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

Figure 1 The optimal dose and duration of lipopolysaccharide stimulation were determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. The cell survival rate was determined after incubation with 0 (saline) and 0.1, 1 and 10 μg/mL lipopolysaccharide (LPS) for 3, 6, 12 and 24 h. ^aP < 0.05, ^bP < 0.01 vs the saline group.

Figure 2 The dose effect of BN52021 on lipopolysaccharide-induced inflammation was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method and Hoechst 33342/propidium iodide staining. MS1 cell activity at A_{490 nm} was significantly decreased 24 h after administration of 10 μg/mL lipopolysaccharide (LPS) vs the control group (^aP < 0.05). Pretreatment with BN52021 for 20 min before incubation with LPS significantly improved the MS1 cell activity at A_{490 nm}vs the group that received LPS treatment only when its concentration reached 50 μmol/L (^cP < 0.05) (A). Pretreatment with 50 μmol/L BN52021 for 20 min before incubation with LPS significantly improved MS1 cell activity vs the LPS + saline group, and the LPS + dimethyl sulfoxide (DMSO) group as determined Hoechst 33342/propidium iodide staining (B, C, D and E). The arrows indicate the apoptosis (short) and necrosis (long) of the cells.

Figure 3 The effect of BN52021 on platelet-activating factor receptor signaling molecules at the mRNA level under lipopolysaccharide-induced inflammation. The mRNA level of adenylate cyclase (AC) (A), G protein-coupled receptor kinases (GRK) (B), phospholipase A₂ (PLA₂) (C), phospholipase Cβ (PLCβ) (D), p38-mitogen-activated protein kinase (p38 MAPK) (E) and protein tyrosine kinase (PTK) (F) was up-regulated after lipopolysaccharide (LPS) stimulation. The up-regulation of AC, GRK, p38 MAPK, PLCβ and PLA₂ mRNA was significantly suppressed by BN52021 except for that of PTK. ^aP < 0.05 vs control; ^dP < 0.01 vs the LPS + dimethyl sulfoxide (DMSO) groups.

Figure 4 The effect of BN52021 on platelet-activating factor receptor signaling molecules at the protein level under lipopolysaccharide-induced inflammation. The protein level of p-adenylate cyclase (p-AC) (A), G protein-coupled receptor kinases (GRK) (B), p-phospholipase A₂ (p-PLA₂) (C), phospholipase Cβ (PLCβ) (D) and p-p38-mitogen-activated protein kinase (p-p38 MAPK) (E) was up-regulated after lipopolysaccharide (LPS) stimulation vs the blank control (^aP < 0.05). The up-regulation of p-AC, p-p38 MAPK, GRK and PLCβ protein levels was significantly suppressed by BN52021. However, p-PLA₂ and phosphorylated protein tyrosine kinase (p-PTK) protein levels were insignificantly up-regulated after LPS stimulation and were not significantly changed by BN52021 (F). ^cP < 0.05, ^dP < 0.01 vs LPS + dimethyl sulfoxide (DMSO) groups.