Dried loquat leaves were purchased from Pure Mind Co. (Daegu, Korea) and LE was prepared as previously described with slight modifications (17). Unless otherwise stated, all chemical reagents were obtained from Sigma-Aldrich (St Louis, MO, USA). Primary antibodies against MyHC (cat. no. sc-20641), MuRF1 (cat. no. sc-32920), MAFbx (cat. no. sc-33782), FoxO1 (cat. no. sc-67140), α-tubulin (cat. no. sc-5286), transcription factor (TF)-IIB (cat. no. sc-225) and ubiquitin (cat. no. sc-271289), and secondary goat anti-rabbit immunoglobulin (Ig)G (cat. no. sc-2004) and anti-mouse IgG conjugated to horseradish peroxidase (cat. no. sc-2031) were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Polyvinylidene difluoride (PVDF) membranes were obtained from EMD Millipore (Bedford, MA, USA).

All animal studies were approved by the Institutional Animal Care Committee of Pusan National University (Busan, Korea) and were performed in accordance with the guidelines for animal experimentation issued by Pusan National University (Busan, Korea). Sprague Dawley rats (six weeks old) were obtained from Samtako (Osan, Korea). The rats were maintained in controlled environmental conditions (23±1°C, 55±5% relative humidity) under a 12-h light/dark cycle with ad libitum access to water and a standard laboratory diet. The animals were randomly divided into five groups of six animals following a two-week acclimation period. Dexamethasone (600 µg/kg body mass) or normal saline were intraperitoneally injected once daily for five consecutive days. The other compounds were administered by oral gavage. The animals were divided into groups based on the treatment type, as follows: Leucine (600 mg/kg/day), ursolic acid (100 mg/kg/day) or LE (50 or 200 mg/kg/day). The leucine and ursolic acid supplementation groups were used as positive controls. At the end of the experimental period (day 5), forelimb grip strength was determined using a Grip Strength Meter equipped with a T-shaped pull bar (Model 1027SR; Columbus Instruments, Columbus, OH, USA). The rats were then sacrificed by decapitation and soleus and gastrocnemius muscles were rapidly removed. Muscle weight was measured immediately and the muscles were divided for biological analysis and histological examination.

The serum creatine kinase (CK) activities were determined using a creatine kinase enzymatic assay kit (Bioo Scientific Corp, Austin, TX, USA) according to manufacturer's instructions. Serum was collected at sacrifice and stored at −80°C until analysis. Briefly, 250 µl CK reagent was added to 5 µl serum sample in a 96-well microplate. The CK activity was immediately measured twice with 5-min time interval at 340 nm using a GENios microplate reader (Tecan, Salzburg, Austria). The activity of CK was calculated by subtracting the initial reading from the second reading and multiplying the absorbance increase by 2,186 (conversion factor) to the obtain CK activity (IU/l).

The medial portions of soleus muscles were fixed in 10% formalin solution for 24 h, routinely embedded in paraffin blocks, transversely sectioned (3 µm) and stained with hematoxylin and eosin (H&E). The muscle histologies were analyzed using an AE-31 light microscope (Motic, Hong Kong, China).

Whole soleus muscle was homogenized in homogenizing buffer containing 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (pH 7.4), 10 mM KCl, 2 mM MgCl₂, 1 mM dithiothreitol, 0.1 mM EDTA, 0.1 mM phenylmethanesulfonylfluoride (PMSF), 20 mM b-glycerophosphate, 20 mM NaF, 2 mM Na₃VO₄, 1 µM pepstatin, 2 µM leupeptin and 5 µM aprotinin. The homogenates obtained were placed on ice for 15 min. Thereafter, NP-40 was added at a final concentration of 1% (v/v) and the lysates were mixed for 15 sec. The nuclei were pelleted by centrifugation at 14,000 × g for 2 min at 4°C. The supernatants were regarded as cytosolic fractions. The nuclear pellets were washed once, suspended in buffer containing 50 mM KCl, 300 mM NaCl, 0.1 mM PMSF, 10% (v/v) glycerol, 20 mM β-glycerophosphate, 20 mM NaF, 2 mM Na₃VO₄, 1 µM pepstatin, 2 µM leupeptin and 5 µM aprotinin, and maintained on ice for 30 min. The suspensions were subsequently centrifuged at 14,000 × g for 10 min at 4°C and the harvested supernatants were regarded as nuclear fractions. The protein concentrations were measured using the bicinchoninic acid method (Pierce™ BCA Protein Assay kit; Life Technologies, Carlsbad, CA, USA) using bovine serum albumin as a standard.

Nuclear or cytosolic proteins (20–100 µg) were boiled for 5 min in gel-loading buffer containing 0.125 M Tris-HCl (pH 6.8), 4% SDS, 10% 2-mercaptoethanol, 20% glycerol and 0.2% bromophenol blue, at a volume ratio of 1:1. The samples containing identical quantities of protein were subsequently separated using 8%–15% SDS-PAGE and transferred using a Bio-Rad Western system (Bio-Rad Laboratories, Hercules, CA, USA) onto PVDF membranes. The membranes were immediately placed in blocking buffer containing 5% non-fat milk in 10 mM Tris (pH 7.5), 100 mM NaCl and 0.1% Tween-20 for 1 h. The membranes were incubated with the following primary antibodies at 4°C for 24 h: Anti-MyHC (1:1,000), anti-MuRF1 (1:1,000), anti-MAFbx (1:1,000), anti-FoxO1 (1:1,000), anti-TF-IIB (1:1,000), anti-ubiquitin (1:1,000), and anti-α-tubulin (1:1,000). The membranes were then washed with TBS-Tween buffer (10 mM Tris, pH 7.5, 100 mM NaCl and 0.1% Tween-20) and incubated with horseradish peroxidase-conjugated secondary antibodies (1:5,000) for 1 h at room temperature. The resulting immunoblots were visualized using Western Bright Peroxide solution (Advansta, Menlo Park, CA, USA) on a Davinch-chemi™ Chemiluminescence Imaging system CAS-400 (Davinch-K, Seoul, Korea).

Homogenized tissue proteins were subjected to immunoprecipitation in a buffer, containing 40 mM Tris-HCl (pH 7.6), 120 mM NaCl, 20 mM β-glycerophosphate, 20 mM NaF, 2 mM Na₃VO₄, 5 mM EDTA, 1 mM PMSF, 0.1% NP-40 containing leupeptin (2 µg/ml), aprotinin (1 µg/ml) and pepstatin A (1 µg/ml). An aliquot of protein extract (200 µg) was incubated with the required primary antibody for 4 h at 4°C and incubated overnight at 4°C with the appropriate protein A/G agarose. Following washing of the immunoprecipitates three times with IP buffer, the pellets were dissolved in gel-loading buffer at a volume ratio of 1:1 and boiled for 5 min. Following cooling, the samples were centrifuged at 2,400 x g for 1 min and the supernatants were used in subsequent SDS-PAGE analysis, followed by immunoblotting.

Total RNA was isolated from tissues using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) and RT-qPCR was performed as previously described (18). Briefly, cDNA was synthesized from RNA using M-MLV Reverse Transcriptase (Promega Corporation, Madison, WI, USA) at the following temperatures: 60 min at 42°C and 2 min at 100°C. cDNA amplification was performed in a PCR master mix containing 1 µl reverse transcription reaction product, 1X PCR buffer (Perkin Elmer, Gaithersburg, MD, USA), 0.2 mM dNTP, 0.25 U Taq polymerase (Perkin Elmer), and 50 ng sense and anti-sense primers (Bioneer Corp., Daejeon, Korea). The primers used were as follows: MuRF1 sense, 5′-TTCATCGAGGCCCTGATCCT-3′ and anti-sense, 5′-CTTGGCTTCCTTCCCCCTTT-3′; MyHC sense, 5′-TGCCAAGACCGTGAGGAATG-3′ and anti-sense, 5′-AATGCATCACAGCTCCCGTG-3′; GAPDH sense, 5′-GGGTGATGCTGGTGCTGAGTATGT-3′ and anti-sense, 5′-AAGAATGGGAGTTGCTGTTGAAGTC-3′. The PCR reactions were performed in a GeneAmp^® PCR system 2400 (Perkin Elmer). The amplified PCR products were electrophoresed on 1% agarose gels, visualized by ethidium bromide staining, and images were captured using the BioSpectrum^® AC Imaging system (UVP, LLC, Upland, CA, USA). The images were imported into TotalLab 1D software (version 12.2; TotalLab Ltd., Newcastle-upon-Tyne, UK) and analyzed.

Values are expressed as the mean ± standard error of the mean and were analyzed using GraphPad Prism (version 5.0; GraphPad Software, Inc., La Jolla, CA, USA). Analysis of variance was used to analyze intergroup differences. Tukey's Multiple Comparison test was used to evaluate differences between values. P<0.05 was considered to indicate a statistically significant difference.

Body weight changes were measured over the five-day experimental period. The dexamethasone-treated groups demonstrated progressive weight loss throughout the experiment compared with the control group (Fig. 1A). To investigate muscle loss following treatment, the weight of soleus and gastrocnemius muscles was measured immediately following biopsy. Dexamethasone significantly decreased the weight of each muscle (Fig. 1B and C) and LE supplementation failed to prevent this muscle weight loss.

To assess the loss of muscle strength by dexamethasone, forelimb grip strength was measured using a grip strength meter. Dexamethasone treatment gradually reduced grip strength during the experimental period (data not shown) and significantly reduced grip strength on experimental day 5 compared with that in the vehicle-treated control group (Fig. 2A). Of note, supplementation with LE effectively prevented this dexamethasone-induced muscle weakness (Fig. 2A). Since the increment of CK in the serum is considered a surrogate marker of muscle damage, particularly for the diagnosis of myocardial infarction, muscular dystrophy and cerebral diseases (19), the activity of serum CK was determined following 5 days of dexamethasone treatment. Dexamethasone significantly increased the serum CK activity compared with that in the vehicle-treated controls (Fig. 2B), while supplementation with LE effectively reduced these dexamethasone-induced increases.

In order to assess the protective effect of LE on dexamethasone-induced muscle damage, histological staining was performed on the medial portions of soleus muscles, using hematoxylin and eosin dyes (H&E). As shown in Fig. 3A, the muscle fibers in the vehicle-treated group were in close contact in muscle bundles and exhibited relatively uniform fiber diameters (Fig. 3A). However, dexamethasone treatment caused severe damage to the muscle bundles and resulted in an atrophic pattern of muscle fibers (Fig. 3B). The proliferation of connective tissue, particularly surrounding the muscle fibers (endomysium) and fiber bundles (perimysium), as well as angulated and degenerated fibers were observed in the soleus muscle of dexamethasone-treated group (Fig. 3B). However, these atrophic damages were ameliorated by supplementation with leucine or ursolic acid (Fig. 3C and D), and to a similar extent by supplementation with LE (Fig. 3E and F). These results suggested that LE supplementation effectively prevented dexamethasone-induced muscle damage and muscle weakness.

Since it was demonstrated that dexamethasone treatment induced the selective loss of the critical sarcomeric protein MyHC, which is important in muscle contraction (12), the present study assessed the protein expression levels of MyHC in soleus muscles. Dexamethasone markedly reduced the protein expression of MyHC, while LE supplementation effectively inhibited this reduction (Fig. 4A). Therefore, the present study aimed to determine whether the observed reduction in MyHC levels was caused by increased protein degradation or decreased protein synthesis. Neither dexamethasone nor supplementation had any effect on the mRNA expression levels of MyHC (Fig. 4B), which indicated that the decline of MyHC levels was not caused by modulation of MyHC protein synthesis. Since muscle degradation is largely due to the ubiquitination of MyHC, which is catalyzed by E3 ligase (12), MyHC ubiquitination was subsequently detected. Dexamethasone significantly increased the ratio of ubiquitinated MyHC compared with total MyHC (Fig. 4C) and this increase was markedly reduced by LE supplementation (Fig. 4C). These results demonstrated that LE supplementation inhibited dexamethasone-induced muscle loss by reducing the ubiquitination of MyHC.

MuRF1 is a specific E3 ligase responsible for the degradation of MyHC (12). It was demonstrated that dexamethasone markedly increased the protein expression levels of MuRF1 (Fig. 5A), which was significantly attenuated by supplementation with LE (Fig. 5A). In addition, the effect of dexamethasone on the nuclear trans-location of the transcription factor FoxO1, which induces the expression of MuRF1, was assessed. Dexamethasone was revealed to induce FoxO1 translocation (Fig. 5B) and LE supplementation reduced this effect. These results suggested that LE supplementation reduces the expression of MuRF1 by inhibiting the nuclear translocation of FoxO1.