independent experiments performed with four replicates for each concentration point. To ensure that treatments were not inducing cytotoxic or apoptotic changes in SN4741 cells, we tested cell viability (CellTiter Blue, Promega) (C) and caspase-3 immunoreactivity (D). The CellTiter-Blue® Assay, which is based on the ability of living cells to convert a redox dye (resazurin; Absorbance^max = 605 nm) into a fluorescent end product (resorufin; Absorbance^max = 573 nm) (viable cells reduce resazurin into resorufin) demonstrated that ghrelin and LPS had no significant effect on viability (C). Similarly, nuclear intensity of caspase-3 was not significantly altered by treatments (D). The data represent the mean ± SEM of two independent experiments with seven replicates for each concentration point. Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparison. P <0.05 regarded as significant (*P <0.05; ***P <0.001; ****P <0.0001 vs LPS).