Fish Collagen Peptides Protect against Cisplatin-Induced Cytotoxicity and Oxidative Injury by Inhibiting MAPK Signaling Pathways in Mouse Thymic Epithelial Cells

Thymic epithelial cells (TECs) account for the most abundant and dominant stromal component of the thymus, where T cells mature. Oxidative- or cytotoxic-stress associated injury in TECs, a significant and common problem in many clinical settings, may cause a compromised thymopoietic capacity of TECs, resulting in clinically significant immune deficiency disorders or impairment in the adaptive immune response in the body. The present study demonstrated that fish collagen peptides (FCP) increase cell viability, reduce intracellular levels of reactive oxygen species (ROS), and impede apoptosis by repressing the expression of Bax and Bad and the release of cytochrome c, and by upregulating the expression of Bcl-2 and Bcl-xL in cisplatin-treated TECs. These inhibitory effects of FCP on TEC damage occur via the suppression of ROS generation and MAPK (p38 MAPK, JNK, and ERK) activity. Taken together, our data suggest that FCP can be used as a promising protective agent against cytotoxic insults- or ROS-mediated TEC injury. Furthermore, our findings provide new insights into a therapeutic approach for the future application of FCP in the prevention and treatment of various types of oxidative- or cytotoxic stress-related cell injury in TECs as well as age-related or acute thymus involution.


Introduction
The thymus is the primary lymphoid organ composed of multiple cell types creating a unique microenvironment for producing immunocompetent T cells from bone marrowderived T cell progenitors/precursors, thereby playing a crucial role in the development of the host adaptive immune system. T cells are not only trained to recognize and eliminate foreign antigens during development in the thymus, but also to tolerate self-antigens through positive and negative selection. This thymic education of T cells is mainly orchestrated by thymic epithelial cells (TECs) that play a pivotal role in the multistep processes, including the homing and clonal expansion, survival, and maturation of the immature T cells [1].

FCP Attenuate Cisplatin-Induced ROS Generation
To measure changes in the cellular levels of ROS in response to cisplatin with or without FCP pretreatment, TECs were pretreated with 0.08% FCP for 24 h followed by cisplatin exposure at 10 µM for 24 h ( Figure 5A,B). Treatment with cisplatin significantly increased the ROS level as detected by fluorescence microscopy using the oxidant-sensing fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) by 34.4% (p < 0.05) compared with the control (Figure 5A,B). Results are presented as the means ± SD of three independent experiments. ** p < 0.01, *** p < 0.001 vs. the control. # p < 0.05, ## p < 0.01, ### p < 0.001 vs. the cisplatin alonetreated group.

FCP Attenuate Cisplatin-Induced ROS Generation
To measure changes in the cellular levels of ROS in response to cisplatin with or without FCP pretreatment, TECs were pretreated with 0.08% FCP for 24 h followed by cisplatin exposure at 10 µM for 24 h ( Figure 5A,B). Treatment with cisplatin significantly increased the ROS level as detected by fluorescence microscopy using the oxidant-sensing fluorescent probe 2 ,7 -dichlorodihydrofluorescein diacetate (DCFH-DA) by 34.4% (p < 0.05) compared with the control ( Figure 5A,B).
Pretreatment with FCP showed a significant decrease in the cisplatin-induced ROS release by 72.1% (p < 0.01) compared with the cisplatin alone-treated group, indicating its restorative effect on the endogenous antioxidant defense mechanism impaired by cisplatin ( Figure 5A,B). Furthermore, treatment with N-acetyl cysteine (NAC), a common ROS scavenger, at a concentration of 5 mM for 2 h, significantly reduced the cisplatin-enhanced ROS level in TECs, by 69.2% (p < 0.001) compared with the cisplatin alone-treated group ( Figure 5A Pretreatment with FCP showed a significant decrease in the cisplatin-induced ROS release by 72.1% (p < 0.01) compared with the cisplatin alone-treated group, indicating its restorative effect on the endogenous antioxidant defense mechanism impaired by cisplatin ( Figure 5A,B). Furthermore, treatment with N-acetyl cysteine (NAC), a common ROS scavenger, at a concentration of 5 mM for 2 h, significantly reduced the cisplatin-enhanced ROS level in TECs, by 69.2% (p < 0.001) compared with the cisplatin alone-treated group ( Figure 5A,B). These findings also indicate that the inhibitory action of FCP on cisplatininduced cytotoxicity in TECs is attributed to its antioxidant effect.

FCP Alleviate Cisplatin-Induced TEC Cytotoxicity through Suppression of MAPK Pathway
MAPK cascades are key signaling pathways that regulate various cellular processes, including stress and inflammatory responses, as well as cell proliferation, differentiation, apoptosis, and motility under both normal and pathological conditions [29,30]. Since cisplatin-induced cell death has been shown to be dependent on the MAPK pathways in several cell types [31], we first tested if cisplatin could activate this pathway in TECs. The results of this study revealed an increase in the expression of p-p38 MAPK, p-JNK, and p-ERK by 57.9% (p < 0.001), 16.8% (p < 0.001), and 21.8% (p < 0.001), in the cisplatin alonetreated group, respectively, than in the control, whereas the expression of total p38 MAPK, JNK, and ERK levels remained unaltered, indicating the activation of p38 MAPK, JNK, and ERK pathways after cisplatin treatment in TECs ( Figure 6). However, these elevated levels of p-p38 MAPK, p-JNK and p-ERK were attenuated by the pretreatment with 0.08% FCP for 24 h, by 37.3% (p < 0.01), 15.3% (p < 0.001), and 58.5% (p < 0.001), respectively, compared with the cisplatin alone-treated group ( Figure 6). The increased ROS level induced by 10 µM cisplatin treatment was significantly attenuated by pretreatment with 0.08% FCP for 24 h and NAC for 2 h. Quantification of staining intensity was measured by ImageJ software. Results are presented as the means ± SD of three independent experiments. * p < 0.05, *** p < 0.001 vs. the control. ## p < 0.01, ### p < 0.001 vs. the cisplatin alone-treated group. Scale bar = 50 µm.

FCP Alleviate Cisplatin-Induced TEC Cytotoxicity through Suppression of MAPK Pathway
MAPK cascades are key signaling pathways that regulate various cellular processes, including stress and inflammatory responses, as well as cell proliferation, differentiation, apoptosis, and motility under both normal and pathological conditions [29,30]. Since cisplatin-induced cell death has been shown to be dependent on the MAPK pathways in several cell types [31], we first tested if cisplatin could activate this pathway in TECs. The results of this study revealed an increase in the expression of p-p38 MAPK, p-JNK, and p-ERK by 57.9% (p < 0.001), 16.8% (p < 0.001), and 21.8% (p < 0.001), in the cisplatin alone-treated group, respectively, than in the control, whereas the expression of total p38 MAPK, JNK, and ERK levels remained unaltered, indicating the activation of p38 MAPK, JNK, and ERK pathways after cisplatin treatment in TECs ( Figure 6). However, these elevated levels of p-p38 MAPK, p-JNK and p-ERK were attenuated by the pretreatment with 0.08% FCP for 24 h, by 37.3% (p < 0.01), 15.3% (p < 0.001), and 58.5% (p < 0.001), respectively, compared with the cisplatin alone-treated group ( Figure 6).
To further explore the role of MAPK pathways in cisplatin-induced cell cytotoxicity, the cells were treated with a selective p38 MAPK inhibitor (SB203580), JNK inhibitor (SP600125), or ERK inhibitor (U0126). Analysis of the cell viability by WST-1 assay after TECs were treated with 0.08% FCP, 10 µM SB203580, SP600125, and U0126 for 24 h, followed by treatment with or without 10 µM cisplatin for 24 h, showed that FCP pretreatment caused a significant reduction of cisplatin-induced cytotoxicity, similar to all MAPK inhibitor pretreatments, whereas SB203580, SP600125 or U0126 alone did not affect cell viability (Figure 7).
Confirmation of the effect of FCP treatment on the cisplatin-induced expression of MAPK was performed using western blot analysis after TECs were treated with 0.08% FCP and 10 µM SB203580, SP600125, and U0126 for 24 h, followed by treatment with or without 10 µM cisplatin for 24 h. Notably, we observed that FCP pretreatment potently repressed To further explore the role of MAPK pathways in cisplatin-induced cell cytotoxicity, the cells were treated with a selective p38 MAPK inhibitor (SB203580), JNK inhibitor (SP600125), or ERK inhibitor (U0126). Analysis of the cell viability by WST-1 assay after TECs were treated with 0.08% FCP, 10 µM SB203580, SP600125, and U0126 for 24 h, followed by treatment with or without 10 µM cisplatin for 24 h, showed that FCP pretreatment caused a significant reduction of cisplatin-induced cytotoxicity, similar to all MAPK inhibitor pretreatments, whereas SB203580, SP600125 or U0126 alone did not affect cell viability (Figure 7). Confirmation of the effect of FCP treatment on the cisplatin-induced expression of MAPK was performed using western blot analysis after TECs were treated with 0.08% FCP and 10 µM SB203580, SP600125, and U0126 for 24 h, followed by treatment with or without 10 µM cisplatin for 24 h. Notably, we observed that FCP pretreatment potently  To further explore the role of MAPK pathways in cisplatin-induced cell cytotoxicity, the cells were treated with a selective p38 MAPK inhibitor (SB203580), JNK inhibitor (SP600125), or ERK inhibitor (U0126). Analysis of the cell viability by WST-1 assay after TECs were treated with 0.08% FCP, 10 µM SB203580, SP600125, and U0126 for 24 h, followed by treatment with or without 10 µM cisplatin for 24 h, showed that FCP pretreatment caused a significant reduction of cisplatin-induced cytotoxicity, similar to all MAPK inhibitor pretreatments, whereas SB203580, SP600125 or U0126 alone did not affect cell viability (Figure 7). Confirmation of the effect of FCP treatment on the cisplatin-induced expression of MAPK was performed using western blot analysis after TECs were treated with 0.08% FCP and 10 µM SB203580, SP600125, and U0126 for 24 h, followed by treatment with or without 10 µM cisplatin for 24 h. Notably, we observed that FCP pretreatment potently Together, these results indicate that FCP acts as a potent inhibitor of MAPK pathways, which exerts a protective effect against cisplatin-induced cytotoxicity via suppression of p38 MAPK, JNK, and ERK pathway in TECs.
Mar. Drugs 2022, 20, x FOR PEER REVIEW 9 of 17 repressed the cisplatin-induced upregulated expression of p-p38 MAPK, p-JNK, and p-ERK, as was the case with MAPK inhibitor pretreatment ( Figure 8A-C). Together, these results indicate that FCP acts as a potent inhibitor of MAPK pathways, which exerts a protective effect against cisplatin-induced cytotoxicity via suppression of p38 MAPK, JNK, and ERK pathway in TECs.

FCP Prevent Cisplatin-Induced ROS Generation by Inhibition of MAPK Signaling
To investigate whether p38 MAPK, JNK and ERK activation is associated with cisplatin-induced oxidative cell injury in TECs, we examined the effect of p38 MAPK, JNK and ERK inhibition on the cisplatin-induced ROS production. We also assessed the protective effect of FCP on cisplatin-induced ROS release in TECs using the DCFH-DA assay. As shown in Figure 9A-C, the exposure of TECs to 10 µM cisplatin for 24 h caused an increase in ROS level, by 29.5% (p < 0.01), 34% (p < 0.001) and 33.8% (p < 0.001) versus the control. However, the enhanced level of ROS induced by cisplatin treatment was significantly declined by the treatment with 10 µM SB203580, 10 µM SP600125 or 10 µM U0126 for 24 h by 25.2% (p < 0.01), 46.3% (p < 0.01), and 29.6% (p < 0.05), respectively, and with 0.08% FCP for 24 h by 37.8% (p < 0.01), 79% (p < 0.001) and 50.3% (p < 0.05), respectively ( Figure 9A-C). jury in TECs, we investigated the effect of NAC on the cisplatin-induced expression of p-p38 MAPK, p-JNK, and p-ERK in TECs. Pretreatment of cells with FCP for 24 h or NAC for 2 h prior to cisplatin treatment completely abolished the cisplatin-induced phosphorylation of p38 MAPK, JNK, and ERK ( Figure 10). These findings indicate that p38 MAPK, JNK and ERK activation is important in cisplatin-induced cellular oxidative stress, and FCP exhibits a strong protective effect against cisplatin-induced oxidative cell injury via the suppression of p38 MAPK, JNK, and ERK pathways in TECs. Results are presented as the means ± SD of three independent experiments. * p < 0.01, ** p < 0.01, *** p < 0.001 vs. the control. # p < 0.05, ### p < 0.001 vs. the cisplatin alone-treated group.

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 Figure 10. Inhibitory effects of FCP and NAC on cisplatin-induced activation of p38 MAPK, JNK, and ERK signaling pathways in TECs. The expression of p38 MAPK, JNK, and ERK was increased in cisplatin-treated TECs, as assessed by Western blot analysis (A). The pretreatment of TECs with FCP or NAC blocked the cisplatin-induced phosphorylation of p38 MAPK, JNK, and ERK. Bar graphs depict relative densitometry quantitation of each protein normalized to β-actin (B). Results are presented as the means ± SD of three independent experiments. * p < 0.01, ** p < 0.01, *** p < 0.001 vs. the control. # p < 0.05, ### p < 0.001 vs. the cisplatin alone-treated group.

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 agent 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 great 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][38][39][40][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 great 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 agreement 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.

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 −1 penicillin, and 100 mg mL −1 streptomycin (all from Gibco, Thermo Fisher Scientific, Waltham, MA, USA) in a humidified atmosphere of 5% CO 2 at 37 • C. Subconfluent cells were harvested with trypsin-EDTA and used for further experiments. Media were replaced every second day.

Cell Viability Assay
After TECs (8 × 10 3 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 reagent (total 10 µL) was added to each well, and cells were incubated for 2 h in a humidified incubator at 37 • C under 5% CO 2 . 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.

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 5 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% CO 2 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.
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).

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.

Conclusions
The findings of the present study demonstrate for the first time that FCP stimulates proliferation, and ameliorates cisplatin-induced cytotoxicity and oxidative stress in TECs. In addition, it was shown that the inhibitory effects of FCP on cytotoxicity are likely associated with suppression of the ROS and MAPK (p38 MAPK, JNK, and ERK) signaling pathways. Therefore, these results suggest that FCP may be a promising protective agent in TEC injury induced by cytotoxicity and oxidative stress elaborated by chemotherapeutic drugs, such as cisplatin and other cytotoxic agents. Furthermore, the data of the current study may provide new insights into the therapeutic approach for the future application of FCP in the prevention and treatment of a variety of the cytotoxic and oxidative stress-mediated injuries in TECs as well as acute or age-related thymic involution.