The Role Of P38 MAPK And ERK In INOS Expression Induced By Lipopolysaccharides

During the pathologic process of sepsis (endotoxemia) and multiple organ dysfunction syndrome (MODS), many types of tissues and cells produce large amounts of nitric oxide (NO) in response to proinflammatory cytokines and lipopolysaccharides (LPS) by expressing inducible isoform of NO synthase (iNOS). Although the involvement of NO and iNOS in systemic inflammatory response or septic shock has been studied extensively, the mechanism of iNOS induction hasn't been completely elucidated until now, since many factors have been shown to affect this process, including the cell type, different tissues, or animal species. The aim of this study is to carry out a series of experiments in vitro and in vivo by using molecular biology and cellular biology method in order to investigate the role of p38 MAPK and p44/42 MAPK (ERK1/2 or ERK) in the iNOS expression of human endothelial cells-ECV304, mice macrophages-RAW264.7 and BALB/c mice treated by LPS.In the cellular level research, mice macrophages-RAW264.7 and HUVEC-ECV304 were used. The NO level in supernatants of cell media was measured by Griess method, iNOS protein was detected by immunofluorescence and Western blot. Total RNA of cells was isolated and analyzed for the magnitude of mRNA expression of iNOS by RT-PCR. p38 MAPK activity was measured by immunokinase assay detection(including immunoprecipitation and autoradiography). ERK activity was determined by immunoprecipitation and Western blot. The results indicated that the activity of p38 MAPK and ERK was increased at 15-20 min in ECV304 and RAW264.7 after LPS stimulation, maintained for about 45 min, and thendescended gradually. Pretreatment with SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfmylphe-nyl)-5-(4-pyridyl) imidazole] and PD98059 [2'-Amino-3'-methoxyflavone] respectively, the activation of p38 and ERK in both cells stimulated by LPS was completely inhibited. It was shown that comparing with those in control group, the expression of iNOS mRNA and protein as well as NO in supernatants of cell media were significantly increased after LPS treatment. The expression of iNOS protein and mRNA and the level of NO in ECV304 treated with LPS were markedly inhibited by both MAPK inhibitors. SB203580 but not PD98059 could decrease the iNOS expression and NO production of RAW264.7 cells. The results indicated that either p38 MAPK or ERK involved in iNOS expression and NO production of ECV304. However, only p38 MAPK was involved in iNOS expression of macrophages-RA W2 64.7.In the tissue level research, the expression of iNOS in different tissues from mice was examined and the effect of SB203580 and PD98059 on iNOS expression of tissues was evaluated. BALB/c mice were used to reproduce endotoxin shock model for the observation of iNOS expression of tissues under different treatment. Mice were anaesthetized and cannulated carotid artery for the measurement of mean arterial pressure (MAP), then treated with LPS, SB203580+LPS and PD98059+LPS. All animals were killed, and then blood samples and tissues were collected. iNOS protein and mRNA of tissues were detected by Western blot and RT-PCR respectively. p38 MAPK activity was measured by immunokinase assay and ERK activity was detected by immunoprecipitation and Western blot. All tissues were stained with HE for pathological observation. The data indicated: (1) Compared with that in normal group, iNOS gene transcription and protein expression of tissues (including heart, liver, spleen, intestines, lungs and kidney) of BALB/c mice were increased in time-dependent and dose-dependent in LPS-treated animals. (2) iNOS was expressed in a small quantity in normal lung tissues. However, basal expression of iNOS was undetectable in other-5-normal organs. The iNOS expression in lungs stimulated by LPS was more early and apparently than in other tissues. (3) The p38 MAPK and ERK activity was increased in lung tissues treated with LPS and pretreatment with SB203580 and PD98059 could completely inhibit p38 and ERK activation respectively, and also could partially inhibit...