Neuroprotective Effects of Acetylcholinesterase Inhibitory Peptides

Nov 30, 2017 - from Anchovy (Coilia mystus) against Glutamate-Induced Toxicity in ... KEYWORDS: anchovy protein hydrolysates, acetylcholinesterase, PC...
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Article Cite This: J. Agric. Food Chem. 2017, 65, 11192−11201

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Neuroprotective Effects of Acetylcholinesterase Inhibitory Peptides from Anchovy (Coilia mystus) against Glutamate-Induced Toxicity in PC12 Cells Tiantian Zhao,†,‡ Guowan Su,†,‡ Shuguang Wang,†,‡ Qi Zhang,†,‡ Jianan Zhang,†,‡ Lin Zheng,†,‡ Baoguo Sun,§ and Mouming Zhao*,†,‡,§ †

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 § Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Technology & Business University (BTBU), Beijing 100048,China

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ABSTRACT: Ameliorations of cholinergic system dysfunction and oxidative stress in neurodegenerative diseases were main approaches to improve memory disorder. Our previous investigation showed that anchovy protein hydrolysate (APH) could attenuate scopolamine-induced memory deficits in mice by regulating acetylcholinesterase (AChE) activity. Therefore, peptides with AChE inhibitory activity in APH were explored and identified in this study, and their possible neuroprotective mechanisms on glutamate induced apoptosis in PC12 were also elucidated. Two peptides with strong AChE inhibitory capacity were identified as Pro-Ala-Tyr-Cys-Ser (PAYCS) and Cys-Val-Gly-Ser-Tyr (CVGSY) by ultraperformance liquid chromatography coupled with tandem mass spectrometry. The AChE inhibitory was 23.68 ± 0.97% and 6.08 ± 0.41%, respectively. Treatment with PAYCS and CVGSY could significantly (p < 0.05) increase cells viability, reduce lactate dehydrogenase release, reactive oxygen species (ROS) production, malondialdehyde content, and the ratio of Bax/Bcl-2 of glutamate-induced apoptosis PC12 cells (82.78 ± 6.58 and 109.94 ± 7.16% of control, respectively) as well as increase superoxide dismutase and GSH-px activities. In addition, both the peptides could inhibit Ca2+ influx but have no effects on mitochondrial membrane potential. Results indicated that AChE inhibitory peptides (PAYCS and CVGSY) possibly protected the PC12 cells against glutamate-induced apoptosis via inhibiting ROS production and Ca2+ influx. PAYCS and CVGSY might be considered as nutraceuticals for alleviating memory deficits. KEYWORDS: anchovy protein hydrolysates, acetylcholinesterase, PC12 cells, oxidative stress, neuroprotection

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INTRODUCTION Memory degeneration and deficits are commonly found in agerelated Alzheimer’s (AD), Parkinson’s disease, and other neurodegenerative diseases.1−3 A growing body of literature suggests that cholinergic system dysfunction is a hallmark of memory deficits.4 As an important transmitter, acetylcholine (Ach) is responsible for the electrical impulses conduction from cells to cells and could be rapidly hydrolyzed by acetylcholinesterase (AChE).5 Therefore, enhancing cholinergic transmission directly by inhibiting AChE has been targeted for alleviating memory defects.6 For example, alpha-lipoic acid was proved to be very effective in increasing the Ach release and decreasing the AChE activity in the hippocampus in bilateral common carotid arteries occlusion-treated rats.7 In addition to cholinergic system dysfunction, apoptosis of neurons also plays a vital role in the development of memory loss.8 Therefore, much effort has been done to explore beneficial agents from natural sources to achieve neuroprotection. Si et al. has reported that socampneoside II could ameliorate H2O2-induced oxidative stress and apoptosis in PC12 cells, which could be beneficial to neurodegenerative diseases.9 Additionally, green tea and its major polyphenol (−)-epigallocatechin-3-gallate (EGCG),10 genistein,11 gallic acid,12 selenium polysaccharide,13 proanthocyanidin, and ellagitannin in berry fruits14 as well as other bioactive © 2017 American Chemical Society

compounds were studied to be neuroprotective that might be partly mediated via antioxidant effects. These investigations on neuroprotective compounds indicated that the neuroprotective compounds might be capable of modulating signaling pathways involved in oxidative stress or inflammation, cell survival, neurotransmission, mitochondrial protection, and further enhancing memory even ameliorating neurodegenerative diseases. Recently, the prevalence of memory-deficits-related diseases and lack of effective pharmaceutical treatment have drawn an urgent need for novel therapeutic methods. Bioactive peptides come from a wide range of sources. Some naturally exist in the natural resources and some can be obtained from the hydrolysates of the animal or plant proteins. These peptides play a key role in regulating the digestive, endocrine, cardiovascular, immune, and nervous systems in human health.15 It is worthy to note that peptides with antioxidant, anti-inflammatory, or other bioactivities could also exhibit neuroprotective capacity in nervous systems. An iron-chelating derivative of peptide NAPVSIPQ could inhibit iron-catalyzed Received: Revised: Accepted: Published: 11192

August 24, 2017 November 29, 2017 November 30, 2017 November 30, 2017 DOI: 10.1021/acs.jafc.7b03945 J. Agric. Food Chem. 2017, 65, 11192−11201

Article

Journal of Agricultural and Food Chemistry

In Vitro Determination of AChE Inhibition. The inhibitory effects of samples on AChE activity were measured according to Ellman’s method24 and Rhee et al.25 with some modifications for peptides. HEPES (pH 8.0, 50 mM) was chosen in the study. The 0.055 U/mL acetylcholinesterase and 7.5 mM ATCI were applied in this investigation. ATCI (30 μL), DTNB (125 μL), HEPES (30 μL), and samples (50 μL, 10 mg/mL) were added in 96-well plate and then incubated for 15 min at 37 °C, and then 30 μL of AChE was added to activate the reaction. The Varioskan Flash spectral scanning multimode reader (Thermo Fisher Scientific, USA) was used to measure the absorbance at 412 nm after 15 min. In this method, the absorbance of samples was recorded as Asample. The absorbance of samples without AChE, which was replaced by HEPES, was recorded as Asample blank. The absorbance of well without samples, which was replaced by HEPES, was recorded as Acontrol. The absorbance of well without samples and AChE, which were replaced by HEPES, was recorded as Acontrol blank. The AChE inhibitory rate was calculated as follows:

hydroxyl radical formation and protect human neuroblastoma cell cultures against H2O2 toxicity.16 Hartwig et al. found that Cerebrolysin had neuroprotective effects in CoCl2-induced cytotoxicity in PC12 cells by decreasing production of superoxides and getting involved in GSK3β pathway.17 Moreover, Chai et al. also obtained two bioactive peptides (Phe-Tyr-Tyr and Asp-Trp) from Benthosema pterotum, which possessed cytoprotection effects on both H2O2-induced apoptosis in SH-SY5Y cells and D-gal-induced memory deficit in mice.18 Therefore, bioactive peptides might play a promising role in alleviating or preventing memory deficits in the aging or age-related neurodegenerative diseases. Our previous investigations showed that anchovy protein hydrolysate (APH) exhibited antioxidative effect in vitro and memory-improving function in scopolamine-induced memory impairment in mice.19 Moreover, further study showed that APH exhibited significantly (p < 0.05) AChE inhibitory activity in brain tissue homogenate of mice and could enhance memory in mice.19,20 However, the specific peptide sequences with AChE inhibitory capacity in APH were still unknown. Moreover, whether the AChE inhibitory peptides could be neuroprotective needed to be explored. Glutamate is an important neurotransmitter in neurons and glial cells and strongly depends on calcium homeostasis and mitochondrial function.21 However, excessive amounts of extracellular glutamate could induce cytotoxicity in neurons and consequently lead to apoptosis.22 PC12 cells have been widely used for neurological studies.23 Thus, the present study was carried out to identify AChE inhibitory peptides in APH and further to determine whether the peptides displayed neuroprotective effects against glutamate-induced cytotoxicity in PC12 cells. Furthermore, the possible underlying mechanisms were also elucidated.

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⎛ A sample − A sample blank ⎞ AchE inhibitory rate (%) = ⎜1 − ⎟ × 100 Acontrol − Acontrol blank ⎠ ⎝ Where Asample, Asample blank, Acontrol, and Acontrol blank represent the absorbance of sample, sample blank, control, and control blank at 412 nm, respectively. Purification of AChE Inhibitory Peptides from APH. To purify and identify the potential AChE inhibitory peptides in APH, Sephadex G-25 gel filtration chromatography, reverse-phase (RP)-HPLC and UPLC−tandem mass spectrometry (MS/MS) were applied. The APH sample was fractionated using Sephadex G-25 gel filtration chromatography (2.6 × 70 cm2) with an ultraviolet detector (STI501Plus) at 220 nm, and six major peaks (Fr.1−6) were collected, pooled, and vacuum-dried using a rotary evaporator (RE-52A, Shanghai Yarong Biochemical Instrument Factory, Shanghai, China) for the AChE inhibitory activity assessment. Then the fractions with stronger inhibitory activity were pooled and further purified by Waters e2695 HPLC with a 2998 PDA detector (Waters Corporation, USA) and a XBridge Prep BEH130 C18 column (10 × 150 mm2, 5 μm, Waters). The elution program was set as 0−1 min, 5% acetonitrile (B) and 95% trifluoroacetic acid (0.1%, v/v) in water (A); 1−35 min, 5− 40% B; 35−36 min, 40% B; 36−40 min, 40−5% B; 40−42 min, 5% B at a flow rate of 1 mL/min. The fractions were collected manually and lyophilized for the assay of AChE inhibitory activity. UPLC−MS/MS Identification and Sequence Analysis. The resultant fraction that exhibited the strongest AChE inhibitory activity was subjected to analytical RP-UPLC for further peptide analysis in an Agilent 1290 infinity machine using an SB-C18 column (2.1 × 50 mm2, RRHD 1.8 μm, Agilent, USA). Five microliters of each peptide sample (1 mg/mL) was loaded for each elution. The flow rate was 0.50 mL/min. Methyl alcohol was used as the mobile phase B, while ultrapure water was used as the mobile phase A. The elution program was set as 0−5 min, 2% B; 5−10 min, 2−10% B; 10−15 min, 10−30% B; 15−20 min, 30−85% B; 20−25 min, 85% B; 25.01−30 min, 2% B. Column temperature was 30 °C. The elution peaks were detected at a wavelength of 220 nm. The identification of the sequence and accurate molecular mass of peptides presented in Fr.4−6 was performed by electrospray ionization-quadrupole time-of-flight micromass spectrometer (ESI-Q-TOF-MS/MS). These data were obtained a Bruker maxis impact ultrahigh resolution mass spectrometer (Bruker Daltonics Inc., Billerica, MA). The mass range was set at 80−1300 m/z in positive ion modes. The quadrupole ion energy was set at 4.0 eV, while the collision inducing dissociation energy was set at 8.0 eV. The parameters for the ESI interface were as follows: 180 °C drying gas temperature, 8.0 L/min drying gas flow, and 1.5 bar ESI nebulizer pressure. Because of the little data of anchovy sequence in UniProtKB, the computer program Data analysis, version 3.0 (Bruker Daltonics Inc., Billerica, MA) was applied to sequence the peptides by manual de novo. Notably, the final resulted peptides molecular weight should match the theory value (error ±0.002 Da). The peptides were chemically synthesized by GL Biochem Ltd. (Shanghai, China) and stored at −20 °C until use.

MATERIALS AND METHODS

Chemicals. Anchovy was purchased from Huangsha aquatic product consumer market in Guangzhou, China. Alcalase 2.4 L was purchased from Novozymes Biotechnology Co. Ltd. (Beijing, China). Papain was purchased from Guangzhou Huaqi Biotechnology Co. Ltd. (Guangzhou, China), and pancreatin was provided by Chongqing Xiangsheng Biological Pharmaceutical Co., Ltd. (Chongqing, China). Cerebrolysin was purchased from Cardinal Health Pharmacy (Guangzhou, China). Acetylthiocholine iodide (ATCI), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), AChE from electric eel, and HEPES were purchased from Sigma Chemical Co. Ltd. (St. Louis, MO, USA). The PC12 cell line (rat pheochromocytoma cells) was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Lactate dehydrogenase (LDH) release assay kit, reactive oxygen species (ROS) detection kit, malondialdehyde (MDA) detection kit, glutathione peroxidase (GSH-px) detection kit, superoxide dismutase (SOD) detection kit, mitochondrial membrane potential (MMP) detection kit, and Fura-2 AM were bought from Beyotime Institute of Biotechnology (Shanghai, China). All the solvents for high-performance liquid chromatography/ultraperformance liquid chromatography (HPLC/UPLC) were of HPLC grade. Other chemicals used were of analytical grade. Preparation of Anchovy Protein Hydrolysate (APH). APH was prepared as previously described.19 The anchovy meat mince was hydrolyzed by a mixture of three proteases (Alcalase 2.4 L, papain and pancreatin) at 55 °C for 8 h and centrifuged at 5000 × g at 4 °C for 20 min. Then the supernatant was collected and spray-dried. The inlet and outlet temperatures of spray drying were 185 and 90 °C, respectively, and the flow rate was 20 mL/min. The spray-dried powder was stored at −20 °C until used. The protein content of APH was >95%. 11193

DOI: 10.1021/acs.jafc.7b03945 J. Agric. Food Chem. 2017, 65, 11192−11201

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Figure 1. (A) Chromatography of APH separated by gel filtration on a Sephadex G-25 column (at 220 nm). (B) AChE inhibitory activity of each fraction; (C) RP-HPLC purification chromatography of Fr.4; (D) AChE inhibitory activity of each subfraction (Fr.4−1−10); (E) mass spectrum of Pro-Ala-Tyr-Cys-Ser (PAYCS, m/z 540.1936 Da); (F) mass spectrum of Cys-Val-Gly-Ser-Tyr (CVGSY, m/z 527.1955 Da); (G) AChE inhibitory capacities of PAYCS and CVGSY; the sample concentration for AChE inhibitory capacity detection was 10 mg/mL. Superscript letters (a, b, c) represent the statistical significances between samples, p < 0.05. Cell Culture and Treatment. PC12 cells were cultured in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum at 37 °C in a humidified atmosphere of 5% carbon dioxide. Cells were seeded in 96-well plates (1 × 105 cells/well). PAYCS and CVGSY derived from anchovy protein hydrolysates were probably used as the health care products, and the long-time use process was accompanied by glutamate or other impairment in the nervous system. The PC12 cell model with adding samples and glutamate at the same time (coincubation) was chosen to assess the protective effects of Cerebrolysin, PAYCS, and CVGSY on glutamate induced toxicity in PC12 cells.26 After being cultured for 24 h, PC12 cells were treated with medium containing 0.5 mg/mL Cerebrolysin (positive control), PAYCS, and CVGSY and 32.5 mM glutamate for another 24 h. In our pre-experiment, glutamate with 32.5 μM treatment for 24 h could lead to 50−60% cell viability of PC12 cells. Therefore, 32.5 μM of the glutamate was chosen as the test concentration. For these experiments, control and blank cultures were also administered the same amount of PRIM 1640 (culture medium) and PBS, respectively. The concentrations of peptides and Cerebrolysin in this experiment were selected according to our preliminary experiment. All manipulations were repeated three or more times under each treatment condition. Cell Viability Assay. After 24 h of incubation, 100 μL of MTT (0.5 mg/mL) was added to each well with additional incubation at 37 °C for 4 h, and the formazan crystals were dissolved in DMSO (150 μL). Absorbance of the formazan solution was measured at 570 nm

with a microplate reader (Thermo Fisher Scientific, USA). Cell survival was expressed as a relative percentage of the untreated control. The toxicity of samples and the selection of glutamate concentration were both conducted by this method. Lactate Dehydrogenase (LDH) Release Assay. LDH can be released from cells with damaged membranes; thus, the LDH level in the culture supernatant is a reflection of cellular injury extent. At the end of treatments, the supernatant was collected and LDH leakage was measured using the assay kit according to the manufacturer’s instructions and the absorbance was measured at a wavelength of 450 nm using a microplate reader (Thermo Fisher Scientific, USA). Morphological Analysis. To observe nuclear changes that occurred during apoptosis, the chromatin specific dye, Hoechst 33258, was used according to Ma et al.27 Briefly, PC12 cells were harvested and washed with PBS three times. Then they were fixed by 4% paraformaldehyde for 20 min at room temperature. After that, the fixed PC12 cells were washed with PBS three times before they were exposed to 5 μg/mL Hoechst 33258 for 15 min at room temperature in the dark. The samples were washed and observed under a confocal laser scanning microscope (Leica TCS-SP5, Germany) at 100× magnification. Determination of SOD, GSH-px, and MDA Levels. After the cell treatment, the medium was removed and the cells were washed thrice with PBS. Cells were collected, centrifuged, and resuspended in PBS (200 μL), dissociated by cell lysis buffer, which consisted of 200 11194

DOI: 10.1021/acs.jafc.7b03945 J. Agric. Food Chem. 2017, 65, 11192−11201

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Journal of Agricultural and Food Chemistry μL of RIPA (radio-immunoprecipitation assay) lysis and 1 mM PMSF (phenylmethanesulfonyl fluoride), and centrifuged for 6 min (13 000 × g). The supernatant was used to measure the GSH-Px, SOD, and MDA activities using assay kit based on the specified manufacturer’s instructions. Measurement of Intracellular ROS. Intracellular ROS were monitored by using the DCFH-DA (2,7-dichlorodihydrofluorescein diacetate) fluorescent probe according to the ROS assay kit. At the end of treatment, cells were incubated with 10 μM DCFH-DA at 37 °C for 30 min and then washed twice with PBS. DCFH could be oxidized to be the strong green fluorescent material-dichlorofluorescein (DCF) at the presence of ROS. The intensity of fluorescent (Ex = 854 nm, Em = 525 nm) was measured using a microplate reader (Thermo Fisher Scientific, USA). Measurement of Intracellular Calcium Concentration of ([Ca2+]i). The concentration of [Ca2+]i was measured with Fluo-2/AM. At the end of treatment, the PC12 cells were collected by tripsin digestion and then incubated with Fluo-2/AM (final concentration 5 μM) for 30 min at 37 °C, and then the fluid of PC12 cells was washed and plated on 96-well assay plates. Additionally, the maximum fluorescence ratio (Rmax) was conducted by adding Triton 100 (0.1%) into the cells to saturate Fura-2 and Ca2+ and then measured the value at 340 and 380 nm to get the ratio (F340/F380). The minimum fluorescence ratio (R min ) was conducted by adding a high concentration of Ca2+ chelating agent (EGTA, ethylene glycol bis (2-aminoethyl ether) tetraacetic acid, final concentration at 5 mM, pH 8.5) into the cells to fully chelate Ca2+ and then measured the value at 340 and 380 nm to get the ratio (F340/F380). Changes of [Ca2+]i were measured using a microplate reader. [Ca2+]i was calculated as follows: [Ca 2 +]i = Kd ×

developed using an Azure c300 Chemiluminescent Western Blot Imaging System (Azure Biosystems, Inc., Dublin, CA, USA). Statistical Analysis. Statistical analysis was performed using the statistical package SPSS 17.0 (SPSS Inc., Chicago, IL) with ANOVA analysis. Duncan post hoc test was employed for comparison of mean values among treatments and for assessment of significant differences (p < 0.05) among treatments. Data were expressed as “means ± standard deviation” of triplicate determinations.

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RESULTS Purification and Identification of Peptides from APH. Cerebrolysin, as a peptidergic drug used clinically in dementia, was chosen as the positive control in this study.17 APH was initially separated into six fractions (Fr.1−Fr.6) by gel filtration chromatography (Figure 1A). The AChE inhibitory capacities of Fr.1−Fr.6 are presented in Figure 1B. Fr.4 showed the highest AChE inhibitory rate (12.06 ± 0.36% at 10 mg/mL). Then Fr.4 was further separated into ten fractions (Figure 1C), and their AChE inhibitory capacities are shown in Figure 1D. Fr.4−6 exhibited the strongest AChE inhibitory capacity (23.30 ± 0.97% at 10 mg/mL) among all the fractions. Therefore, Fr.4−6 was collected and further analyzed by UPLC−MS/MS. Among 12 peptides that were identified, two AChE inhibitory peptides, PAYCS (m/z 540.1936 Da) and CVGSY (m/z 527.1955 Da), were investigated (Figure 1E,F). They exhibited AChE inhibitory capacity with 23.68 ± 0.97% and 6.08 ± 0.41% (Figure 1G), respectively. Effects of PAYCS and CVGSY on Cell Cytotoxicity. According to our previous pre-experiment, 32.5 mM glutamate could lead to 40−50% cell death and 0.5 mg/mL sample could be better for neuroprotection without cytoxicity. Therefore, the proper parameters were applied in this study. MTT results showed that all the peptides did not show cytotoxicity in PC12 cells (Figure 2A). Additionally, the viability of cells exposed to 32.5 mM glutamate for 24 h was 49.38 ± 2.34% of the control value. Figure 2B presents that glutamate-induced cell viability reduction in PC12 cells was attenuated by treatment with Cerebrolysin, PAYCS, or CVGSY. The cell viability of Cerebrolysin, PAYCS, or CVGSY treated groups was raised to 60.38 ± 1.69%, 64.19 ± 3.19%, and 65.08 ± 2.44% of the control value, respectively. Effects of PAYCS and CVGSY on LDH Release. LDH release is considered as an indicator of cellular impairment. Treatment with glutamate resulted in increasing LDH release into the medium, which was 569.52 ± 33.26% of the control group (Figure 2C). Interestingly, treatment of PC12 cells with Cerebrolysin, PAYCS or CVGSY at 0.5 mg/mL significantly (p < 0.05) suppressed LDH leakage. Moreover, CVGSY showed the highest inhibition effect, followed by Cerebrolysin and PAYCS. Effects of PAYCS and CVGSY on Morphology of Cell Nuclei. The protective effects of PAYCS and CVGSY on glutamate-induced cytotoxicity in PC12 cells were also confirmed by morphological observations. Under inverted light microscope, glutamate caused marked morphological changes (nuclear condensation, membrane blebbing, nuclear fragmentation, and apoptotic bodies) in PC12 cells, while Cerebrolysin, PAYCS, or CVGSY at 0.5 mg/mL mitigated such morphological features (Figure 3). Effects of PAYCS and CVGSY on Intracellular ROS, MDA Contents, SOD Activity, and GSH-px Activities. As shown in Figure 4A, the intracellular ROS generation in glutamate treated group was 3.42-times higher than that of the

R − R min F × min R max − R Fmax

Where Kd (224 nM) represents the dissociation constant of the reaction between Fura-2 and Ca2+. The Rmax and Rmin values were determined according to the method mentioned above. The Fmin and Fmax values were the Fura-2 fluorescence intensity (F380) measured at 380 nm excitation wavelength when Ca2+ was zero and saturated, respectively. Measurement of MMP (ΔΨm). When ΔΨm is high, 5,5′,6,6′tetrachloro-1,10,3,30-tetraethylbenzimidazol-carbocyanine iodide (JC1) could be gathered in the mitochondrial matrix leading to the formation of polymer (J-aggregates), which can produce red fluorescence. While when ΔΨm is low, JC-1 could not be gathered in the mitochondrial matrix resulting in the JC-1 monomer (monomer), which can produce green fluorescence. Therefore, it can be used as a sensitive measure of changes in mitochondrial membrane potential.28 The detection was conducted according to the assay kit. Briefly, at the end of treatment, cells were incubated with JC1 (working solution) for 30 min at 37 °C and then were centrifuged for 4 min at 600 × g. Cell sample was collected and washed twice with PBS followed by refloating and analyzing with a Thermo Scientific Lumina fluorescence spectrophotometer (Thermo Fisher Scientific Co., USA). The ratio of monomer and aggregate fluorescence intensity was calculated for the ΔΨm change. Western Blotting. To analyze the Bax and Bcl-2 protein expression, the cytosolic fractions were prepared by cell lysis. The protein concentration was determined using the BCA method, after which equal amounts of protein (25 μg) were electrophoresed. Following electrophoresis, the proteins were transferred from the gel to a PVDF membrane using an electric transfer system. Nonspecific binding was blocked with 5% skim milk in Tris buffered saline with Tween (TBST) buffer (5 mM Tris-HCl, pH 7.6, 136 mM NaCl and 0.1% Tween-20) for 1.5 h. The blots were incubated with antibodies against Bcl-2 (1:500), Bax (1:500) and actin (1:1000) overnight at 4 °C, after which they were washed three times with 1 × TBST. The blots were incubated for 1 h at room temperature with a 1:1000 dilution of horseradish peroxidase-labeled antirabbit or antimouse IgG. Then the blots were washed 3 times with 1× TBST before they were 11195

DOI: 10.1021/acs.jafc.7b03945 J. Agric. Food Chem. 2017, 65, 11192−11201

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Effects of PAYCS and CVGSY on Intracellular Ca2+ Concentration. It was reported that glutamate toxicity involves peroxide production, which contributes to loss of Ca2+ homeostasis.29 As shown in Figure 5A, exposure to glutamate for 24 h resulted in an obvious increase of fluorescence intensity of [Ca2+]i (1003.32 ± 115.36 nM), while in glutamate treated PC12 cells, it was markedly downregulated by Cerebrolysin, PAYCS, or CVGSY (0.5 mg/mL) treatment, which was 13.69 ± 4.16, 5.69 ± 4.16 1.95, and 5.02 ± 1.52 nM, respectively. Effects of PAYCS and CVGSY on MMP (ΔΨm). MMP collapse has been implicated in several models of apoptosis. To examine glutamate-induced apoptosis and the effect of PAYCS and CVGSY on apoptosis, MMP was measured using JC-1 staining method.27 The results revealed that MMP was partly lost when PC12 cells were exposed to glutamate. However, the ratio of aggerates and monomer was not significantly rescued by treatment with Cerebrolysin, PAYCS, or CVGSY for 24 h in glutamate injured PC12 cells (Figure 5B). Effects of PAYCS and CVGSY on Protein Expression of Bcl-2 and Bax. The inhibitory effect of PAYCS and CVGSY on glutamate-induced apoptosis was assessed by measuring changes in the levels of apoptosis-related protein expressions using Western blotting method. As shown in Figure 6, Bax upregulation and Bcl-2 down-regulation expression was observed in the glutamate treated group as compared with control group. PAYCS and CVGSY treatment significantly reversed the Bax and Bcl-2 expression levels (p < 0.05), while Cerebrolysin could not significantly up-regulate the expression of Bcl-2 (p > 0.05). The Bax/Bcl-2 ratio after glutamate treatment was 1.6-fold higher than that of the control group. Treatment with Cerebrolysin, PAYCS, or CVGSY altered the Bax/Bcl-2 ratio to 77.67 ± 4.08%, 82.78 ± 6.58%, and 109.94 ± 7.16%, when compared with that of the control group, respectively.

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DISCUSSION Recently, the existence of peptides with biological activities derived from foods has attracted a lot of attention for showing beneficial effects for health. Marine fish-derived bioactive peptides might be involved in various biological functions (e.g., angiotensin-I-converting enzyme inhibition, antioxidant, immunomodulatory, antimicrobial, anticoagulant, and matrix metalloproteinases-1 inhibition activities) based on their structural properties, amino acid composition, sequences, and nutrient utilization.30,31 In our previous study, hydrolysates derived from Anchovy protein were proved to be capable of regulating the mRNA expression of choline acetyltransferase and AChE activity to combat memory-impairment in mice.19 Therefore, APH was chosen for the further purification of potential AChE inhibitory peptides in the hydrolysates. Cerebrolysin displays neurotrophic activity and could protect primary neurons from glutamate-induced excitotoxicity.17 Thus, Cerebrolysin was chosen as the positive control in this study. AChE inhibitory results showed that PAYCS exhibited higher AChE inhibitory capacity than CVGSY and Cerebrolysin. This might be related to the different amino acid compositions and sequence in peptides. Further, we aimed to investigate the possible protective effects of PAYCS and CVGSY on the glutamate-induced toxicity in cultured PC12 cells and elucidate the underlying mechanism. Glutamate is a neurotransmitter mediating excitatory synaptic responses. However, excessive content of

Figure 2. Effects of PAYCS and CVGSY on glutamate-induced oxidative stress in PC12 cells. (A) Cell viability after treatment with PAYCS and CVGSY (0.5 mg/mL); (B) cell viability after treatment with PAYCS and CVGSY (0.5 mg/mL) and 32.5 mM glutamate for 24 h; (C) LDH leakage in PC12 cells. △, p < 0.05 versus control group; ∗, p < 0.05 versus glutamate group; #, significant differences among sample groups, p < 0.05.

control group. PAYCS and CVGSY (0.5 mg/mL) could significantly reduce ROS production to 266.12 ± 5.21% and 282.83 ± 2.71%, respectively (p < 0.05). However, Cerebrolysin only showed slight inhibitory capacity (324.28 ± 3.68%). Cell death caused by ROS overproduction is accompanied by a lipid peroxide increment and activity of enzymes changes. As shown in Figure 4B−D, after PC12 cells were exposed to glutamate, the decreased activities of SOD and GSH-px and elevated contents of MDA were observed. Treatment of PC12 cells with Cerebrolysin, PAYCS, or CVGSY (0.5 mg/mL) caused a marked increase in the SOD activity from 59.95 ± 2.86 U/mg prot in glutamate treated cells to 70.49 ± 0.39, 73.98 ± 0.32, and 75.01 ± 0.92 U/mg prot, respectively (Figure 4B). Likewise, GSH-Px were significantly up-regulated in all treated cell groups compared with the glutamate treated group (Figure 4 C). Furthermore, treatment with Cerebrolysin, PAYCS, and CVGSY significantly decreased the up-regulated levels of MDA (Figure 4D). 11196

DOI: 10.1021/acs.jafc.7b03945 J. Agric. Food Chem. 2017, 65, 11192−11201

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Figure 3. Morphological analysis of nuclear chromatins in PC-12 cells as determined by Hoechst 33258 staining (100×).

Figure 4. Effects of PAYCS and CVGSY (0.5 mg/mL) on the glutamate-induced (A) intracellular ROS accumulation; (B) SOD activity downregulation; (C) GSH-px activity down-regulation; (D) MDA levels overproduction. △, p < 0.05 versus control group; ∗, p < 0.05 versus glutamate group; #, significant differences among sample groups, p < 0.05.

extracellular glutamate is related to the neuronal disorders.32 It is worthy to note that oxidative stress and glutamate receptormediated neurotoxicity are two main mechanisms involved with

glutamate neurotoxicity.29 The oxidative stress related to glutamate neurotoxicity is mediated by ROS production, mitochondrial dysfunction, and several apoptosis-related deaths 11197

DOI: 10.1021/acs.jafc.7b03945 J. Agric. Food Chem. 2017, 65, 11192−11201

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Figure 5. Effects of PAYCS and CVGSY (0.5 mg/mL) on the (A) glutamate-induced intracellular Ca2+ influx and (B) MMP loss. [Ca2+]i was detected with a fluorescence spectrophotometer. PAYCS and CVGSY (0.5 mg/mL) could inhibit the [Ca2+]i increment. JC-1 mmonomer and aggregates were measured by fluorescence spectrophotometer. The ratio of aggregates intensity and monomer intensity of JC-1 could be used to represent the change of MMP. △, p < 0.05 versus control group; ∗, p < 0.05 versus glutamate group; #, significant differences among sample groups, p < 0.05.

Figure 6. Effects of PAYCS and CVGSY on the expression of Bax and Bcl-2 in PC12 cells were determined after the treatment of cells with PAYCS and CVGSY (0.5 mg/mL) and 32.5 mM of glutamate for 24 h. Quantitative analysis of the expression of (A) Bcl-2 and (B) Bax. (C) Determination of Bax and Bcl-2. (D) Determination ratio of values of Bax/Bcl-2 were. △, p < 0.05 versus control group; ∗, p < 0.05 versus glutamate group; #, significant differences among sample groups, p < 0.05.

signaling pathways.33 Because glutamate receptor-mediated cytotoxicity is related to the activation of glutamate receptors (NMDA receptors), it leads to the massive influx of Ca2+ and cell death.27 The neuroprotective effects of PAYCS and CVGSY on glutamate-induced cytotoxicity in PC12 cells were investigated next. Sample toxicity results demonstrated that PAYCS and CVGSY (0.5 mg/mL) did not affect the viability of PC12 cells (Figure 2A), and the decreased cell viability resulted from glutamate treatment was significantly attenuated by PAYCS and CVGSY treatment (Figure 2B). Additionally, the leakage of

LDH in culture medium was detected after being exposed to glutamate, indicating that cell membrane integrity was destroyed, which could also be ameliorated by treatment with PAYCS and CVGSY (Figure 2C). Also, Cerebrolysin, as a positive control could reverse the damage induced by glutamate. CVGSY showed better LDH leakage alleviation than that of Cerebrolysin and PAYCS. Similarly, Boshra and Atwa had investigated reversed effect of Cerebrolysin on LDH leakage in the ischemic myocardial tissue.34 Additionally, another cell apoptosis indicator was the morphological of nuclear chromatins, which was investigated. When PC12 cells 11198

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reductions in intracellular Ca2+ that were observed by the treatment with PAYCS and CVGSY involved with the NMDA receptor remains to be determined. Mitochondrial dysfunctions, glutamate excitotoxicity, and sequential apoptosis are tightly in relation to the pathogenesis of neurodegenerative disease like AD.21 The Bcl-2 family members (pro- and antiapoptotic) are the key regulators of the mitochondrial related apoptosis.47 Therefore, the ratio of pro and antiapoptotic members was crucial to predict the apoptotic fate of the cell.48 Our results indicated that glutamate significantly up- and down-regulated the expression of Bax and Bcl-2 proteins in PC12 cells, respectively (Figure 6). Reversal of these trends by PAYCS and CVGSY treatment was found. Thus, we could conclude that PAYCS and CVGSY protected PC12 cells from glutamate-induced apoptosis, which may be through the inhibition of mitochondrial apoptotic pathway. In summary, two AChE inhibitory pentapeptides, sequenced as PAYCS and CVGSY, were identified from APH. The subsequent study demonstrated that PAYCS and CVGSY exhibited neuroprotective effects on glutamate-induced toxicity in PC12 cells through inhibiting ROS production and Ca2+ influx. The different protective effects of PAYCS and CVGSY might be partly related to the differences in the amino acid compositions and sequences. The AChE inhibitory peptides, PAYCS and CVGSY, may be potential neuroprotective compounds for protecting against apoptosis induced by glutamate cytotoxicity. Further study is needed to elucidate its apoptosis cascade signaling and the underlying molecular mechanisms as well as the absorption and transport mechanisms.

were exposed to glutamate, the morphological features of nuclear chromatins were obviously observed after staining. The nuclear condensation, membrane blebbing, nuclear fragmentation and apoptotic bodies were observed. These changes could be partially reversed by PAYCS and CVGSY as well as Cerebrolysin treatment (Figure 3). One of the main features of neuronal damage, in particular of glutamate-induced cells toxicity is the production of ROS.21 The accumulation of ROS was related to the cell death, which could result in oxidative injury through lipid peroxidation, protein deficit, DNA damage, and mitochondrial dysfunction.35 Additionally, multiple reports have proved that antioxidants could alleviate or delay oxidative stress induced apoptosis in cells.36,37 Such antioxidants are reported to reverse oxidative stress by upregulating the endogenous antioxidant defense systems levels like glutathione peroxidase. Results (Figure 4A) showed that glutamate treatment significantly induced ROS accumulation in cells, while cells treated with PAYCS and CVGSY exhibited a significant reduction of ROS production compared with the glutamate treated cells (p < 0.05). This effect could be partly due to the free radicals quenching ability of Tyr and Cys in PAYCS and CVGSY. The hydrogen donors of the reactivity phenolic structure in Tyr and sulydryl group in Cys could be responsible for their free radical scavenging capacity in damaged PC12 cells.38,39 Apart from ROS accumulation, glutamate treatment could also induce a significant decrease in the activities of SOD and GSH-px as well as a significant increase in the level of MDA in PC12 cells. These parameters could be reversed by the treatment of PAYCS and CVGSY, which indicated that PAYCS and CVGSY acted as antioxidants to protect PC12 cells against oxidative damage. Furthermore, PAYCS showed better inhibitory effects on intracellular ROS content and MDA content as well as better up-regulation functions on SOD and GSH-px activities than CVGSY and Cerebrolysin. Previous studies reported that glutamate can result in an increase of the cytoplasmic Ca2+ concentration, which is caused by ROS formation. Furthermore, Ca2+ accumulation could induce ROS generation.40,41 Additionally, mitochondria, as a very effective Ca2+ buffer, could intake massive amounts of cytosolic Ca2+ at the expense of MMP, resulting in MMP loss.28 Moreover, the disruption of MMP causes apoptosis-related factors release, which leads to nuclear condensation, and generates secondary ROS, which eventually results in apoptosis.27,42 In this study, glutamate led to Ca2+ influx and MMP loss, while PAYCS and CVGSY treatment significantly prevented the influx of Ca2+ (p < 0.05), whereas the MMP disruption was not significantly ameliarated by PAYCS, CVGSY, and Cerebrolysin treatment. The injuries appeared in neuronal cells might be predominantly induced by excessive influx of calcium into neurons through ionic channels triggered, which was activated by the glutamate receptors.43 Additionally, calcium overload resulted in proteolytic enzymes over stimulation, lipid peroxidation, free-radical formation, and other injuries in cells.44−46 The obtained results showed that PAYCS and CVGSY could significantly decrease the elevated level of intracellular Ca2+ and decrease the overproduction of MDA levels (which was related to the lipid peroxidation and oxidative stress) induced by glutamate in neuronal cells (p < 0.05). The inhibition of lipid peroxidation and oxidative stress might be an explanation for the inhibitory effects of PAYCS and CVGSY on Ca2+ influx and the neuroprotection effects on glutamate induced toxicity in PC12 cells. However, whether the

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +86 20 87113914. Fax: +86 20 87113914. ORCID

Mouming Zhao: 0000-0003-0221-3838 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors gratefully acknowledge the Strategic Emerging Industry Key Scientific and Technological Program of Guangdong Province (Nos. 2012A020800002 and 2012A080800014), the Science and Technology Program of Guangzhou, China (2012Y2-00012), and State Key Research and Development Plans (Project Nos. 2017YFD0400200 and 2017YFD0400100) for their financial support.

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REFERENCES

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