Roles of BN52021 in platelet-activating factor pathway in inflammatory MS1 cells

To determine the effects of BN52021 on platelet-activating factor receptor (PAFR) signaling molecules under lipopolysaccharide (LPS)-induced inflammatory conditions in MS1 cells.

MS1 cells (a mouse pancreatic islet endothelial cell line) were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mmol/L glutamine and 100 μg/mL penicillin/streptomycin in 5% CO2 at 37 °C. After growth to confluency in media, the cells were processed for subsequent studies. The MS1 cells received 0, 0.1, 1 and 10 μg/mL LPS in this experiment. The viability/proliferation of the cells induced by LPS was observed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric assay. Apoptosis and necrosis of the cells under the inflammatory condition described previously were observed using Hoechst 33342-propidium iodide staining. Adenylate cyclase (AC), phospholipase A2 (PLA2), phospholipase Cβ (PLCβ), protein tyrosine kinase (PTK), G protein-coupled receptor kinases (GRK) and p38-mitogen-activated protein kinase (p38 MAPK) mRNA in the PAFR signaling pathway were measured by real-time polymerase chain reaction. The protein expression level of phosphorylated AC (p-AC), phosphorylated PLA2 (p-PLA2), phosphorylated PTK (p-PTK), phosphorylated p38 MAPK (p-p38 MAPK), PLCβ and GRK was measured using Western blotting analysis.

The activity of MS1 cells incubated with different concentrations of LPS for 6 h decreased significantly in the 1 μg/mL LPS group (0.49 ± 0.10 vs 0.67 ± 0.13, P < 0.05) and 10 μg/mL LPS group (0.44 ± 0.10 vs 0.67 ± 0.13, P < 0.001), but not in 0.1 μg/mL group. When the incubation time was extended to 12 h (0.33 ± 0.05, 0.32 ± 0.03 and 0.25 ± 0.03 vs 0.69 ± 0.01) and 24 h (0.31 ± 0.01, 0.29 ± 0.03 and 0.25 ± 0.01 vs 0.63 ± 0.01), MS1 cell activity decreased in all LPS concentration groups compared with the blank control (P < 0.001). BN52021 significantly improved the cell activity when its concentration reached 50 μmol/L compared with the group that received LPS treatment alone, which was consistent with the results obtained from fluorescence staining. The mRNAs levels of AC (4.02 ± 0.14 vs 1.00 ± 0.13), GRK (2.63 ± 0.03 vs 1.00 ± 0.12), p38 MAPK (3.87 ± 0.07 vs 1.00 ± 0.17), PLA2 (3.31 ± 0.12 vs 1.00 ± 0.12), PLCβ (2.09 ± 0.08 vs 1.00 ± 0.06) and PTK (1.85 ± 0.07 vs 1.00 ± 0.11) were up-regulated after LPS stimulation as compared with the blank control (P < 0.05). The up-regulated mRNAs including AC (2.35 ± 0.13 vs 3.87 ± 0.08), GRK (1.17 ± 0.14 vs 2.65 ± 0.12), p38 MAPK (1.48 ± 0.18 vs 4.30 ± 0.07), PLCβ (1.69 ± 0.10 vs 2.41 ± 0.13) and PLA2 (1.87 ± 0.11 vs 2.96 ± 0.08) were significantly suppressed by BN52021 except for that of PTK. The level of p-AC (1.11 ± 0.12 vs 0.65 ± 0.08), GRK (0.83 ± 0.07 vs 0.50 ± 0.03), PLCβ (0.83 ± 0.16 vs 0.50 ± 0.10) and p-p38 MAPK (0.74 ± 0.10 vs 0.38 ± 0.05) was up-regulated after LPS stimulation as compared with the blank control (P < 0.05). The up-regulated proteins, including p-AC (0.65 ± 0.15 vs 1.06 ± 0.14), GRK (0.47 ± 0.10 vs 0.80 ± 0.06), PLCβ (0.47 ± 0.04 vs 0.80 ± 0.19) and p-p38 MAPK (0.30 ± 0.10 vs 0.97 ± 0.05), was significantly suppressed by BN52021, but p-PLA2 and p-PTK protein level were not suppressed.

BN52021 could effectively inhibit LPS-induced inflammation by down-regulating the mRNA and protein levels of AC, GRK, p38 MAPK, PLA2 and PLCβ in the PAFR signaling pathway.