Suppression of NF-κB signaling by andrographolide with a novel mechanism in human platelets: regulatory roles of the p38 MAPK-hydroxyl radical-ERK2 cascade.

Andrographolide, a novel nuclear factor-κB (NF-κB) inhibitor, is isolated from leaves of Andrographis paniculata. Platelet activation is relevant to a variety of coronary heart diseases. Our recent studies revealed that andrographolide possesses potent antiplatelet activity by activating the endothelial nitric oxide synthase (eNOS)-NO-cyclic GMP pathway. Although platelets are anucleated cells, they also express the transcription factor, NF-κB, that may exert non-genomic functions in platelet activation. Therefore, we further investigated the inhibitory roles of andrographolide in NF-κB-mediated events in platelets. In this study, NF-κB signaling events, including IKKß phosphorylation, IκBα degradation, and p65 phosphorylation, were time-dependently activated by collagen in human platelets, and these signaling events were attenuated by andrographolide (35 and 75 µM). ODQ and KT5823, respective inhibitors of guanylate cyclase and cyclic GMP-dependent kinase (PKG), strongly reversed andrographolide-mediated inhibition of platelet aggregation, relative [Ca(2+)]i mobilization, and IKKß, and p65 phosphorylation. In addition, SB203580 (an inhibitor of p38 MAPK), but not PD98059 (an inhibitor of ERKs), markedly abolished IKKß and p65 phosphorylation. SB203580, NAC (a free-radical scavenger), and BAY11-7082 (an inhibitor of NF-κB) all diminished ERK2 phosphorylation, whereas PD98059, BAY11-7082, and NAC had no effects on p38 MAPK phosphorylation. Furthermore, SB203580, but not BAY11-7082 or PD98059, reduced collagen-induced hydroxyl radical ((·)HO) formation. KT5823 also markedly reversed andrographolide-mediated inhibition of p38 MAPK and ERK2 phosphorylation, and hydroxyl radical formation in platelets. In conclusion, this study demonstrated that andrographolide may involve an increase in cyclic GMP/PKG, followed by inhibition of the p38 MAPK/(·)HO-NF-κB-ERK2 cascade in activated platelets. Therefore, andrographolide may have a high therapeutic potential to treat thromboembolic disorders and may also be considered for treating various inflammatory diseases.

Andrographolide enhances nuclear factor-kappaB subunit p65 Ser536 dephosphorylation through activation of protein phosphatase 2A in vascular smooth muscle cells.

Recent studies have demonstrated that transcription factor nuclear factor (NF)-κB inhibition may contribute to the protective anti-inflammatory actions of andrographolide, an abundant component of plants of the genus Andrographis. However, the precise mechanism by which andrographolide inhibits NF-κB signaling remains unclear. We thus investigated the mechanism involved in andrographolide suppression of NF-κB signaling in rat vascular smooth muscle cells (VSMCs) exposed to proinflammatory stimuli, LPS, and IFN-γ. Andrographolide was shown to suppress LPS/IFN-γ-induced inducible nitric-oxide synthase and matrix metalloprotease 9 expression in rat VSMCs. Andrographolide also inhibited LPS/IFN-γ-induced p65 nuclear translocation, DNA binding activity, p65 Ser(536) phosphorylation, and NF-κB reporter activity. However, IKK phosphorylation and downstream inhibitory κBα phosphorylation and degradation were not altered by the presence of andrographolide in LPS/IFN-γ-stimulated VSMCs. These andrographolide inhibitory actions could be prevented by selective inhibition of neutral sphingomyelinase and protein phosphatase 2A (PP2A). Furthermore, andrographolide was demonstrated to increase ceramide formation and PP2A activity in VSMCs and to inhibit neointimal formation in rat carotid injury models. These results suggest that andrographolide caused neutral sphingomyelinase-mediated ceramide formation and PP2A activation to dephosphorylate p65 Ser(536), leading to NF-κB inactivation and subsequent inducible nitric-oxide synthase down-regulation in rat VSMCs stimulated by LPS and IFN-γ.