Andrographolide, a Novel NF- κ B Inhibitor, Induces Vascular Smooth Muscle Cell Apoptosis via a Ceramide-p47phox-ROS Signaling Cascade

Abstract

Atherosclerosis is linked with the development of many cardiovascular complications. Abnormal proliferation of vascular smooth muscle cells (VSMCs) plays a crucial role in the development of atherosclerosis. Accordingly, the apoptosis of VSMCs, which occurs in the progression of vascular proliferation, may provide a beneficial strategy for managing cardiovascular diseases. Andrographolide, a novel nuclear factor- κ B inhibitor, is the most active and critical constituent isolated from the leaves of Andrographis paniculata. Recent studies have indicated that andrographolide is a potential therapeutic agent for treating cancer through the induction of apoptosis. In this study, the apoptosis-inducing activity and mechanisms in andrographolide-treated rat VSMCs were characterized. Andrographolide significantly induced reactive oxygen species (ROS) formation, p53 activation, Bax, and active caspase-3 expression, and these phenomena were suppressed by pretreating the cells with N-acetyl-L-cysteine, a ROS scavenger, or diphenylene iodonium, a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) inhibitor. Furthermore, p47phox, a Nox subunit protein, was phosphorylated in andrographolide-treated rat VSMCs. However, pretreatment with 3-O-methyl-sphingomyelin, a neutral sphingomyelinase inhibitor, significantly inhibited andrographolide-induced p47phox phosphorylation as well as Bax and active caspase-3 expression. Our results collectively demonstrate that andrographolide-reduced cell viability can be attributed to apoptosis in VSMCs, and this apoptosis-inducing activity was associated with the ceramide-p47phox-ROS signaling cascade.

Figure 1

Chemical structure of andrographolide (Andro).

Figure 2

The role of ROS in andrographolide-reduced cell viability in rat VSMCs. (a) Rat VSMCs were treated with 50 μM andrographolide for the indicated periods. Cells were harvested, and the formation of ROS was examined using flow cytometric analysis of DCF-DA-stained cells, as described in Section 2. (b) Cells were pretreated with a vehicle or 1 mM NAC for 30 min before being treated with 50 μM andrographolide for 48 h; cell viability was subsequently determined using an MTT assay. The results shown are representative of 4 independent experiments. The data are presented as the mean ± SEM (error bars: **P < 0.01 and ***P < 0.001, compared with the control group, and ^### P < 0.001, compared with the group treated only with andrographolide).

Figure 3

Nox-mediated redox signaling in andrographolide-induced ROS formation. (a) Cells were pretreated with a vehicle, 1 mM NAC, or 10 μM DPI for 30 min before being treated with 50 μM andrographolide for 10 min, and the production of ROS was examined using flow cytometric analysis of DCF-DA-stained cells, as described in Section 2. (b) Cells were treated with 50 μM andrographolide for the indicated periods. Cells were harvested, and the phosphorylation of p47phox was examined using immunoblotting. The data are presented as the mean ± SEM (error bars: *P < 0.05, **P < 0.01, and ***P < 0.001, compared with the control group, and ^## P < 0.01 and ^### P < 0.001, compared with the group treated only with andrographolide).

Figure 4

Effects of ROS scavengers on andrographolide-stimulated p53 activation, Bax, and active caspase-3 expression in rat VSMCs. (a) Cells were transiently transfected with PG-13-luc and Renilla-luc for 48 h. After transfection, the cells were pretreated with a vehicle, 1 mM NAC, or 10 μM DPI for 30 min before being treated with 50 μM andrographolide for another 24 h. A PG13-luciferase assay was subsequently conducted. Cells were pretreated with a vehicle, 1 mM NAC, or 10 μM DPI for 30 min before being treated with 50 μM andrographolide for 48 hr, and the expression of Bax (b) and active caspase-3 (c) was examined using immunoblotting. The data are presented as the mean ± SEM (error bars: **P < 0.01 and ***P < 0.001, compared with the control group, and ^# P < 0.05, ^## P < 0.01, and ^### P < 0.001, compared with the group treated only with andrographolide).

Figure 5

The role of ceramide signaling in andrographolide-induced p47phox phosphorylation, Bax, and active caspase-3 expression in rat VSMCs. Cells were pretreated with a vehicle or 30 μM 3-OMS for 30 min before being treated with 50 μM andrographolide for 10 min (a), or 48 h (b and c). The extent of p47phox phosphorylation (a), Bax (b), or active caspase-3 expression (c) was examined. The data are presented as the mean ± SEM (error bars: *P < 0.05, **P < 0.01, and ***P < 0.001, compared with the control group, and ^## P < 0.01 and ^### P < 0.001, compared with the group treated only with andrographolide).

Figure 6

Hypothetical scheme of the signal pathways in andrographolide-induced rat VSMC apoptosis. Andrographolide stimulated the ceramide-mediated signal events, resulting in the activation of the p47phox-ROS cascade, ultimately stimulating active caspase-3 expression and VSMC apoptosis. Nox produces superoxide (O₂ ⁻), followed by the induction of H₂O₂. H₂O₂ is capable of inducing DNA damage to cause p53 activation, which can lead Bax and active caspase-3 expression. nSMase: neutral sphingomyelinase; SM: sphingomyelinase; Nox: NADPH oxidase.