Andrographolide inhibits nuclear factor-κB activation through JNK-Akt-p65 signaling cascade in tumor necrosis factor-α-stimulated vascular smooth muscle cells

Abstract

Critical vascular inflammation leads to vascular dysfunction and cardiovascular diseases, including abdominal aortic aneurysms, hypertension, and atherosclerosis. Andrographolide is the most active and critical constituent isolated from the leaves of Andrographis paniculata, a herbal medicine widely used for treating anti-inflammation in Asia. In this study, we investigated the mechanisms of the inhibitory effects of andrographolide in vascular smooth muscle cells (VSMCs) exposed to a proinflammatory stimulus, tumor necrosis factor-α (TNF-α). Treating TNF-α-stimulated VSMCs with andrographolide suppressed the expression of inducible nitric oxide synthase in a concentration-dependent manner. A reduction in TNF-α-induced c-Jun N-terminal kinase (JNK), Akt, and p65 phosphorylation was observed in andrographolide-treated VSMCs. However, andrographolide affected neither IκBα degradation nor p38 mitogen-activated protein kinase or extracellular signal-regulated kinase 1/2 phosphorylation under these conditions. Both treatment with LY294002, a phosphatidylinositol 3-kinase/Akt inhibitor, and treatment with SP600125, a JNK inhibitor, markedly reversed the andrographolide-mediated inhibition of p65 phosphorylation. In addition, LY294002 and SP600125 both diminished Akt phosphorylation, whereas LY294002 had no effects on JNK phosphorylation. These results collectively suggest that therapeutic interventions using andrographolide can benefit the treatment of vascular inflammatory diseases, and andrographolide-mediated inhibition of NF-κB activity in TNF-α-stimulated VSMCs occurs through the JNK-Akt-p65 signaling cascade, an IκBα-independent mechanism.

Figure 1

Chemical structure of andrographolide (Andro).

Figure 2

Effects of andrographolide on iNOS expression in TNF-α-stimulated VSMCs. (a) Photomicrograph showing the primary cultured rat aortic VSMCs (magnification ×100). (b) The VSMCs were treated with PBS (resting group) or pretreated with andrographolide (20 and 50 μM) or an equal volume of DMSO (solvent control) for 20 min, and TNF-α (10 ng/mL) was subsequently added for 24 h. The iNOS protein level was evaluated as described in Section 2.**P < 0.01 compared with the resting group; ^## P < 0.01 compared with the TNF-α group. The data are presented as the mean ± SEM (n = 3).

Figure 3

Effects of andrographolide on p38MAPK, ERK1/2, JNK, and Akt signaling pathways in TNF-α-stimulated VSMCs. The VSMCs were treated with PBS (resting group) or pretreated with andrographolide (20 and 50 μM) or an equal volume of DMSO (solvent control) for 20 min, and TNF-α (10 ng/mL) was subsequently added for 10 min. (a) p38MAPK phosphorylation, (b) ERK1/2 phosphorylation, (c) JNK phosphorylation, and (d) Akt phosphorylation were evaluated as described in Section 2.*P < 0.05 and **P < 0.01 compared with the resting group; ^# P < 0.05 and ^### P < 0.001 compared with the TNF-α group. The data are presented as the mean ± SEM (n = 3).

Figure 4

Effects of andrographolide on IκBα degradation and p65 activation in TNF-α-stimulated VSMCs. The VSMCs were treated with PBS (resting group) or pretreated with andrographolide (20 and 50 μM) or an equal volume of DMSO (solvent control) for 20 min, and TNF-α (10 ng/mL) was subsequently added for 30 min. (a) IκBα degradation and (b) p65 phosphorylation were evaluated as described in Section 2.*P < 0.05 and ***P < 0.001 compared with the resting group; ^# P < 0.05 compared with the TNF-α group. The data are presented as the mean ± SEM (n = 3).

Figure 5

Regulatory effects of various signal inhibitors on p65 activation and Akt and JNK phosphorylation in TNF-α-stimulated VSMCs. The VSMCs were treated with PBS (resting group) or pretreated with LY294002 (10 μM), SP600125 (10 μM), or an equal volume of DMSO (solvent control) for 20 min, and TNF-α (10 ng/mL) was subsequently added for 10 min ((b) and (c)) or 30 min (a). (a) p65 phosphorylation, (b) Akt phosphorylation, and (c) JNK phosphorylation were evaluated as described in Section 2.*P < 0.05 and **P < 0.01 compared with the resting group; ^# P < 0.05 compared with the TNF-α group. The data are presented as the mean ± SEM (n = 3).

Figure 6

Diagram of the hypothetical inhibitory mechanism of andrographolide-induced effects in TNF-α-stimulated VSMCs. TNF-α triggers the expression of iNOS through IκBα-(IκBα-p65) and IκBα-independent (JNK-Akt-p65) pathways.