Light brown EAP powder was supplied by Glucan Corporation (Busan, South Korea) and was stored at 4°C. EAP consisted of 13% β-1, 3/1,6-glucan and 40% β-glucans, as determined using previously described analytical methods (45,46,53). Oxymetholone (50 mg tablet; Celltrion, Incheon, South Korea) was used as a reference drug; tablets were ground and were also stored at 4°C protected from light. Ground 50 mg oxymetholone tablets were dissolved at a 15 mg/ml concentration (5 mg/ml oxymetholone) in deionized distilled water. EAP was dissolved at 40 mg/ml in deionized distilled water.

A total of 60 adult male SPF/ICR mice (6 weeks old), weighing 27–30 g were obtained from orient Bio, Inc. (Seongnam, South Korea). After 10 days of acclimatization, the 48 mice that were well acclimatized in the laboratory environment (8 mice per group; a total of 6 groups) were used in the present study. The mice were maintained in polycarbonate cages (n=4–5 mice/cage) in a humidity (40–45%)- and temperature (20–25°C)-controlled room under a 12-h light/dark cycle. Normal rodent pellets (cat. no. 38057; Purina Feed, Seongnam, South Korea) and water were provided ad libitum during acclimation.

Three doses of EAP (100, 200 and 400 mg/kg) were administered orally in a volume of 10 ml/kg, once a day for 24 days; EAP treatment was initiated 2 weeks prior to DEXA treatment. In addition, 50 mg/kg oxymetholone was administered orally, in a similar manner to EAP. EAP was dissolved at 10, 20 or 40 mg/ml in distilled water, and was administered orally in a volume of 10 ml/kg body weight using a zonde needle attached to a 1 ml syringe. Ground 50 mg oxymetholone tablets were also dissolved in distilled water at 15 mg/ml (5 mg/ml as oxymetholone) and administered orally at 10 ml/kg, which was equivalent to 150 mg/kg (50 mg/kg as oxymetholone). The dosage of oxymetholone was selected based on previous efficacy tests in mice (26,33–35). Doses of 100, 200 and 400 mg/kg EAP were selected based on previously reported in vivo efficacy tests of EAP (45,46). In the present study, catabolic muscle atrophy was initiated by subcutaneous treatment with 1 mg/kg DEXA, once a day for 10 days, according to previously reported methods (16,26). Water-soluble DEXA (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was dissolved in saline at 1.5 mg/ml (0.1 mg/ml DEXA) and was subcutaneously injected into the cervical dorsal region in a volume of 10 ml/kg, equivalent to 15 mg/kg (1 mg/kg as DEXA itself). An equal volume of deionized distilled water, instead of oxymetholone or EAP, was orally administered in the DEXA control and intact vehicle groups, and an equal volume of saline, instead of DEXA, was injected subcutaneously into the intact vehicle control group. The present study was conducted in accordance with international regulations of the usage and welfare of laboratory animals, and was approved by the Institutional Animal Care and use Committee, Daegu Haany University (Gyeongsan, South Korea; approval no. DHU2016-051, May 27, 2016).

Body weight (g) was measured 1 day prior to, the day of, and 1, 7, 14, 19, 23 and 24 days after treatment administration using an electronic balance (Precisa Gravimetrics AG, Dietikon, Switzerland). The gain in body weight during the 14 days of pretreatment, the 10 days of DEXA treatment and the total 24-day treatment periods was measured to decrease individual differences, according to equation 1, where BW indicates body weight:

During 14 days of pretreatment= BW at 14 days after initial administration - BW at first administration (Eq. 1a).

The thickness of the left hind calf was measured 1 day prior to, the day of, and 1, 7, 14, 19, 23 and 24 days after treatment administration using electronic digital calipers (Mitutoyo, Tokyo, Japan), similar to previous studies (26,35). Gastrocnemius muscle thickness in the left hind limb was measured following muscle exposure after sacrifice (all mice were sacrificed at the end of the 24-day period; liver, kidney, pancreas, calf muscle mass and gastrocnemius muscle tissues were collected following sacrifice), in order to decrease variability from the surrounding tissues. Gastrocnemius muscle thickness was measured according to the method used to measure calf thickness; alterations in calf thickness (mm) during 14 days of pretreatment, 10 days of DEXA treatment and the total 24-day treatment period were measured to reduce individual differences, according to equation 2, where CT indicates calf thickness:

During 14 days of pretreatment = CT at 14 days after initial administration - CT at first administration (Eq. 2a).

A total of 1 h after the last dose of oxymetholone, vehicle or EAP was administered (10 days after the initial DEXA treatment), the calf muscle strengths of individual mice were measured as tensile strengths using a computerized testing machine (SV-H1000, Japan Instrumentation System Co., Ltd., Tokyo, Japan) in Newtons (N) according to established methods (26,35). Briefly, animals were restrained in the machine using two separate 1-0 silk suture ties on the chest and left ankle, and the peak tensile loads were documented as calf muscle strengths during knee angle reach of 0° (10–20-mm distance).

After gastrocnemius muscle thickness was measured following sacrifice, the gastrocnemius muscles were separated carefully from the tibia and fibula bones. The weights of individual gastrocnemius muscles were measured in g (absolute wet-weights) using an electronic balance, and to reduce the differences from individual body weights, relative weights (% of body weights) were calculated according to body weight at sacrifice and absolute weight, following equation 3.

To obtain sera for biochemical analysis, blood samples were collected on the day of sacrifice using a separation tube, and were then centrifuged at 600 × g for 10 min at ambient temperature. Separated serum samples were stored at −150°C in an ultra-deep freezer until further analysis. Serum creatine, creatine kinase (CK) and lactate dehydrogenase (LDH) levels were measured using an auto analyzer (Dri-Chem NX500i; FUJIFILM Medical Systems U.S.A., Inc., Stamford, CT, USA).

Following muscle mass measurements, gastrocnemius muscles were separated and the malondialdehyde (MDA), glutathione (GSH) and reactive oxygen species (ROS) contents, and superoxide dismutase (SOD) and catalase (CAT) enzyme activities were assessed in individual muscles. Separated gastrocnemius muscles were weighed and homogenized in ice-cold 0.01 M Tris-HCl (pH 7.4), after which they were centrifuged at 12,000 × g for 15 min at ambient temperature, as described previously (54). Muscle tissue homogenates were stored at −150°C in an ultra-deep freezer until analysis. The degree of gastrocnemius muscle lipid peroxidation was measured by assessing MDA values using the thiobarbituric acid test at 525 nm using a UV/vis spectrometer (Optizen POP; Mecasys Co., Ltd., Daejeon, South Korea) (55). The total protein contents were measured using the Lowry method (56), whereas bovine serum albumin (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used as a standard. ROS level analyses were performed using 2′,7′-dichlorofluorescein diacetate fluorescent dye as a probe and fluorescence density was measured at 490/520 nm according to the manufacturer’s protocol (Cellular Reactive oxygen Species Detection assay kit; ab113851; Abcam, Cambridge, MA, USA); the measured optical density values were corrected to the protein contents of samples and were expressed as RFu/μg⁻¹ protein (57). In addition, prepared homogenates were mixed with 0.1 ml 25% trichloroacetic acid (EMD Millipore, Billerica, MA, USA) and were then centrifuged at 800 × g for 40 min at 4°C. GSH contents were measured at 412 nm using 2-nitrobenzoic acid (Sigma-Aldrich; Merck KGaA), and were expressed as mg/g⁻¹ tissue (58). H₂O₂ decomposition in the presence of CAT was estimated at 240 nm (59). CAT activity was defined as the amount of enzyme required to decompose 1 nM H₂O₂ per min, at 25°C and pH 7.8, and the results are expressed as U/mg⁻¹ protein. Furthermore, SOD activity was measured at 560 nm according to a protocol previously described by Sun et al (60), and was expressed as U/mg⁻¹ protein. One unit of SOD enzymatic activity is equal to the amount of enzyme that diminishes the initial absorbance of nitroblue tetrazolium by 50% during 1 min.

Total RNA was extracted from gastrocnemius muscles using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to previous studies (9,26,35,61). The RNA concentration and quality were determined using a CFX96™ Real-Time PCR Detection system using iTaq™ SYBR-Green (both from Bio-Rad Laboratories, Inc., Hercules, CA, USA). The samples were treated with recombinant DNase I (DNA-free DNA removal kit; Ambion, Austin, TX, USA) to remove possible DNA contamination. RNA was reverse-transcribed using the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) according to the manu facturer’s protocol. The PCR cycling conditions were as follows: Initial pre-denaturation of 95°C for 1 min, denaturation for 15 sec, annealing of 55–65°C for 20 sec and extension of 72°C for 30 sec. A total of 50 cycles were performed. 18S ribosomal RNA was used as an internal control. PCR primer sequences are listed in Table I. For quantitative analysis, the intact control muscle tissue was used as the control, and the relative expression of Atrogin-1, MuRF 1, PI3K p85α, Akt1, Adenosine A1R, TRPV4, Myostatin and SIRT1 was calculated using the 2^−ΔΔCt method (62).

Samples from gastrocnemius muscles were separated and fixed in 10% neutral buffered formalin, embedded in paraffin wax, sectioned (3–4 μm), and stained with Sirius red for collagen fibers or hematoxylin and eosin for general histopathology (63,64). Histopathological profiles were observed under a light microscope (Eclipse 80i; Nikon Corporation, Tokyo, Japan). Mean muscle fiber diameters (μm/fiber) and collagen fiber-occupied regions (%/mm²) in muscle bundles were calculated using an automated image analyzer (iSolution FL, version 9.1; Brooke Anco Corporation, Cicero, NY, USA) in gastrocnemius muscle samples, according to previous studies (9,15,21,26,35,63) with some modifications.

Following deparaffinization of gastrocnemius muscle histological sections, citrate buffer antigen retrieval was conducted as previously described (26,35,65). Briefly, a staining dish containing 10 mM citrate buffer (pH 6.0) was preheated at 95–100°C in a water bath. Slides were immersed in the staining dish and incubated for 20 min prior to turning off the water bath. The staining dish was placed at room temperature and the slides were allowed to cool for 20 min. Subsequently, sections were immunostained using the avidin-biotin complex (ABC) method, to detect caspase-3, poly (ADP-ribose) polymerase (PARP), nitrotyrosine, 4-hydroxynonenal (4-HNE), inducible nitric oxide synthase (iNOS) and myostatin expression (Table II) according to previous studies (26,35). Briefly, endogenous peroxidase activity was blocked by incubation in methanol and 0.3% H₂O₂ for 30 min at ambient temperature, and non-specific binding was blocked with normal horse serum blocking solution (1:100; Vector Laboratories, Inc., Burlingame, CA, USA) for 1 h at ambient temperature in a humidified chamber. Slides were incubated with primary antibodies (Table II) overnight at 4°C in a humidified chamber, and were then incubated with biotinylated universal secondary antibody [1:50; Vectastain Elite ABC kit (PK-6200); Vector Laboratories, Inc.] and ABC reagents (1:50; Vectastain Elite ABC kit, Vector Laboratories, Inc.) for 1 h at room temperature in a humidified chamber. Finally, sections were treated with a peroxidase substrate kit (Vector Laboratories, Inc.) for 3 min at room temperature. All of the sections were rinsed in 0.01 M PBS three times between steps. Cells or muscle fibers that exhibited >20% immunoreactivity with each antibody were considered positive, and the mean numbers of caspase-3, PARP, nitrotyrosine, 4-HNE, iNOS and myostatin-immunoreactive fibers, as dispersed in 1 mm² of muscle bundles, were counted using an image analysis process described by Kim et al (26,35) with some modifications. The histopathologist was blinded to the group distribution when performing the analysis.

All numerical values are expressed as the means ± SD of 8 mice. Multiple comparison tests for different dose groups were conducted. Variance homogeneity was examined using the Levene test (66). If the Levene test indicated no significant deviation from variance homogeneity, data were analyzed by one-way analysis of variance followed by least-significant differences multi-comparison test to determine which pairs of group comparisons were significantly different. In cases where significant deviations from variance homogeneity were observed with the Levene test, the non-parametric Kruskal-Wallis H-test was used. When a significant difference was observed with the Kruskal-Wallis H test, the Mann-Whitney U test was conducted to determine the specific pairs of group comparisons that were significantly different. Statistical analyses were conducted using SPSS 14K for Windows software (SPSS Inc., Chicago, IL, USA) (67). Statistical significances were set at P<0.01 and P<0.05. Percent changes between intact vehicle and DEXA control groups were calculated to assess the severities of catabolic muscle atrophy induced, and the percent changes between the DEXA control and test material-treated mice were calculated, in order to understand the efficacy of the test substances according to the following equations 4 and 5: Percentage change compared to the intact vehicle control group ( % ) = [ Data of DEXA control − Data of intact vehicle control Data of intact vehicle control ] × 100 (Eq. 4). Percentage change compared to the DEXA control group ( % ) = [ Data of test material treated mice − Data of DEXA control Data of DEXA control ] × 100 (Eq. 5).

Significant decreases (P<0.01) in body weight were demonstrated in the DEXA control mice compared with in the intact control mice from 5 days after initial DEXA treatment to sacrifice. Accordingly, body weight during the 10 days of DEXA treatment, and after the total 24-day experimental period, was significantly decreased (P<0.01) in the DEXA control mice compared with in the intact vehicle control group. However, these decreases in body weight were significantly inhibited (P<0.01) by treatment with oxymetholone and all three doses of EAP (100, 200 and 400 mg/kg) from 5 days after initial DEXA treatment to sacrifice. In addition, body weight after 10 days of DEXA treatment, and after the total 24-day experimental period, was significantly increased (P<0.01) in the oxymetholone- and EAP-treated mice compared with in the DEXA control group. Anyway, no test material treatment-related alterations in body weight were detected compared with intact vehicle or DEXA control mice in this experiment. Treatment with EAP (100, 200 and 400 mg/kg) exhibited dose-dependent inhibitory effects on DEXA-induced decreases in body weight, in particular 400 mg/kg EAP exhibited favorable inhibitory activities on DEXA-induced decreases in body weight, which were comparable with the effects of 50 mg/kg oxymetholone (Table III and Fig. 1).

Significant decreases (P<0.01) in calf thickness were demonstrated in the DEXA control mice compared with in the intact control mice from 19 days after initial administration of the test substances to the day of sacrifice. Accordingly, calf thickness alterations after 10 days of DEXA treatment, and after the total 24-day test substance administration period, were also significantly decreased (P<0.01) in the DEXA control mice compared with in the intact vehicle controls. However, 5 days after the initial DEXA treatment, these decreases in calf thickness were significantly inhibited (P<0.01) by treatment with the three doses of EAP, and calf thickness during the 10 days of DEXA treatment, and the total 24-day test substance administration period, were also significantly increased (P<0.01) in these groups compared with in the DEXA control group. Furthermore, 50 mg/kg oxymetholone-treated mice also exhibited significant increases (P<0.01) in calf thickness from 5 days after the initial DEXA treatment, and also exhibited significant increases (P<0.01) in calf thickness during the 10 days of DEXA treatment and the total 24-day test substance administration period. A dose of 400 mg/kg EAP exhibited favorable inhibitory activities on DEXA-induced decreases in calf thickness, which were comparable with the effects of 50 mg/kg oxymetholone (Table IV and Fig. 2).

Significant decreases (P<0.01) in gastrocnemius muscle thickness following muscle exposure were observed in the DEXA control mice compared with in the intact vehicle control mice. However, significant increases (P<0.01) in gastrocnemius muscle thickness were detected in the mice treated with oxymetholone and all three doses of EAP compared with in the DEXA control group. EAP (100, 200 and 400 mg/kg) exhibited dose-dependent inhibitory effects on DEXA-induced decreases in gastrocnemius muscle thickness. In particular, 400 mg/kg EAP exhibited favorable inhibitory activities on gastrocnemius muscle thickness, which were comparable with the effects of 50 mg/kg oxymetholone (Figs. 3 and 4).

Significant decreases (P<0.01) in relative weights and absolute wet weights of gastrocnemius muscle mass were demonstrated in the DEXA control mice compared with in the intact vehicle control mice. However, significant increases (P<0.01) in gastrocnemius muscle weights were observed in the oxymetholone-treated and 100, 200 and 400 mg/kg EAP-treated mice compared with in the DEXA control group. EAP doses (100, 200, and 400 mg/kg) exhibited dose-dependent inhibitory effects on the DEXA-induced decreases in gastrocnemius muscle weights; in particular, 400 mg/kg EAP exhibited favorable inhibitory activities on gastrocnemius muscle weight, which were comparable with the effects of 50 mg/kg oxymetholone (Fig. 5).

Significant decreases (P<0.01) in the tensile strength of calf muscles were demonstrated in the DEXA control mice compared with in the intact vehicle control mice. However, significant increases (P<0.01) in calf muscle strength were observed in oxymetholone-treated and 200 and 400 mg/kg EAP-treated mice compared with in the DEXA control group. In addition, 100 mg/kg EAP-treated mice exhibited non-significant increases in calf muscle strength compared with in the DEXA control mice. EAP (100, 200 and 400 mg/kg) exhibited dose-dependent inhibitory effects on DEXA-induced decreases in calf muscle strength; in particular, 400 mg/kg EAP exhibited favorable inhibitory activities on decreases in calf muscle strength, which were comparable with the effects of 50 mg/kg oxymetholone (Fig. 6).

Significant increases (P<0.01) in serum CK and creatine levels, and decreases in serum LDH levels, were demonstrated in the DEXA control mice compared with in the intact vehicle control mice. However, significant decreases (P<0.05) in serum CK and creatine levels were observed in oxymetholone- and EAP-treated mice compared with in the DEXA control group, alongside significant increases (P<0.05) in serum LDH levels. EAP (100, 200, and 400 mg/kg) exhibited dose-dependent inhibitory effects on DEXA-induced increases in serum CK and creatine levels, and decreases in serum LDH levels. In particular, 400 mg/kg EAP exhibited favorable inhibitory activities on serum CK and creatine level elevations, and decreases in serum LDH levels, which were comparable with the effects of 50 mg/kg oxymetholone (Table V).

Significant increases (P<0.01) in MDA levels were observed in the DEXA control group compared with in the intact control group. However, the elevations in MDA levels were significantly (P<0.01) and dose-dependently decreased following treatment with EAP. Gastrocnemius muscle lipid peroxidation in oxymetho-lone-treated mice was also significantly decreased (P<0.01) compared with in the control mice. In particular, 400 mg/kg EAP exhibited favorable inhibitory activities on DEXA-induced increases in muscle lipid peroxidation, which were comparable with the effects of 50 mg/kg oxymetholone (Table VI).

Significant increases (P<0.01) in muscle ROS content were observed in the DEXA control group compared with in the intact control group. However, elevated ROS levels were significantly and dose-dependently decreased (P<0.01) following treatment with EAP. In addition, gastrocnemius muscle ROS levels were significantly (P<0.01) inhibited in 50 mg/kg oxymetholone-treated mice compared with in the DEXA control mice. In particular, 400 mg/kg EAP exhibited favorable inhibitory activities on DEXA-induced muscle ROS elevations, which were comparable with the effects of oxymetholone (Table VI).

Significant decreases (P<0.01) in the levels of the endogenous antioxidant, GSH, were detected in the DEXA control group compared with in the intact control group. However, these decreases in muscle GSH were significantly (P<0.05) inhibited following 24 days of oral treatment with oxymetholone, and 100, 200 and 400 mg/kg EAP. EAP increased gastrocnemius muscle GSH content in a dose-dependent manner compared with in the DEXA control mice. In particular, 400 mg/kg EAP exhibited favorable inhibitory activities on DEXA-induced decreases in muscle GSH content, which were comparable with the effects of oxymetho-lone (Table VI).

Significant decreases (P<0.01) in the activity levels of the endogenous antioxidant enzyme, SOD, were detected in the DEXA control group compared with in the intact control group. However, significant increases (P<0.01) in SOD activity were observed in oxymetholone-treated, and 100, 200 and 400 mg/kg EAP-treated mice compared with in the DEXA control mice. EAP exerted dose-dependent increases on SOD activity in gastrocnemius muscles compared with in the DEXA control mice. In particular, 400 mg/kg EAP exhibited favorable inhibitory activities on DEXA-induced decreases in SOD activity levels, which were comparable with the effects of 50 mg/kg oxymetholone (Table VI).

Significant decreases (P<0.01) in the activity levels of the endogenous antioxidant enzyme, CAT, were detected in the DEXA control group compared with in the intact control group. However, these decreases in muscle CAT activity were significantly and dose-dependently inhibited (P<0.01) following 24 days of oral treatment with EAP. Gastrocnemius muscle CAT activity levels in 50 mg/kg oxymetholone-treated mice were also significantly increased (P<0.01) compared with in the DEXA control mice. In particular, 400 mg/kg EAP exhibited favorable inhibitory activities on DEXA-induced decreases in muscle CAT activity levels, which were comparable with the effects of 50 mg/kg oxymetholone (Table VI).

Significant alterations (P<0.01) in the mRNA expression levels of atrogin-1, MuRF1, PI3K, Akt1, A1R, TRPV4, myostatin and SIRT1 were detected in the gastrocnemius muscles of the DEXA control group compared with in the intact control group. However, these alterations in muscle atrogin-1, MuRF1, PI3K, Akt1, A1R, TRPV4, myostatin and SIRT1 expression were significantly reversed (P<0.05), in a dose-dependent manner, by treatment with EAP. In addition, the mRNA expression levels of atrogin-1, MuRF1, PI3K, Akt1, A1R, TRPV4, myostatin and SIRT1 in gastrocnemius muscle tissues, were significantly reversed in 50 mg/kg oxymetholone-treated mice (P<0.01) compared with in the DEXA control mice. In particular, 400 mg/kg EAP exhibited favorable activities on DEXA-induced alterations in muscle atrogin-1, MuRF1, PI3K, Akt1, A1R, TRPV4, myostatin and SIRT1 mRNA expression, which were comparable with the effects of 50 mg/kg oxymetholone (Table VII).

Marked alterations associated with catabolic muscle atrophy, including focal fibrosis in muscle bundles, microvacuolation and diminished muscle fibers, were induced by treatment with DEXA in the control mice. Accordingly, significant decreases (P<0.01) in mean muscle fiber diameters and increases in collagen fiber-occupied region percentages in muscle bundles were detected in the DEXA control mice compared with in the intact control mice. However, these DEXA treatment-associated catabolic alterations were significantly (P<0.05) and dose-dependently decreased following treatment with EAP. The muscle atrophy-associated alterations were also significantly inhibited (P<0.01) in 50 mg/kg oxymetholone-treated mice compared with in the DEXA control mice. In particular, 400 mg/kg EAP exhibited favorable inhibitory activities on DEXA-induced decreases in mean muscle fiber diameters and increases in collagen fiber-occupied regions in muscle bundles, which were comparable with the effects of oxymetholone (Table VIII and Fig. 7).