Figure 1.
Backbone of the synthesized polyenylpyrroles.
Figure 2.
Effect of polyenylpyrrole derivatives on the expression of inflammatory mediators in LPS-stimulated RAW 264.7 macrophages.
In (A) and (C), the cells (5 × 105/ml; 1 ml) were incubated for 30 min with 2.5-40 µM compound 1h, 1i, or 1n or DMSO (vehicle), then LPS (1 µg/ml) was added and incubation continued for 24 h, then NO (A) or IL-6 or TNF-α (C) in the culture medium was assayed by the Griess reaction or ELISA, respectively. In (B), cells (5 × 105/ml; 1 ml) were pretreated for 30 min with 2.5-40 µM compound 1h or DMSO, then LPS (1 µg/ml) was added and incubation continued for 24 h, then expression of iNOS and COX-2 was measured by Western blotting. The fold increase is the intensity of the band of interest divided by that of the actin band normalized to the corresponding value for the 0 LPS/0 inhibitor control. In (A) and (C), the data are expressed as the mean ± SD for three separate experiments, while, in (B), the results are representative of those obtained in three different experiments and the histogram shows the quantification expressed as the mean ± SD for these 3 experiments. *, **, and *** indicate a significant difference at the level of p < 0.05, p < 0.01, or p < 0.001, respectively, compared to the DMSO/LPS group.
Figure 3.
Effect of compound 1h on LPS-induced secretion of IL-6 and TNF-α by J774A.1 macrophages, peritoneal macrophages, and JAWSII dendritic cells.
(A) J774A.1 macrophages, (B) peritoneal macrophages, or (C) JAWSII dendritic cells (all 4 × 105/ml; 1 ml) were incubated for 30 min with 10-40 µM compound 1h or DMSO, then LPS (1 µg/ml) was added and incubation continued for 24 h, then IL-6 levels (left panels) and TNF-α levels (right panels) in the culture medium were measured by ELISA. The data are expressed as the mean ± SD for three separate experiments. *, **, and *** indicate a significant difference at the level of p < 0.05, p < 0.01, or p < 0.001, respectively, compared to the DMSO/LPS group.
Figure 4.
Effect of compound 1h on NLRP3 inflammasome activation in LPS+ATP-activated J774A.1 macrophages.
(A) J774A.1 macrophages (1 × 106/ml; 1 ml) or (B) peritoneal macrophages (1 × 105/ml; 1 ml) were incubated with 10-40 µM compound 1h or DMSO for 30 min, then LPS (1 µg/ml) was added and incubation continued for 5.5 h, then the cells were stimulated with ATP (5 mM) for an additional 30 min, then IL-1β in the culture medium was measured by ELISA (A, upper panel; B) and levels of active caspase-1 (p10) (A, lower panel) measured by Western blotting. In (C) and (D), J774A.1 macrophages (1 × 106/ml; 1ml) were incubated with LPS (1 µg/ml) for 5.5 h, then with 10-40 µM compound 1h or DMSO for 30 min in the continued presence of LPS, followed by stimulation with ATP (5 mM) for an additional 30 min, then IL-1β levels (C, upper panel) and IL-6 levels (D) in the culture medium were measured by ELISA and levels of active caspase-1 (p10) were measured by Western blotting (C, lower panel). In A and C, the fold increase is the intensity of the p10 band divided by that of the p45 band normalized to the corresponding value for the 0 LPS/0 inhibitor control. In (E), J774A.1 macrophages (1 × 106/ml; 1 ml) were incubated for 30 min with DMSO or 1-40 µM compound 1h, then LPS (1 µg/ml) was added and incubation continued for 6 h, then expression of NLRP3 and proIL-1β was measured by Western blotting. The fold increase is the intensity of the band of interest divided by that of the actin band normalized to the corresponding value for the 0 LPS/0 inhibitor control. In the ELISA studies, the data are expressed as the mean ± SD for three separate experiments, while, in the Western blot studies, the results shown are representative of those obtained in three different experiments and the histogram shows the quantification expressed as the mean ± SD. *, **, and *** indicate a significant difference at the level of p < 0.05, p < 0.01, and p < 0.001, respectively, compared to the DMSO/LPS/ATP group (A, B), LPS/DMSO/ATP group (C, D), or the DMSO/LPS group (E).
Figure 5.
Effect of compound 1h on ROS production and MAPK phosphorylation in LPS-activated macrophages.
In (A), RAW 264.7 macrophages (5 × 105/ml; 1 ml) were incubated for 30 min with compound 1h (20 µM), N-acetyl cysteine (NAC; 10 mM), or DMSO (vehicle), then 2’, 7’-dichlorofluorescein diacetate (2 µM) was added for 30 min, followed by LPS (1 µg/ml) stimulation for the indicated time, then ROS levels were measured by detection of the mean fluorescence intensity (MFI) of the fluorophore carboxyl-DCF and expressing this value relative to that at time zero. In (B), RAW 264.7 macrophages (5 × 105/ml; 1 ml) were incubated for 30 min with compound 1h (20 µM) or DMSO, then LPS (1 µg/ml) was added and incubation continued for 0-60 min, then phosphorylation of ERK1/2, JNK1/2, and p38 was analyzed by Western blotting and expressed relative to actin expression and as a fold increase compared to the control group at 0 time. In (C), J774A.1 macrophages (5 × 105/ml; 1 ml) were incubated for 30 min with 10-40 µM compound 1h or DMSO, then LPS (1 µg/ml) was added and incubation continued for 20 min, then phosphorylation of ERK1/2, JNK1/2, and p38 was analyzed as in B. In (A), the data are expressed as the mean ± SD for three separate experiments, while, in (B) and (C), the results are representative of those obtained in three different experiments. * indicates a significant difference at the level of p < 0.05 compared to the DMSO/LPS group.
Figure 6.
Effect of compound 1h on NF-κB activation in LPS-activated macrophages.
(A) RAW 264.7 macrophages or (B) J774A.1 macrophages (both 5 × 105/ml; 1 ml) were incubated for 30 min with 10-40 µM compound 1h or DMSO, then LPS (1 µg/ml) was added and incubation continued for 20 min, then levels of phosphorylated and total IKK-α and IκB-α were measured by Western blotting. (C) RAW 264.7 macrophages or (D) J774A.1 macrophages (both 5 × 105/ml; 1 ml) were treated as in A and B, then nuclear translocation of NF-κB was analyzed by ELISA. (E) RAW-BlueTM cells (5 × 105/ml; 1 ml) were incubated for 30 min with 2.5-40 µM compound 1h or DMSO, then LPS (1 µg/ml) was added and incubation continued for 24 h, then SEAP activity was measured by the QUANTI-BlueTM assay and expressed as a percentage of that in the absence of compound 1h. In (A) and (B), the results are representative of those obtained in three different experiments. In (C-E), the data are expressed as the mean ± SD for three separate experiments. *, **, and *** indicate a significant difference at the level of p < 0.05, p < 0.01, and p < 0.001, respectively, compared to the DMSO/LPS group.
Figure 7.
Effect of compound 1h on ROS production and PKC-α phosphorylation in ATP-activated macrophages.
In (A), J774A.1 macrophages (1 × 106/ml; 1 ml) were incubated with LPS (1 µg/ml) for 6 h, then with compound 1h (20 µM), the NADPH oxidase inhibitor DPI (25 µM), or DMSO (vehicle) for 30 min in the continued presence of LPS, then 2’,7’-dichlorofluorescein diacetate (2 µM) was added for 30 min, followed by ATP (5 mM) for the indicated time, then ROS levels were determined by measuring the mean fluorescence intensity (MFI) of the fluorophore carboxyl-DCF and expressing this value relative to that at time zero. In (B), J774A.1 macrophages (1 × 106/ml; 1 ml) were incubated with compound 1h (20 µM), DPI (25 µM), or DMSO (vehicle) for 30 min, then LPS (1 µg/ml) was added for 6 h; the cells were then incubated with 2’,7’-dichlorofluorescein diacetate (2 µM) for 30 min, then with ATP (5 mM) for the indicated time and ROS levels were measured by detection of the fluorescence intensity of the fluorophore carboxyl-DCF and expressed relative to that at time zero. In (C), LPS-primed J774A.1 macrophages (1 × 106/ml; 1 ml) were incubated for 30 min with 20 µM compound 1h or DMSO (vehicle) followed by ATP (5 mM) stimulation for 0-60 min, then phosphorylation of PKC-α was analyzed by Western blotting and expressed as the fold increase measured as the intensity of the PKC-α band divided by that of the actin band normalized to the corresponding value for DMSO at 0 minutes. In (A) and (B), the data are expressed as the mean ± SD for three separate experiments, while, in (C), the results are representative of those obtained in three different experiments and the histogram shows the quantification expressed as the mean ± SD. * indicates a significant difference at the level of p < 0.05 compared to the DMSO/ATP group.
Figure 8.
Proposed anti-inflammatory mechanism of compound 1h in LPS+ATP-activated macrophages.