## 4 Discussion
Thus, the anti-muscle-atrophy effect of the BuOH fraction is likely mediated by inhibiting FoxO3\(\alpha\) activation. These results suggest that the BuOH fraction of ISE can prevent the development of muscle atrophy. Although ISE significantly reduced LPS-induced MAFbx and MuRF1 mRNA and protein expression in C2C12 myotubes, the effect on FoxO3a expression was minimal. However, the BuOH fraction significantly attenuated the expression of MAFbx and MuRF1 through inhibiting the expression of FoxO3a. The reason for the different effects of ISE and BuOH fractions could be due to the higher TPC and total antioxidant capacity of the BuOH fraction compared to ISE. Antioxidants can potentially prevent skeletal muscle atrophy by inhibiting reactive oxygen species (ROS) production [12]. High cellular ROS levels increase the expression of proteins required for the ubiquitin-proteasome system [12]. Furthermore, studies have shown that strong antioxidant properties and high polyphenol content are closely related to inhibition of muscle atrophy-related ubiquitin ligases. Polyphenols, such as catechin and quercetin, have been shown to prevent clinorotation-induced expression of MAFbx and MuRF1 in C2C12 myotubes [23]. Rutin, one of the flavonoids, prevented dexamethasone-induced muscle loss in C2C12 myotubes and mouse models by controlling FOXOO3-dependent signaling [24]. Didrovaltrate, an active compound of _Valeriana fauriei_, has ROS scavenging activity and has inhibited dexamethasone-induced muscle atrophy in C2C12 myotubes and C57 BL/6 mice [25]. The higher TPC and antioxidant capacity of the BuOH fraction compared to ISE may contribute to the repressed expression of muscle-specific E3 ubiquitin ligases genes by the BuOH fraction in LPS-induced C2C12 myotubes. Thus, the study to find bioactive compounds of the BuOH fraction from ISE with potent muscle atrophy effects is warranted to test this possibility. Inflammation is an important factor that causes skeletal muscle atrophy [14]. Pro-inflammatory cytokines can trigger muscle atrophy mediated by the activating ubiquitin-proteasome and NF-\(\kappa\)B/MAPK signaling pathways [26; 27]. MAPKs including ERK, c-JunN-terminal kinase, and p38 regulate MAFbx and MuRF1 expression of muscles under oxidative stress and inflammatory conditions. In particular, elevated phosphorylation of p38 and ERK induces overexpression of MAFbx and MuRF1 in C2C12 myotubes [28]. Also, studies have demonstrated that systemic administration of LPS can stimulate the expression of MAFbx and MuRF1 through an increase of NF-kB activity and its downstream inflammatory mediator, such IL-6, IL-1\(\beta\), and COX2 [26; 29]. In the present study, we observed that ISE and the BuOH fraction significantly decreased the expression of pro-inflammatory cytokines and inflammation-inducible enzymes in LPS-induced C2C12 myotubes. Also, ISE and the BuOH fraction noticeably attenuated the LPS-stimulated increase in protein expression of phosphorylation of p38 and ERK in C2C12 myotubes. Thus, the decreased mRNA expression of IL-6, IL-1\(\beta\), iNOS, and COX2 by ISE and the BuOH fraction is mediated through inhibition of the p38/ERK MAPK signaling pathway. This can contribute to the suppressed expression of muscle atrophy-related ubiquitin ligases by ISE and the BuOH fraction in LPS-induced C2C12 myotubes. Also, since oxidants produced by infiltrating immune cells can directly cause muscle damage, the effect of ISE and the BuOH fraction on muscle atrophy is likely accompanied by the antioxidant and anti-inflammatory effects of ISE and BuOH fraction.