2.3.1. Effect of DGS on the Proliferation and Migration of HUVECs

The CCK-8 assay was performed to evaluate the cell viability after the treatment with DGS. As illustrated in Figure 3A, DGS significantly increased the viability of HUVECs. Six concentration groups of DGS were used to treat the HUVECs for 24 h. The mean cell survival rate of the 1 µg/mL DGS group was 116%, which did not show a proliferative effect compared with the control (Ctrl) group (100%). After treatment with DGS, the cell survival rates were 129.0% (*p* < 0.01), 127.4% (*p* < 0.01), 126.3% (*p* < 0.01), 128.0% (*p* < 0.01), and 124.3% (*p* < 0.01) in the five concentration groups of 2, 4, 8, 16, and 32, respectively, in comparison with the control group. These results indicate that DGS significantly promotes the proliferation of HUVECs.

Endothelial cell migration is essential for angiogenesis. Therefore, the effect of DGS on the migration of HUVECs was evaluated using wound-healing assays. As shown in Figure 3A,B, VEGF significantly increased the migration of endothelial cells to the wound healing zone after injury (*p* < 0.0001). Treatment of HUVECs with DGS revealed that DGS significantly promoted the migration of endothelial cells toward the middle of the scratch and promoted scratch healing.

**Figure 3.** Effect of DGS on HUVECs cells in vitro: (**A**) An CCK-8 assay was carried out to measure HUVECs viability. (**B**) Effect of different concentrations of DGS on the migration of HUVECs cells. Results are presented as the mean ± SEM. (**C**) The healing area of the wound at 0 and 24 h were photographed by microscopy. The red dashed box represents the area counted after migration. Scale bar: 100 µm. (**D**) The migration of HUVECs in Transwell migration assays. Scale bar: 100 µm. (**E**) DGS promoted tube formation of HUVECs. Scale bar: 100 µm. (**F**) Quantification of the number of migrated cells. (**G**) Quantitative analysis of branch points for tube formation assays. (**H**) Quantitative analysis of capillary length for tube formation assays. Values are expressed as the mean ± SEM. ns *p* < 0.05 vs. Control, \* *p* < 0.05 vs. Control, \*\* *p* < 0.01 vs. Control, \*\*\* *p* < 0.001 vs. Control, \*\*\*\* *p* < 0.0001 vs. Control.

The formation of blood vessels rests on the proliferation and migration of endothelial cells. Therefore, in addition to the above wound healing assay that examined the lateral migration of the endothelial cells, the transwell assay that investigated the longitudinal migration of the cells after the treatment with DGS was performed. As presented in Figure 3D,F, the migration of HUVECs was significantly increased compared with the control after treatment with VEGF-positive drugs. Moreover, DGS significantly increased the ability of HUVECs to migrate toward the backside of the membrane. Compared with the Control group (0.6568 HPF), the mean values of the number of cells migrating to the back of the membrane with 4, 8, and 16 µg/mL of DGS were 1.198 (*p* > 0.05), 1.763 (*p* < 0.01), and 1.946 (*p* < 0.001) HPF, respectively.

Angiogenesis was evaluated in terms of endothelial cell migration. Matrix gel was used to evaluate the ability of DGS to promote tube formation in HUVECs. Furthermore, the ability to promote tube formation was assessed based on the capillary length and the number of branching points. HUVECs treated with VEGF resulted in a mean number of branch points and a mean capillary length of 52.80 (*p* < 0.01) and 8144 µm (*p* < 0.05), respectively, compared with the control group; the mean value of the number of branch points for the three concentrations after the treatment of HUVECs with DGS control were 49.91, 65.75, and 50.64, respectively, and the lumen lengths were 8417, 10,568, and 8098, respectively (Figure 3E,G,H).
