Neuroprotection of round scad (Decapterus maruadsi) hydrolysate in glutamate-damaged PC12 cells: Possible involved signaling pathways and potential bioactive peptides

Journal of Functional Foods 64 (2020) 103690

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Neuroprotection of round scad (Decapterus maruadsi) hydrolysate in glutamate-damaged PC12 cells: Possible involved signaling pathways and potential bioactive peptides

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Qi Zhanga,b, Guowan Sua,b, Tiantian Zhaoa,b, Baoguo Sunc, Lin Zhenga,b,⁎, Mouming Zhaoa,b,c,⁎ a

School of Food Science and Engineering, South China University of Technology, Guangzhou 510640, China Guangdong Food Green Processing and Nutrition Regulation Technologies Research Center, Guangzhou 510650, China c Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Technology & Business University, Beijing 100048, China b

ARTICLE INFO

ABSTRACT

Keywords: Neuroprotection Hydrolysate Oxidative stress Nrf2 Molecular docking

Neuroprotective effects of round scad hydrolysate (RSH) on neurotoxicity induced by glutamate in PC12 cells and the involved signaling pathways were explored in this study. In addition, the potential bioactive peptides in RSH were separated and identified by UPLC-QTOF-MS/MS. Results showed that pretreatment with RSH at 0.11.0 mg/mL could dose-dependently enhance cell viability, alleviate mitochondrial dysfunction and inhibit apoptosis in glutamate-damaged PC12 cells, indicating that RSH could exert neuroprotection against glutamate neurotoxicity. Several signaling pathways including Bax/Bcl-2-related apoptotic pathway, Nrf2-mediated oxidative stress response pathway and CREB-related neuronal survival pathway were involved in the neuroprotection. Furthermore, 23 peptides with potential neuroprotection were identified, and Tyr- and Trp-containing peptides might be potent contributors to the neuroprotection. Molecular docking studies indicated that these peptides such as PPW might directly bind to Keap1 to modulate nuclear factor-E2-related factor (Nrf2) pathway. Thus, RSH can be used as a potential neuroprotective ingredient for preventing neurodegenerative diseases.

1. Introduction Alzheimer's disease (AD), the most common neurodegenerative disease, is characterized by progressive cognitive dysfunction and memory impairments that ultimately result in incapacity and death in patients (Copple, Jaeschke, & Klaassen, 2010). The pathogenesis of AD is complex and many factors such as the amyloid β peptides, disorders of neurotransmitters and tau protein can influence neurodegeneration through oxidative stress, inflammation and synaptic dysfunction (Agostinho, Cunha, & Oliveira, 2010). Importantly, oxidative stress can result in neuronal damage, mitochondrial dysfunction and apoptosis, which eventually leads to cell death. Although current clinical drugs, such as acetylcholinesterase inhibitors and NMDA receptor antagonists, can alleviate cognition deficits, many of them have serious side effects, and no treatment is available to stop the progressive loss of neurons (Godyń, Jończyk, Panek, & Malawska, 2016). Therefore, studies for the discovery of novel safe agents responsible for neuroprotection and prevention of early neurodegeneration are still urgently needed. Recently, food-derived strategies have received increasing attention to protect against oxidative stress in neurodegenerative diseases.

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Diverse chemicals (e.g., polysaccharides (Olatunji et al., 2016), polyphenols and peptides (Wang et al., 2018) displayed neuroprotective actions. In response to cell death, several signaling pathways are activated such as mitogen-activated protein kinase (MAPK) and phosphoinositide 3 kinase (PI3k)/protein kinase B (Akt) pathway that are responsible for cell survival and proliferation (Kim & Choi, 2015; Zhong et al., 2018). Akt is a crucial survival factor that modulates multiple neurotrophins and growth factors to prevent inflammatory and apoptotic cellular injury (Zhao, Li, & Maiese, 2005). The PI3K/Akt signaling pathway could be regulated by multiple compounds, resulting in mitigating oxidative stress and exerting neuroprotective effects in cells and in vivo (Yoo, Lee, Sok, Ma, & Kim, 2017). Additionally, AD patients were observed with the alteration of antioxidant enzymes and glutathione levels as well as oxidation of lipids and protein (Ansari & Scheff, 2010). The impaired nuclear factor-E2-related factor (Nrf2) defense pathway is also closely linked to neurodegeneration (Lee, Song, Huh, Oh, & Kim, 2018). Under oxidative stress, reactive oxygen species (ROS) or inhibitors for Keap1-Nrf2 interaction can promote the release of Nrf2, thus facilitating Nrf2 nuclear translocation to bind to antioxidant response element (ARE) and consequently increasing cytoprotective

Corresponding authors at: School of Food Science and Engineering, South China University of Technology, Guangzhou 510640, China. E-mail addresses: [email protected] (L. Zheng), [email protected] (M. Zhao).

https://doi.org/10.1016/j.jff.2019.103690 Received 29 September 2019; Received in revised form 17 November 2019; Accepted 17 November 2019 Available online 24 November 2019 1756-4646/ © 2019 Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

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genes expressions (Buendia et al., 2016). Furthermore, Nrf2 is also involved in the regulation of diverse types of cytoprotective proteins including neurotrophins, anti-apoptotic and anti-inflammatory proteins (Buendia et al., 2016). Neuroprotection exerted by food-derived compounds was achieved by activating Nrf2 pathway (Ren et al., 2018). Therefore, the Nrf2 pathway has been proposed to be an emerging target in the treatment of neurodegenerative disorders. Bioactive peptides derived from food are beneficial for health with a variety of effective biological activities (e.g., antioxidant, anti-inflammation, anti-fatigue and anti-acetylcholinesterase). Importantly, acting as antioxidants and anti-apoptosis molecules, bioactive peptides or protein hydrolysates are considered to be potent and promising neuroprotectants for neuronal survival via modulating signaling pathways involved in redox state, mitochondrial dysfunction and cell death (Wang et al., 2018; Zhao et al., 2017). Peptides could also inhibit neuroinflammation and thus suppress cognition decline in various animal models such as scopolamine-induced amnesia mice and sleep-deprived rats (Wang et al., 2018; Zhao et al., 2017, 2019). Notably, small molecular peptides below pentapeptides might be absorbed across intestine in an intact form and transported by bloodstream to target organs after oral administration (Karaś, Jakubczyk, Szymanowska, Złotek, & Zielińska, 2017; Shen & Matsui, 2017). Dipeptides such as YP, which permeated blood-brain barrier in an intact form, targeted to brain regions that were involved in memory and cognition in mice (Nwachukwu, Alashi, Zahradka, & Aluko, 2019; Tanaka et al., 2019). These observations suggested that bioactive peptides or protein hydrolysates were potential candidates for the treatment of neuronal damage and memory deficits in vivo or in vitro. However, it is still unclear about the underlying cellular and molecular mechanisms on neuroprotection, especially for related signaling pathways. Particularly, studies on the mechanisms of peptides exerting neuroprotection via Nrf2 pathways were limited. Moreover, only a few studies have reported the specific sequence of potential neuroprotective peptides (Shimizu et al., 2018; Zhao et al., 2019). Therefore, it is necessary to extensively elucidate the mechanisms of peptides against oxidative stress and apoptosis in neuronal cells and their potential peptide sequences. Round scad hydrolysate (RSH) has been reported to have strong free radical scavenging and antioxidant activities (Jiang et al., 2014). It is worthwhile to investigate the neuroprotective effects of RSH and its peptides fraction on alleviating oxidative stress. Therefore, in this report, the neuroprotective effects of RSH on neurotoxicity induced by glutamate in PC12 cells were explored and the involved signaling pathways were also studied. In addition, the potential bioactive peptides of RSH were separated based on their viability-promotion in vitro and identified by UPLC-QTOF-MS/MS. Finally, the potential mechanisms of peptides on neuroprotection underlying the Nrf2 pathway were evaluated by molecular docking studies with Keap1.

for UPLC were of HPLC grade, and other reagents were of analytical grade.

2. Materials and methods

2.6. Determination of ROS level

2.1. Materials

The level of intracellular ROS was determined by DCFH-DA protocol assay. After appropriate treatment, cells (104 cells/well in 96-well plate) were incubated with DCFH-DA (10 μL) for 30 min at 37 °C in the dark and then washed thrice with PBS. The DCF fluorescence was measured by the microplate reader at a wavelength of 488 nm for excitation and 530 nm for emission.

2.2. Preparation of RSH The minced round scad meat was mixed with distilled water at a ratio of 1:3 (w/v). The suspension was adjusted to pH 7.5, and then it was hydrolyzed at 55 °C with a mixture of protamex and pancreatin at an enzyme: substrate ratio of 0.08% and 0.15%, respectively. The reaction was terminated after 8 h of hydrolysis by heating at 100 °C for 15 min. The hydrolysate was then centrifuged at 9000g for 20 min. The supernatant was collected and stored at −20 °C until utilization. 2.3. Cell culture and treatment PC12 rat adrenal pheochromocytoma cell line was acquired from the National Infrastructure of Cell Line Resources (Shanghai, China). The cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 mg/mL streptomycin in a humidified atmosphere of 5% CO2 at 37 °C. PC12 cells were seeded in a transparent 96-well plate of 104 cells/well. After 24 h, the cells were pretreated with samples for 24 h, followed by treatment with 32.5 mM glutamate for another 24 h. 2.4. Cell viability assay and lactate dehydrogenase (LDH) release assay After the treatment, cells (104 cells/well in 96-well plate) were incubated with MTT for 4 h in the dark at 37 °C, and then the medium was removed and 150 μL of DMSO was added. The mixture was incubated for 10 min in the dark and the absorbance values were measured at 570 nm by a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). PC12 cells (105 cells/mL) were grown in 24-well plate, after the cell treatment, the medium was collected and the supernatant was used for the assay of extracellular LDH release according to the manufacturer’s instructions in the assay kit (Beyotime Biotechnology Institute, China). The absorbance was measured at 450 nm with the microplate reader. The results were represented as a percentage of the control group. 2.5. Hoechst staining The morphological changes of PC12 cells were observed by Hoechst 32,258 staining. Cells (105 cells/mL) were cultured in chamber slides in 6-well plate, after appropriate treatment, cells were stained with 5 μg/ mL Hoechst 33,258 for 20 min at 37 °C in the dark, and then the cells were washed and observed using the fluorescence microscope (Leica TCS-SP5, Germany).

Round scad meat (22.75% w/w protein) was obtained from a fish market in Guangzhou, China. Cerebrolysin was purchased from Cardinal Health Pharmacy (Guangzhou, China). Glutamate and methylthiazolyldiphenyl-tetrazolium bromide (MTT) were purchased from Sigma Chemical Co. Ltd (St. Louis, MO, USA). LY294002 (LY), SP600125 (SP), SB203580 (SB) and PD98059 (PD) were purchased from Beyotime Biotechnology Institute, China. Antibodies for detecting Bax, Bcl-2, tropomyosin-related kinase B (TrkB), β-actin, cAMP-response element binding (CREB), p-CREB and brain-derived neurotrophic factor (BDNF) were purchased from Abcam (Cambridge, UK). The primary antibodies for Nrf2, Keap1, NAD(P)H quinone oxidoreductase-1 (NQO1), heme oxygenase-1 (HO-1) and GAPDH were purchased from Proteintech (Chicago, IL, USA). Antibodies for Akt and pAkt were obtained from Wanleibio (Shenyang, China). All reagents used

2.7. Determination of activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx) and the level of malondialdehyde (MDA) The PC12 cells (105 cells/mL) were cultured in 100 mm culture dish, after appropriate treatment, the medium was removed, and the cells were washed and lysed with RAPI buffer for 30 min at 4 °C. Then, the mixture was centrifuged at 13000g for 6 min. The obtained supernatant was used to measure the GPx and SOD activities and MDA levels according to the kits (Beyotime Biotechnology Institute, China). 2

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2.8. Determination of mitochondrial membrane potential (MMP) and intracellular calcium (Ca2+) level

were detected at 220 nm. The injection volume was 2 μL. Collision energies for the mass range of m/z 100–1300 were provided at 10–65 V. Peptide sequence was carried out by de novo sequencing by DataAnalysis (Version 4.4, Bruker Daltonics) along with searching by a Mascot search engine (Matrix Sciences, UK). The probability of identified peptides will be bioactive or toxic were further predicted using PeptideRanker (http://distilldeep.ucd.ie/PeptideRanker/) and ToxinPred (http://crdd.osdd.net/raghava/toxinpred/index.html), respectively.

The level of MMP was determined according to the method of Zhao et al (Zhao et al., 2017). Briefly, PC12 cells (105 cells/mL) were grown in 6-well plate, after treatment, cells were loaded with JC-1 (Beyotime Biotechnology Institute, China) at 37 °C for 30 min in the dark, and then the cells were collected and washed twice and the fluorescence intensity was measured using a microplate reader. The MMP was represented as the ratio of aggregate and monomer. The level of intracellular calcium was determined by Fluo-2/AM according to Zhao et al. (2017) with some modifications. Briefly, PC12 cells (105 cells/mL) were grown in 6-well plate, after appropriate treatment, the cells were incubated with 5 μM Fluo-2/AM for 30 min in the dark at 37 °C. Then the cells were washed and resuspended, and the fluorescence intensity was detected on a microplate reader.

2.12. Molecular docking The binding affinities between peptides and target protein were determined by molecular docking. X-ray crystal structure of protein Keap1 (PDB code: 5FNQ) was retrieved from Protein Data bank (www.rcsb.org). The 3D structures and energy minimization of peptides were constructed in ChemBio3D Ultra 14.0. The ligand, receptor and docking parameters were performed using AutoDock Tools. AutoDock Vina was applied for processing with docking parameters and docking, and the predicted affinity of the peptides for binding to the Keap1 was expressed as Vina scores (kcal/mol). PyMol 1.7 was used for molecular visualization and analysis.

2.9. RT-qPCR and western blot analysis The PC12 cells (105 cells/mL) were cultured in 100 mm culture dish. After treatment, total RNA of cell was extracted and the RT-qRNA assay was applied to detect the mRNA level of NQO1 and HO-1. Total cell lysates were obtained by an RPAI lysis buffer containing protease inhibitors and cocktail (Servicebio, Wuhan, China). Nuclear and cytosolic proteins were prepared and then lysed according to the nuclear extraction kit (Solarbio, Beijing, China). All proteins were determined by BCA protein kits (Beyotime) prior to being separated by SDS-PAGE, followed by being transferred to a PVDF membrane. After being blocked with 5% non-fatted milk in TBST for 1.5 h, the membrane was incubated with primary antibodies overnight at 4 °C. The membrane was washed four times with TBST, and then incubated with corresponding secondary antibodies for 1 h. The protein blots were visualized by an ECL detection reagent and exposed to the Azure c300 Chemiluminescent Western Blot Imaging System (Azure Biosystems, Inc, Dublin, CA, USA), and the intensity of the band was quantified by Image J software.

2.13. Statistical analysis A one-way analysis with a variance at P < 0.05 followed by Duncan’s test was carried out to analyze the statistical significance of results using SPSS (version 19.0, Chicago, IL, USA). All data are presented as means ± SD. 3. Results 3.1. Neuroprotective effects of RSH in glutamate-damaged PC12 cells RSH (0–2.5 mg/mL) was proved to be non-toxic in PC12 cells using MTT assay (Fig. S1). Glutamate with a concentration of 32.5 mM was used as a modeling concentration in this study and could reduce the cell viability of PC12 cells to 50.80 ± 3.42% (Fig. S2). Cerebrolysin, a clinical peptidergic drug for memory decline, was used as a positive control at 0.5 mg/mL in this study. After incubation with RSH (0.1–1.0 mg/mL) prior to glutamate challenge, the cell viability suppressed by glutamate was enhanced by RSH in a dose-dependent manner (P < 0.05, Fig. 1A). The release of LDH, an important hallmark of the cell membrane integrity and cell damage, was measured. Results revealed that pretreatment with RSH could obviously decrease the excessive release of LDH induced by glutamate (Fig. 1B). Moreover, pretreatment with RSH attenuated the features of apoptosis including nucleus condensation and morphological nuclear size changes (Fig. 1C).

2.10. Separation of RSH with cutoff ultrafiltration membrane and Sephadex G-15 RSH was separated with 10 kDa, 5 kDa, 3 kDa and 1 kDa cutoff ultrafiltration membrane to obtain five fractions (U1-U5): molecular weight (MW) > 10 kDa, MW 5–10 kDa, MW 3–5 kDa, MW 1–3 kDa and MW < 1 kDa, respectively. The fraction with the highest cell viability was further separated with Sephadex G-15 gel filtration chromatography (2.6 × 70.0 cm) at a flow rate of 1.5 mL/min. The elution peaks were detected at 220 nm, and six fractions (S1-S6) were collected and lyophilized for the measurement of cell viability of PC12 cells induced by glutamate and further study.

3.2. Possible signaling pathways involved in neuroprotection of RSH 3.2.1. Effects of RSH on apoptosis in PC12 cells MMP, a prominent indicator of early cell apoptosis which presents the integrity of the mitochondria membrane, was detected to determine the protection of RSH on apoptosis. As shown in Fig. 2A, the rapid loss of MMP was observed in PC12 cells after treatment with glutamate, which was partially enhanced by RSH in a dose-dependent manner. Considering that mitochondria are one of the largest calcium pools in cells, we further monitored the level of intracellular Ca2+. Results showed that glutamate increased the intracellular Ca2+ levels to 5.7fold of the normal group, which was partially attenuated by RSH dosedependently (Fig. 2B). Furthermore, exposure to glutamate resulted in a marked up-regulation of Bax and down-regulation of Bcl-2, with a concomitant increase of the ratio of Bax/Bcl-2 (Fig. 2C-D). These alterations were almost completely reversed and returned to the level of control by treatment with RSH at a low concentration. Additionally, RSH with a concentration above 0.5 mg/mL could significantly

2.11. Identification of peptide by UPLC-QTOF-MS/MS and sequence analysis Peptides identification was performed according to the method by Zheng et al with some modifications (Zheng et al., 2019). Briefly, the Waters Acquity UPLC I-Class system (Waters Corporation, USA) was coupled with an Impact II QTOF MS system (Bruker Daltonics, Germany). The Acquity UPLC HSS T3 column (i.d. 2.1 × 100 mm, 1.8 μm, Waters, Milford, MA) was used for peptide separation. The MS system was equipped with an electrospray ionization (ESI) source (Bruker Daltonics, Germany). The fraction with the highest viability-promotion was filtered through a 0.22 μm filter prior to auto-injection. Mobile phase A was 0.1% formic acid in Milli-Q water, and mobile B was acetonitrile. The sample was eluted at a flow rate of 0.2 mL/min and separated as follows: 0–1 min: 100% A; 1–9 min: 100–40% A; 9–10 min, 40% A; 10–13 min, 40–100% A; 13–15 min, 100% A. The fraction peaks 3

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Fig. 1. Effects of RSH on the cell injury and damage induced by glutamate in PC12 cells. Cells were pretreated with different concentrations of RSH (0.1–1.0 mg/mL) for 24 h prior to the exposure of glutamate. (A) Cell viability, n = 4. (B) LDH release, n = 3. (C) Hoechst 33,258 staining under the fluorescence microscope (200×). Different letters among groups represent the significant differences, P < 0.05.

decrease the Bax/Bcl-2 ratio when compared with the control. Moreover, the anti-apoptotic effect of RSH was more effective than that of Cerebrolysin at the same concentration.

in the glutamate-treated group (Fig. 4C). Moreover, treatment with RSH significantly up-regulated the nuclear level of Nrf2 and down-regulated the cytoplasmic level of Nrf2 (P < 0.05, Fig. 4D) compared with those of control. Incubation of RSH in cells with or without glutamate led to the accumulation of nuclear Nrf2, which was 3.0- and 7.8-fold higher than that of the control, respectively. We subsequently sought to determine which kinase had a critical role in the activation of Nrf2/HO-1 pathway. PC12 cells were incubated with PD (Erk inhibitor), SP (JNK inhibitor), SB (p38 inhibitor) and LY (Akt inhibitor) in the combination of RSH for 24 h before glutamate challenge, respectively. As shown in Fig. 4B, co-treatment with Akt inhibitor (LY294002) in cells could partially abolish the viability-enhancing effects of RSH. It indicated that activation of Akt might play the main role in the neuroprotection of RSH. Therefore, the effects of Akt inhibitor on the expression of Nrf2 were evaluated next. Fig. 4E showed that incubation with LY294002 could inhibit RSH-induced phosphorylation of Akt, thereby influencing the expression of Nrf2.

3.2.2. Effects of RSH on glutamate-induced ROS production in PC12 cells As illustrated in Fig. 3A, exposure to glutamate led to a nearly 4-fold accumulation of ROS compared to that in the control group. Furthermore, this increase of ROS was dramatically decreased by the pretreatment with RSH in a dose-dependent manner (P < 0.05). Our results showed an 80% increase of MDA level in glutamate-treated PC12 cells was observed, whereas RSH dose-dependently reduced the level of MDA and almost completely restored it at a concentration above 0.5 mg/mL (Fig. 3B). Pretreatment with RSH also markedly enhanced the activities of SOD and GPx in a dose-dependent manner as compared with those in glutamate-damaged cells (Fig. 3C and D). 3.2.3. Effects of RSH on the regulation of Akt/Nrf2 pathway Since RSH exerted protective and antioxidant actions, the effect of RSH on Nrf2-mediated pathways in PC12 cells was investigated. Our results revealed that glutamate treatment significantly suppressed the mRNA levels of HO-1 and NQO1, which were up-regulated in the presence of RSH in PC12 cells with or without glutamate (Fig. 4A). RSH could also obviously enhance the expression of Nrf2, HO-1 and NQO1

3.2.4. Effects of RSH on BDNF/CREB/TrkB pathway Considering that the phosphorylation of CREB is crucial to cell survival (Zhong et al., 2018), the effects of RSH on the expression of pCREB were further investigated. Results showed that glutamate significantly promoted the phosphorylation of CREB, which might be a 4

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Fig. 2. Effects of RSH on glutamate-induced mitochondrial dysfunction and apoptosis. Cells were pretreated with different concentrations of RSH (0.1–1.0 mg/mL) for 24 h prior to the exposure of glutamate. (A) Effects of RSH on MMP loss and (B) intracellular Ca2+ levels in glutamate-damaged PC12 cells. Cell lysates were analyzed using western blotting. The protein expression of (C) Bax and (D) Bcl-2 and (E) the corresponding densitometric analysis. (F) The ratio of Bax and Bcl-2 expression. Different letters represent significant differences, n = 3, P < 0.05.

cellular defense response to stressful stimuli induced by glutamate (Lonze & Ginty, 2002). In contrast, RSH could significantly promote the phosphorylation of CREB both in normal and glutamate-treated conditions. In this study, glutamate also markedly suppressed the expression of BDNF and TrkB, which were significantly up-regulated by treatment with RSH both in basal and stressed conditions (Fig. 4F).

ultrafiltration membrane. As shown in Fig. 5A, peptides with MW below 3 kDa (U4 and U5) revealed higher cell viability than those above 3 kDa. Then, U5 fraction with the highest viability-promotion among all fractions was further separated into six fractions by Sephadex G-15 which is widely used for separating peptides with MW below 1500 Da. Cells treated with S4 fraction displayed the highest cell viability (66.38 ± 1.05%) among all fractions, which was higher than that of RSH (Fig. 5C). Therefore, S4 fraction was collected and further analyzed by UPLC-QTOF-MS/MS. Using de novo sequencing and Mascot searching, 23 peptides from S4 fraction were identified and their physicochemical characteristics were listed in Table 1. Here, these peptides fell within an observed m/z ranging from 229.1547 to 749.3945, mostly di-, tri- and tetra-peptide, which were characterized with aromatic

3.3. Potential bioactive peptides with neuroprotection within RSH 3.3.1. Separation and identification of peptides It is widely accepted that molecular weight has a critical role in the biological activities of peptides (Lorenzo et al., 2018). Thus, in the present study, RSH was initially separated into five fractions by 5

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Fig. 3. Effects of RSH (0.1–1.0 mg/mL) on (A) ROS production, (B) the content of MDA and the activities of (C) SOD and (D) GPx. Bars with different letters among samples indicate significant differences at P < 0.05, n = 3.

amino acids (particularly for Tyr and Trp) at C-terminal, and these peptides contained 50–100% hydrophobic amino acids in the sequence. In addition, all peptides were predicted to be non-toxic, and 21 peptides were evaluated as promising bioactive peptides with a score > 0.8 via PeptideRanker.

4. Discussion Oxidative stress caused by excessive accumulation of ROS in mitochondria is involved in neuronal damage and death, leading to the pathological process of various neuron diseases including AD and Parkinson’s disease (Gu, Chauhan, & Chauhan, 2014). Therefore, antioxidants and mitochondrial-targeting agents have been considered to be prospective in neurodegenerative diseases (Hroudová, Singh, & Fišar, 2014). There is rising attention in bioactive peptides which help prevent neuronal disorders due to their high biological activities, high membrane penetration abilities and easy absorption compared with other small molecules (Vlieghe, Lisowski, Martinez, & Khrestchatisky, 2010). Studies revealed that bioactive peptides can exert neuroprotection and primarily function by their antioxidant activities and inhibition of mitochondrial dysfunction and apoptosis (Wang et al., 2018; Zhao et al., 2017). Nonetheless, the underlying molecular mechanisms of bioactive peptides remain unclear, especially for neurotrophic signaling and the Nrf2-antioxidant signaling pathway. This study was designed to investigate the neuroprotective mechanisms of RSH on oxidative stress and cell death induced by glutamate in PC12 cells. Our findings revealed that RSH alleviated oxidative stress and mitochondrial dysfunction caused by glutamate via the Nrf2-mediated pathway and CREB-related pathway, and thus repaired the damaged cellular defense system. Glutamate, a predominant excitatory neurotransmitter in the central nervous system, plays a pivotal part in excitatory synaptic transmission, neuronal development and adult neuroplasticity (Cassano et al., 2015). However, excessive glutamate can lead to excitotoxicity

3.3.2. Molecular docking simulations Keap1-Nrf2 interaction plays a role in the activation of Nrf2 (Buendia et al., 2016). Thus, peptides within the S4 fraction that may be potential Nrf2 activators to directly inhibit Keap1-Nrf2 interaction were recognized using molecular docking. A high absolute value of the affinity represented a high probability for peptides binding with the receptor protein. As shown in Table 1, our results revealed that most peptides with Trp and Tyr in sequence docked with Keap1 binding site with powerful calculated affinities below −8.3 kcal/mol. PPW displayed the highest score among all peptides with the strongest affinity of −10.4 kcal mol−1, followed by IGPW with an affinity of −10.0 kcal mol−1. PPW docked well within the hydrophobic active site of Keap1 in kelch domain (Fig. 6A-B). Some interactions were observed between Keap1 and PPW (Fig. 6C-D). There were eight conventional hydrogen bonds of PPW with Keap1, including two hydrogen bonds with Val-606 (bond length, 2.5 Å and 2.3 Å, respectively), as well as bonds with Leu-365, Val-418, Gly-367, Val-512, Ile-559 and Val-465, with a distance of 2.1 Å, 2.4 Å, 2.4 Å, 2.5 Å, 2.4 Å and 2.9 Å, respectively. Meanwhile, the pyrrole ring of Pro (in PPW) contributed to the alkyl interactions with Val-512. In addition, Gly-417 and Gly-605 performed carbon-hydrogen bonds towards to PPW. 6

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Fig. 4. Effects of RSH on the BDNF/TrkB/CREB and Nrf2 signaling pathway. PC12 cells were pretreated with RSH (0.5 mg/mL) for 24 h before glutamate challenge (A, C, D and F) or incubated with RSH and inhibitors of protein kinases including PD (40 μM), SP (5 μM), SB (20 μM) and LY (10 μM) before glutamate challenge (B, E). (A) Levels of mRNA were determined by RT-qPCR, n = 3. (B) Cell viability of PC12 cells was measured using MTT assay, n = 4. (C) The expressions of Keap1, total Nrf2, HO-1 and NQO1 protein were detected by western blot and the bands were quantified, n = 3. (D) The western blot results for Nrf2 (nuclear and cytoplasm) were detected and quantified, n = 3. (E) The expressions of Nrf2, p-Akt and Akt were detected and quantified, n = 3. (F) The expressions of TrkB, p-CREB, CREB and BDNF were detected and quantified, n = 3. Bars with different letters indicate statistical significance, P < 0.05.

which is associated with glutamate receptor and oxidative stress, triggering caspase-dependent or caspase-independent apoptosis death, a prominent feature of AD (Cassano et al., 2015; Olatunji et al., 2016). Our results showed that RSH could dose-dependently reverse the

glutamate-induced cell viability reduction and LDH release in PC12 cells, indicating that RSH may exert neuroprotection against glutamate neurotoxicity. Furthermore, excessive glutamate can also result in mitochondria dysfunction and release of apoptosis factor. Notably, RSH 7

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Bax/Bcl-2 pathway in cells under oxidative stress (Xu et al., 2018). A study reported that a high Trp diet also suppressed aging-related hippocampus apoptosis by downregulating caspase-3 and Bax and upregulating Bcl-2 (Musumeci et al., 2017). It suggested that RSH abundant in Trp might affect the apoptosis by regulating the pro- and antiapoptotic molecules. Mitochondria, as the major sites of endogenous ROS production, also play important roles in modulating cellular reduction-oxidation potential and free radical scavenging capacity (Cassano et al., 2015; Gu et al., 2014). Excessive glutamate caused overproduction of ROS and the increase of MMP, while treatment with RSH could markedly attenuate these features, positively impacting on redox state. Consistently, RSH could also reduce the elevation of MDA, a biomarker in some neurodegenerative diseases, indicating that the neuroprotection of RSH was connected with the inhibition of lipid peroxidation. Also, previous studies reported that the consequences of Ca2+ overload include the deactivation of antioxidant enzyme activities such as SOD and GPx (Yang, Sykora, Wilson, Mattson, & Bohr, 2011), which was also observed in glutamate-damaged cells in this study and could be improved by RSH. Taken together, RSH appeared to inhibit oxidative stress induced by glutamate via reducing ROS and controlling lipid peroxidation as well as GPx- and SOD-related mechanisms. Nrf2 has a central role in the activity and induction of cytoprotective genes including antioxidant enzymes and phase II antioxidant genes, which regulate the redox homeostasis, inflammation and enhance cellular antioxidant capacity (Lu et al., 2019). Therefore, Nrf2 activators or Nrf2-Keap1 protein-protein interaction (PPI) inhibitors are recognized as promising therapeutics for many diseases including neurodegeneration, aging and cancer (Lee et al., 2018). Antioxidants can promote the nuclear translocation of Nrf2 which activates antioxidative enzymes, thereby counteract oxidative stress and enhance the antioxidative signaling in neurodegenerative-related models (Lee et al., 2018; Ren et al., 2018). Here, our results demonstrated that RSH significantly promoted the nuclear translocation of Nrf2 and enhanced the expression of HO-1 and NQO1 both in normal and glutamate-treated cells. These results corroborated the effects of RSH in activating the Nrf2/ARE pathway, which was consistent with the above results that RSH elevated the activities of SOD and GPx, two target genes of Nrf2. As far as we know, our findings showed, for the first time, that RSH exerted neuroprotection against glutamate-induced neurotoxicity within PC12 cells, in part, through the activation of Nrf2-mediated pathway. Multiple kinases such as PI3K/Akt and MAPK may be involved in the activation and accumulation of nuclear Nrf2 to coordinate the antioxidative defense systems (Buendia et al., 2016). PI3K/Akt signaling is also crucial to cell survival and death, while dephosphorylation of Akt is connected with neuronal damage in neurodegeneration (Zhong et al., 2018). In our study, the viability-promotion and activation of Nrf2/HO1 pathway by RSH could be partially neutralized by co-treatment with Akt inhibitor LY294002 in glutamate-damaged PC12 cells. It indicated that the activation of Akt in RSH appeared to function in modulating oxidative stress induced by glutamate. Evidence also revealed that GSK3β, a downstream target of PI3K/Akt, could phosphorylate Nrf2 to down-regulate ARE activity (Reddy et al., 2015). Thus, Akt-mediated Nrf2 accumulation may be modulated by directly increasing the phosphorylation of Nrf2 and/or inactivating GSK-3β, which should be evaluated in further study. Other kinases might also be involved in the activation of the Nrf2/HO-1 pathway, this warrants further studies. Moreover, activation of Akt signaling is also critical for the anti-apoptosis pathway by activating Bcl-2 to protect against neurotoxicity (Li et al., 2018; Yoo et al., 2017), which was consistent with the elevated pAkt in addition with Bcl-2 expression in RSH-treated cells. PI3K/Akt signaling also modulates inflammation independent of Nrf2 to maintain homeostasis (Reddy et al., 2015). Collectively, it demonstrated that the Akt/Nrf2 signaling was one of the relevant mechanisms of neuroprotection of RSH.

Fig. 5. Effects of fractions (0.5 mg/mL) derived from RSH by ultrafiltration membrane separation on the loss of cell viability in glutamate-damaged PC12 cells. U1-U5: MW > 10 kDa, MW 5–10 kDa, MW 3–5 kDa, MW 1–3 kDa and MW < 1 kDa. The above fraction with the highest cell viability was further separated on a Sephadex G-15 column, and the chromatography profile of U5 fraction from RSH is shown in (B). Cell viability after treatment with fractions of RSH was detected by MTT assay and presented in (A) and (C), respectively. Different letters indicate statistical significance, P < 0.05, n = 4.

obviously decreased the elevated MMP and Ca2+ influx induced by glutamate (Fig. 2), indicating that RSH may have potential efficacy on the inhibition of mitochondrial dysfunction and apoptosis. RSH could also lead to a dramatic decrease in Bax/Bcl-2 ratio after glutamate challenge, which corroborated the neuroprotection of RSH in apoptosis. It was consistent with the previous study that bioactive peptides and protein hydrolysates could regulate apoptosis-related pathways such as 8

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Table 1 Peptides Identified from the S4 fraction of Round Scad Hydrolysate. No

Retention time (min)

Observed (m/z)

Expected (m/z)

Sequence

Peptide ranker response

Affinity (kcal/mol)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23*

8.9 9.8 9.2 8.4 8.5 9 9.2 7.6 10.5 9.1 8.8 6.2 7.7 8.5 4.4 10.5 9 8.2 7.7 7.5 6.1 6.6 5.6

399.2031 472.2558 426.2024 359.1712 373.1867 432.2238 389.2181 329.1493 431.2654 749.3945 361.1868 333.1559 319.1402 302.1498 303.1452 497.2390 318.1810 304.1652 306.1444 292.1288 292.1291 229.1548 515.2170

399.2027 472.2554 426.2023 359.1713 373.1870 432.2241 389.2183 329.1496 431.2652 749.3981 361.1870 333.1557 319.1401 302.1499 303.1452 497.2395 318.1812 304.1652 306.1448 292.1292 292.1292 229.1547 1029.4611

PPW I(L)GPW YPF GPW APW I(L)NW AI(L)W FY I(L)I(L)W FVAIGGW VGW WQ NW PW FH AYPF I(L)W VW TW SW WS I(L)P WCPFSRSF

0.99 0.95(0.97) 0.98 0.99 0.97 0.79(0.87) 0.85(0.93) 0.98 0.82(0.95) 0.76 0.85 0.91 0.93 0.99 0.95 0.95 0.94(0.99) 0.8 0.81 0.93 0.89 0.58(0.80) 0.97

−10.4 −10.0(−9.9) −9.8 −9.3 −9.2 −9.1(−9.2) −8.8(−9.2) −8.9 −8.9(−8.8) −8.8 −8.8 −8.7 −8.6 −8.6 −8.5 −8.5 −8.4(−8.5) −8.4 −8.4 −8.4 −8.3 −6.8(−7.0) −6.6

*

Peptide with 2 charge (H+).

CREB, another transcription factor that plays a crucial role in neuronal survival, growth and proliferation, can be a target for neuroprotectant in degenerative diseases (Lonze & Ginty, 2002). The phosphorylation of CREB is also associated with neurotrophins, neuronal signaling transmitter and plasticity (Ju, 2019). BDNF, the target of

CREB, activates TrkB and thereby promotes cell survival against oxidative stress (Han et al., 2014). Furthermore, evidence also revealed that the activation of TrkB/CREB/BDNF pathway is an important mechanism of neuroprotection in glutamate-treated HT-22 cells (Yoo et al., 2017). Studies have reported that administration of pro-BDNF

Fig. 6. The molecular docking interaction of PPW with Keap1. PPW is displayed as a white stick model. The front (A) and top (B) view of the docking mode between PPW and Keap1. (C) The docked pose of PPW in the active site of Keap1, yellow line indicated the hydrogen bonds interacted with Keap1. (D) Map of 2D interaction of PPW with active site residues of Keap1. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) 9

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Fig. 7. Schematic model of neuroprotection of RSH on glutamate-induced neurotoxicity.

peptides or food-derived peptides such as SKKY, IKRG, GR and VHCC could display neurogenic and neurotropic and maintain neuronal survival to promote neurogenesis (Cardenas-Aguayo, Kazim, GrundkeIqbal, & Iqbal, 2013; Ju, 2019; Shimizu et al., 2018). Positively charged amino acids in peptide sequences such as Lys and Arg may contribute to binding with TrkB and His contributes to neurogenesis and maintains the integrity of the blood-brain barrier (Shimizu et al., 2018; Song et al., 2018). The branched-chain amino acid also participates in many processing including biosynthesis, metabolism and absorption (Ju, 2019). Trp promoted 5-HT neurotransmission and may thereby modulate the BDNF system, and it also restored the phosphorylation of CREB (2017; Musumeci et al., 2015). In this study, RSH could activate the CREB and promote the phosphorylation of CREB under both basal and stressed conditions. RSH could also up-regulate the expression of BDNF and TrkB in stressed conditions. We speculated that Leu (Ile), His and Trp in peptide sequence from RSH may play a role in regulating the CREBmediated pathway. Moreover, the cell damage induced by glutamate may be due, in part, to oxidative stress, which led to a deterioration of CREB that was partially restored by treatment with RSH through regulating TrkB/CREB/BDNF pathway. We also aimed to investigate the potential specific sequence of neuroprotective peptides in RSH. After separation and identification, S4 fraction with the highest viability-promotion was found to contain a high content of aromatic/hydrophobic amino acids in the peptide sequence, particularly for Tyr and Trp, which may be main contributors for the neuroprotection. Tyr- and Trp-containing dipeptides can serve as electron/hydrogen donors to display strong radical scavenging activities and exerted a viability-promotion effect in vitro. (Wang et al., 2018; Zheng, Zhao, Dong, Su, & Zhao, 2016; Zheng, Zhao, Xiao, Zhao, & Su, 2016). Trp-containing peptides such as WY and WM could be delivered to the brain and thereby inhibit neuroinflammation and regulate microglial activity and dopamine system in vivo (Ano et al., 2019). Apart from that, peptides with hydrophobic amino acids increase the lipo-solubility of peptides and thereby enhance the permeability to the cell membrane and blood-brain barrier to interact with target organs (Rajapakse, Mendis, Byun, & Kim, 2005; Tanaka et al., 2019). Therefore, it was speculated that peptides containing Tyr and Trp as well as hydrophobic amino acids may contribute to the neuroprotection of RSH. Our results, in addition to the fact of these peptides were nontoxic, provided a hypothesis to further explore their application for the treatment of neurological disorders in vivo. RSH could activate the Nrf2 pathway by regulating the Akt

pathway, a Keap1-independent mechanism. Our results also showed that there was no significance among all the groups in the expression of Keap1 (Fig. 4C), which was consistent with the effects of sesamol on the Nrf2/Keap1 pathway in H2O2-damaged cells (Ren et al., 2018). However, the Nrf2 cell defense pathway can also be regulated by Keap1dependent mechanisms such as the direct modification of Keap1 or interference on Nrf2-Keap1 interaction (Lee et al., 2018). Compared to Nrf2 activators, Nrf2-Keap1 PPI inhibitors such as small molecules can directly disrupt Nrf2-Keap1 interaction via non-covalent interactions to activate Nrf2 and thereby provide protection against oxidative stress (Lu et al., 2019). In this study, the results of molecular docking showed that Trp- or Tyr-containing peptides displayed strong affinities towards Keap1 owing to strong hydrogen bond and hydrophobic interaction, which was consistent with the activation of Nrf2 treated with RSH. It further confirmed that Trp- and Tyr-containing peptides could be potent peptides for neuroprotection. Consistently, a previous study also reported that peptides such as DDK and DWW, which exerted viabilitypromotion and up-regulated the activities of antioxidant defense enzymes, may directly interfere with Keap1 via hydrogen-bond to modulate Nrf2 pathway (Li et al., 2017). In addition, benzene rings of aromatic amino acids (such as Tyr and Trp) in peptides are more favorable for binding to specific target sites. It was speculated that peptides from RSH such as PPW may directly bind to Keap1 and thus interfere with Nrf2-Keap1 interaction to promote the accumulation and nuclear translocation of Nrf2 and further up-regulate its target gene expression. It is necessary to further substantiate the effects of these peptides on neuroprotection and Keap1-Nrf2 interaction in the future. Furthermore, the addition of non-polar amino acids such as Pro (P), Gly (G), Ala (A), Leu (L) and Ile (I) at N-terminal changed the hydrophobicity of peptide, which might make it easier to interact with the active site of Keap1, by comparing PW, XPW (X means P, G, I(L), A, etc.) and I(L)GPW, etc. It indicated that non-polar amino acids at the Nterminal might promote the aromatic amino acid-containing peptide directly interact with Keap1. 5. Conclusion Peptides derived from round scad were demonstrated to exert neuroprotection against glutamate-induced neurotoxicity in PC12 cells. It could dose-dependently inhibit cell damage by reducing ROS and inhibit apoptosis via regulating Bax/Bcl-2 pathway in PC12 cells. RSH could also promote cell survival via TrkB/CREB/BDNF and Akt/Nrf2 10

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pathways under both normal and stressed conditions (as depicted in Fig. 7), and Nrf2 signaling could be used as a target for peptides to modulate redox state. Additionally, small molecular peptides (MW < 1 kDa) played a key role in the neuroprotection and 23 novel peptides were identified. Tyr- and Trp-containing peptides may contribute to neuroprotection. Molecular docking analysis revealed that the neuroprotection of these peptides such as PPW might be owing to the direct binding with Keap1 by hydrogen bond and hydrophobic interaction to interfere Nrf2-Keap1 interaction and thus modulate Nrf2 pathway. Future assessment of these peptides both in vitro and in vivo is of concern as this may help the application of novel dietary supplements or functional ingredients for neuroprotection.

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6. Ethics Statements File Our research did not include any human subjects and animal experiments. CRediT authorship contribution statement Qi Zhang: Methodology, Formal analysis, Data curation, Visualization, Investigation, Writing - original draft. Guowan Su: Conceptualization, Funding acquisition. Tiantian Zhao: Software, Visualization, Investigation, Funding acquisition. Baoguo Sun: Project administration. Lin Zheng: Validation, Formal analysis, Supervision, Writing - review & editing. Mouming Zhao: Supervision, Resources, Project administration, Writing - review & editing, Funding acquisition. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments The work was supported by the State Key Research and Development Plans (No. 2017YFD0400201), Guangdong Province for Science and Technology Innovative Young Talents (No. 2016TQ03N728), Guangzhou Science and Technology Plan Project (No. 201604020122) and the Fundamental Research Funds for the Central Universities (No. x2skD2192510). Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jff.2019.103690. References Agostinho, P., Cunha, R. A., & Oliveira, C. (2010). Neuroinflammation, oxidative stress and the pathogenesis of Alzheimer’s disease. Current Pharmaceutical Design, 16(25), 2766–2778. Ano, Y., Yoshino, Y., Kutsukake, T., Ohya, R., Fukuda, T., Uchida, K., ... Nakayama, H. (2019). Tryptophan-related dipeptides in fermented dairy products suppress microglial activation and prevent cognitive decline. Aging, 11(10), 2949–2967. Ansari, M. A., & Scheff, S. W. (2010). Oxidative stress in the progression of Alzheimer disease in the frontal cortex. Journal of Neuropathology and Experimental Neurology, 69(2), 155–167. Buendia, I., Michalska, P., Navarro, E., Gameiro, I., Egea, J., & León, R. (2016). Nrf2-ARE pathway: An emerging target against oxidative stress and neuroinflammation in neurodegenerative diseases. Pharmacology and Therapeutics, 157, 84–104. Cardenas-Aguayo, M. D. C., Kazim, S. F., Grundke-Iqbal, I., & Iqbal, K. (2013). Neurogenic and neurotrophic effects of BDNF peptides in mouse hippocampal primary neuronal cell cultures. Plos One, 8(1), e53596. https://doi.org/10.1371/journal.pone. 0053596. Cassano, T., Pace, L., Bedse, G., Lavecchia, M. A., Marco, D. F., Gaetani, S., & Serviddio, G. (2015). Glutamate and mitochondria: Two prominent players in the oxidative stressinduced neurodegeneration. Current Alzheimer Research, 13(2), 185–197. Copple, B. L., Jaeschke, H., & Klaassen, C. D. (2010). Oxidative stress and the

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