Inhibition of vascular smooth muscle cell proliferation by Gentiana lutea root extracts

Abstract

Gentiana lutea belonging to the Gentianaceae family of flowering plants are routinely used in traditional Serbian medicine for their beneficial gastro-intestinal and anti-inflammatory properties. The aim of the study was to determine whether aqueous root extracts of Gentiana lutea consisting of gentiopicroside, gentisin, bellidifolin-8-O-glucoside, demethylbellidifolin-8-O-glucoside, isovitexin, swertiamarin and amarogentin prevents proliferation of aortic smooth muscle cells in response to PDGF-BB. Cell proliferation and cell cycle analysis were performed based on alamar blue assay and propidium iodide labeling respectively. In primary cultures of rat aortic smooth muscle cells (RASMCs), PDGF-BB (20 ng/ml) induced a two-fold increase in cell proliferation which was significantly blocked by the root extract (1 mg/ml). The root extract also prevented the S-phase entry of synchronized cells in response to PDGF. Furthermore, PDGF-BB induced ERK1/2 activation and consequent increase in cellular nitric oxide (NO) levels were also blocked by the extract. These effects of extract were due to blockade of PDGF-BB induced expression of iNOS, cyclin D1 and proliferating cell nuclear antigen (PCNA). Docking analysis of the extract components on MEK1, the upstream ERK1/2 activating kinase using AutoDock4, indicated a likely binding of isovitexin to the inhibitor binding site of MEK1. Experiments performed with purified isovitexin demonstrated that it successfully blocks PDGF-induced ERK1/2 activation and proliferation of RASMCs in cell culture. Thus, Gentiana lutea can provide novel candidates for prevention and treatment of atherosclerosis.

Figure 1. IC₅₀ values for the G. lutea extract (1 mg/ml) on smooth muscle cells.

A) Primary cultures of rat aortic smooth muscle cells (RASMCs), B) Rat aortic smooth muscle cell line A7r5 and C) Human aortic smooth muscle cell line ATCC-CRL-1999.

Figure 2. Effect of G. lutea extract (1 mg/ml) on proliferation and apoptosis.

A) Effect of extract on PDGF-BB (20 ng/ml, 24 hours) induced proliferation of primary cultures of rat aortic smooth muscle cells (RASMCs), B) A7r5 and C) percentage of apoptotic RASMCs in response to experimental treatments. *P<0.05 and ***P<0.001 versus control and †P<0.05 and †††<0.001 versus PDGF treatment.

Figure 3. Cell cycle analysis of RASMCs.

A) Representative flow cytogram depicting cells in various stages of cell cycle upon treatment with PDGF-BB (20 ng/ml) in presence and absence of G. lutea extract, and B) Bar graph summarizing data for four independent experiments. ***P<0.001 versus control, ††<0.01 and †††<0.001 versus PDGF treatment.

Figure 4. Effect of PDGF-BB (20 ng/ml) on ERK1/2 and NO signaling.

A) Time course of ERK1/2 activation in response to PDGF, B&C) Time course of generation of intracellular nitric oxide in response to PDGF, D) Activation of eNOS through phosphorylation of Ser¹¹⁷⁷ residue in response to PDGF and E) PDGF-induced expression of iNOS. Bar graphs summarize data for a minimum of four independent experiments. *P<0.05, **P<0.01 and ***P<0.001 versus control.

Figure 5. Role of ERK1/2-NO axis on PDGF-induced proliferation of RASMCs and effect of G lutea on PDGF induced NO index.

A) Effect of ERK inhibitor 328000 (1 µmole/L) on PDGF induced cell proliferation, B) Effect of NOS inhibitor L-NAME (10 µmole/L) on PDGF induced cell proliferation, C) Effect of G lutea extract on generation of cellular NO in response to PDGF. *P<0.05 versus control and †<0.05, ††<0.01 and †††<0.001 versus corresponding PDGF treatment.

Figure 6. Effect of G lutea extract on PDGF-induced cell signaling in RASMCs.

A) PDGFR-β phosphorylation, B) ERK1/2 activation, C) Phosphorylation of IKKα and D&E) Representative blot and bar graph indicating expression of cyclin D1, PCNA and iNOS for a minimum of three independent experiments. *<0.05, **P<0.01 and ***P<0.001 versus control and ††<0.01 and †††<0.001 versus PDGF treatment.

Figure 7. Effect of isovitexin on PDGF- induced RASMC proliferation.

A) Docking analysis revealing binding pocket of isovitexin on MEK1, B) Effect of isovitexin (0.5–10 µmol/L) on PDGF induced ERK1/2 activation and cell cycle progress and C) Effect of isovitexin on PDGF induced cell proliferation measured through alamar blue assay. Bar graphs summarize data for a minimum of three independent experiments. **P<0.01 versus control and ††<0.01 and †††<0.001 versus PDGF treatment.

Figure 8. Summary figure depicting the pathway blocked by G lutea extract to prevent PDGF-induced RASMC proliferation.