Andrographolide, a Novel NF-κB Inhibitor, Inhibits Vascular Smooth Muscle Cell Proliferation and Cerebral Endothelial Cell Inflammation

Abstract

Background: Aberrant vascular smooth muscle cell (VSMC) proliferation and cerebral endothelial cell (CEC) dysfunction contribute significantly in the pathogenesis of cardiovascular diseases. Therefore, inhibition of these cellular events would be by candidate agents for treating these diseases. In the present study, the mechanism of anti-proliferative and anti-inflammatory effects of andrographolides, a novel nuclear factor-κB inhibitor, was investigated in VSMC and CEC cells.

Methods: VSMCs and CECs were isolated from rat artery and mouse brain, respectively, and cultured before experimentation. The effect of andro on platelet-derived growth factor-BB (PDGF-BB) induced VSMC cell proliferation was evaluated by cell number, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The expression of extracellular signal regulated kinase 1/2 (ERK1/2), proliferating cell nuclear antigen (PCNA), and the effects on lipopolysaccharide (LPS)-induced inducible nitric oxide synthase (iNOS) and, cyclooxygenase-2 (COX2) were detected by Western blotting.

Results: Andro significantly inhibited PDGF-BB (10 ng/ml) induced cell proliferation in a concentration (20-100 μM) dependent manner, which may be due to reducing the expression of ERK1/2, and by inhibiting the expression of PCNA. Andro also remarkably diminished LPS-induced iNOS and COX2 expression.

Conclusions: The results of this study suggested that the effects of andro against VSMCs proliferation and CECs dysfunction may represent a promising approach for treatment of vascular diseases.

Key words: Andrographolide; CECs; COX2/iNOS; ERK/PCNA; LPS; PDGF-BB; VSMCs.

Figure 1

Effects of andrographolide on cell proliferation in vascular smooth muscle cells (VSMCs) stimulated by platelet-derived growth factor-BB (PDGF-BB). (A) VSMCs (2 × 10⁴ cells/well) were treated with PBS (resting) or pretreated with andro (20 and 50 mM), or an isovolumetric solvent control (0.1% DMSO), followed by the addition of PDGF-BB (10 ng/ml). Cell numbers were evaluated by an MTT assay as described under Materials and Methods. (B) VSMCs (2 × 105 cells/dish) were treated with PBS (resting) or pretreated with solvent control (0.1% DMSO) or 20-100 μM andro, followed by the addition of PDGF-BB (10 ng/ml). Compiled statistical data are shown on the right. * p < 0.01 compared to the resting (PBS treatment) group; ^# p < 0.01; ^† p < 0.001, compared to the PDGF-BB group. Data are presented as the mean ± SEM (N = 5).

Figure 2

Effects of andrographolide on proliferating cell nuclear antigen (PCNA) and signal-regulated kinase (ERK)1/2 phosphorylation in platelet-derived growth factor-BB (PDGF-BB) stimulated vascular smooth muscle cell (VSMC)s. VSMCs (2 × 105 cells/dish) were treated with PBS (resting) or pretreated with a solvent control (0.1% DMSO) or andro (20, 50 and 100 μM), followed by the addition of PDGF-BB (10 ng/ml) to trigger (A) PCNA and (B) ERK1/2 phosphorylation. * p < 0.001, compared to the resting (PBS treatment) group; ^# p < 0.05; ^† p < 0.01 and ^§ p < 0.001, compared to the PDGF-BB group. Data are presented as the means ± SEM (N = 5).

Figure 3

LPS-induced expressions of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in cerebral endothelial cell (CEC)s. (A) Morphology of primary cerebral endothelial cell. Cerebral endothelial cells (5 × 10⁵ cells in 6-well plates) were treated with vehicle (0.5% DMSO) or various concentrations of lipopolysaccharide (LPS) (10, 20, and 50 μg/ml) for 24 hr. Cell lysates were obtained and analyzed for (B) COX-2 and (C) iNOS protein expression by Western blotting. α-tubulin normalized to the resting condition. Data are shown as the mean ± SEM of three independent experiments.* p < 0.05, ^# p < 0.01 and ^† p < 0.001, compared with the resting group.

Figure 4

Effects of andrographolide on LPS-induced expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in cerebral endothelial cell (CEC)s. Cerebral endothelial (2 × 10⁵) cells were pretreated with the vehicle (DMSO, 0. 1%, v/v) or andro (10 and 20 mM) for 3 hr and then stimulated by lipopolysaccharide (LPS) (50 μg/mL) for 24 h. Cell lysates were obtained and analyzed for (A) COX-2 and (B) iNOS protein expressions by Western blotting. a-tubulin is used as an internal control. Data are shown as the mean ± SEM of three independent experiments. * p < 0.001 compared with the resting group, ^# p < 0.001, compared with the vehicle group.