Protease-activated receptor 1 challenges human lung epithelial cells to produce MCP-1 / 第三军医大学学报

ABSTRACT

Objective To investigate the actions of PAR1 agonists and thrombin on the secretion of monocyte chemoattractant protein (MCP)-1 from human lung epithelial cells. Methods A549 cells were cultured in a 12-well culture plate. The challenge was performed by addition of various concentrations of PAR1 agonist peptides SFLLR and its reverse peptides RLLFS, thrombin or thrombin inhibitor named hirudin into each well, respectively. After 2 h or 16 h, the reactions were terminated by removal of the supernatant from each well. A sandwich ELISA was used to determine the levels of MCP-1 in supernatants. Results Following 16 h incubation, SFLLR could induce concentration-dependent secretion of MCP-1. The maximum release of MCP-1 was nearly 12-fold more than baseline release. The reverse PAR1 agonists had little effects on MCP-1 release. Thrombin could induce concentration-dependent secretion of MCP-1. As low as 3 000 U/L thrombin could induce MCP-1 release from epithelial cells, and the maximum of accumulated release of MCP-1 was observed with 10 000 U/L thrombin, which was 5-fold more than baseline release. Thrombin inhibitor hirudin could inhibit thrombin induced secretion of MCP-1. The time course showed that the actions of PAR1 agonist peptides SFLLR and thrombin initiated at 2 h and reached their peak at 16 h. Conclusion PAR1 agonist peptides and thrombin are potent secretogogue of MCP-1 release from cultured human lung epithelial cells, and PAR1 antagonists and thrombin inhibitor may possess the ability to inhibit airway inflammation.