Objective: To understand the role of p38 mitogen-activated protein kinase (p38MAPK) in the expression of inducible nitric oxide synthase (iNOS) and NO production in human endothelial cells under the stimulation by lipopolysaccharide (LPS).
Methods: NO level in the supernatant of the cell culture media was measured with Griess method, and iNOS protein and mRNA expressions by the cells were detected with immunofluorescence analysis and reverse transcriptase-polymerase chain reaction (RT-PCR) respectively. Immunoprecipitation assay was employed to examine p38 MAPK activity.
Results: It was shown that in comparison with the basal level of iNOS expression in cultured endothelial cells line ECV304, iNOS mRNA and protein expressions were significantly increased in the cells after LPS stimulation. In response to LPS treatment, obvious enhancement of p38 MAPK activity in ECV304 took place after the stimulation, with the peak level occurring at 15 min that maintained for approximately 45 min before gradual decline. When treated with SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl) imidazole], a highly specific inhibitor of p38 MAPK, significant inhibition of LPS-induced iNOS protein and mRNA expressions was observed.
Conclusions: p38 MAPK plays an important role in iNOS expression and NO production in ECV304 cells, and, inhibition of the signal transduction pathway may consequently be an effective approach to reduce the production of iNOS and other cytokines for the treatment of septic shock.