Linoleic Acid Attenuates Denervation-Induced Skeletal Muscle Atrophy in Mice through Regulation of Reactive Oxygen Species-Dependent Signaling

Abstract

Muscle atrophy is a major muscle disease, the symptoms of which include decreased muscle volume leading to insufficient muscular support during exercise. One cause of muscle atrophy is the induction of oxidative stress by reactive oxygen species (ROS). This study aimed to identify the antioxidant mechanism of linoleic acid (LA) in muscle atrophy caused by oxidative stress. H₂O₂ has been used to induce oxidative stress in myoblasts in vitro. C2C12 myoblasts treated with H₂O₂ exhibited decreased viability and increased ROS synthesis. However, with LA treatment, the cells tended to recover from oxidative effects similar to those of the control groups. At the molecular level, the expression of superoxide dismutase 1 (SOD1), Bax, heat shock protein 70 (HSP70), and phosphorylated forkhead box protein O1 was increased by oxidative stress, causing apoptosis. LA treatment suppressed these changes. In addition, the expression of MuRF1 and Atrogin-1/MAFbx mRNA increased under oxidative stress but not in the LA-treated group. Sciatic denervation of C57BL/6 mice manifested as atrophy of the skeletal muscle in micro-computed tomography (micro-CT). The protein expression levels of SOD1, HSP70, and MuRF1 did not differ between the atrophied muscle tissues and C2C12 myoblasts under oxidative stress. With LA treatment, muscle atrophy recovered and protein expression was restored to levels similar to those in the control. Therefore, this study suggests that LA may be a candidate substance for preventing muscle atrophy.

Keywords: antioxidant; linoleic acid; muscle atrophy; oxidative stress; sciatic denervation.

Figure 1

Linoleic acid prevents oxidative stress-induced cell death in C2C12 myoblasts. C2C12 myoblasts were seeded, and the medium was then replaced with serum-free DMEM containing LA (0–200 mM) for 24 h. After pre-incubation, C2C12 myoblasts were treated with 1 mM H₂O₂ for 2 h. The cell viability was assessed by using the EZ-Cytox cell viability kit. Absorbance at 450 nm was measured using a microplate reader. (A) Effect of LA on H₂O₂-induced cell viability in C2C12 myoblasts. The cell viability was calculated according to the following equation: cell viability (%) = [(absorbance of the H₂O₂-treated sample/absorbance of the H₂O₂-untreated control) × 100]. Each value indicates the mean ± SD (n = 5). (B) Effect of LA on apoptosis by oxidative stress in C2C12 myoblasts. C2C12 myoblasts were fixed with 4% para-formaldehyde in PBS for 30 min and mounted using a mounting medium with DAPI. Imaging data were acquired on a confocal microscope. DAPI staining was detected at wavelengths of 358 and 461 nm. (C) Quantitating apoptotic cells. Apoptotic cells were measured using the Image J software. Apoptosis was calculated according to the following equation: apoptosis (%) = [(number of apoptotic cells/number of total cells) × 100]. Each value indicates the mean ± SD (n = 4). * p < 0.05 and *** p < 0.001. vs. H₂O₂ alone. The effect of LA was compared with that of 2 mM N-acetyl-l-cysteine (NAC).

Figure 2

Linoleic acid decreases MuRF1 and Atrogin-1/MAFbx mRNA expression in oxidative stress-induced C2C12 myoblasts. C2C12 myoblasts were cultured with serum-free DMEM containing LA (0–200 µM). Cell lysis and mRNA extraction were performed using Tri-reagent. cDNA was synthesized according to the manufacturer’s instructions. mRNA expression was analyzed by using SYBR Green 1 and a LightCycler ® 96 instrument. (A) MuRF1 mRNA expression. Each value indicates the mean ± SD (n = 6). (B) Atrogin-1/MAFbx mRNA expression. Each value indicates the mean ± SD (n = 6). * p < 0.05, ** p < 0.01, *** p < 0.001 vs. H₂O₂ alone. The effect of LA was compared with that of 2 mM NAC.

Figure 3

Linoleic acid decreases intracellular ROS synthesis in oxidative stress-induced C2C12 myoblasts. For the estimation of intracellular ROS, DCF was detected using confocal microscopy. (A) The effect of LA on intracellular ROS in C2C12 myoblasts identified using a confocal microscope. C2C12 myoblasts were pre-incubated in serum-free DMEM with LA (0–200 µM) for 24 h, and the medium was replaced with 1 mM H₂O₂ for 2 h. After incubation, C2C12 myoblasts were treated with 10 µM DCF-DA in PBS for 30 min, fixed with 4% para-formaldehyde in PBS for 30 min, and mounted using a mounting medium with DAPI. DCF was detected at wavelengths of 485 and 535 nm. DAPI was detected at wavelengths of 358 and 461 nm. The effect of LA was compared with that of 2 mM NAC. (B) Quantitating intracellular ROS synthesis. A graph showing average intensity of fluorescence in various GFPs, observed with a confocal laser scanning microscope in the C2C12 myoblast. Data are represented as mean ± SD (n = 3). ** p < 0.01, *** p < 0.001 vs. H₂O₂ alone.

Figure 4

Effect of linoleic acid on intracellular protein and ceramide level of oxidative stress-induced C2C12 myoblasts. C2C12 myoblasts were seeded and cultured in serum-free DMEM with or without 200 mM LA. Protein expression level as analyzed by SDS-PAGE. Cell lysis and protein extraction were conducted using the PRO-PREP protein extraction kit. (A) Effect of LA on SOD1, Bcl-2, Bax, HSP70 and p-FoxO1 protein expression level by oxidative stress in C2C12 myoblasts. (B) Relative density SOD1, Bcl-2, Bax, HSP70 and p-FoxO1 protein expression level. Each value indicates the mean ± SD (n = 3). (C) Effect of LA on ceramide levels under H₂O₂-induced oxidative stress in C2C12 myoblasts. Ceramide levels as analyzed by HPTLC. Cells were lysed in 0.2 N NaOH. Lipids were extracted using a methanol/chloroform/1 M NaCl solution. Lipids were separated using a silica gel TLC plate and analyzed by HPLC. Each value indicates the mean ± SD (n = 3). ** p < 0.01, *** p < 0.001 vs. H₂O₂ alone. The effect of LA was compared with that of 2 mM NAC.

Figure 5

Linoleic acid treatment prevents denervation-induced muscle atrophy in C57BL/6 mice. (A) Muscle atrophy was induced in C57BL/6 mice by sciatic denervation. Evaluation of muscle volume and analysis of two-dimensional (2D) and three-dimensional (3D) models were conducted 21 days after sciatic denervation. LA (0–5 mg/kg) was administered by intramuscular injection. (B) Effects of LA on disuse atrophy by sciatic denervation in mice. (C) Evaluation of muscle volume after sciatic denervation. Evaluation of muscle volume after treatment with LA (0–5 mg/kg). Each value indicates the mean ± SD (n = 6); * p < 0.05, ** p < 0.01 vs. sciatic denervation alone.

Figure 6

Linoleic acid decreases SOD1, HSP70, and MuRF1 protein expression in sciatic denervation-induced muscle atrophy of C57BL/6 mice. Muscle atrophy was induced in C57BL/6 mice by sciatic denervation. Protein expression level as analyzed by SDS-PAGE. Lysis of muscle tissue and protein extraction was performed using the PRO-PREP protein extraction kit. (A) Effect of LA on SOD1, HSP70, and MuRF1 protein expression level by sciatic denervation-induced muscle atrophy in mice. (B) Relative density of SOD1, HSP70, and MuRF1 protein expression level. Each value indicates the mean ± SD (n = 6); ** p < 0.01, *** p < 0.001 vs. sciatic denervation alone.

Figure 7

Mechanism of action for linoleic acid on muscle atrophy induced by oxidative stress in vitro and in vivo.