Chemicals, reagents and kits were obtained from commercial sources as follows: S-Rg3 (Urchem Sinopharm Chemical Reagent Co., Ltd.), dexamethasone (DEX)and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich; Merck KGaA), mouse skeletal muscle-derived C2C12 myoblasts (American Type Culture Collection), fetal bovine serum (FBS; Clark BioScience), horse serum (Gibco; Thermo Fisher Scientific, Inc.), antibodies against cleaved caspase-3 (cat. no. 9661), phosphorylated-AMPK (p-AMPK; cat. no. 2535), AMPK (cat. no. 2532), Bcl-2 (cat. no. 3498), Bax (cat. no. 2772), FoxO3 (cat. no. 2497), histone H3 (cat. no. 4499) and β-tubulin (cat. no. 2146) were obtained from Cell Signaling Technology, Inc.; atrogin-1 antibody (cat. no. ab168372) and total OXPHOS Rodent WB Antibody Cocktail (anti-complex I, II, III, IV, V; cat. no. ab110413) were obtained from Abcam; antibody specific for muscle-specific RING finger 1 (MuRF1; cat. no. bs-2539R) was purchased from BIOSS. Chemiluminescence reagents was purchased from Santa Cruz Biotechnology.

C2C12 myoblasts were cultivated via incubation at 37°C in an atmosphere containing 5% CO₂ with humidification in medium containing Dulbecco's modified Eagle's medium (DMEM) with 25 mM glucose, 10% FBS, 100 µg/ml streptomycin and 100 units/ml penicillin until 70–80% confluence. Next, myoblasts were seeded at 7.5×10⁴ cells/well in 6-well plates or at 5×10³ cells/well in 96-well plates. Myoblast fusion to form C2C12 myotubes was induced by culturing cells for 5 days in DMEM containing 2% horse serum and 25 mM glucose (26).

MTT assays were conducted to monitor cell viability (27,28) as follows: C2C12 myotubes treated for 24 to 48 h with various concentrations of DEX (0, 25, 50, 100 or 200 µM) (29) or with S-Rg3 (0, 0.02, 0.2 or 2 µM) and/or DEX (200 µM) for 24 h were incubated with MTT (0.5 mg/ml) for 4 h. All treatments were conducted at 37°C. Next, formazan dissolved in 150 µl DMSO was added to wells then measurements of absorbance levels of wells were taken at a wavelength of 490 nm at room temperature. Calculations of cell viability were performed that generated results as percentages of viable cells relative to vehicle control.

After C2C12 myotubes were treated with various concentrations of DEX (0, 25, 50, 100 or 200 µM) for 24 h, 50 µM DEX for 0, 3, 6, 12 or 24 h or S-Rg3 (0, 0.02, 0.2 or 2 µM) and/or DEX (50 µM) for 6 h, all at 37°C, C2C12 myotubes were lysed using ATP lysis buffer composed of 0.5% Triton X-100, 100 mM glycine, pH 7.4 then lysates were centrifuged at 15,000 × g for 10 min at 4°C (30). Supernatants were collected and an ATP Bioluminescent Assay kit (Promega Corporation) was used to measure intracellular ATP levels in supernatants on ice according to the manufacturer's instructions.

Basal respiration rates of mitochondria were measured via previously reported methods using a kit (MitoXpress^® Xtra Oxygen Consumption Assay; Agilent Technologies, Inc.) (31). In brief, C2C12 myoblasts previously seeded into 96-well microplates (Corning, Inc.) were incubated for 5 day to allow them to differentiate. C2C12 myotubes were then treated with various concentrations of DEX (0, 25, 50, 100 or 200 µM) for 24 h, 50 µM DEX for 0, 3, 6, 12 or 24 h or S-Rg3 (0, 0.02, 0.2 or 2 µM) and/or DEX (50 µM) for 6 h, all at 37°C. OCRs were measured using a plate reader at room temperature (Cytation 5; BioTek Instruments, Inc.).

C2C12 myotubes were treated with 50 µM DEX with or without 0.2 µM S-Rg3 for 6 h at 37°C. After cells were fixed by 2-h immersion in 2.5% glutaraldehyde fixative at 4°C, they were post-fixed in 1% OsO₄ at room temperature for 2 h, then dehydrated in an ascending alcohol series and embedded in Epon resin. Using a microtome (Leica UC7; Leica Microsystems, Inc.), ultrathin sections (60–80 nm) were sliced from embedded cell blocks, then sections were stained with 2% uranyl acetate and lead citrate at room temperature for 15 min each, then examined using an electron microscope (Tecnai G2 20 TWIN, FEI; Thermo Fisher Scientific, Inc.). To measure DEX-induced changes in mitochondrial number and area, images were processed using Image-Pro Plus 6.0 software (Media Cybernetics, Inc.) and data were analyzed by a statistician who had no access to the images (blind data analysis).

C2C12 myotubes were treated with 50 µM DEX and 0, 0.02, 0.2 or 2 µM S-Rg3 for 6 h at 37°C. A JC-1 fluorescent probe (Beyotime Institute of Biotechnology) was used to estimate the effect of S-Rg3 on mitochondrial membrane potential using the manufacturer's instructions provided with the probe, but with modifications (32). After 5-days culture of C2C12 myoblasts seeded at 7.5×10⁴ cells/well in 6-well plates, cells were treated for 6 h with S-Rg3 and DEX, each at various concentrations. Cells were next incubated in the dark with JC-1 stain for 20 min at 37°C then stained cells were immediately analyzed using flow cytometry (FACScan; BD Biosciences).

C2C12 myoblasts seeded at a density of 7.5×10⁴ cells/well into 6-well plates were induced with DMEM containing 2% horse serum and 25 mM glucose for 5 days, then treated with 200 µM DEX for 24 h and collected on ice. After extraction of total RNA using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols, cDNA was generated from total RNA (1 µg) via a reverse transcription kit (cat. no. KR118-02; Tiangen Biotech Co., Ltd.) on ice, then a real-time PCR system (Eppendorf) was used to conduct qPCR. The following thermocycling conditions were used for the qPCR: Initial denaturation at 95°C for 5 min; followed by 40 cycles of 95°C for 15 sec, 60°C for 30 sec and 72°C for 30 sec. Labeling of amplification products generated via qPCR was achieved using SYBR Green Master Mix (Takara Biotechnology Co., Ltd.) and gene-specific primers. Primer sequences were as follows: MuRF1, 5′-TGGAAACGCTATGGAGAACC-3′ (forward) and 5′-ATTCGCAGCCTGGAAGATG-3′ (reverse); Atrogin-1, 5′-CTGGCAGCAGCAGCTGAATAG−3′ (forward) and 5′-CACATGCAGGTCTGGGGCTGC−3′ (reverse); actin, 5′-AGGCCCAGAGCAAGAGAGGTA−3′ (forward) and 5′-CCATGTCGTCCCAGTTGGTAA−3′ (reverse). Amplification products generated from housekeeping gene actin mRNA were used to normalize the levels of the other products. Relative mRNA expression was calculated based on the 2^−∆∆Cq method (33).

C2C12 myotubes were treated with 50 or 200 µM DEX and 0, 0.02, 0.2 or 2 µM S-Rg3 for 6 or 24 h at 37°C. After washing cells twice with phosphate-buffered saline, they were suspended in RIPA lysis buffer (Beyotime Institute of Biotechnology) and allowed to lyse for 30 min at 4°C. Nuclear and cytoplasmic proteins were isolated separately using a BestBio BB-3102 kit (BestBio). Protein concentration was determined using a BCA Protein Assay kit (Beyotime Institute of Biotechnology). Lysates (30 µg total/lane) were separated via 12% SDS-PAGE followed by electro-transfer to polyvinylidene fluoride (PVDF) membranes. After blocking of membranes in 5% bovine serum albumin (cat. no. 4240; BioFroxx) at room temperature for 1 h, membranes were incubated overnight with 1:1,000 dilution of primary antibody at 4°C followed by two washes and a 1-h incubation with 1:5,000 of horseradish peroxidase-conjugated goat anti-rabbit IgG (cat. no.) BA1054 and goat anti-mouse IgG (cat. no. BA1050) secondary antibodies (Boster Biological Technology) at room temperature. Proteins were visualized via chemiluminescence (ProteinSimple), then imaged using a FluorChem HD2 system (ProteinSimple). Densitometry was performed using AlphaView SA software 3.4.0.0 (ProteinSimple) (34).

All statistical analyses were performed with GraphPad Prism 7 (GraphPad Software, Inc.) and using an unpaired Student's t-test or one-way analysis of variance (ANOVA) followed by Tukey's post-hoc tests. Data are expressed as the mean ± standard deviation of three independent experimental repeats. P<0.05 was considered to indicate a statistically significant difference.

Results of experiments whereby C2C12 myotubes received treatment with various doses of DEX (25, 50, 100 and 200 µM) for 24 and 48 h revealed no effect on cell viability at DEX concentrations below 200 µM (Fig. 1A). Meanwhile, increased levels of two muscle atrophy marker proteins, MuRF1 and atrogin-1, were observed in C2C12 myotubes treated with 200 µM DEX (Fig. 1B). Next, measurements of C2C12 myotube OCR and intracellular ATP level were performed to reveal the relationship between muscle atrophy and mitochondrial function. Subsequently it was observed that 24-h DEX treatment (50–200 µM DEX) led to ATP deprivation (Fig. 1C) and reduced OCR (Fig. 1D). Next, C2C12 myotubes were treated with DEX (50 µM) for various lengths of time, with the results revealing that 6-h DEX treatment of C2C12 myotubes led to ATP deprivation (Fig. 1E) and OCR reduction (Fig. 1F). When considered together, these results demonstrated that damage to mitochondria preceded myotube atrophy, since 50 µM DEX treatment of C2C12 myotubes for only 6 h led to mitochondrial dysfunction, while 24-h treatment with 200-µM DEX was necessary to induce myotube atrophy. Therefore, to generate a mitochondrial damage model the present study employed 6-h administration of 50 µM DEX and for the myotube atrophy model administered 24-h treatment with 200 µM DEX.

After verifying that DEX-induced myotube atrophy had occurred as the present study's model for skeletal muscle atrophy, effects of S-Rg3 treatment on DEX-induced injury were evaluated. DEX treatment alone decreased C2C12 myotube cell viability to 68.68±1.21%, while S-Rg3 treatment restored C2C12 myotube cell viability in a dose-dependent manner (Fig. 2A). The expression levels of muscle cell-specific major muscle atrophy-associated E3 ubiquitin ligases atrogin-1 and MuRF1 were examined to assess S-Rg3 effects on DEX-induced skeletal muscle cell atrophy. Notably, DEX increased expression of both atrogin-1 and MuRF1 proteins in C2C12 myotubes, while reduced expression of the two proteins was observed following S-Rg3 treatment (Fig. 2B-D). The effect of S-Rg3 on apoptosis caused by DEX was also tested. The expression levels of apoptosis-related proteins, such as cleaved caspase-3, Bcl-2 and Bax were examined by western blot analysis. The expression of cleaved caspase-3, which involved in the activation cascade of caspases responsible for apoptosis execution was decreased by S-Rg3 treatment for 24 h in DEX-induced C2C12 myotubes, and the ratio of the antiapoptotic protein Bcl-2 and proapoptotic protein Bax was increased following treatment with S-Rg3 for 24 h (Fig. 2E-F).

Previous studies conducted using a C2C12 skeletal muscle cell line have demonstrated that mitochondrial morphological changes occur during mitophagy, a necessary cellular process for removing damaged mitochondria (35,36). Using mitochondrial volume as a morphological indicator, DEX-induced mitochondrial injury was analyzed in C2C12 myotubes using electron microscopy (Fig. 3A). Following DEX treatment, relatively large mitochondrial structural changes were observed, including swollen and broken mitochondria or the absence of mitochondria. These changes were ameliorated by treatment with S-Rg3 as evidence that S-Rg3 restored mitochondrial function (Fig. 3B and C).

To reveal whether an association exists between DEX-induced muscle atrophy and mitochondrial dysfunction, intracellular ATP levels and OCRs were measured in DEX-injured C2C12 myotubes. Ultimately, low ATP levels were observed following treatment with DEX that were increased following subsequent S-Rg3 treatment (Fig. 4A). In addition, S-Rg3 treatment reversed DEX-induced mitochondrial respiratory dysfunction (Fig. 4B), reversed DEX-induced reduction of mitochondrial membrane potential (an early sign of apoptosis in C2C12 myotubes) (Fig. 4C) and reversed DEX-induced reductions of JC-1 aggregate levels from below control levels (as revealed via staining followed by flow cytometry) to levels above levels of untreated DEX-injured cells (Fig. 4D). Finally, as an additional assessment of whether mitochondrial dysfunction was induced by DEX, expression levels of key mitochondrial respiratory electron transport chain subunit proteins of complexes II, III and V were measured and significantly reduced expression levels of all key subunit proteins following DEX treatment were observed; these levels were increased following addition of S-Rg3 (Fig. 4E and F).

A critical cellular energy sensor, AMPK (37), is regulated by the intracellular AMP to ATP ratio, with robust activation of AMPK induced by ATP deprivation (38). In the present study, AMPK phosphorylation increased markedly following 6-h DEX treatment (Fig. 5A and B). In addition, increased nuclear and decreased cytoplasmic levels of transcription factor protein FoxO3, a known master regulator of muscle atrophy-associated E3 ligases MuRF1 and atrogin-1 (39), were observed. These changes were reversed by S-Rg3 treatment, which decreased AMPK phosphorylation (Fig. 5A and B) and reduced FoxO3 nuclear translocation (Fig. 5C and D). Therefore, these data suggested that the AMPK/FoxO3 pathway is involved in the protective effect of S-Rg3 on mitochondrial dysfunction.