Inhibitory Effect of Ginsenoside Rg1 on Vascular Smooth Muscle Cell Proliferation Induced by PDGF-BB Is Involved in Nitric Oxide Formation

Abstract

Ginsenoside Rg1 (Rg1) has been reported to suppress the proliferation of vascular smooth muscle cells (VSMCs). This study aimed to observe the role of nitric oxide (NO) in Rg1-antiproliferative effect. VSMCs from the thoracic aorta of SD rats were cultured by tissue explant method, and the effect of Rg1 (20 mg·L(-1), 60 mg·L(-1), and 180 mg·L(-1)) on platelet-derived growth factor-BB (PDGF-BB)-induced proliferation was evaluated by MTT assay. The cell cycle was analyzed by flow cytometry. For probing the mechanisms, the content of NO in supernatant and cGMP level in VSMCs was measured by nitric oxide kit and cGMP radio-immunity kit, respectively; the expressions of protooncogene c-fos and endothelial NO synthase (eNOS) mRNA in the VSMCs were detected by real-time RT-PCR; the intracellular free calcium concentration ([Ca2(+)](i)) was detected with Fura-2/AM-loaded VSMCs. Comparing with that in normal group, Rg1 180 mg·L(-1) did not change the absorbance of MTT and cell percent of G(0)/G(1), G(2)/M, and S phase in normal cells (P > 0.05). Contrarily, PDGF-BB could increase the absorbance of MTT (P < 0.01) and the percent of the S phase cells but decrease the G(0)/G(1) phase cell percent in the cell cycle, accompanied with an upregulating c-fos mRNA expression (P < 0.01), which was reversed by additions of Rg1(20 mg·L(-1), 60 mg·L(-1), and 180 mg·L(-1)). Rg1 administration could also significantly increase the NO content in supernatant and the cGMP level in VSMCs, as well as the eNOS mRNA expression in the cells, in comparison of that in the group treated with PDGF-BB alone (P < 0.01). Furthermore, Rg1 caused a further increase in the elevated [Ca(2+)](i) induced by PDGF-BB. It was concluded that Rg1 could inhibit the VSMC proliferation induced by PDGF-BB through restricting the G(0)/G(1) phase to S-phase progression in cell cycle. The mechanisms may be related to the upregulation of eNOS mRNA and the increase of the formation of NO and cGMP.

Figure 1

Effect of Rg1 on PDGF-BB-induced VSMC proliferation (MTT assay, $\bar{x}\pm s$, n = 7). The VSMCs were cultured for 24 h. Normal: vehicle; N+Rg1: Rg1 180 mg·L⁻¹; model: PDGF-BB 25 μg·L⁻¹; Rg1-L: Rg1 20 mg·L⁻¹ + PDGF-BB 25 μg·L⁻¹; Rg1-M: Rg1 60 mg·L⁻¹ +PDGF-BB 25 μg·L⁻¹; Rg1-H: Rg1 180 mg·L⁻¹ + PDGF-BB 25 μg·L⁻¹. Data were mean ± S.E.M. ^##Significant difference from normal control at P < 0.01* significant difference from model control at P < 0.05, and ** significant difference from model control at P < 0.01.

Figure 2

Effect of Rg1 on the VSMC cell cycle in the presence of PDGF-BB ($\bar{x}\pm s$, n = 4 ~ 6,%). A: representative results of flow cytometry measurements to determine the cell-cycle stages of VSMCs; B:percentage of cells in each phase of the cell cycle. Normal: vehicle; N+Rg1: Rg1 180 mg·L⁻¹; model : PDGF-BB 25 μg·L⁻¹; Rg1-L : Rg1 20 mg·L⁻¹ + PDGF-BB 25 μg·L⁻¹; Rg1-M : Rg1 60 mg·L⁻¹ +PDGF-BB 25 μg·L⁻¹; Rg1-H : Rg1 180 mg·L⁻¹ +PDGF-BB 25 μg·L⁻¹.Data were mean ± S.E.M. ^##Significant difference from normal control at P < 0.01; ** significant difference from model control at P < 0.01.

Figure 3

Effects of Rg1 on the changes of expressions of c-fos and eNOS mRNA induced by PDGF-BB in VSMC ($\bar{x}\pm s,n=3$); real-time RT-PCR analysis of c-fos, and eNOS mRNA were performed after 24 h of PDGF-BB and Rg1 actions in VSMCs. Normal : vehicle; model : PDGF-BB 25 μg·L⁻¹; Rg1-L : Rg1 20 mg·L⁻¹ + PDGF-BB 25 μg·L⁻¹; Rg1-M : Rg1 60 mg·L⁻¹ +PDGF-BB 25 μg·L⁻¹; Rg1-H : Rg1 180 mg·L⁻¹ +PDGF-BB 25 μg·L⁻¹. Data were mean ± S.E.M. ^##Significant difference from normal control at P < 0.01; ** significant difference from model control at P < 0.01.

Figure 4

Effects of Rg1 on the NO content in supernatant (a) and cGMP level in cultured VSMCs (b) in the presence of PDGF-BB. VSMCs were cultured for 24 h, the NO content in supernatant and cGMP level in the cells were determined with the corresponding Kits. Normal: vehicle; model: PDGF-BB 25 μg·L⁻¹; Rg1-L : Rg1 20 mg·L⁻¹ + PDGF-BB 25 μg·L⁻¹; Rg1-M : Rg1 60 mg·L⁻¹ +PDGF-BB 25 μg·L⁻¹; Rg1-H : Rg1 180 mg·L⁻¹ +PDGF-BB 25 μg·L⁻¹. Data were mean ± S.E.M. ^##Significant difference from normal control at P < 0.01; ** significant difference from model control at P < 0.01.

Figure 5

Effect of Rg1 on the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) of VSMCs. The [Ca²⁺]_i was detected after 3 minutes of PDGF-BB addition with Fura-2/AM loaded VSMCs, and Rg1 administrations were performed before 30 minutes of the addition of PDGF-BB. Normal : vehicle; model : PDGF-BB 25 μg·L⁻¹; Rg1-L : Rg1 20 mg·L⁻¹ + PDGF-BB 25 μg·L⁻¹; Rg1-M : Rg1 60 mg·L⁻¹ +PDGF-BB 25 μg·L⁻¹; Rg1-H : Rg1 180 mg·L⁻¹ + PDGF-BB 25 μg·L⁻¹. Data were mean ± S.E.M. ^#Significant difference from normal control at P < 0.05; *significant difference from model control at P < 0.05, **Significant difference from model control at P < 0.01.