## 2 Results
### Effect on Antioxidant Enzymes in HG-Stimulated \(\beta\)-Cells  
HG is known to decrease the activity of antioxidant enzymes in diabetic patients [28], we examined in this study whether SOE could maintain the activity of antioxidant enzymes. Figure 3A,C indicate the protective effects of SOE on the activity of intracellular antioxidant enzymes, including catalase, GPx and SOD, in HG-stimulated \(\beta\)-cells. HG treatment alone dramatically decreased the activities of catalase, GPx, and SOD. The higher the glucose concentration, the lower the activity of these enzymes. At 50 and 100 mM glucose, the catalase activity decreased to 70.6% and 48.4%, respectively; the GPx activity decreased to 72.6% and 59.8%, respectively; the SOD activity decreased to 68.6% and 57.2%, respectively. However, when SOE was also included in the culture, as shown in the results in Figure 3A-C, SOE treatment increased the activity of these three enzymes in the HG-stimulated \(\beta\)-cells, and the level of increase was in relation to the increase of SOE concentration. When SOE concentration was at 250 \(\upmu\)g/mL, the activity of these enzymes was restored to near the normal level.  
Figure 2: Effect of SOE on the formation of ROS in RIN-m5f \(\beta\)-cells under HG-induced conditions. (**A**) Changes in the intracellular ROS levels indicated by the DCFH\({}_{2}\)-DA fluorescence intensity. (**B**) Fluorescent quantitation. The \(\beta\)-cells were cultivated in 96-well plates at a density of 1 \(\times\) 10\({}^{5}\) cells/well for 24 h, and the indicated concentration of SOE was added under the stimulation of 50 mM or 100 mM glucose. After 24 h treatment, the cells were washed with PBS and incubated with 5 \(\upmu\)M DCF-DA for another 30 min at room temperature. Fluorescence of this compound was measured by a microplate reader. The experimental data were calculated from the results of five repeated experiments. The statistically significant differences compared with the vehicle (group 0) are expressed as ** \(p\) < 0.01, and *** \(p\) < 0.001. The positive control was 50 \(\upmu\)M Metformin.  
Reduced glutathione (GSH) is an antioxidant present in various types of cells. As shown in Figure 3D, glucose concentration at 50 or 100 mM significantly reduced the intracellular GSH content to 62.6% and 54.4%, respectively. The SOE treatment significantly increased the GSH content concentration-dependently.  
To understand whether the increase in the activity of these three antioxidant enzymes was due to the activation of enzyme or to the promotion of enzyme protein production by SOE, we determined the protein expression of these three enzymes in the RIN-m5f cells using Western blotting. Results in Figure 4 indicate that the protein expression of catalase, GPx, and SOD decreased to 68%, 60%, and 38%, respectively, in cells stimulated with 100 mM glucose. However, when SOE was included in the culture, the protein expression of these three enzymes increased significantly, and the increase was in a concentration-dependent manner. Thus, the increase in the activity of antioxidant enzymes (shown in Figure 3) should be due to the promotion of the formation of these enzymes stimulated by SOE.