Ginsenoside Rg3 stereoisomers differentially inhibit vascular smooth muscle cell proliferation and migration in diabetic atherosclerosis

Abstract

Ginsenoside 20(R/S)-Rg3, as a natural peroxisome proliferator-activated receptor gamma (PPARγ) ligand, has been reported to exhibit differential biological effects. It is of great interest to understand the stereochemical selectivity of 20(R/S)-Rg3 and explore whether differential PPARγ activation by Rg3 stereoisomers, if it exists, could lead to differential physiological outcome and therapeutic effects in diabetic atherosclerosis. Here, we investigated the binding modes of 20(R/S)-Rg3 stereoisomers in the PPARγ ligand-binding domain (PPARγ-LBD) using molecular modelling and their effects on smooth muscle cell proliferation and migration induced by advanced glycation end products (AGEs). The results revealed that 20(S)-Rg3 exhibited stronger antiproliferative and antimigratory effects due to stronger PPARγ activation. To validate the in vitro results, we used a mice model with diabetic atherosclerosis and obtained that 20(S)-Rg3 markedly reduced the plaque size secondary to reducing the proliferation and migration of VSMCs, while the plaques were more stable due to improvements in other plaque compositions. The results shed light on the structural difference between Rg3 stereoisomers that can lead to significant differential physiological outcome, and the (S)-isomer seems to be the more potent isomer to be developed as a promising drug for diabetic atherosclerosis.

Keywords: PPARγ; Rg3; atherosclerosis; ginsenoside; stereoisomer.

Figure 1

Simulation and calculation of the binding modes of 20(R/S)‐Rg3 in models R2 and S1. A, Superposition of 20(R/S)‐Rg3 in models R2 and S1 after simulation. The LBD in model S1 is shown as a green ribbon and that in model R2 is shown as a blue ribbon. 20(S)‐Rg3 in model S1 is shown as green ball‐and stick models, and 20(R)‐Rg3 in model R2 is shown as blue sticks. Tyr473, Ser342, Glu343 and His449 in model S1 are shown as green wires and those in model R2 are shown as blue wires. The hydrogen bonds in model S1 are shown as orange dotted lines, and those in model R2 are shown as black dotted lines. B, EDA projections onto the first 2 eigenvectors of models S1 and R2 involved in those of the apo‐bound, GW9662‐bound, LT160‐bound and LT127‐bound systems. The calculation was based on the last 10‐ns equilibrated trajectories. C, D, PPARγ transcriptional activity in transfected 293T cells with and without AGEs incubation (n = 3). Values are presented as the mean ± SEM. * P < .05, ** P < .01, *** P < .001 vs control; ##P < .01, ###P < .001 vs the 20(R)‐Rg3 groups

Figure 2

Differential effects of the 20(R/S)‐Rg3 stereoisomers on VSMC proliferation. A, Cell Counting Kit‐8 analysis of the cell viability of VSMCs (n = 5). ###P < .001 vs control; * P < .05, ** P < .01, *** P < .001 vs the AGEs group; †P < .05, ††P < .01 vs the 20(R)‐Rg3 groups. B, Fluorescence microscopy of EdU staining. Nuclei were counterstained with DAPI (blue); red staining indicates cells undergoing proliferation. The EdU‐positive index was expressed as a percentage (positive/total cell number) (n = 5). Scale bar: 10 μm. * P < .05, ** P < .01, *** P < .001. All quantitative data are means ± SEM

Figure 3

Effects of the 20(R/S)‐Rg3 stereoisomers on cell cycle progression and cell cycle related‐protein expression in AGE‐stimulated VSMCs. A, Flow cytometry analysis of the cell cycle. The relative percentage of cells in different cell cycle phases are reported, while the percentage of apoptotic events was ignored (n = 3). B, Western blot analysis of cyclin D1, cyclin E and PCNA protein expression. The bands are quantified by densitometric analysis. Protein expression of cyclin D1 and PCNA was normalized to β‐actin, and expression of cyclin E was normalized to GADPH (n = 5, 4, and 5 for cyclin D1, cyclin E and PCNA, respectively). The results are expressed as the mean values ± SEM. * P < .05, ** P < .01, *** P < .001

Figure 4

Differential effects of the 20(R/S)‐Rg3 stereoisomers on the migration of AGE‐treated VSMCs. A, Transwell migration assay. Percentage of migrated cells relative to the control are quantified (n = 5). B, Western blot analysis of MMP2 and MMP9 protein expression after 48 h of stimulation. The bands are quantified by densitometric analysis, and protein expression was normalized to GADPH (n = 4 and 5 for MMP2 and MMP9, respectively). C, Gelatin zymography analysis of MMP2 and MMP9 activity in the medium after 48 h of stimulation. Relative activity was quantified by densitometric analysis (n = 3; respectively). The values shown represent means ± SEM of independent assays. * P < .05, ** P < .01, *** P < .001

Figure 5

Effects of the 20(R/S)‐Rg3 stereoisomers on early atherogenesis in diabetic mice. A,B, En face analysis of aortas. Atherosclerotic lesions were identified by Oil Red O staining (n = 3). The ratio of the atherosclerotic lesion area to the total vessel area is quantified, indicating level of atherogenesis. C, E, Haematoxylin and eosin staining of aortic sinus cryosections; the ratio of total atherosclerotic lesion area to aorta lumen area are quantified (n = 3). Scale bar: 100 μm. D, F, Representative immunohistochemical α‐SMA staining and quantification of the plaque smooth muscle cell content (n = 3). Scale bar: 50 μm. The data are expressed as the mean ± SEM * P < .05, ** P < .01, *** P < .001

Figure 6

Differential effects of the 20(R/S)‐Rg3 stereoisomers on proliferation of VSMCs within plaques. A, Co‐immunofluorescence staining of the aortic root for VSMCs (anti‐α‐SMA antibody, red) and proliferation marker PCNA (green) and a bar graph summarizing the results (n = 3). Scale bar: 20 μm. B, Western blot analysis of cyclin D1, cyclin E and PCNA protein expression within plaques. The bands are quantified by densitometric analysis. Protein expression of cyclin D1 and PCNA was normalized to β‐actin, and expression of cyclin E was normalized to GADPH (n = 3, 4, and 4 for cyclin D1, cyclin E and PCNA, respectively). The results are expressed as the mean values ± SEM. * P < .05, ** P < .01, *** P < .001

Figure 7

Differential effects of the 20(R/S)‐Rg3 stereoisomers on MMP expression within plaques. A, Representative immunohistochemical MMP2 and MMP9 staining and quantification analysis (n = 3, respectively). Scale bar: 50 μm. B, Western blot analysis of MMP2 and MMP9 protein expression within plaques. The bands are quantified by densitometric analysis, and protein expression was normalized to GADPH (n = 3, respectively). The results are expressed as the mean values ± SEM. * P < .05, ** P < .01, *** P < .001