After being washed (10 mM Tris, 100 mM NaCl, and 0.1% Tween 20), selleck kinase inhibitor membranes were incubated with a peroxidase-conjugated IgG antibody (Bio-Rad) according to each primary antibody used. Immunocomplexes were detected using an enhanced horseradish peroxidase-luminol chemiluminescence system
(ECLPlus, Amersham) and subjected to autoradiography (Hyperfilm ECL, Amersham). Signals on the immunoblot were quantified with Scion Image software. In the same membrane, α-actin protein expression was determined (1:10,000 anti-α-actin antibody, Sigma–Aldrich) and its content was used as an internal control for the experiments. Data are presented as mean ± SEM, unless otherwise specified. Concentration–response curves were analyzed by two-way ANOVA followed by the Bonferroni post hoc test. Selleck ON1910 For comparisons between two means, the unpaired Student’s t-test was used and one-way ANOVA was used to compare three or more means.
Values of p < 0.05 were considered significantly different. The statistical analysis was performed using the GraphPad Prism version 4.0 (GraphPad Software Corp., USA). The endothelium-dependent relaxation evoked by acetylcholine was significantly impaired in pulmonary arterial rings from PM2.5-exposed rats compared to control rats (Fig. 1A). However, the relaxation response induced by the NO donor sodium nitroprusside was not changed (Fig. 1B). Pulmonary
arteries from PM2.5-exposed animals showed an enhanced hydroethidine-fluorescence signal compared to the control group (Fig. 2A and B). PEG-SOD incubation reduced this fluorescence in arteries from PM2.5-exposed animals to control levels (Fig. 2A and B). Protein expression of Cu/Zn- and Mn-SOD in the pulmonary arteries was enhanced by PM2.5 compared to the control group (Fig. 2C and D), while EC-SOD protein expression did not change in this artery (Fig. 2E). IL-1β (Fig. 3A) and IL-6 (Fig. 3B) protein expression were not modified by Avelestat (AZD9668) PM2.5 exposure in the pulmonary artery, while TNF-α protein expression was significantly enhanced as compared to filtered air-exposed rats (Fig. 3C). In addition, PM2.5 significantly reduced eNOS protein expression in pulmonary arteries compared to the control group (Fig. 3D). There was a significant positive correlation between eNOS protein expression and the maximal relaxation evoked by acetylcholine in pulmonary arteries (Fig. 4A), while TNF-α protein expression negatively correlates with acetylcholine-induced maximal relaxation (Fig. 4B). These data suggest that endothelial dysfunction present in the pulmonary arteries of PM2.5-exposed rats is strongly associated with reduced NO synthesis and vascular inflammation.