# _Applied_
## 3 Results
### SBE Increases the Protein Levels of Nrf2, HO-1, SOD2, CAT, and GPx  
H\({}_{2}\)O\({}_{2}\) has been generally used to cause ROS release. In order to counteract intracellular ROS, the antioxidant enzyme defense system including SOD2, CAT, and GPx is activated [28]. To determine whether PC12 cells were protected from oxidative stress by SBE, we induced oxidative stress via treatment with H\({}_{2}\)O\({}_{2}\) in PC12 cells, and then added SBE to PC12 cells. To investigate the effect of SBE on the protein levels of an antioxidant-related factor (Nrf2) and enzymes (HO-1, SOD, CAT, and GPx), we performed western blotting. Nrf2 is a key transcription factor that regulates antioxidant-related gene expression. The activation of Nrf2 leads to an induction of antioxidant enzymes, such as HO-1, SOD, CAT, and GPx [28]. As shown in Figure 4A, SBE (50, 100, and 200 \(\upmu\)g/mL) increased the levels of Nrf2 (1.35-, 2.13-, and 3.87-fold, respectively, relative to H\({}_{2}\)O\({}_{2}\)-treated cells) in a concentration-dependent manner. The level of HO-1 in cells treated with SBE (50, 100, and 200 \(\upmu\)g/mL) increased in a concentration-dependent manner (1.14-, 1.19-, and 1.26-fold, respectively, relative to the H\({}_{2}\)O\({}_{2}\)-treated cells). SBE (50, 100, 200 \(\upmu\)g/mL) increased the protein levels of SOD2 (0.74-, 0.79-, and 1.42-fold, respectively, relative to H\({}_{2}\)O\({}_{2}\)-treated cells) and CAT (1.28-, 1.57-, and 1.68-fold, respectively, relative to H\({}_{2}\)O\({}_{2}\)-treated cells). The level of GPx in cells treated with SBE (50, 100, 200 \(\upmu\)g/mL) was increased 1.20-, 1.19-, and 1.44-fold, respectively, relative to the H\({}_{2}\)O\({}_{2}\)-treated cells. Figure 4: _Cont._ \begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Factors** & **DPPH 1** & **ABTS\({}^{+}\)1** & **Isoorientin** & **Caffeic Acid** & **ROS 2** \\ \hline
**DPPH 1** & 1.000 & & & & \\
**ABTS\({}^{+}\)1** & 0.933 ** & 1.000 & & & \\
**Isoorientin** & 0.995 *** & 0.959 ** & 1.000 & & \\
**Caffeic acid** & 0.889 * & 0.902 * & 0.893 * & 1.000 & \\
**ROS 2** & \(-\)0.932 ** & \(-\)0.887 * & \(-\)0.925 ** & \(-\)0.768 & 1.000 \\ \hline \hline \end{tabular} \({}^{1}\)DPPH and ABTS\({}^{+}\) radical scavenging activities were indicated as mg ascorbic acid (AA) equivalent per g. 2 ROS, reactive oxygen species generation in PC12 cells. Statistical significance was decided by Pearson’s correlation coefficient, * \(p<0.05\), ** \(p<0.01\), *** \(p<0.001\).  
\end{table} 
Table 3: Correlations among antioxidant activities, isoorientin content, caffeic acid content, and ROS generation in PC12 cells. Figure 4: **(A)** SBE (50, 100, and 200 \(\mathrm{\SIUnitSymbolMicro\mathrm{g}}\)/mL) increased the levels of antioxidant-related proteins in H\({}_{2}\)O\({}_{2}\)-treated PC12 cells. **(a-e)** Nrf2, HO-1, SOD2, CAT, and GPx levels determined by Western blotting with densitometry. **(B)** SBE (50, 100 and 200 \(\mathrm{\SIUnitSymbolMicro\mathrm{g}}\)/mL) increased the levels of antioxidant-related proteins in non-treated PC12 cells. **(a–e)** Nrf2, HO-1, SOD2, CAT, and GPx levels determined by Western blotting with densitometry. Values are presented as mean \(\pm\) SD from three experimental replicates. Significance was statistically defined with small letters at \(p\) < 0.05. Protein band quantification was conducted using ImageJ J software. All protein values were divided by the relative \(\beta\)-actin values in their respective lanes. To confirm whether SBE has an impact on the protein levels of an antioxidant-related factor (Nrf2) and enzymes (HO-1, SOD, CAT, and GPx) in normal conditions, PC12 cells without a H\({}_{2}\)O\({}_{2}\) stimulus were assessed using western blotting. As shown in Figure 4B, SBE (50, 100, and 200 \(\upmu\)g/mL) raised the expression levels of Nrf2 and HO-1. SBE (50, 100, and 200 \(\upmu\)g/mL) caused concentration-dependent increases in the levels of Nrf2 (1.20-, 1.24-, and 1.39-fold, respectively, relative to the non-treated cells).