Pyropia yezoensis protein protects against TNF‑α‑induced myotube atrophy in C2C12 myotubes via the NF‑κB signaling pathway

Abstract

The protein extracted from red algae Pyropia yezoensis has various biological activities, including anti‑inflammatory, anticancer, antioxidant, and antiobesity properties. However, the effects of P. yezoensis protein (PYCP) on tumor necrosis factor‑α (TNF‑α)‑induced muscle atrophy are unknown. Therefore, the present study investigated the protective effects and related mechanisms of PYCP against TNF‑α‑induced myotube atrophy in C2C12 myotubes. Treatment with TNF‑α (20 ng/ml) for 48 h significantly reduced myotube viability and diameter and increased intracellular reactive oxygen species levels; these effects were significantly reversed in a dose‑dependent manner following treatment with 25‑100 µg/ml PYCP. PYCP inhibited the expression of TNF receptor‑1 in TNF‑α‑induced myotubes. In addition, PYCP markedly downregulated the nuclear translocation of nuclear factor‑κB (NF‑κB) by inhibiting the phosphorylation of inhibitor of κB. Furthermore, PYCP treatment suppressed 20S proteasome activity, IL‑6 production, and the expression of the E3 ubiquitin ligases, atrogin‑1/muscle atrophy F‑box and muscle RING‑finger protein‑1. Finally, PYCP treatment increased the protein expression levels of myoblast determination protein 1 and myogenin in TNF‑α‑induced myotubes. The present findings indicate that PYCP may protect against TNF‑α‑induced myotube atrophy by inhibiting the proinflammatory NF‑κB pathway.

Keywords: Pyropia yezoensis protein; aging; myotube atrophy; sarcopenia; tumor necrosis factor‑α.

Figure 1.

Effects of TNF-α and PYCP on the cytotoxicity of C2C12 myotubes. (A) The cell viability of C2C12 myotubes in the presence of 0, 12.5, 25, 50, and 100 µg/ml PYCP for 24 h. (B) The cell viability of C2C12 myotubes treated with 25, 50, and 100 µg/ml PYCP for 24 h and cotreated with 20 ng/ml TNF-α for 24 h.*P<0.05 vs. control untreated cells; ^#P<0.05 vs. TNF-α-only treated cells. PYCP, Pyropia yezoensis protein; TNF-α, tumor necrosis factor-α.

Figure 2.

Effects of PYCP on myotube diameter in TNF-α-treated C2C12 myotubes. Representative images and quantification of myotube diameters are shown for C2C12 myotubes treated with 20 ng/ml TNF-α and PYCP (25, 50, or 100 µg/ml) for 48 h. Images were captured at ×20 magnification (scale bar, 50 µm). Data are presented as the mean ± standard deviation of three independent experiments. *P<0.05 vs. untreated cells; ^#P<0.05 vs. TNF-α-only treated cells. PYCP, Pyropia yezoensis protein; TNF-α, tumor necrosis factor-α.

Figure 3.

Effects of PYCP on the production of intracellular ROS in TNF-α-treated C2C12 myotubes. C2C12 myotubes were treated with 20 ng/ml TNF-α and PYCP (25, 50, or 100 µg/ml) for 48 h. Intracellular ROS production was measured using DCF-DA and fluorescence intensity analysis. Data are presented as the mean ± standard deviation of three independent experiments. *P<0.05 vs. untreated cells; ^#P<0.05 vs. TNF-α-only treated cells. PYCP, Pyropia yezoensis protein; ROS, reactive oxygen species; TNF-α, tumor necrosis factor-α.

Figure 4.

Effects of PYCP on the expression of TNF-R1 in TNF-α-treated C2C12 myotubes. TNF-R1 protein expression levels were detected by western blot analysis. GAPDH was used as an internal standard. Data are presented as the mean ± standard deviation of three independent experiments. *P<0.05 vs. untreated cells; ^#P<0.05 vs. TNF-α-only treated cells. PYCP, Pyropia yezoensis protein; TNF-R1, TNF receptor-1; TNF-α, tumor necrosis factor-α.

Figure 5.

Effects of PYCP on the activation and translocation of NF-κB/p65 in TNF-α-treated C2C12 myotubes. (A) p-IκBα, IκBα, and NF-κB/p65 protein expression levels of cytosolic fractions were detected by western blot analysis. (B) NF-κB/p65 levels were measured in the nuclear fractions by western blot analysis. β-actin and lamin B1 were used as internal controls for the cytosolic and nuclear fractions, respectively. Data are presented as the mean ± standard deviation of three independent experiments. *P<0.05 vs. untreated cells; ^#P<0.05 vs. TNF-α-only treated cells. PYCP, Pyropia yezoensis protein; NF-κB, nuclear factor-κB; TNF-α, tumor necrosis factor-α; p-, phosphorylated; IκBα, inhibitor of κB α.

Figure 6.

Effects of PYCP on the ubiquitin-proteasome system in TNF-α-treated C2C12 myotubes. (A) The atrogin-1/MAFbx and MuRF1 protein expression levels were detected by western blot analysis. GAPDH was used as an internal standard. (B) 20S proteasome activity was assessed by detecting AMC in cell lysates after cleavage from the AMC-tagged peptide LLVY. Data are presented as the mean ± standard deviation of three independent experiments. *P<0.05 vs. untreated cells; ^#P<0.05 vs. TNF-α-only treated cells. PYCP, Pyropia yezoensis protein; TNF-α, tumor necrosis factor-α; MAFbx, muscle atrophy F-box; MuRF1, muscle RING-finger protein-1; AMC, 7-amino-4-methylcoumarin.

Figure 7.

Effects of PYCP on the production of IL-6 in TNF-α-treated C2C12 myotubes. C2C12 myotubes were treated with 20 ng/ml TNF-α and PYCP (25, 50, or 100 µg/ml) for 48 h. IL-6 in the culture media was measured using ELISA. Data are presented as the mean ± standard deviation of three independent experiments. *P<0.05 vs. untreated cells; ^#P<0.05 vs. TNF-α-only treated cells. PYCP, Pyropia yezoensis protein; IL-6, interleukin-6; TNF-α, tumor necrosis factor-α.

Figure 8.

Effects of PYCP on the expression of MyoD and myogenin in TNF-α-treated C2C12 myotubes. MyoD and myogenin protein expression levels were detected by western blot analysis. GAPDH was used as an internal standard. Data are presented as the mean ± standard deviation of three independent experiments. *P<0.05 vs. untreated cells; ^#P<0.05 vs. TNF-α-only treated cells. PYCP, Pyropia yezoensis protein; MyoD, myoblast determination protein 1; TNF-α, tumor necrosis factor-α.