Antifatigue Effects of Antrodia cinnamomea Cultured Mycelium via Modulation of Oxidative Stress Signaling in a Mouse Model

Antrodia cinnamomea, a folk medicinal mushroom, has numerous biological effects. In this study, we aim to assess whether the antifatigue effects of A. cinnamomea mycelia (AC) and its underlying mechanisms are related to oxidative stress signaling using behavioral mouse models and biochemical indices detection. Mice were orally treated with AC at doses of 0.1, 0.3, and 0.9 g/kg for three weeks. AC had no effect on the spontaneous activities of mice indicating its safety on central nervous system. Furthermore, results obtained from weight-loaded forced swimming test, rotary rod test, and exhausted running test confirmed that AC significantly enhanced exercise tolerance of mice. Biochemical indices levels showed that these effects were closely correlated with inhibiting the depletion of glycogen and adenosine triphosphate stores, regulating oxidative stress-related parameters (superoxide dismutase, glutathione peroxidase, reactive oxygen species, and malondialdehyde) in serum, skeletal muscle, and liver of mice. Moreover, the effects of AC may be related with its regulation on the activations of AMP-activated protein kinase, protein kinase B, and mammalian target of rapamycin in liver and skeletal muscle of mice. Altogether, our data suggest that the antifatigue properties of AC may be one such modulation mechanism via oxidative stress-related signaling in mice.


Autonomic activities test.
Thirty minutes after AC administration, mice were placed individually in the autonomic activities instrument (ZZ-6, Chengdu Taimeng Science Technology Co., Ltd., Chengdu, China). Locomotor activities and enabled vertical movements, including jumping, horizontal movements and walking and running, were counted.

Weight-loaded forced swimming Test (FST). Thirty minutes after AC
administration, test was carried out with mice loaded with a lead block (10% of bodyweight) attached to their tails in water maintained at 25±2˚C. Mice loss coordinated movements and failure to swim to the surface within 10 sec were used to measure their exhaustion. The time of exhaustive swimming was recorded.

Rota-rod test (RRT).
Before the formal test, mice were trained twice on rota-rod at 15 rpm for 60 s to adapt to the instrument Chengdu Taimeng Science Technology Co.,Ltd.,Chengdu,China). And then, mice were placed inside a rota-rod spinning and allowed to run at speed of 20 rpm until they were exhausted and dropped from the rod. The total running time was recorded.

Exhaustive running test (ERT). Thirty minutes after AC administration, mice
were trained twice on the runway at 20 rpm for 5 min to adapt to the treadmill Chengdu Taimeng Science Technology Co.,Ltd.,Chengdu,China).
Exhaustion was determined by failuring to return to the runway within 15 sec and lossing dynamism of movements. The exhaustive running time was recorded.

Samples collection and biochemical indexes measurement
Thirty minutes after the final AC administration, half of mice (n=12/group; half male and female) were separately placed in the swimming pond (diameter 20 cm, depth 50 cm, and temperature 25±2°C) and swam for 60 min without loads. Another half of mice (n=12/group; half male and female) were not received any treatment during 60 min. Blood, liver and skeletal muscle were collected and immediately placed in -80°C.
Levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum, levels of glycogen in serum, liver and skeletal muscle were determined according to the protocol recommended by the commercial diagnostic kits purchased from Nanjing Jiancheng Institute of Biotechnology Co. Ltd. (Nanjing, China).

Western blot
Liver and skeletal muscle tissues were homogenized, lysed and measured total protein concentration by BCA protein assay kit (Merck Millipore, Germany). Samples containing 40 μg of total protein were loaded and separated by 12% SDS-PAGE (Bio-Rad, USA), transferred to nitrocellulose membrane (0.45 µm; Millipore, USA), and blocked for more than 4 h with 5% bovine serum albumin (BSA)/TBS buffer.
Immunoblotting was detected using primary antibodies including phosphor (P)-Akt

Statistical analysis
Data were expressed as mean ±S.E.M.. A one-way analysis of variance (ANOVA) was used to detect statistical significance followed by post-hoc multiple comparisons (Dunn's test) using SPSS 16.0 software (IBM corporation, Armonk, USA). P values less than 0.05 were considered significant.

Effects of AC on mouse exercise capacity
Behaviors were measured to investigate the effect of AC on mouse exercise capacities.
Compared to control mice, three-week AC treatment brought no significant differences in numbers of standing and activities indicating its safety on mice (P > 0.05, F = 0.03-1.47; Table.1s). FST, RRT and ERT are classic rodent models to evaluate the capacity of antifatigue. AC at doses of 0.3 and 0.9 g/kg strongly enhanced exercise capacity of mice in ERT and FST (P < 0.05, Table.1s). 0.9 g/kg of AC prolonged over 35% of exhaustive time than that of control mice in ERT (53.9 min vs. 39.8 min; P < 0.01, F = 10.23; Table.1s). In FST, 0.3 and 0.9g/kg of AC resulted in over 66% enhancement on swimming time than that of control mice (261.5 s and 252.8 s vs. 152.4 s; P < 0.01; F = 10.85 and 11.15; Table.1s). The retention time in rotating exercise was significantly increased by AC at 0.1 g/kg (6.4 min vs. 3.9 min; P < 0.05; F = 5.09; Table.1s).

Effects of AC on liver function
The levels of ALT and AST in the serum are considered as a biochemical marker for assessing liver function, which were examined to explore effect of AC on hepatic function. Compared to non-excise mice, 60-min swimming failed to influence the levels of ALT and AST in serum (P > 0.05, F = 0.18-1; Table.2s). In mice without swimming, AC at 0.3 g/kg strongly reduced the levels of ALT (5.89 IU/L vs. 10.31 IU/L; P < 0.05, F = 5.63) and AST (18.27 IU/L vs. 29.83 IU/L; P < 0.05, F = 4.43) ( Table.2s). In mice with 60-min swimming, AC at 0.3 g/kg showed similar reductive effects on the serum levels of ALT (6.97 IU/L vs. 12.27 IU/L; P < 0.05, F = 6.5) and AST (18.77 IU/L vs. 28.74 IU/L; P < 0.05, F = 5.86) ( Table.2s).

Effects of AC on levels of glycogen
Depletion of glycogen is the primary factor in fatigue and exhaustion during exercise.