## 4 Discussion  
ROS can oxidize cellular components, thereby threatening cellular integrity. Some defense mechanisms protect cells from oxidative stress, and such mechanisms involve the clearance of specific proteins via the ubiquitin-proteasome system, upregulation of antioxidant production, and clearance of damaged organelles [17]. These defense systems are activated under excessive ROS conditions, leading to cell death via diverse signaling pathways and cascades [18]. Many previous studies have reported that natural compounds exhibit protective effects against oxidative stress. In addition, the inhibition of ROS production and accumulation by antioxidants may be effective in preventing oxidative DNA damage and cell death [19; 20]. In the present study, the protective effect of LE against H2O2-induced oxidative stress and its mechanism was investigated in C2C12 cells. The results of this investigation indicate that LE can significantly reduce H2O2-induced DNA damage and apoptosis associated with the inhibition of ROS production (Figures 2 and 3). The stress sensor AMPK can prevent ROS accumulation and alleviate oxidative stress [21] and also inhibits nicotinamide adenine dinucleotide phosphate consumption by phosphorylating ACC, which is a key downstream target. Therefore, AMPK is considered  
Figure 10: Effect of loquat leaf extract (LE) on C2C12 myoblast differentiation. The pre-differentiation step involved incubation for 2 days in differentiation medium that included 2% horse serum. Then, LE was added to the differentiation medium to induce myoblasts differentiation for 3 days: (**a**) Representative images of C2C12 cells. Cells were cultured for 3 days in differentiation medium, with or without LE. Representative results of three independent experiments are shown; (**b**) Effect of LE on myotube diameter after 3 days of differentiation. Data indicate mean \(\pm\) SD values (\(n\) = 3; \"\" \(p\) \(<\) 0.001 vs. control cells; \(p\) \(<\) 0.05 and \({}^{\#}\)\(p\) \(<\) 0.01 vs. differentiated control cells); (**c**) Expression levels of MyoD and myogenin. Western blot analysis was performed using \(\beta\)-actin as a loading control. Representative results of three independent experiments are shown. MyoD, myogenic differentiation 1.  
a cell survival factor under conditions of oxidative stress [22; 23]. Remarkably, the AMPK pathway shares noticeable crosstalk with antioxidant responses of Nrf [24]. Under normal physiological conditions, Nrf joins Keap1 in the cytoplasm, is ubiquitinated by the Cullin-3-Keap1 E3 ubiquitin ligase complex and is degraded through the ubiquitin proteasome pathway. When cells are exposed to oxidative stress, Nrf is released from Keap1 and migrates to the nucleus, where it promotes the transcriptional activation of ARE-responsive genes [25]. In the present study, LE activated AMPK and initiated AMPK under H2O2 stimulation conditions. However, the LE-mediated reversal of H2O2-induced cytotoxicity was eliminated by pretreatment with compound C, an AMPK inhibitor, which suggests that the activation of AMPK is involved in the protective effect of LE against oxidative stress-mediated cytotoxicity (Figure 5). As shown in Figure 7, LE treatment upregulated p-Nrf2 (Ser40) and HO-1 and downregulated Keap1, in a concentration-dependent manner. In addition, the expression levels of p-Nrf2 (Ser40) and HO-1 remarkably increased and the expression level of Keap1 noticeably diminished in the LE pretreatment group. However, these effects were completely abrogated by the AMPK inhibitor compound C, which suggested that LE activates the Nrf/HO-1 signaling pathway and is affected by AMPK. In addition, the inhibitory effects of LE on H2O2-mediated ROS induction, DNA damage, and apoptosis were completely abolished by compound C. Together, these results suggest that LE protects against oxidative stress-induced cellular damage via AMPK activation (Figures 6 and 8).