**3. Discussion**

The results of the present study indicated that FCP derived from tilapia scales exhibited antioxidant and cytoprotective effects on mouse TECs against oxidative stress provoked by cisplatin exposure. In addition, our findings showed increased cellular viability accompanied by FCP-mediated antioxidant activity based on a diminished level of ROS in the cytosol. Furthermore, this study also provides the first molecular evidence for elucidating the function of FCP serving as a cytoprotective agen<sup>t</sup> against TEC damage by cisplatin. Thus, it is proposed that FCP may offer protective effects on TECs against cytotoxic and oxidative stress-induced cellular injury caused by various types of noxious stimuli.

Cisplatin is a highly reactive molecule that exerts its cytotoxic effects mainly through the formation of covalent DNA adducts [32]. In addition, it stimulates the production of intracellular ROS in several types of cells, including hepatocytes [33], pulmonary alveolar cells [34], renal proximal tubule epithelial cells [35], and intestinal epithelial cells [36]. Our findings are consistent with previous studies showing that treatment with antioxidants alleviates the toxic effects of cisplatin, indicating an essential role of oxidative stress in the pathogenesis of cisplatin-induced cell injury in several different types of organs [33,34].

Marine organisms are important sources of bioactive compounds with potential therapeutic applications. In particular, fish collagen-derived peptides are of considerable interest and have drawn grea<sup>t</sup> attention recently due to their bioactive functions [37]. Fish collagen has been shown to exhibit microbicidal, anti-inflammatory and anti-skin-aging activities, as

well as wound healing and tissue regeneration [37–41]. Despite much having been learnt about the diverse bioactivities of FCP on multiple cell types [42,43]; there is a paucity of information on the biological effects of FCP on TECs. Antioxidant properties of peptides from the diverse sources of fish collagen, such as skin from cod, hoki, and pollock have been demonstrated in many different cell types, such as liver cells [44,45], fibroblasts [46], macrophages [47,48], and keratinocytes [49]. In addition, it was also revealed that FCP enhances the viability of human lung fibroblasts damaged by oxygen radicals [46]. In accordance with these results, the present study showed that FCP from tilapia scales has potent antioxidative and cytoprotective effects on TECs.

Antioxidants, widely used as ingredients in dietary supplements to improve health in sectors of the food and beverage manufacturing industry, have been studied for their potential in the prevention or treatment of several human diseases, such as cardiovascular diseases, diabetes, metabolic syndrome, neurodegenerative disorders, cancer and agerelated diseases [50,51]. In addition, they are also used as food preservatives for preventing lipid oxidation. Although synthetic antioxidants, such as butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) have been extensively used due to their high stability, low costs, and wide availability, health risks including carcinogenicity, are of grea<sup>t</sup> concern [52]. Thus, there is a growing trend toward replacing synthetic antioxidants with natural antioxidants in the food processing industry [53,54].

It is well documented that ROS generated endogenously or in response to environmental stress have long been implicated in cellular injury, which causes cell death, especially triggered by the dysregulation of the pro- or anti-apoptotic pathways, and tissue damage leading to the development of many diseases [55]. The present study demonstrated that FCP acts as a potent suppressor of TEC apoptosis induced by cisplatin treatment by promoting Bcl-2 and Bcl-xL expression and inhibiting Bad and Bax expression and cytochrome-c release. Taken together, these findings indicate that the amelioration of cisplatin-induced cytotoxicity by FCP in TECs is mediated by their antioxidant and anti-apoptotic properties. The discovery of the protective mechanisms of FCP for repairing cellular injuries induced by oxidative stress and activation of apoptotic cell death pathway in human TECs would advocate the use of FCP for the prevention and treatment of many clinical conditions linked to excessive ROS generation and perturbation in the apoptotic balance in TECs. This is particularly important because oxidative- or cytotoxic stress-mediated injury in TECs can be a significant problem in many clinical settings, and linked to the induction of acute thymic involution, that may cause compromised thymopoietic capacity in TECs, leading to a severe and clinically significant immune deficiency disorder or dysfunction of the adaptive immunity, and causing the body to be unable to generate appropriate immune responses against invading pathogens.

The present study also demonstrated that FCP can promote the proliferation of TECs. In agreemen<sup>t</sup> with our study, Liu and Sun [56], also observed the growth-promoting effect of tilapia FCP on rat bone marrow mesenchymal stem cells. In addition, our previous study suggested that nanofibrous scaffolds containing tilapia FCP contribute to the enhancement of mouse TEC proliferation [57]. Furthermore, Liu et al. [58] showed that bovine collagen peptide compounds promote the proliferation and differentiation of MC3T3-E1 preosteoblasts. These investigations, therefore, corroborate that tilapia FCP exhibit significant growth promotion properties in several types of cells.

MAPK signal transduction pathways are involved in the regulation of a wide variety of fundamental cellular processes, such as cell growth, differentiation, survival, apoptosis, migration, inflammation, and environmental stress responses [59]. To determine the role of the p38 MAPK, JNK, and ERK in cisplatin-induced ROS production and the signaling pathway in TECs, the expression levels of p38 MAPK, JNK, and ERK were analyzed by DCFH-DA, cell proliferation, and western blot assays after treatment with NAC, SB203580, SP600125, U0126, and FCP. Consequently, the cisplatin-elicited p38 MAPK, JNK, and ERK activation was abolished by SB203580, SP600125, and U0126 as well as FCP and NAC, suggesting that cisplatin-induced oxidative stress injury in TECs is mediated by p38 MAPK, JNK and ERK and that FCP, similarly to NAC, notably ameliorates cisplatin-induced oxidative stress in TECs by blocking p38 MAPK, JNK, and ERK activation. In addition, the cisplatin-induced cytotoxic responses were also significantly blocked by SB203580, SP600125, and U0126 as well as FCP. Taken together, these data indicate that FCP plays a critical role in protecting various cytotoxic and oxidative stresses in TECs by repressing the activation of MAPK signal transduction pathways.

### **4. Materials and Methods**

### *4.1. Cell Culture and Reagents*

Mouse thymic cortical epithelial reticular cells (1308.1) were provided by Dr. Barbara B. Knowles (The Jackson Laboratory, Bar Harbor, ME, USA). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Hyclone, GE Healthcare Life Sciences, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS), 100 IU mL−<sup>1</sup> penicillin, and 100 mg mL−<sup>1</sup> streptomycin (all from Gibco, Thermo Fisher Scientific, Waltham, MA, USA) in a humidified atmosphere of 5% CO2 at 37 ◦C. Subconfluent cells were harvested with trypsin-EDTA and used for further experiments. Media were replaced every second day.

Cisplatin, <sup>2</sup>,7-dichlorodihydrofluorescein diacetate (DCFH-DA), N-acetyl-L-cysteine (NAC), <sup>4</sup>,6-diamidino-2-phenylindole (DAPI), and bicinchoninic acid (BCA) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against ERK, phospho-ERK (*p*-ERK), JNK, phospho-JNK (p-JNK), p38 MAPK, phospho-p38 MAPK (p-p38 MAPK), cytochrome-c, and cyclin D1 were supplied by Cell Signaling Technology (Cambridge, MA, USA). The antibodies against Bcl-2, Bcl-xL, Bax, Bad, and CDK1 were obtained from Abcam (Cambridge, UK). Additionally, an antibody against β-actin was bought from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The p-p38 MAPK inhibitor (SB203580), p-JNK/MAPK inhibitor (SP600125), and p-ERK/MAPK inhibitor (U0126) were purchased by Tocris Bioscience (Ellisville, MO, USA). FCP extracted from tilapia were provided by Geltech (Busan, Korea), and their physicochemical properties were described in our previous study [49]. All other reagents and compounds used were supplied from Sigma-Aldrich.

### *4.2. Cell Viability Assay*

After TECs (8 × 10<sup>3</sup> cells/well) in 96-well flat-bottom culture plates (SPL Life Sciences, Pocheon, Korea) were treated with the indicated doses of FCP for 24 h with or without cisplatin. The cell viability was determined using the colorimetric WST-1 conversion assay (EZ-Cytox assay kit, Daeil Lab Service, Seoul, Korea). A WST-1 reagen<sup>t</sup> (total 10 μL) was added to each well, and cells were incubated for 2 h in a humidified incubator at 37 ◦C under 5% CO2. The absorbance of the formazan dye, generated by the reaction of dehydrogenase with WST-1 in the metabolically active cells, was measured using a microplate reader (Tecan, Männedorf, Switzerland) at 450 nm according to the manufacturer's instructions, and the percent cell viability was calculated. The experiments were performed in triplicate.

### *4.3. Measurement of ROS*

The effect of FCP on the cisplatin-induced generation of ROS in TECs was detected by DCFH-DA, a ROS-sensitive fluorescent probe, under a fluorescent microscope. Cellpermeable DCFH-DA is non-fluorescent, but in the presence of ROS, when this dye is oxidized, it is converted to a highly fluorescent 2,7-dichlorofluorescein (DCF) [60]. TECs (1 × 10<sup>5</sup> cells/well) in 6-well culture plates were treated with 0.08% FCP for 24 h before treatment with cisplatin (10 μM) for 24 h. After removing the medium from wells, the cells were washed with phosphate buffered saline and then incubated with 10 μM DCFH-DA in fresh serum-free medium for 30 to 40 min in a humidified incubator at 37 ◦C with 5% CO2 under dark conditions. The labeled cells were observed with an epi-fluorescence microscope (BX50, Olympus, Tokyo, Japan). Photomicrographs were acquired digitally at 1360 × 1024 pixel resolution with an Olympus DP70 digital camera. Furthermore, the DCF fluorescence was measured using a fluorescent microplate reader (SpectraMax M2e, BioTek, Winooski, VT, USA) at 495–529 nm. To minimize the possible photo-oxidation of

the probe and or photo-reduction of DCF, the plates were covered with aluminum foil to shield the probe from light.

### *4.4. Western Blot Analysis*

To determine protein expression levels, TECs (8 × 10<sup>5</sup> cells/dish), after reaching 70–80% confluency in 60 mm culture dishes (SPL Life sciences), were treated with 0.08% FCP for 24 h before treatment with cisplatin (10 μM) for 24 h. Cells from each set of experiments were harvested and washed twice in cold Tris-buffered saline (TBS, 20 mM Tris-HCl, 150 mM NaCl, pH 7.4). For the western blot analysis, cells were lysed in 100 μL RIPA cell lysis buffer with EDTA (GenDEPOT, Barker, TX, USA) containing a protease inhibitor mixture (Roche, Basel, Switzerland). Samples were kept on ice for 30 min, vortexing briefly (15 s) every 2–3 min. Then, the lysates were centrifuged at 14,000 RPM for 30 min at 4 ◦C, and the protein concentration was measured using a BCA protein assay (Sigma-Aldrich). Equal amounts of protein samples were heated for 10 min at 70 ◦C in Bolt LDS sample buffer (Invitrogen, Waltham, MA, USA) and separated by 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE, Invitrogen) at 200 V for 25 min, using a Mini-Protean III system (Bio-Rad, Hercules, CA, USA). Proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (GE Healthcare Life science) at 20 V for 1 h. The nonspecific binding was blocked with 3% bovine serum albumin (BSA) in TBS buffer containing 0.1% Tween 20 (TBST buffer), incubated with the indicated primary antibodies at a dilution of 1:500–1:2000 with 5% BSA in TBST overnight at 4 ◦C with anti-p38 MAPK, anti-p-p38 MAPK, anti-JNK, anti-p-JNK, anti-ERK, anti-p-ERK, anti-Bax, anti-cytochrome-c, anti-Bcl-2, anti-Bcl-xL, anti-Bad, anti-cyclin D1, anti-CDK1, and anti-βactin (Supplementary Table S1).

On the following day, the membrane was washed with TBST buffer thrice and incubated with secondary antibodies, namely, anti-rabbit IgG HRP conjugate (Cell Signaling Technology) and anti-mouse IgG HRP conjugate (Cell Signaling Technology), at a dilution of 1:10,000 with 3% BSA in TBST for 1 h at room temperature. Subsequently, the membrane was washed thrice with TBST. Immunoreactivity was detected with enhanced chemiluminescence (ECL, Super Signal West Pico Chemiluminescent Substrate kit, Pierce, Rockford, IL, USA) according to the manufacturer's instructions. Images were captured and quantified using a LAS-3000 imaging system (Fujifilm, Tokyo, Japan).

### *4.5. Statistical Analysis*

The results of the present study were expressed as the mean ± SD under all conditions. Statistical analysis was performed using a two-tailed Student's *t*-test. Statistically significant differences were considered at *p* < 0.05.
