OBJECTIVETo investigate the role of REMP2 derived from limulus anti-lipopolysaccharide factor in neutralizing endotoxin in vitro and its antibacterial activity.

METHODS(1) REMP2 and PMB in the concentrations of 100.00, 10.00, 1.00, 0.10, 0.01 micromol/L were respectively mixed with LPS (lEU/mL), with PMB as positive control. The LPS concentrations in different specimens were determined by routine method, and the neutralizing percentage was respectively calculated. (2) After adding isotonic saline (NS), the final concentrations of REMP2 and PMB were 10, 20, 40, 80 micromol/L, and the concentration of LPS was 100 microg/L. The murine monocytic macrophages were stimulated with LPS, then cultured with REMP2 and PMB, with NS in culture as negative control. The content of tumor necrosis factor (TNF)-alpha was determined by ELISA kit. (3) The morphologic changes of Escherichia coli. was observed under electron microscope at 10, 20 and 40 minutes after addition of REMP2 to Escherichia coli suspension (with terminal concentration of REMP2 at 40 micromol/L).

RESULTSThere were no significant difference in endotoxin-neutralizing percentages between PMB and REMP2 in concentrations of 0.10, 10.00, 100.00 micromol/L (P > 0.05). The contents of TNF-alpha were 1175 +/- 162, 859 +/- 122, 645 +/- 142, 489 +/- 102 ng/L, respectively,after treatment of 10, 20, 40, 80 micromol/L REMP2, which were obviously lower than that of NS (3463 +/- 218 ng/L, P < 0.01). Under transmission electron microscope, the outer and interior membranes of Escherichia coli were obscure and rough, bacterial bodies were swollen with vacuoles in cytoplasm after treatment with REMP2.

CONCLUSIONREMP2 has ability of neutralizing endotoxin and also antibacterial activity.