## 4 Discussion  
Since the regulation of inflammation and oxidative stress is essential for muscle atrophy, seaweeds with potent anti-inflammatory and antioxidant activity are an attractive natural resource for the management of muscle atrophy [14, 16]. Studies have demonstrated IS exerts  
Figure 4: Effects of ISE and the BuOH fraction of ISE on the expression of pro-inflammatory cytokines and inflammation-inducible enzymes in LPS-treated C2C12 myotubes. C2C12 myotubes were treated with LPS (1 ug/mL) and the (**a**) ISE or (**b**) BuOH fraction of ISE (50, 75, and 100 ug/mL) for 24 h. The mRNA expression of IL-6, IL-1\(\beta\), iNOS, and COX2 was determined using RT-PCR. GAPDH was used as the internal controls. Bars with a different letter are significantly different from the control (\(p<0.05\)). Figure 5: Effects of ISE and BuOH on the protein expression of inflammation-inducible enzymes and phosphorylation of p38 and ERK in LPS-induced C2C12 myotubes. C2C12 myotubes were treated with LPS (1 ug/mL) and ISE, or the BuOH fraction of ISE (50, 75, and 100 ug/mL) for 24 h. (**a**) The protein expression of iNOS and COX2 was analyzed by Western blot analysis. \(\beta\)-Actin was employed as a loading control. (**b**) The protein expression of p-938 and t-938 was analyzed by Western blot analysis. Phosphorylation p38 was calculated as a ratio of p-p38/t-p38 and expressed relative to LPS. (**c**) The protein expression of p-ERK and t-ERK was analyzed by Western blot analysis. Phosphorylation ERK was calculated as a ratio of p-ERK/t-ERK and expressed relative to LPS. anti-inflammatory and antioxidant properties. ISE and its active compounds suppressed pro-inflammatory cytokines and nitric oxide production by inhibiting the MAPK/nuclear factor \(\kappa\)B (NF-\(\kappa\)B) signaling pathway in LPS-induced RWA 264.7 macrophages [7; 17]. Also, IS methanol extract showed high hydrogen peroxide and DPPH free radical scavenging activity [10; 18]. ISE has inhibitory effects on inflammatory cytokines and oxidative stress and therefore likely exert muscle atrophy effects in LPS-induced C2C12 myotubes. In this study, we found that ISE and the BuOH fraction exert anti-muscle atrophy effects in C2C12 myotubes by attenuating LPS-induced expression of muscle-specific E3 ubiquitin ligases and pro-inflammatory through inhibition of the p38/ERK MAPK signaling pathway, which was accompanied by high TPC and total antioxidant activity. Skeletal muscle atrophy depends on the relative balance between protein degradation and synthesis [19]. When muscle atrophy occurs, protein breakdown exceeds protein synthesis. Protein synthesis is mainly regulated through activation of the mTOR pathway. The mTOR signaling pathway is mediated by mTORC1 (mTOR complex 1), which regulates protein synthesis, and mTOR complex 2 (mTORC2), which regulates cell survival and metabolism. mTORC1 is activated through the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB/Akt) pathway to increase protein synthesis by phosphorylating the downstream targets, 70 kDa ribosomal protein S6 kinase 1 (70S6K1) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). On the other hand, the primary regulatory mechanism of protein degradation in skeletal muscle is the ubiquitin-proteasome pathway [19]. Muscle-specific E3 ubiquitin ligases, such as MAFbx and MuRF1, are instrumental in the processes underlying protein degradation and are highly stimulated by the transcription factor FoxO3\(\alpha\)[20; 21]. In skeletal muscle cells, muscle-specific E3 ubiquitin ligases are activated by dephosphorylation of FoxO3\(\alpha\), which influences its transcriptional activity [22]. In this study, the BuOH fraction attenuated the LPS-induced mRNA expression of MAFbx and MuRF1 in C2C12 myotubes by significantly suppressing the transcription factor FoxO3a. In addition, the BuOH fraction increased myotube diameter against LPS-induced myotube atrophy, which might be associated with the suppression of MAFbx and MuRF1 expression by the BuOH fraction.