Original Article
Copyright ©2013 Baishideng Publishing Group Co., Limited. All rights reserved.
World J Gastroenterol. Jul 7, 2013; 19(25): 3969-3979
Published online Jul 7, 2013. doi: 10.3748/wjg.v19.i25.3969
Roles of BN52021 in platelet-activating factor pathway in inflammatory MS1 cells
Shi-Hai Xia, Xiao-Hui Xiang, Kai Chen, Wei Xu
Shi-Hai Xia, Xiao-Hui Xiang, Kai Chen, Wei Xu, Department of Hepatopancreatobiliary and Splenic Medicine, Affiliated Hospital of the Logistics University of the Chinese People’s Armed Police Force, Tianjin 300162, China
Author contributions: Xiang XH and Chen K contributed equally to this work; Xia SH contributed to the conception of this study; Xia SH, Xiang XH, Chen K and Xu W designed the research strategy and performed the experiments; Xia SH and Xiang XH performed the data analysis and interpretation; Xiang XH, Chen K and Xu W prepared the manuscript; Xia SH revised the manuscript.
Supported by The National Natural Science Foundation of China, No. 81173393; the Natural Science Foundation of Tianjin City, Grant No. 12YFJZJC00800; the Scientific Research Foundation for PhD grant to Xia SH, No. WYB201010; and the Innovation Team Program (WHTD201310) from the Logistics University of the Chinese People’s Armed Police Force
Correspondence to: Shi-Hai Xia, MD, PhD, Department of Hepatopancreatobiliary and Splenic Medicine, Affiliated Hospital of the Logistics University of the Chinese People’s Armed Police Force, 220 Chenglin Road, Hedong District, Tianjin 300162, China. xshhcx@sina.com
Telephone: +86-22-60577731 Fax: +86-22-24370605
Received: December 19, 2012
Revised: June 5, 2013
Accepted: June 8, 2013
Published online: July 7, 2013
Processing time: 209 Days and 4.9 Hours
Abstract

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

METHODS: 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.

RESULTS: 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.

CONCLUSION: 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.

Keywords: BN52021; Platelet-activating factor receptor; Signaling pathway; Inflammation; Pancreatitis

Core tip: Microcirculatory disorder is considered to be one of the possible mechanisms of pathogenesis of severe acute pancreatitis (SAP). Platelet-activating factor (PAF) is known to mediate microcirculatory disturbance and inflammation. Although BN52021, a PAF receptor antagonist, has demonstrated significant treatment effects on SAP, its mechanism has not been elucidated in detail. In this study, we examined the signaling molecules of the PAF receptor pathway to evaluate whether BN52021 has any influence on the inflammatory effects induced by lipopolysaccharide in MS1 cells, hoping to elucidate the mechanism underlying microcirculatory disturbances in the pathogenesis of SAP in vitro.