Ziziphus nummularia Inhibits Inflammation-Induced Atherogenic Phenotype of Human Aortic Smooth Muscle Cells

Abstract

Cardiovascular disease (CVD) continues to be the leading cause of death worldwide. Atherosclerosis is a CVD characterized by plaque formation resulting from inflammation-induced insults to endothelial cells, monocytes, and vascular smooth muscle cells (VSMCs). Despite significant advances, current treatments for atherosclerosis remain insufficient, prompting the search for alternative modalities, including herbal medicine. Ziziphus nummularia is an herb commonly used in the amelioration of symptoms associated with many health conditions such as cold, diarrhea, cancer, and diabetes. However, its effect on the inflammation-induced behavior of VSMCs remains unknown. In this study, we sought to determine the effect of the ethanolic extract of Z. nummularia (ZNE) on TNF-α-induced phenotypic changes of human aortic smooth muscle cells (HASMCs). The treatment of HASMCs with ZNE decreased cell proliferation, adhesion to fibronectin, migration, and invasion. ZNE treatment also caused a concentration- and time-dependent reduction in the TNF-α-induced expression of matrix metalloproteases MMP-2 and MMP-9, NF-κB, and cell adhesion molecules ICAM-1 and VCAM-1. Furthermore, ZNE decreased the adhesion of THP-1 monocytes to HASMCs and endothelial cells in a concentration-dependent manner. These data provide evidence for the anti-inflammatory effect of Ziziphus nummularia, along with potential implications for its use as an agent that could ameliorate inflammation-induced atherogenic phenotype of VSMCs in atherosclerosis.

Figure 1

ZNE decreases VSMC proliferation in a concentration- and time-dependent manner as demonstrated by trypan blue cell count at the indicated time intervals (a) and the overall metabolic activity measured by CellTiter-Glo luminescent cell viability assay (b). Viability values are calculated as % of the corresponding vehicle control value and represented as mean ± SEM of three replicates. Statistical significance was tested using 2-way ANOVA followed by Dunnett's post-hoc test. ∗ denotes P < 0.05 compared to that of the vehicle control, while # indicates P < 0.05 compared to the effect of the same concentration of ZNE at 24 hours.

Figure 2

ZNE decreases VSMC migration in a concentration-dependent manner. (a) Representative photomicrographs of the effect of 100 and 150 μg/ml of ZNE on VSMC migrating to the lower chamber in transwell migration assay. (b) Mean ± SEM of % control values for VSMC migration to the lower chamber (n = 3 replicates). (c) Representative photomicrographs of the effect of 100 and 150 μg/ml of ZNE on VSMC scratch wound healing at 24 hours postscratch. (d) Mean ± SEM of % control values of the residual scratch wound area (n = 3 replicates). Statistical significance was tested with ANOVA followed by Tuckey's post-hoc test. ∗ denotes P < 0.05 on the indicated comparisons.

Figure 3

ZNE decreases VSMC adhesion to fibronectin in a concentration-dependent manner. (a) Representative photomicrographs of the effect of 100 and 150 μg/ml of ZNE on VSMC adhesion to fibronectin. (b) Invasion data summary represented as mean ± SEM of % corresponding control values for VSMC adhesion (n = 3 replicates). Statistical significance was tested with ANOVA followed by Tuckey's post-hoc test. ∗ denotes P < 0.05 on the indicated comparisons.

Figure 4

The effect of ZNE on VSMC invasion. (a) The effect of 100 and 150 μg/ml of ZNE on VSMC invasion. Values represented are mean ± SEM of % corresponding control values for VSMC invasion into Matrigel (n = 3 replicates). Treatment with increasing concentrations of ZNE is associated with graded inhibition of both spontaneous (b, d) and TNF-α-evoked (c, e) MMP-2 and MMP-9 levels in VSMCs. Data represented are mean ± SEM of % MMP level in the corresponding vehicle control treatment (n = 3 replicates). For (b, d), statistical significance was tested with 2-way ANOVA followed by Dunnett's post-hoc test for the effect of concentration at the same exposure time (∗ denotes P < 0.05 compared to that of the vehicle control) and Sidak's post-hoc test for the effect of time at the same concentration (# denotes P < 0.05 compared to that of the corresponding concentration at 24 hours). For (c, e), statistical significance was tested with ANOVA followed by Tuckey's post-hoc test. ∗ denotes P < 0.05 compared to that of the MMP level evoked by TNF-α in the absence of ZNE, while # denotes P < 0.05 compared to that of the MMP level of the vehicle control.

Figure 5

Treatment with increasing concentrations of ZNE leads to a graded decrease in TNF-α-evoked adhesion of THP-1 cells to VSMCs (a) and HUVECs (b). Values represented are mean ± SEM of % THP-1 cell adhesion upon treatment with the corresponding vehicle control (n = 3 replicates). Statistical significance was tested with ANOVA followed by Tuckey's post-hoc test. ∗ denotes P < 0.05 compared to that of the THP-1 cell adhesion evoked by TNF-α in the absence of ZNE, while # denotes P < 0.05 compared to that of the cell adhesion seen upon treatment with the vehicle control.

Figure 6

Gradual reduction in TNF-α-evoked adhesion molecule expression ((a), VCAM-1; (b), ICAM-1) on HUVECs detected by cell-surface ELISA upon treatment with increased concentrations of ZNE. Values represented are mean ± SEM of % adhesion molecule expression observed upon treatment with the corresponding vehicle control (n = 3 replicates). Statistical significance was tested with ANOVA followed by Tuckey's post-hoc test. ∗ denotes P < 0.05 compared to that of the adhesion molecule expression evoked by TNF-α in the absence of ZNE, while # denotes P < 0.05 compared to that of the expression observed upon treatment with the vehicle control.

Figure 7

TNF-α-evoked NF-κB expression in HUVECs transfected with a luciferase-expressing NF-κB promotor is decreased by ZNE treatment in a concentration-dependent manner. Values represented are mean ± SEM of % promotor units recorded upon treatment with the corresponding vehicle control (n = 3 replicates). Statistical significance was tested with ANOVA followed by Tuckey's post-hoc test. ∗ denotes P < 0.05 compared to that of the expression evoked by TNF-α in the absence of ZNE, while # denotes P < 0.05 compared to that of the expression observed upon treatment with the vehicle control.