cyclic-gmp and andrographolide

Topics: Anti-Inflammatory Agents; Carbon Monoxide; Cell Hypoxia; Cell Line; Cyclic GMP; Diterpenes; Dual-Specificity Phosphatases; Endothelin-1; Heme Oxygenase-1; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Mitogen-Activated Protein Kinase Phosphatases; p38 Mitogen-Activated Protein Kinases; Thionucleotides

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 IKKβ 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 IKKβ, and p65 phosphorylation. In addition, SB203580 (an inhibitor of p38 MAPK), but not PD98059 (an inhibitor of ERKs), markedly abolished IKKβ 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.

Topics: Blood Platelets; Collagen; Cyclic GMP; Diterpenes; eIF-2 Kinase; Gene Expression Regulation; Humans; Hydroxyl Radical; I-kappa B Kinase; Mitogen-Activated Protein Kinase 1; NF-kappa B; p38 Mitogen-Activated Protein Kinases; Platelet Activation; Platelet Aggregation Inhibitors; Signal Transduction

Andrographolide is a novel NF-κB inhibitor from the leaves of Andrographis paniculata. Platelet activation is relevant to a variety of thrombotic diseases. However, no data are available concerning the effects of andrographolide in platelet activation. The aim of this study was to examine the mechanisms of andrographolide in preventing platelet activation. Andrographolide (25-75 μΜ) exhibited a more potent activity of inhibiting platelet aggregation stimulated by collagen. Andrographolide inhibited collagen-stimulated platelet activation accompanied by relative Ca(2+) mobilization; thromboxane A(2) formation; and phospholipase C (PLC)γ2, protein kinase C (PKC), mitogen-activated protein kinase (MAPK), and Akt phosphorylation. Andrographolide markedly increased cyclic GMP, but not cyclic AMP levels. Andrographolide also stimulated endothelial nitric oxide synthase (eNOS) expression, NO release, and vasodilator-stimulated phosphoprotein (VASP) phosphorylation. ODQ, an inhibitor of guanylate cyclase, markedly reversed the andrographolide-mediated inhibitory effects on platelet aggregation, p38 MAPK and Akt phosphorylation, and the andrographolide-mediated stimulatory effect on VASP phosphorylation. Furthermore, a PI3 kinase inhibitor (LY294002) but not a PKC inhibitor (Ro318220) significantly diminished p38 MAPK phosphorylation; nevertheless, a p38 MAPK inhibitor (SB203580) and LY294002 diminished PKC activity stimulated by collagen. Andrographolide also reduced collagen-triggered hydroxyl radical (OH([Symbol: see text])) formation. In vivo studies revealed that andrographolide (22 and 55 μg/kg) is effective in reducing the mortality of ADP-induced acute pulmonary thromboembolism and significantly prolonged platelet plug formation in mice. This study demonstrates for the first time that andrographolide possesses a novel role of antiplatelet activity, which may involve the activation of the eNOS-NO/cyclic GMP pathway, resulting in the inhibition of the PI3 kinase/Akt-p38 MAPK and PLCγ2-PKC cascades, thereby leading to inhibition of platelet aggregation.

Topics: Adenosine Diphosphate; Animals; Blood Platelets; Calcium; Collagen; Cyclic GMP; Diterpenes; Humans; Mice; NF-kappa B; Nitric Oxide; Nitric Oxide Synthase Type III; Platelet Activation; Platelet Aggregation Inhibitors; Protein Kinase Inhibitors; Protein Kinases; Pulmonary Embolism; Thromboxane A2