Effects of Fish Meat-derived Peptide and Dipeptides on Dexamethasone-induced Fatigue in Mice

In patients with inflammatory diseases, exogenous glucocorticoids have become the most common cause of drug-induced muscle wasting. In this study, we showed that isoleucine-arginine (IR) and arginineisoleucine (RI) are the main dipeptides with antioxidant activity in fish meat-derived peptide extract (FMDP). To investigate the anti-fatigue effect of FMDP and the two dipeptides (IR or RI), dexamethasone (DEX)-treated mice performed a weighted forced swimming test. Despite no change in body weight, the shortened swim time after DEX administration returned to baseline levels following the administration of FMDP, IR, and RI. However, the swim time of naive mice cannot be extended with the administration of FMDP, IR, or RI. Our data suggest that FMDP, IR, and RI may have beneficial effects on DEX-induced fatigue in mice. Nevertheless, further research is required to determine the mechanism through which FMDP reduces fatigue.


Introduction
According to the data collated by the World Health Organization (WHO) in 2018, Japan has one of the longest lifespans in the world [1]. The aging Japanese population has become a serious problem due to the growing prevalence of lifestyle-related diseases such as cancer, diabetes, and hypertension. According to a report by the Ministry of Health, Labour, more than 10 million people suffered from hypertension in Japan prior to the year 2014.
Corticosteroids (glucocorticoids) are hormones that are secreted from the adrenal cortex and are responsible for homeostasis. Since corticosteroids have strong antiinflammatory and immunosuppressive effects, they are widely used for the treatment of various inflammatory and autoimmune diseases. More than 60 years they were first incorporated into clinical applications, the details of the molecular mechanisms behind the action of corticosteroids are still unknown. Furthermore, side effects often become a problem during treatment with corticosteroids, which is why there are continued efforts to separate the therapeutic actions from the side. Although glucocorticoids are highly effective, some of the adverse effects they may produce include hyperglycemia, decreased immune function, and skeletal muscle weakness [7]. Administering high doses of glucocorticoids also causes muscular atrophy in humans and animals [8,9]. With the increasing use of glucocorticoids to treat inflammatory diseases, exogenous glucocorticoids have become the most common cause of drug-induced muscle wasting, which is a clinical problem [10]. Recently, Huang et al indicated that dexamethasone (DEX) elevates oxidative stress markers, protein carbonyl levels and malondialdehyde content (lipid peroxidation) in skeletal muscle which resulted in mitochondrial dysfunction [11].
Marine fish are often consumed as a source of food, and many potential nutraceutical and medicinal products have been found in a large number of marine fish species. Consequently, muscle proteins, peptides, collagen, gelatin, oil, and bones from fish have yielded bioactive compounds. Certain peptides, such as imidazole, have an anti-fatigue effect. Imidazole is the generic name for a dipeptide containing carnosine and anserine. It exists within the skeletal muscles of organisms that move for a long time such as migratory birds, bonito, tuna, and other migratory fish [12]. Therefore, imidazole dipeptide has promise as a treatment for fatigue. To identify new bioactive compounds against steroid-induced fatigue, we examined the effects of fish meat-derived peptide extract (FMDP) in this study.

Fractionation of the Fish Meat Sample
The FMDP (1.0 g) was dissolved in water (100 mL) and centrifuged at 5,000 x g for 5 min. The supernatant was purified with hexanes (100 mL  2) and ethyl acetate (100 mL  2) to remove lipids. The water phase was evaporated prior to fractionation with high-performance liquid chromatography (HPLC) fraction. FMDP was provided as an in-kind gift from Suzuhiro Co. Ltd.
HPLC was performed using a Shimadzu 20A series HPLC system, which consisted of a pump, autoinjector, column oven, UV detector, and a fraction collector. The water phase fraction was separated using an Inertsil ODS 3 column (150  4.6 mm, 5 m) at a flow rate of 0.5 mL/min and a constant temperature of 40°C. The chromatography was performed with a solvent gradient using 0.1% aqueous formic acid (solvent A) and methanol containing 0.1% formic acid (solvent B). The gradient started with 5% solvent B, was increased to 20% within 10 min, increased to 90% for 1 min, was kept 3 min to wash a column, decreased 5% within 1 min, and equilibrated for 5 min.

Electron Spin Resonance Measurement of Hydroxyl Radical
The analysis of hydroxyl radicals (•OH) was carried out using an electron spin resonance (ESR) spectrometer (JES-RE1X, JEOL Co., Tokyo, Japan). The ESR spectrum was measured using the following parameters: a microwave frequency of 9.43 GHz, a magnetic field of 335.5 ± 5 mT, a power level of 9 mW, a modulation of 100 kHz, a time constant of 0.03 s, and a sweep time of 30 s. The spectra of the samples were scanned to record the signal intensities (peak-to-peak heights).

Animals
Male ICR mice (6 weeks old, 20-23 g) were used for all experiments. All procedures were approved by the Animal Care Committee at Hoshi University (Tokyo, Japan).

Treatment Protocol
The following treatments were administered orally at Days 0,1, and 2: 100 and 300 mg/kg FMDP, 1 and 3 mg/kg dipeptide (arginine-isoleucine; RI or isoleucine-arginine; IR) or the vehicle control (0.5% carboxymethyl cellulose; CMC). Naï ve mice did not receive any treatment. Mice were given a single subcutaneous (s.c.) injection of water-soluble DEX (15 mg/kg, Sigma Aldrich) once a day for three days, and saline was used as the vehicle control. On Days 0, 1, and 2, the 300 mg/kg FMDP, 3 mg/kg dipeptide (arginine-isoleucine; RI or isoleucine-arginine; IR) or the vehicle control (saline) were administered orally 15 min prior to DEX treatment.

Weighted Forced Swimming Test
Sixty minutes after treatment with FMDP, RI, and IR on Day 0 and Day 2, the mice were placed individually in a swimming pool filled with water (24 ± 1°C) to a depth of 15 cm with a lead sheath (3% of the mouse's body weight) attached to the root of the tail. The swim time was recorded as the moment when the physical strength of the mouse was exhausted and it could not swim to the surface.

Statistical Analysis
Data are expressed as mean ± SEM and statistical analyses were performed using GraphPad Prism 5 Statistical Software program (GraphPad Software for Science, San Diego, CA). Groups were analyzed via analysis of variance followed by Bonferroni and Dunn post hoc tests. A P value less than 0.05 was considered statistically significant.

Fractionation of FMDP by HPLC
Fish meat is contained in several types of amino acids and dipeptides [13]. The fish meat was purified with hexanes and ethyl acetate to remove lipids before the fish meat was fractionated using revised phase HPLC for the antioxidant activity assay. Each prepared sample was assessed for antioxidant activity via ESR. Although the organic phase fraction (FP-2 Hex and FP-3 EA) did not have antioxidant activity, the water phase (FP-5) had a strong antioxidant activity (Figure 1). Purified fish meat samples (FP-5) were analyzed using MS/MS and the spectra from the samples are shown in Figure 2. We obtained a mass spectrum of 278.4 as a protonated ion. We estimated RI or IR from the mass spectrum and fragmented ion. FP-5 was fractionated using HPLC. These HPLC fractional samples were assessed for antioxidant activity (Figure 3A), and antioxidant activity was detected in Fractions 1, 2, and 5. We obtained mass spectra for all of the fractions ( Figure 3B). We assumed that Fractions 1 and 2 contained RI or IR because the protonated ion was 288.3 and fragment ions were 115.9 (Ile-H 2 O) and 174.9 (Arg), respectively.  Quantitative analysis of RI and IR was performed using hydrophilic interaction chromatography with tandem mass spectrometry (HILIC/MS/MS). We decided RI and IR peak using those standard solutions.

Effects of FMDP, IR, and RI on the Body Weights of Mice
The body weights were not changed after treatment of 100, 300 mg/kg FMDP, 1 and 3 mg/kg IR and RI compared with vehicle treatment ( Figure 4A and Figure 4B). However, body weight was decreased after treatment with 15 mg/kg DEX. The DEX-induced loss of body weight was not changed after treatment with FMDP, IR, and RI ( Figure 4C).
We performed a weighted forced swimming test to investigate the anti-fatigue effects of FMDP, IR, and RI. The ratio of the swim time on Day 2 divided by the swim time of Day 0 was used as an index of fatigue. The swim time of normal mice was not affected by the administration of FMDP, IR, or RI compared to the vehicle-treated group. (Figure 5A and Figure 5B). However, the administration of DEX once per day on Days 0-2 significantly reduced the swim time on Day 2. The DEX-induced decrease in swim time was significantly abolished following the administration of FMDP. Furthermore, IR or RI treatment produced a tendency toward increased swim time after the DEX-induced decrease in swim time ( Figure 5C).

Discussion
Although many aspects of fatigue have been studied for a long time, no appropriate method has been established for the optimal and objective assessment of fatigue. The forced swim test is often used in mouse studies to measure fatigue [14][15][16]. In this study, we hypothesized that IR and RI are the peptides with the highest antioxidant activity in FMDP. However, the therapeutic mechanisms of IR and RI dipeptides on reducing fatigue are still unclear. In the present study, mice swam to fatigue, and the change in swim time after the administration of FMDP and dipeptides (IR and RI) was evaluated. This study suggests that reduction in swim time that is observed after DEX administration may be recovered by administration of FMDP, IR, and RI. However, these substances cannot increase the swim time of naive mice.
During DEX-induced muscular atrophy, there is an increase in muscle degradation and an inhibition of muscle synthesis, which leads to an atrophic state of the muscle tissue [17]. By suppressing Akt phosphorylation, mTORC1 is suppressed, and downstream proteins such as P70S6K and elf4E are suppressed, which reduces overall protein synthesis. [18,19,20]. The degradation process is triggered by increased transcription of REDD1 and REDD2 (regulated in development and DNA damage responses 1/2), which increase the activity of the myostatin/Smad2/3 pathway [17]. REDD1/2 are related to the stress response and are activated by glucocorticoid administration and increased ROS generation [21,22,23]. However, oxidative stress mainly promotes via the activation of p38 mitogen-activated protein kinase, which subsequently induces the expression of atrogin-1, MuRF1 and the autophagy-lysosome system [24,25]. Moreover, it also has been reported that DEX-induced muscle atrophy is partially caused by mitochondrial dysfunction mediated by the PGC-1α/TFAM and PGC-1α/mfn2 signaling pathways [11]. The mechanism through which FMDP suppresses DEX-induced fatigue is not clarified in this study. Therefore, further research is necessary to identify this mechanism.

Conclusions
Our data suggest that FMDP, IR, and RI may help reduce on DEX-induced fatigue. However, comprehensive chemical and pharmacological research is required to determine the mechanism through which FMDP affects DEX-induced fatigue.