Urotensin II promotes secretion of LTB4 through 5-lipoxygenase via the UT-ROS-Akt pathway in RAW264.7 macrophages

Figures

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  Figure 1. UII promotes LTB4 release into the supernatant of RAW264.7 cells. A – Effect of UII (10–8 M) on LTB4 production after the indicated incubation times. The cells were left untreated (control) or treated with UII for different durations. B – Effect of 24 h treatments with different concentrations of UII on LTB4 production. The cells were left untreated (control) or treated with UII for 24 h at the indicated concentrations Values are the means of the LTB4 protein concentration in pg/ml ± SD. The data are from three independent experiments and are expressed as the mean ± SEM. *P < 0.05 vs. control; **p < 0.01 vs. control.

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  Figure 2. UII promotes 5-lipoxygenase (5-LO) expression in RAW264.7 macrophages. A – Time course of UII-stimulated 5-LO expression determined by real-time PCR. The cells were incubated with 10–8 M UII for the indicated times. B – Concentration-response relationship of UII with 5-LO expression in RAW264.7 macrophages via UT determined by real-time PCR. The cells were incubated with different concentrations of UII for 24 h or pretreated with urantide (10 μM) for 1 h prior to stimulation with UII (10–8 M) for 24 h. C – Time course of UII-stimulated 5-LO expression determined by western blot analysis. D – Concentration-response relationship of UII with 5-LO expression in RAW264.7 macrophages via UT determined by western blot analysis UA – urantide. The data are from three independent experiments and are expressed as the mean ± SEM. *P < 0.05 compared with control; **p < 0.01 compared with control; #p < 0.01 compared with the UII (10–8 M) group.

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  Figure 3. Involvement of ROS in UII-induced 5-LO expression and LTB4 release in RAW264.7 macrophages. A – Effect of the different inhibitors on the UII-stimulated generation of ROS. The cells were stimulated with UII (10–8 M) for 2 h after pretreatment with urantide (10 μM), NAC (10 mM) and DPI (10 μM) for 1 h and then incubated for 30 min with DCFH-DA (10 μM). B – Effect of NAC and DPI on UII-induced 5-LO mRNA expression determined by real-time PCR. The cells were stimulated with UII (10–8 M) for 24 h after pretreatment with NAC (10 mM) and DPI (10 μM) for 1 h. C – Effect of NAC and DPI on UII-induced 5-LO protein expression determined by western blot analysis. D – Effect of different inhibitors on LTB4 release in the presence of UII (10–8 M) for 24 h UA – urantide. The data are from three independent experiments and are expressed as the mean ± SEM. *P < 0.05 compared with control; **p < 0.01 compared with control; #P < 0.01 compared with the UII (10–8 M) group.

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  Figure 4. Akt mediates UII-induced expression of 5-LO and LTB4 release in RAW264.7 macrophages. A – UII induces Akt phosphorylation in RAW264.7 macrophages at the indicated time points. The cells were exposed to 10–8 M UII for 0 to 60 min. B, C – Effect of the different inhibitors on UII-induced 5-LO expression. The cells were pretreated with PD98059 (10 μM), SB203580 (10 μM) and LY294002 (10 μM) for 1 h prior to stimulation with UII (10–8 M) for 24 h. 5-LO mRNA expression (C) and protein level (B) were then detected by real-time PCR and western blot analysis, respectively. D – Effect of different inhibitors on LTB4 release in the presence of UII (10–8 M) for 24 h UA – urantide, PD – PD98059, SB – SB203580, LY – LY294002. The data are from three independent experiments and are expressed as the mean ± SEM. *P < 0.01 compared with control; #p < 0.01 compared with the UII (10–8 M) group.

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  Figure 5. Effects of different inhibitors on the phosphorylation level of Akt. The cells were stimulated with UII (10–8 M) for 10 min after pretreatment with urantide (10 μM), NAC (10 mM), DPI (10 μM), and LY294002 (10 μM) for 1 h. Total and phosphorylated Akt levels were determined by western blot analysis UA – urantide, LY – LY294002. The data are from three independent experiments and are expressed as the mean ± SEM. *P < 0.01 compared with control; #P < 0.01 compared with the UII (10–8 M) group.