## 4 Discussion
Most C2C12 cells exposed to H2O2 undergo apoptosis accompanied by mitochondrial damage, and it is well known that apoptotic proteins, such as cytochrome \(c\), are released into the cytoplasm during this process [26; 27]. Cytoplasmic release of these proteins initiate the intrinsic apoptotic pathway and is controlled by members of the Bcl-2 family, which is composed of anti-apoptotic and pro-apoptotic proteins [28; 29]. Bcl-2 proteins control the permeability of the mitochondrial outer membrane, thereby regulating the release of cytochrome \(c\) into the cytoplasm [29; 30]. Therefore, to assess the inhibitory effect of LE on mitochondrial dysfunction, MMP and cytochrome \(c\) levels were evaluated. The results show that the loss of MMP and cytoplasmic cytochrome \(c\) level were significantly increased in H2O2-treated cells (Figure 4). However, LE pretreatment inhibited MMP reduction and increased mitochondrial cytochrome \(c\) levels, which indicated that LE can prevent oxidative stress-induced mitochondrial damage. Moreover, the reduction of anti-apoptotic protein Bcl-2 level by H2O2 was reversed by LE pretreatment, likely by blocking caspase-3 activity, thereby inhibiting the degradation of substrate proteins, like PARP (Figure 3d). However, the inhibitory effect of LE on H2O2-mediated mitochondrial dysfunction was abrogated by compound C (Figure 9). According to previous studies, antioxidant and anti-inflammatory effects of LE have been reported [31; 32]. In addition, several studies have reported that muscle cell differentiation including C2C12 cells is induced through antioxidant activity [14; 33; 34]. Therefore, we additionally studied the effect of LE on C2C12 cell differentiation along with the study of the mechanism, which is a major topic of apoptosis. The process of skeletal muscle creation (i.e., myogenesis [35]) is a basic physiological process related to muscle growth and regeneration [36]. MRF factors, such as MyoD, myogenic factor 5 (Myf5), myogenin, and myogenic regulatory factor 4 (MRF4) regulate the myogenic differentiation of the muscle lineage at multiple points and, thus, regulate the determination and differentiation of skeletal muscle cells during embryogenesis and postnatal myogenesis [37]. The findings of the present study indicate that LE promotes cell elongation and that the diameter of the resulting myotubes increased in a concentration-dependent manner, as did MyoD and myogenin expression levels (Figure 10). As myogenesis progresses, MRF family gene expression levels are greatly increased [38]. High levels of Myf5 expression result in the formation of primitive muscle structures, including committed muscle cells [39], and subsequent MyoD gene expression is initiated in the dermomyotome [37; 40]. Two of these elements are muscle-determining genes and key factors in the activation of myogenic programs in both muscle and non-muscle cells [41]. Unlike MyoD and Myf5, myogenic is the main regulator of muscle differentiation initiation, as demonstrated by the lower number of skeletal muscle fibers in myogenin-deficient mice [42]. The role of MRF4 is more complex during myogenesis. In mice, MRF4 is closely linked to the Myf5 gene, initiated in myotomes after Myf5 expression, and upregulated at the differentiated myocyte stage. LE has been reported to contain unsolic acid (UA) as its main active ingredient [43; 44]. UA was found to reduce muscle atrophy and stimulate muscle hypertrophy in vivo study [45]. Moreover, another previous study revealed that the LE supplements inhibited dexamethasone-induced reduction of muscle strength in Sprague Dawley rats [46]. In addition, a randomized, double-blind, placebo-controlled trial of 54 healthy adults investigated the effect of LE on human strength and muscle, and found that female subjects in the LE group had significantly better right-hand grip strength than subjects in the control group [47]. Accordingly, those results suggest the possibility of using LE as a therapeutic agent for the prevention of skeletal muscle atrophy and sarcopenia.