## 3 Results
### Effect of LE on ROS Generation and DNA Damage  
The mechanism underlying the protective effect of LE against H\({}_{2}\)O\({}_{2}\)-induced cytotoxicity was evaluated. Levels of ROS were increased by 41.68% within 1 h of H\({}_{2}\)O\({}_{2}\) treatment (Figure 2a) but were also 13.49% lower in cells pretreated with 10 \(\mathrm{\SIUnitSymbolMicro g}\)/mL LE. Figure 1: Effect of LE on H\({}_{2}\)O\({}_{2}\)-induced cytotoxicity in C2C12 cells. Cells were treated with various concentrations of LE (**a**) or H\({}_{2}\)O\({}_{2}\) (**b**) for 24 h or pre-treated, with or without the indicated diverse concentrations of LE for 1 h prior to exposure to 0.8 mM H\({}_{2}\)O\({}_{2}\) for 24 h (**c**,**d**); (**a**–**c**) Cell viability was assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Data indicate mean \(\pm\) SD values (\(n=3\); * \(p<0.05\), ** \(p<0.01\), and *** \(p<0.001\) vs. control cells; ** \(p<0.01\) and *** \(p<0.001\) vs. H\({}_{2}\)O\({}_{2}\)-treated cells); (**d**) Effect of LE on morphological changes in H\({}_{2}\)O\({}_{2}\)-treated cells. Representative results from three independent experiments are shown. Next, the effect of LE on H\({}_{2}\)O\({}_{2}\)-mediated DNA damage was evaluated. Figure 2b shows the results of the comet assay conducted to assess the protective effect of LE against H\({}_{2}\)O\({}_{2}\) induced DNA damage. In addition, the extents of DNA damage were manually measured and are shown in Figure 2c. Figure 2: Effect of LE on H\({}_{2}\)O\({}_{2}\)-induced ROS generation and DNA damage in C2C12 cells. Cells were pretreated with LE for 1 h and treated, with or without 0.8 mM H\({}_{2}\)O\({}_{2}\), for 1 h (**a**) or 24 h (**b**), (**c**), and (**d**). (**a**) Intracellular ROS levels were measured using 2′,7′-dichlorofluorescein diacetate (DCF-DA) staining and flow cytometry. Representative results of three independent experiments are shown; (**b**) DNA damage was evaluated using a 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); (**d**) H2 AX and \(\gamma\)-H2 AX levels analysis of cell lysates. 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.  
was evaluated by flow cytometry with Annexin V-FITC and PI double staining, DAPI staining, agarose gel electrophoresis analysis, and Western blot analysis. Annexin V-FITC and PI double staining indicated that H\({}_{2}\)O\({}_{2}\) exposure increased apoptosis to 40.6%, whereas 10 ug/mL LE pretreatment reduced it to 9.41% (Figure 3a). Control cells had perfect nuclei, whereas H\({}_{2}\)O\({}_{2}\)-treated cells exhibited noticeable chromatin condensation (Figure 3b). However, such morphological changes were significantly reduced by 10 ug/mL LE pretreatment, and the concentration dependence of this effect was confirmed by agarose gel electrophoresis (Figure 3c). The mechanism underlying the anti-apoptotic effect of LE was then investigated by evaluating the effect of LE on H\({}_{2}\)O\({}_{2}\)-induced changes in the expression of apoptosis regulatory proteins. The expression levels of Bcl-2 and pro-caspase-3 were reduced by H\({}_{2}\)O\({}_{2}\) treatment, whereas the level of cleaved PARP increased, and both changes were ameliorated by LE pretreatment, in a concentration-dependent manner (Figure 3d). However, Bax expression was not significantly affected by either H\({}_{2}\)O\({}_{2}\) or LE pretreatment, which suggested that LE inhibited H\({}_{2}\)O\({}_{2}\)-induced apoptosis in C2C12 cells. Figure 3: Effect of LE on H\({}_{2}\)O\({}_{2}\)-induced apoptosis in C2C12 cells.