2.3.2. NO and VEGF Release and Protein Expression of DGS

Compared with the control group, the NO level was increased in the high concentration group of DGS (16 µg/mL) (*p* < 0.05), but there was no difference in NO secretion between 4 µg/mL DGS and 8 µg/mL DGS groups (*p* > 0.05). In ELISA, compared with control, the secretion of 8 µg/mL DGS and 16 µg/mL VEGFA treatment groups compared to the control group (*p* < 0.05, *p* < 0.05) (Figure 4A,B).

**Figure 4.** Regulation of NO, VEGF, and related proteins by DGS: (**A**) Effect of DGS on NO levels. (**B**) Effect of DGS on VEGFA levels. (**C**) Western blot results. (**D**–**G**) are the results of statistical analysis of VEGFR2/GAPDH, p-Akt/Akt, p-Erk1/2/Erk1/2, and p-eNOS/eNOS, respectively. Data are presented as the mean ± SEM from at least three independent experiments. \* *p* < 0.05 vs. Control, \*\* *p* < 0.01 vs. Control.

The molecular mechanisms associated with the promotion of angiogenesis by DGS were elucidated. Further experiments to explore the pathways by which DGS enhanced the proliferation and migration of HUVECs. As shown in Figure 4C,D, DGS significantly

upregulated VEGFR2, thereby activating the phosphorylation of proteins related to downstream signaling pathways. As illustrated in Figure 4C,E, the level of Akt phosphorylation was significantly elevated after DGS treatment, and the value of p-Akt/Akt was statistically different compared with that of the control group (*p* < 0.01). Furthermore, Erk phosphorylation level was found to be significantly elevated and statistically different after DGS treatment (*p* < 0.05, *p* < 0.01) (Figure 4C,F). Finally, vasodilation-related proteins were examined, which revealed that the phosphorylation levels of eNOS were also significantly elevated, with statistical differences (*p* < 0.01, *p* < 0.01) (Figure 4C,G). The increase in phosphorylation of VEGFR2 and eNOS was consistent with the increased secretion of VEGFA and NO in the cell cultures described above. These results suggest that DGS activates the VEGFA/VEGFR2/Akt/Erk/eNOS signaling pathway to promote angiogenesis and vasodilation.
