Pyropia yezoensis peptide PYP1‑5 protects against dexamethasone‑induced muscle atrophy through the downregulation of atrogin1/MAFbx and MuRF1 in mouse C2C12 myotubes

Abstract

Skeletal muscle atrophy refers to the decline in muscle mass and strength that occurs under various conditions, including aging, starvation, cancer and other cachectic diseases. Muscle atrophy caused by aging, known as sarcopenia, primarily occurs after 50 years of age. Muscle atrophy‑related genes, including atrogin1/muscle atrophy F‑box (MAFbx) and muscle RING finger 1 (MuRF1), are expressed early in the muscle atrophy process, and their expression precedes the loss of muscle mass. The present study investigated the potential anti‑atrophic effects of the Pyropia yezoensis peptide PYP1‑5. The MTS assay did not detect cytotoxic effects of PYP1‑5 on C2C12 mouse myoblast cells. Subsequently, the anti‑atrophic effects of PYP1‑5 on skeletal muscle cells was examined by treating C2C12 myotubes with 100 µM dexamethasone (DEX) and/or 500 ng/ml PYP1‑5 for 24 h. Compared with the control, myotube diameter was reduced in DEX‑treated cells, whereas PYP1‑5 treatment protected against DEX‑induced muscle atrophy. MAFbx and MuRF1 protein and mRNA expression levels were detected by western blot analysis and reverse transcription‑quantitative polymerase chain reaction, respectively. The results demonstrated that PYP1‑5 significantly reduced the expression of atrogin1/MAFbx and MuRF1. Therefore, data from the present study suggest that PYP1‑5 inhibits the expression of atrogin1/MAFbx and MuRF1 in C2C12 cells, and these characteristics may be of value in the development of anti‑atrophy functional foods.

Figure 1.

Purification of Pyropia yezoensis peptide with Shiseido Capcell Pak C18 column chromatography and mass spectrometry analysis identified a 1,622 Da compound from P. yezoensis, which was named PYP1-5.

Figure 2.

Differentiation of C2C12 myoblasts into myotubes. (A) Representative images of myoblasts prior to induction and on days 1, 2, 4, 5 and 6 following incubation with 2% fetal bovine serum. Magnification, ×100. (B) Myotube diameters and (C and D) myogenin protein expression levels were compared across the differentiation stages. Results are presented as the mean ± standard deviation of three independent experiments. *P<0.05 vs. corresponding control group (D-0). CON, control; D, day; UD, untreated differentiation, D-0.

Figure 3.

Effects on MuRF1 and MAFbx protein expression in C2C12 myoblasts treated with various concentrations of DEX. (A) Western blot and (B) densitometric analysis of MuRF1 and atrogin1/MAFbx protein expression levels following treatment with 1, 10, 50 or 100 µM DEX. Results are expressed as the mean ± standard deviation of three independent experiments. *P<0.05 vs. corresponding control group. (C) Cells were treated with 100 µM DEX and cell morphology was analyzed (original magnification, ×100). CON, control; DEX, dexamethasone; MAFbx, muscle atrophy F-box; MuRF1, muscle RING finger 1.

Figure 4.

Effects of PYP1-5 on C2C12 myotube viability. Cells (1.5×10⁴ cells/well) were seeded in 96-well plates in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Following 24 h incubation, cells were serum-starved for 4 h and treated with PYP1-5 at the indicated concentrations for 24 h. Results are presented as the mean ± standard deviation of three independent experiments. *P<0.05 vs. corresponding control group. PYP1-5, N-terminal 15 residues of Pyropia yezoensis peptide PYP1-5.

Figure 5.

Effects of PYP1-5 and/or DEX on differentiated C2C12 myotubes. (A) Representative images of C2C12 myotubes following the various DEX and PYP1-5 treatments. (B) Comparison of myotube diameters among the four groups. Results are presented as the mean ± standard deviation of three independent experiments. *P<0.05 vs. corresponding control group; ^#P<0.05 vs. corresponding DEX only treatment group. CON, control; DEX, dexamethasone; PYP1-5, N-terminal 15 residues of Pyropia yezoensis peptide PYP1-5.

Figure 6.

Inhibition of MuRF1 and atrogin1/MAFbx expression in C2C12 myotubes treated with DEX (100 µM) and/or PYP1-5 (500 ng/ml) for 24 h. MuRF1 an atrogin1/MAFbx Protein expression levels were analyzed by (A) western blot and (B) densitometric analysis; GAPDH was used to normalize expression. (C) mRNA expression levels were quantified by reverse transcription-quantitative polymerase chain reaction. Results are presented as the mean ± standard deviation of three independent experiments. *P<0.05 and **P<0.01 vs. corresponding control group; ^#P<0.05 and ^##P<0.01 vs. corresponding only DEX treatment group. CON, control; DEX, dexamethasone; MAFbx, muscle atrophy F-box; MuRF1, muscle RING finger 1; PYP1-5, N-terminal 15 residues of Pyropia yezoensis peptide PYP1-5.