Levels of p65 was also determined in nuclear fractions. β-actin was used as a control for equal loading. Data are the summary of averaged relative density units measured in 3 independent experiments.

*p < 0.05 compared with control. Effects of MAPK inhibitors on PCN-induced NF-κB signaling activation To determine whether MAPKs mediate PCN-activated NF-κB signaling pathway, we used PCN (50 μM) to stimulate U937 cells with or without pretreatment with MAPK and NF-κB inhibitors: SB 203580 (50 μM), PD98059 (50 μM) and PDTC 200 μM for 1 h. Cell proteins were collected at 30 min and NF-κB p65 protein translocation was detected by Western blotting. The results showed that there was abundant cytosol distribution of NF-κB p65 before stimulation. All the indicated blockers were able to reduce the localization Daporinad mw of SRT1720 in vitro NF-κB p65 in the cytosol (Figure 9). These data suggest that SB203580 and PD98059 can effectively inhibit PCN-induced NF-κB signaling activation. Therefore, it could be concluded that the activation of p38 and ERK MAPKs are signaling events that lie upstream of NF-κB activation. Figure 9 Effects of MAPK inhibitors on PCN-induced NF- κB signaling pathway. U937 cells were stimulated with PCN at 50 μM for the time periods indicated with or without pretreatment by MAPK and NF-κB inhibitors:

SB 203580 (50 μM), PD98059 (50 μM) and PDTC (200 μM) for 1 h. Cell proteins were then collected and NF-κB p65 protein expression was detected with Western Vitamin B12 blotting. Discussion The National Nosocomial Infection Surveillance indicates that P. aeruginosa is the

second most common cause of nosocomial pneumonia after Staphylococcus aureus[28]. Ventilator-associated pneumonia (VAP) caused by P. aeruginosa is a severe complication of intensive care, with mortality rates of 34 to 48% [28–30]. Therefore, it is critical to study the pathogenesis of P. aeruginosa. In recent years, with the development of technologies such as the gene chip and the protein chip, and the clarification of the genome sequence of the P. aeruginosa strain, it has been found that many elements such as pro-inflammatory cytokines, antimicrobial peptides, complements and epithelial cell receptors and their signal transduction systems (TLR2, 4, 5, CFTR, GM1, and its downstream NF-κB) participate in host defense and immune response induced by P. aeruginosa. It has also been found that P. aeruginosa components (flagella and pili) and virulence factors (such as the density-sensing system, type secretion system, toxins, alginate and cell toxin) play important roles in the pathogenesis [2, 16]. Among them, most P. aeruginosa strains secrete PCN (N-methyl-1-hydroxyphenazine), the pigment that gives blue-green color to the bacterial colonies [4].