## 3 Results
### Effect of LE on Nrf/HO-1 Signaling  
Because Nrf has been reported to play an important role in protection against oxidative stress through the ARE-mediated expression of phase II antioxidant enzymes [15], the role of the Nrf/HO-1 pathway in the observed LE-induced antioxidant and cytoprotective  
Figure 6: AMPK-dependent effect of LE on H\({}_{2}\)O\({}_{2}\)-induced ROS generation and DNA damage in C2C12 cells. Cells were pretreated using 5 \(\upmu\)M compound C, with or without 10 \(\upmu\)g/mL LE, for 1 h, followed by 0.8 mM H\({}_{2}\)O\({}_{2}\) for 1 h (**a**) or 24 h (**b–d**). (**a**) After staining with DCF-DA, DCF fluorescence was measured using flow cytometry. Representative results of three independent experiments are shown; (**b**) DNA damage was measured using the comet assay. DNA damage is observed in the comet’s tail, while intact DNA remains in the comet’s head. Representative results of three independent experiments are shown; (**c**) DNA damage score. Data indicate mean \(\pm\) SD values (\(n\) = 3; *** \(p\) < 0.001 vs. control cells; *** \(p\) < 0.001 vs. H\({}_{2}\)O\({}_{2}\)-treated cells; \({}^{588}\)\(p\) < 0.001 vs. H\({}_{2}\)O\({}_{2}\) and LE treated cells); (**d**) Cellular proteins were prepared and H2 AX and \(\gamma\)-H2 AX protein levels were analyzed. Western blot analysis was performed using \(\beta\)-actin as a loading control. Representative results of three independent experiments are shown. H2 AX, H2A histone family member X.  
activities was investigated. LE treatment significantly enhanced the expression of HO-1, a target protein downstream of Nrf2, p-Nrf2 (Ser40), but not Nrf2, in a concentration-dependent manner, and decreased the expression of Keap1 [16], a negative regulator of Nrf2, in a concentration-dependent manner (Figure 7a). The expression level of Nrf2 was slightly elevated by H\({}_{2}\)O\({}_{2}\) treatment but reduced when pretreated with LE (Figure 7b), whereas that of p-Nrf2 (Ser40) was unaffected by H\({}_{2}\)O\({}_{2}\) treatment but increased when either treated with LE alone or pre-treated with LE. In contrast, Keap1 expression level was reduced by LE treatment and increased by H\({}_{2}\)O\({}_{2}\) treatment, and the H\({}_{2}\)O\({}_{2}\)-mediated increase was significantly reduced by LE treatment. Meanwhile, HO-1 expression level was elevated by LE treatment, reduced by H\({}_{2}\)O\({}_{2}\) treatment, and elevated in cells pretreated with LE. Next, the effect of AMPK on the Nrf2/HO-1 signaling pathway in the protective effect of LE against H\({}_{2}\)O\({}_{2}\)-mediated cytotoxicity in C2C12 cells was investigated using the AMPK inhibitor compound C. The expression level of Nrf2 in C2C12 cells was slightly increased by H\({}_{2}\)O\({}_{2}\) treatment but reduced with LE pretreatment and when treated with compound C (Figure 7c). The expression levels of p-Nrf2 (Ser40) and HO-1 were increased by H\({}_{2}\)O\({}_{2}\) with LE pretreatment but reduced upon treatment with compound C. In addition, Keap1  
Figure 7: Effect of LE on Nrf2/HO-1 signaling in C2C12 cells: (**a**) Effect of LE on the Nrf2, p-Nrf2 (Ser40), Keap1, and HO-1 levels of total cell lysates. Western blot analysis was performed using \(\beta\)-actin as a loading control. Representative results of three independent experiments are shown; (**b**) Cells were pretreated with LE for 1 h and then stimulated in the presence or absence of 1 mM H\({}_{2}\)O\({}_{2}\) for 24 h. Representative results of three independent experiments are shown. Cellular proteins were prepared and Nrf2, p-Nrf2 (Ser40), Keap1, and HO-1 protein levels analyzed. Western blot analysis was performed using \(\beta\)-actin as a loading control. Representative results from three independent experiments are shown; (**c**) Cells were pretreated with 5 uM compound C, with or without 10 ug/mL LE, for 1 h and then treated with 0.8 mM H\({}_{2}\)O\({}_{2}\) for 24 h. Whole-cell lysates were prepared and Nrf2, p-Nrf2 (Ser40), Keap1, and HO-1 expression profiles were confirmed.