Silymarin Ameliorates Diabetes-Induced Proangiogenic Response in Brain Endothelial Cells through a GSK-3 β Inhibition-Induced Reduction of VEGF Release

Abstract

Diabetes mellitus (DM) is a major risk factor for cardiovascular disease. Additionally, it was found to induce a dysfunctional angiogenic response in the brain that was attributed to oxidative stress. Milk thistle seed extract (silymarin) has potent antioxidant properties, though its potential use in ameliorating diabetes-induced aberrant brain angiogenesis is unknown. Glycogen synthase kinase-3β is a regulator of angiogenesis that is upregulated by diabetes. Its involvement in diabetes-induced angiogenesis is unknown. To evaluate the potential of silymarin to ameliorate diabetes-induced aberrant angiogenesis, human brain endothelial cells (HBEC-5i) were treated with 50 μg/mL advanced glycation end (AGE) products in the presence or absence of silymarin (50, 100 μM). The angiogenic potential of HBEC-5i was evaluated in terms of migration and in vitro tube formation capacities. The involvement of GSK-3β was also evaluated. AGE significantly increased the migration and tube formation rates of HBEC-5i by about onefold (p = 0.0001). Silymarin reduced AGE-induced migration in a dose-dependent manner where 50 μM reduced migration by about 50%, whereas the 100 μM completely inhibited AGE-induced migration. Similarly, silymarin 50 μg/mL blunted AGE-induced tube formation (p = 0.001). This effect was mediated through a GSK-3β-dependent inhibition of VEGF release. In conclusion, silymarin inhibits AGE-induced aberrant angiogenesis in a GSK-3β-mediated inhibition of VEGF release.

Figure 1

The effect of silymarin on the migratory potential of untreated HBEC-5i. Human brain endothelial cells were treated with a range of silymarin concentrations, and the migration rate was measured. Representative images of the migrated cells (a) and a quantification of the migratory potential (b). Silymarin did not alter the migration rate of endothelial cells. Data are presented as mean ± SEM; ∗ indicates a significant difference from control. n = 3.

Figure 2

Advanced glycation end products induce a proangiogenic response in endothelial cells. Endothelial cells were treated with 50 μg/mL of AGE, and the migration and tube formation rates of the cells were assessed. AGE increased the migration rate of endothelial cells as well as the rate of tube formation in a time-dependent manner. Representative images of the migrated cells (a) and a quantification of the migratory potential (b). Representative images of the tube formation potential of endothelial cells (c) and its quantification (d). ∗ indicates a significant difference from control. Data are presented as mean ± SEM. n = 3.

Figure 3

Silymarin inhibited diabetes-induced proangiogenic response in brain endothelial cells. Advanced glycation end products (50 μg/mL) increased the migration rate of hBMECs. Treatment with silymarin inhibited AGE-induced brain endothelial cell migration in a dose-dependent manner. Representative images of the migrated cells (a) and a quantification of the migratory potential (b). Similarly, silymarin inhibited AGE-induced angiogenic response in human brain endothelial cells in a dose-dependent manner. Representative images of the tube formation potential of endothelial cells (c) and its quantification (d). Data are presented as mean ± SEM, n = 3–6. ^∗Significantly different as compared to control; ^$significantly different as compared to AGE; ^#significantly different as compared to AGE + 50 μg/mL-treated cells.

Figure 4

Silymarin inhibits diabetes-induced proangiogenic response in a GSK-3β inhibition-dependent reduction of VEGF release. Treatment with silymarin or GSK-3β inhibition using 10 nM of SB-216763 reduced AGE-induced migration in HBEC-5i. Cotreatment with both GSK inhibitor and silymarin resulted in a comparable inhibition to what is achieved in either of them (a). Treatment with AGE induced a onefold increase in VEGF release. This increase was blunted with silymarin cotreatment. GSK inhibition blunted AGE-induced VEGF release and did not alter silymarin-induced VEGF release inhibition (b). n = 3. ^∗Significantly different as compared to control; ^$significantly different as compared to AGE.