Neuroprotective Effects of Acetylcholinesterase Inhibitory Peptides

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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, Bao-Guo Sun, and Mouming Zhao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03945 • Publication Date (Web): 30 Nov 2017 Downloaded from http://pubs.acs.org on December 6, 2017

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Journal of Agricultural and Food Chemistry

Neuroprotective effects of acetylcholinesterase inhibitory peptides from anchovy (Coilia mystus) against glutamate-induced toxicity in PC12 cells Tiantian Zhaoab, Guowan Suab, Shuguang Wangab, Qi Zhangab, Jianan Zhangab, Lin Zhengab, Baoguo Sunc, Mouming Zhaoabc* a

School of Food Science and Engineering, South China University of Technology, Guangzhou 510640, China b

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 (BTBU)，Beijing 100048,China.

Running title: Neuroprotective peptides from anchovy protein

Corresponding author Mouming Zhao Tel/Fax: +86 20 87113914 E-mail: [email protected]

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Abstract

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Ameliorations of cholinergic system dysfunction and oxidative stress in

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neurodegenerative diseases were main approaches to improve memory disorder. Our

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previous investigation showed that anchovy protein hydrolysate (APH) could

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attenuate

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acetylcholinesterase (AChE) activity. Therefore, peptides with AChE inhibitory

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activity in APH were explored and identified in this study, and their possible

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neuroprotective mechanisms on glutamate induced apoptosis in PC12 were also

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elucidated. Two peptides with strong AChE inhibitory capacity were identified as

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Pro-Ala-Tyr-Cys-Ser (PAYCS) and Cys-Val-Gly-Ser-Tyr (CVGSY) by UPLC-MS/MS.

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The AChE inhibitory was 23.68 ± 0.97% and 6.08 ± 0.41%, respectively. Treatment

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with PAYCS and CVGSY could significantly (p < 0.05) increase cells viability, reduce

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LDH release, ROS production, MDA content and the ratio of Bax/Bcl-2 of

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glutamate-induced apoptosis PC 12 cells (82.78 ± 6.58 and 109.94 ± 7.16% of control,

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respectively), as well as increase SOD and GSH-px activities. In addition, both the

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peptides could inhibit Ca2+ influx but have no effects on MMP. Results indicated that

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AChE inhibitory peptides (PAYCS and CVGSY) possibly protected the PC12 cells

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against glutamate-induced apoptosis via inhibiting ROS production and Ca2+ influx.

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PAYCS and CVGSY might be considered as nutraceuticals for alleviating memory

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deficits.

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Keywords Anchovy protein hydrolysates; Acetylcholinesterase; PC 12 cells;

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Oxidative stress; Neuroprotection

scopolamine-induced

memory

deficits

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in

mice

by

regulating

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Introduction

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Memory degeneration and deficits are commonly found in age-related

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Alzheimer’s (AD), Parkinson's disease and other neurodegenerative diseases.1-3 A

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growing body of literature suggests that cholinergic system dysfunction is a hallmark

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of memory deficits.4 As an important transmitter, acetylcholine (Ach) is responsible

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for the electrical impulses conduction from cells to cells and could be rapidly

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hydrolyzed by acetylcholinesterase (AChE).5 Therefore, enhancing cholinergic

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transmission directly by inhibiting AChE has been targeted for alleviating memory

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defects.6 For example, alpha-lipoic acid was proved to be very effective in increasing

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the Ach release and decreasing the AChE activity in the hippocampus in bilateral

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common carotid arteries occlusion-treated rats.7

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In addition to cholinergic system dysfunction, apoptosis of neurons also plays a

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vital role in the development of memory loss.8 Therefore, much effort has been done

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to explore beneficial agents from natural sources to achieve neuroprotection. Si et al.

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has reported that socampneoside II could ameliorate H2O2-induced oxidative stress

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and apoptosis in PC12 cells which could be beneficial to neurodegenerative diseases.9

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Additionally, green tea and its major polyphenol (-)-epigallocatechin-3-gallate

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(EGCG),10 genistein,11 gallic acid,12 selenium polysaccharide,13 proanthocyanidin and

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ellagitannin in berry fruits14 as well as other bioactive compounds were studied to be

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neuroprotective which might be partly mediated via antioxidant effects. These

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investigations on neuroprotective compounds indicated that the neuroprotective

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compounds might be capable of modulating signaling pathways involved in oxidative

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stress or inflammation, cell survival, neurotransmission, mitochondrial protection and

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further enhancing memory even ameliorating neurodegenerative diseases.

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Recently, the prevalence of memory-deficits-related diseases and lack of



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effective pharmaceutical treatment have drawn an urgent need for novel therapeutic

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methods. Bioactive peptides come from a wide range of sources. Some naturally exist

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in the natural resources and some can be obtained from the hydrolysates of the animal

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or plant proteins. These peptides play a key role in regulating the digestive, endocrine,

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cardiovascular, immune, and nervous systems in human health.15 It is worthy to note

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that peptides with antioxidant, anti-inflammatory or other bioactivities could also

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exhibit neuroprotective capacity in nervous systems. An iron-chelating derivative of

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peptide NAPVSIPQ could inhibit iron-catalyzed hydroxyl radical formation and

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protect human neuroblastoma cell cultures against H2O2 toxicity.16 Hartwig et al.

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found that Cerebrolysin had neuroprotective effects in CoCl2-induced cytotoxicity in

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PC12 cells by decreasing production of superoxides and getting involved in GSK3β

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pathway.17 Moreover, Chai et al. also obtained two bioactive peptides (Phe-Tyr-Tyr

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and Asp-Trp) from Benthosema pterotum, which possessed cytoprotection effects on

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both H2O2-induced apoptosis in SH-SY5Y cells and D-gal-induced memory deficit in

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mice.18 Therefore, bioactive peptides might play a promising role in alleviating or

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preventing memory deficits in the ageing and/or age-related neurodegenerative

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diseases.

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Our previous investigations showed that anchovy protein hydrolysate (APH)

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exhibited antioxidative effect in vitro and memory-improving function in

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scopolamine-induced memory impairment in mice.19 Moreover, further study showed

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that APH exhibited significantly (p < 0.05) AChE inhibitory activity in brain tissue

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homogenate of mice and could enhance memory in mice.19-20 However, the specific

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peptide sequences with AChE inhibitory capacity in APH were still unknown.

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Moreover, whether the AChE inhibitory peptides could be neuroprotective or not

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needed to be explored.

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Glutamate is an important neurotransmitter in neurons and glial cells, and

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strongly depends on calcium homeostasis and mitochondrial function.21 However,

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excessive amounts of extracellular glutamate could induce cytotoxicity in neurons and

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consequently lead to apoptosis.22 PC12 cells have been widely used for neurological

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studies.23 Thus, the present study was carried out to identify AChE inhibitory peptides

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in APH and further to determine whether the peptides displayed neuroprotective

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effects against glutamate-induced cytotoxicity in PC 12 cells. Furthermore, the

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possible underlying mechanisms were also elucidated.

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Materials and methods

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Chemicals

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Anchovy was purchased from Huangsha aquatic product consumer market in

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Guangzhou, China. Alcalase 2.4 L was purchased from Novozymes Biotechnology Co.

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Ltd. (Beijing, China). Papain was purchased from Guangzhou Huaqi Biotechnology

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Co. Ltd. (Guangzhou, China) and pancreatin was provided by Chongqing Xiangsheng

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Biological Pharmaceutical Co., Ltd. (Chongqing, China). Cerebrolysin was purchased

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from Cardinal Health Pharmacy (Guangzhou, China). Acetylthiocholine iodide

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(ATCI), 5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB), AChE from electric eel and

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HEPES were purchased from Sigma Chemical Co. Ltd (St. Louis, MO, USA). The

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PC12 cell line (rat pheochromocytoma cells) was purchased from the Cell Bank of the

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Chinese Academy of Sciences (Shanghai, China). Lactate dehydrogenase (LDH)

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release assay kit, reactive oxygen species (ROS) detection kit, malondialdehyde

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(MDA) detection kit, glutathione peroxidase (GSH-px) detection kit, superoxide

95

dismutase (SOD) detection kit, mitochondrial membrane potential (MMP) detection

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kit and Fura-2 AM were bought from Beyotime Institute of Biotechnology (Shanghai,

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China). All the solvents for HPLC/UPLC were of HPLC grade. Other chemicals used



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were of analytical grade.

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Preparation of anchovy protein hydrolysate (APH)

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APH was prepared as previously described.19 The anchovy meat mince was

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hydrolyzed by a mixture of three proteases (Alcalase 2.4 L, papain and pancreatin) at

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55 °C for 8 h and centrifuged at 5,000 ×g at 4 °C for 20 min. Then the supernatant

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was collected, spray-dried. The inlet and outlet temperature of spray drying was 185

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and 90 oC, respectively and the flow rate was 20 mL/min. The spray-dried powder

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was stored at -20 °C until used. The protein content of APH was > 95%.

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In vitro determination of AChE inhibition

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The inhibitory effects of samples on AChE activity were measured according to

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Ellman’s method24 and Rhee et al.25 with some modifications for peptides. HEPES

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(pH 8.0, 50 mM) was chosen in the study. The 0.055 U/mL acetylcholinesterase and

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7.5 mM ATCI were applied in this investigation. ATCI (30 µL), DTNB (125 µL),

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HEPES (30 µL) and samples (50 µL, 10 mg/mL) were added in 96-well plate and then

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incubated for 15 min at 37 oC, and then 30 µL AChE was added to activate the

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reaction. The Varioskan Flash spectral scanning multimode reader (Thermo Fisher

114

Scientific, USA) was used to measure the absorbance at 412 nm after 15 min. In this

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method, the absorbance of samples was recorded as Asample. The absorbance of

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samples without AChE which was replaced by HEPES was recorded as Asample blank.

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The absorbance of well without samples which was replaced by HEPES was recorded

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as Acontrol. The absorbance of well without samples and AChE which were replaced by

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HEPES was recorded as Acontrol blank. The AChE inhibitory rate was calculated as

120

follows:

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Where Asample, Asample blank, Acontrol and Acontrol blank represent the absorbance of sample,

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sample blank, control and control blank at 412 nm, respectively.

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Purification of AChE inhibitory peptides from APH

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To purify and identify the potential AChE inhibitory peptides in APH, Sephadex

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G-25 gel filtration chromatography, RP-HPLC and UPLC-MS/MS were applied. The

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APH sample was fractionated using Sephadex G-25 gel filtration chromatography

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(2.6 × 70 cm) with an ultraviolet detector (STI-501Plus) at 220 nm and six major

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peaks (Fr.1-6) were collected, pooled and vacuum-dried using a rotary evaporator

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(RE-52A, Shanghai Yarong Biochemical Instrument Factory, Shanghai, China) for

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the AChE inhibitory activity assessment. Then the fractions with stronger inhibitory

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activity were pooled and further purified by Waters e2695 HPLC with a 2998 PDA

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detector (Waters Corporation, USA) and a XBridgeTM Prep BEH130 C18 column

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(10×150 mm, 5 µm, Waters). The elution program was set as 0-1 min, 5% acetonitrile

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(B) and 95% trifluoroacetic acid (0.1%, v/v) in water (A); 1-35 min, 5-40% B; 35-36

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min, 40% B; 36-40 min, 40-5% B; 40-42 min, 5% B at a flow rate of 1 mL/min. The

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fractions were collected manually and lyophilized for the assay of AChE inhibitory

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activity.

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UPLC-MS/MS identification and sequence analysis

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The resultant fraction that exhibited the strongest AChE inhibitory activity was

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subjected to analytical RP-UPLC for further peptide analysis in an Agilent 1290

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infinity machine using an SB-C18 column (2.1×50 mm, RRHD 1.8 µm, Agilent,

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USA). Five microliters of each peptide sample (1 mg/mL) were loaded for each

144

elution. The flow rate was 0.50 mL/min. Methyl alcohol was used as the mobile phase

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B while ultra-pure water was used as the mobile phase A. The elution program was

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set as 0-5 min, 2% B; 5-10 min, 2-10% B; 10-15 min, 10-30% B; 15-20 min, 30-85%



147

B; 20-25 min, 85% B; 25.01-30 min, 2% B. Column temperature was 30 oC. The

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elution peaks were detected at a wavelength of 220 nm. The identification of the

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sequence and accurate molecular mass of peptides presented in Fr.4-6 was performed

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by electrospray ionization- quadrupole time-of-flight micromass spectrometer

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(ESI-Q-TOF-MS/MS). These data were obtained a Bruker maxis impact ultra-high

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resolution mass spectrometer (Bruker Daltonics Inc., Billerica, MA). The mass range

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was set at 80-1300 m/z in positive ion modes. The quadrupole ion energy was set at

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4.0 eV while the collision inducing dissociation energy was set at 8.0 eV. The

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parameters for the ESI interface were as follows: 180 oC drying gas temperature, 8.0

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L/min drying gas flow and 1.5 bar ESI nebulizer pressure. Because of the little data of

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anchovy sequence in UniProtKB, the computer program Data analysis, version 3.0

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(Bruker Daltonics Inc., Billerica, MA) was applied to sequence the peptides by

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manual de novo. Notably, the final resulted peptides molecular weight should match

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the theory value (error ± 0.002 Da). The peptides were chemically synthesized by GL

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Biochem Ltd. (Shanghai, China) and stored at -20 °C until use.

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Cell culture and treatment

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PC12 cells were cultured in RPMI 1640 medium supplemented with 10% (v/v)

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fetal bovine serum at 37 oC in a humidified atmosphere of 5% carbon dioxide. Cells

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were seeded in 96-well plates (1×105 cells/well). PAYCS and CVGSY derived from

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anchovy protein hydrolysates were probably used as the health care products, and the

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long-time use process was accompanied by glutamate or other impairment in the

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nervous system. The PC12 cell model with adding samples and glutamate at the same

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time (coincubation) was chosen to assess the protective effects of Cerebrolysin,

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PAYCS and CVGSY on glutamate induced toxicity in PC12 cells.26 After being

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cultured for 24 h, PC12 cells were treated with medium containing 0.5 mg/mL


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Cerebrolysin (positive control), PAYCS and CVGSY and 32.5 mM glutamate for

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another 24 h. In our pre-experiment, glutamate with 32.5 µM treatment for 24 h could

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lead to 50-60% cell viability of PC12 cells. Therefore, 32.5 µM of the glutamate was

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chosen as the test concentration. For these experiments, control and blank cultures

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were also administered the same amount of PRIM 1640 (culture medium) and PBS,

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respectively. The concentrations of peptides and Cerebrolysin in this experiment were

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selected according to our preliminary experiment. All manipulations were repeated

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three or more times under each treatment condition.

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Cell viability assay

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After 24 h of incubation, 100 µL of MTT (0.5 mg/mL) was added to each well

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with additional incubation at 37 °C for 4 h, and the formazan crystals were dissolved

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in DMSO (150 µL). Absorbance of the formazan solution was measured at 570 nm

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with a microplate reader (Thermo Fisher Scientific, USA). Cell survival was

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expressed as a relative percentage of the untreated control. The toxicity of samples

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and the selection of glutamate concentration were both conducted by this method.

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Lactate dehydrogenase (LDH) release assay

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LDH can be released from cells with damaged membranes, thus the LDH level in

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the culture supernatant is a reflection of cellular injury extent. At the end of treatments,

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the supernatant was collected and LDH leakage was measured using the assay kit

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according to the manufacturer’s instructions and the absorbance was measured at a

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wavelength of 450 nm using a microplate reader (Thermo Fisher Scientific, USA).

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Morphological analysis

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To observe nuclear changes that occurred during apoptosis, the chromatin

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specific dye, Hoechst 33258, was used according to Ma et al.27 Briefly, PC12 cells



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were harvested and washed with PBS for 3 times. Then they were fixed by 4%

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paraformaldehyde for 20 min at room temperature. After that, the fixed PC12 cells

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were washed with PBS for 3 times before they were exposed to 5 µg/mL Hoechst

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33258 for 15 min at room temperature in the dark. The samples were washed and

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observed under a confocal laser scanning microscope (Leica TCS-SP5, Germany) at

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100× magnification.

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Determination of SOD, GSH-px and MDA levels

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After the cell treatment, the medium was removed and the cells were washed

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thrice with PBS. Cells were collected, centrifuged, and re-suspended in PBS (200 µL),

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dissociated

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(radio-immunoprecipitation assay) lysis and 1mM PMSF (phenylmethanesulfonyl

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fluoride), and centrifuged for 6 min (13,000 ×g). The supernatant was used to

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measure the GSH-Px, SOD and MDA activities using assay kit based on the specified

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manufacturer’s instructions.

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Measurement of intracellular ROS

by cell lysis

buffer which was consist of 200 µL RIPA

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Intracellular ROS was monitored by using the DCFH-DA (2,7-Dichlorodi

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-hydrofluorescein diacetate) fluorescent probe according to the ROS assay kit. At the

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end of treatment, cells were incubated with 10 µM DCFH-DA at 37 oC for 30 min,

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and then washed twice with PBS. DCFH could be oxidized to be the strong green

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fluorescent material-dichlorofluorescein (DCF) at the presence of ROS. The intensity

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of fluorescent (Ex=854 nm, Em=525 nm) was measured using a microplate reader

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(Thermo Fisher Scientific, USA).

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Measurement of intracellular calcium concentration of ([Ca2+]i)

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The concentration of [Ca2+]i was measured with Fluo-2/AM. At the end of


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treatment, the PC12 cells were collected by tripsin digestion and then incubated with

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Fluo-2/AM (final concentration 5 µM) for 30 min at 37 oC, and then the fluid of PC

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12 cells was washed and plated on 96-well assay plates. Additionally, the maximum

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fluorescence ratio (Rmax) was conducted by adding Triton 100 (0.1%) into the cells to

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saturate Fura-2 and Ca2+ and then measured the value at 340 and 380 nm to get the

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ratio (F340/F380). The minimum fluorescence ratio (Rmin) was conducted by adding a

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high concentration of Ca2+ chelating agent (EGTA, ethylene glycol bis (2-aminoethyl

227

ether) tetraacetic acid, final concentration at 5mM, pH 8.5) into the cells to fully

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chelate Ca2+ and then measured the value at 340 and 380 nm to get the ratio (F340/F380).

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Changes of [Ca2+]i were measured using a microplate reader. [Ca2+]i was calculated as

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follows: ሾCaଶା ሿ௜ = K ௗ ×

ܴ − ܴ௠௜௡ ‫ܨ‬௠௜௡ × ܴ௠௔௫ − ܴ ‫ܨ‬௠௔௫

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Where Kd (224 nM) represents the dissociation constant of the reaction between

233

Fura-2 and Ca2+. The Rmax and Rmin were determined according to the method

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mentioned above. The Fmin and Fmax was the Fura-2 fluorescence intensity (F380)

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measured at 380 nm excitation wavelength when Ca2+ was zero and saturated,

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respectively.

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Measurement of MMP (∆Ψm)

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When

∆Ψm

is

high,

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5,5’,6,6’-tetrachloro-1,10,3,30-tetraethylbenzimidazol-carbocyanine

240

could be gathered in the mitochondrial matrix leading to the formation of polymer

241

(J-aggregates) which can produce red fluorescence. While when ∆Ψm is low, JC-1

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could not be gathered in the mitochondrial matrix resulting in the JC-1 monomer

243

(monomer) which can produce green fluorescence. Therefore, it can be used as a


iodide

(JC-1)


244

sensitive measure of changes in mitochondrial membrane potential.28 The detection

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was conducted according to the assay kit. Briefly, at the end of treatment, cells were

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incubated with JC-1 (working solution) for 30 min at 37 oC, and then were

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centrifuged for 4 min at 600 ×g. Cell sample was collected and washed twice with

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PBS followed by re-floating and analyzing with a Thermo Scientific Lumina

249

fluorescence spectrophotometer (Thermo Fisher Scientific Co., USA) The ratio of

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monomer and aggerate fluorescence intensity was calculated for the ∆Ψm change.

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Western blotting

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To analyze the Bax and Bcl-2 protein expression, the cytosolic fractions were

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prepared by cell lysis. The protein concentration was determined using the BCA

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method, after which equal amounts of protein (25 µg) were electrophoresed.

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Following electrophoresis, the proteins were transferred from the gel to a PVDF

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membrane using an electric transfer system. Non-specific binding was blocked with 5%

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skim milk in Tris buffered saline with Tween (TBST) buffer (5 mM Tris-HCl, pH 7.6,

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136 mM NaCl and 0.1% Tween-20) for 1.5 h. The blots were incubated with

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antibodies against Bcl-2 (1:500), Bax (1:500) and actin (1:1000) overnight at 4 oC,

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after which they were washed 3 times with 1 × TBST. The blots were incubated for 1

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h at room temperature with a 1:1000 dilution of horseradish peroxidase-labeled

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anti-rabbit or anti-mouse IgG. Then the blots were washed 3 times with 1× TBST

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before they were developed using an Azure c300 Chemiluminescent Western Blot

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Imaging System™ (Azure Biosystems, Inc., Dublin, CA, USA).

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Statistical analysis

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Statistical analysis was performed using the statistical package SPSS 17.0 (SPSS

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Inc., Chicago, IL) with ANOVA analysis. Duncan post-hoc test was employed for


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comparison of mean values among treatments and for assessment of significant

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differences (p < 0.05) among treatments. Data were expressed as “means ± standard

270

deviation” of triplicate determinations.

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Results

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Purification and identification of peptides from APH

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Cerebrolysin, as a peptidergic drug used clinically in dementia, was chosen as

274

the positive control in this study.17 APH was initially separated into six fractions

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(Fr.1-Fr.6) by gel filtration chromatography (Fig. 1A). The AChE inhibitory capacities

276

of Fr.1-Fr.6 are presented in Fig. 1B. Fr.4 showed the highest AChE inhibitory rate

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(12.06 ± 0.36% at 10 mg/mL). Then Fr.4 was further separated into ten fractions (Fig.

278

1C) and their AChE inhibitory capacities are showed in Fig 1D, Fr.4-6 exhibited the

279

strongest AChE inhibitory capacity (23.30 ± 0.97% at 10 mg/mL) among all the

280

fractions. Therefore, Fr.4-6 was collected and further analyzed by UPLC-MS/MS.

281

Among twelve peptides which were identified, two AChE inhibitory peptides, PAYCS

282

(m/z 540.1936 Da) and CVGSY (m/z 527.1955 Da), were investigated (Fig.1E/F).

283

They exhibited AChE inhibitory capacity with 23.68 ± 0.97% and 6.08 ± 0.41%

284

(Fig.1G), respectively.

285

Effects of PAYCS and CVGSY on cell cytotoxicity

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According to our previous pre-experiment, 32.5 mM glutamate could lead to

287

40-50% cell death and 0.5 mg/mL sample could be better for neuroprotection without

288

cytoxicity. Therefore, the proper parameters were applied in this study. MTT results

289

showed that all the peptides did not show cytotoxicity in PC12 cells (Fig.2A).

290

Additionally, the viability of cells exposed to 32.5 mM glutamate for 24 h was 49.38

291

± 2.34% of the control value. Fig. 2B presents that glutamate-induced cell viability



292

reduction in PC12 cells was attenuated by treatment with Cerebrolysin, PAYCS or

293

CVGSY. The cell viability of Cerebrolysin, PAYCS or CVGSY treated groups was

294

raised to 60.38 ± 1.69%, 64.19 ± 3.19% and 65.08 ± 2.44% of the control value,

295

respectively.

296

Effects of PAYCS and CVGSY on LDH release

297

LDH release is considered as an indicator of cellular impairment. Treatment with

298

glutamate resulted in increasing LDH release into the medium, which was 569.52 ±

299

33.26% of the control group (Fig. 2C). Interestingly, treatment of PC12 cells with

300

Cerebrolysin, PAYCS or CVGSY at 0.5 mg/mL significantly (p < 0.05) suppressed

301

LDH leakage. Moreover, CVGSY showed the highest inhibition effect, followed by

302

Cerebrolysin and PAYCS.

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Effects of PAYCS and CVGSY on the morphology of cell nuclei

304

The protective effects of PAYCS and CVGSY on glutamate-induced cytotoxicity

305

in PC12 cells were also confirmed by morphological observations. Under inverted

306

light microscope, glutamate caused marked morphological changes (nuclear

307

condensation, membrane blebbing, nuclear fragmentation and apoptotic bodies) in

308

PC12 cells, while Cerebrolysin, PAYCS or CVGSY at 0.5 mg/mL mitigated such

309

morphological features (Fig. 3).

310

Effects of PAYCS and CVGSY on intracellular ROS，MDA contents, SOD activity

311

and GSH-px activities

312

As shown in Fig. 4A, the intracellular ROS generation in glutamate treated group

313

was 3.42 times higher than that of the control group. PAYCS and CVGSY (0.5

314

mg/mL) could significantly reduce ROS production to 266.12 ± 5.21% and 282.83 ±

315

2.71%, respectively (p < 0.05). However, Cerebrolysin only showed slight inhibitory


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capacity (324.28 ± 3.68%).

317

Cell death caused by ROS overproduction is accompanied by a lipid peroxide

318

increment and activity of enzymes changes. As shown in Fig. 4 B, C and D, after

319

PC12 cells were exposed to glutamate, the decreased activities of SOD and GSH-px

320

and elevated contents of MDA were observed. Treatment of PC12 cells with

321

Cerebrolysin, PAYCS or CVGSY (0.5 mg/mL) caused a marked increase in the SOD

322

activity from 59.95 ± 2.86 U/mg prot in glutamate treated cells to 70.49 ± 0.39, 73.98

323

± 0.32 and 75.01± 0.92 U/mg prot, respectively (Fig. 4 B). Likewise, GSH-Px were

324

significantly up-regulated in all treated cell groups compared with the glutamate

325

treated group (Fig. 4 C). Furthermore, treatment with Cerebrolysin, PAYCS and

326

CVGSY significantly decreased the up-regulated levels of MDA (Fig. 4 D).

327

Effects of PAYCS and CVGSY on intracellular Ca2+ concentration

328

It was reported that glutamate toxicity involves peroxide production, which

329

contributes to loss of Ca2+ homeostasis.29 As shown in Fig. 5A, exposure to glutamate

330

for 24 h resulted in an obvious increase of fluorescence intensity of [Ca2+]i (1003.32 ±

331

115.36 nM). While in glutamate treated PC 12 cells, it was markedly down-regulated

332

by Cerebrolysin, PAYCS or CVGSY (0.5 mg/mL) treatment, which was 13.69 ± 4.16,

333

5.69 ± 4.16 1.95 and 5.02 ± 1.52 nM, respectively.

334

Effects of PAYCS and CVGSY on MMP (∆Ψm)

335

MMP collapse has been implicated in several models of apoptosis. To examine

336

glutamate-induced apoptosis and the effect of PAYCS and CVGSY on apoptosis,

337

MMP was measured using JC-1 staining method.27 The results revealed that MMP

338

was partly lost when PC12 cells were exposed to glutamate. However, the ratio of

339

aggerates and monomer was not significantly rescued by treatment with Cerebrolysin,



Page 16 of 34

340

PAYCS or CVGSY for 24 h in glutamate injured PC12 cells (Fig. 5 B).

341

Effects of PAYCS and CVGSY on the protein expression of Bcl-2 and Bax

342

The inhibitory effect of PAYCS and CVGSY on glutamate-induced apoptosis

343

was assessed by measuring changes in the levels of apoptosis-related protein

344

expressions using western blotting method. As shown in Fig.6, Bax upregulation and

345

Bcl-2 down-regulation expression was observed in the glutamate treated group as

346

compared with control group. PAYCS and CVGSY treatment significantly reversed

347

the Bax and Bcl-2 expression levels (p < 0.05). While, Cerebrolysin could not

348

significantly up-regulate the expression of Bcl-2 (p > 0.05). The Bax/Bcl-2 ratio after

349

glutamate treatment was 1.6-fold higher than that of the control group. Treatment with

350

Cerebrolysin, PAYCS or CVGSY altered the Bax/Bcl-2 ratio to 77.67 ± 4.08%，82.78

351

± 6.58% ， 109.94 ± 7.16%, when compared with that of the control group,

352

respectively.

353

Discussion

354

Recently, the existence of peptides with biological activities derived from foods

355

has attracted a lot of attention for showing beneficial effects for health. Marine

356

fish-derived bioactive peptides might be involved in various biological functions (e.g.,

357

angiotensin-I-converting

358

antimicrobial, anticoagulant and matrix metalloproteinases-1 inhibition activities)

359

based on their structural properties, amino acid composition, sequences, and nutrient

360

utilization.30-31 In our previous study, hydrolysates derived from Anchovy protein

361

were proved to be capable of regulating the mRNA expression of choline

362

acetyltransferase and AChE activity to combat memory-impairment in mice.19

363

Therefore, APH was chosen for the further purification of potential AChE inhibitory

enzyme

inhibition,

antioxidant,


immunomodulatory,

Page 17 of 34


364

peptides in the hydrolysates. Cerebrolysin displays neurotrophic activity and could

365

protect primary neurons from glutamate-induced excitotoxicity.17 Thus, Cerebrolysin

366

was chosen as the positive control in this study. AChE inhibitory results showed that

367

PAYCS exhibited higher AChE inhibitory capacity than CVGSY and Cerebrolysin.

368

This might be related to the different amino acid compositions and sequence in

369

peptides.

370

Further, we aimed to investigate the possible protective effects of PAYCS and

371

CVGSY on the glutamate-induced toxicity in cultured PC12 cells and elucidate the

372

underlying mechanism. Glutamate is a neurotransmitter mediating excitatory synaptic

373

responses. However, excessive content of extracellular glutamate is related to the

374

neuronal disorders.32 It is worthy to note that oxidative stress and glutamate

375

receptor-mediated neurotoxicity are two main mechanisms involved with glutamate

376

neurotoxicity.29 The oxidative stress related to glutamate neurotoxicity is mediated by

377

ROS production, mitochondrial dysfunction and several apoptosis-related deaths

378

signaling pathways.33 Because glutamate receptor-mediated cytotoxicity is related to

379

the activation of glutamate receptors (NMDA receptors), it leads to the massive influx

380

of Ca2+ and cell death.27 The neuroprotective effects of PAYCS and CVGSY on

381

glutamate-induced cytotoxicity in PC12 cells were investigated next.

382

Sample toxicity results demonstrated that PAYCS and CVGSY (0.5 mg/mL) did

383

not affect the viability of PC12 cells (Fig. 2A), and the decreased cell viability

384

resulted from glutamate treatment was significantly attenuated by PAYCS and

385

CVGSY treatment (Fig. 2B). Additionally, the leakage of LDH in culture medium

386

was detected after being exposed to glutamate, indicating that cell membrane integrity

387

was destroyed, which could also be ameliorated by treatment with PAYCS and

388

CVGSY (Fig. 2C). Also, Cerebrolysin, as a positive control could reverse the damage



389

induced by glutamate. CVGSY showed better LDH leakage alleviation than that of

390

Cerebrolysin and PAYCS. Similarly, Boshra and Atwa had investigated reversed

391

effect of Cerebrolysin on LDH leakage in the ischemic myocardial tissue.34

392

Additionally, another cell apoptosis indicator was the morphological of nuclear

393

chromatins which was investigated. When PC12 cells were exposed to glutamate, the

394

morphological features of nuclear chromatins were obviously observed after staining.

395

The nuclear condensation, membrane blebbing, nuclear fragmentation and apoptotic

396

bodies were observed. These changes could be partially reversed by PAYCS and

397

CVGSY as well as Cerebrolysin treatment (Fig. 3).

398

One of the main features of neuronal damage, in particular of glutamate-induced

399

cells toxicity is the production of ROS.21 The accumulation of ROS was related to the

400

cell death which could result in oxidative injury through lipid peroxidation, protein

401

deficit, DNA damage and mitochondrial dysfunction.35 Additionally, multiple reports

402

have proved that antioxidants could alleviate or delay oxidative stress induced

403

apoptosis in cells.36-37 Such antioxidants are reported to reverse oxidative stress by

404

upregulating the endogenous antioxidant defense systems levels like glutathione

405

peroxidase. Results (Fig. 4A) showed that glutamate treatment significantly induced

406

ROS accumulation in cells, while cells treated with PAYCS and CVGSY exihibited a

407

significant reduction of ROS production compared with the glutamate treated cells (p

408

< 0.05). This effect could be partly due to the free radicals quenching ability of Tyr

409

and Cys in PAYCS and CVGSY. The hydrogen donors of the reactivity phenolic

410

structure in Tyr and sulydryl group in Cys could be responsible for their free radical

411

scavenging capacity in damaged PC12 cells.38-39 Apart from ROS accumulation,

412

glutamate treatment could also induce a significant decrease in the activities of SOD

413

and GSH-px, as well as a significant increase in the level of MDA in PC12 cells.


Page 18 of 34

Page 19 of 34


414

These parameters could be reversed by the treatment of PAYCS and CVGSY, which

415

indicated that PAYCS and CVGSY acted as antioxidants to protect PC12 cells against

416

oxidative damage. Furthermore, PAYCS showed better inhibitory effects on

417

intracellular ROS content and MDA content, as well as better up-regulation functions

418

on SOD and GSH-px activities than CVGSY and Cerebrolysin.

419

Previous studies reported that glutamate can result in an increase of the

420

cytoplasmic Ca2+ concentration which is caused by ROS formation. Furthermore,

421

Ca2+ accumulation could induce ROS generation.40-41 Additionally, mitochondria, as a

422

very effective Ca2+ buffer, could intake massive amounts of cytosolic Ca2+ at the

423

expense of MMP, resulting in MMP loss.28 Moreover, the disruption of MMP causes

424

apoptosis-related factors released which leads to nuclear condensation, and generates

425

secondary ROS, eventually results in apoptosis.27,42 In this study, glutamate led to

426

Ca2+ influx and MMP loss, while PAYCS and CVGSY treatment significantly

427

prevented the influx of Ca2+ (p < 0.05), whereas the MMP disruption was not

428

significantly ameliarated by PAYCS, CVGSY and Cerebrolysin treatment. The

429

injuries appeared in neuronal cells might be predominantly induced by excessive

430

influx of calcium into neurons through ionic channels triggered which was activated

431

by the glutamate receptors.43 Additionally, calcium overload resulted in proteolytic

432

enzymes over stimulation, lipid peroxidation, free-radical formation and other injuries

433

in cells.44-46 The obtained results showed that PAYCS and CVGSY could significantly

434

decrease the elevated level of intracellular Ca2+ and decrease the overproduction of

435

MDA levels (which was related to the lipid peroxidation and oxidative stress) induced

436

by glutamate in neuronal cells (p < 0.05). The inhibition of lipid peroxidation and

437

oxidative stress might be an explanation for the inhibitory effects of PAYCS and

438

CVGSY on Ca2+ influx and the neuroprotection effects on glutamate induced toxicity



439

in PC12 cells. However, whether the reductions in intracellular Ca2+ which were

440

observed by the treatment with PAYCS and CVGSY involved with the NMDA

441

receptor remains to be determined.

442

Mitochondrial dysfunctions, glutamate excitotoxicity and sequential apoptosis

443

are tightly in relation to the pathogenesis of neurodegenerative disease like AD.21 The

444

Bcl-2 family members (pro- and anti-apoptotic) are the key regulators of the

445

mitochondrial related apoptosis.47 Therefore, the ratio of pro and anti-apoptotic

446

members was crutial to predict the apoptotic fate of the cell.48 Our results indicated

447

that glutamate significantly up- and down-regulated the expression of Bax and Bcl-2

448

proteins in PC12 cells, respectively (Fig.6). Reversal of these trends by PAYCS and

449

CVGSY treatment were found. Thus, we could conclude that PAYCS and CVGSY

450

protected PC12 cells from glutamate-induced apoptosis, which may be through the

451

inhibition of mitochondrial apoptotic pathway.

452

In summary, two AChE inhibitory pentapeptides, sequenced as PAYCS and

453

CVGSY were identified from APH. The subsequent study demonstrated that PAYCS

454

and CVGSY exhibited neuroprotective effects on glutamate-induced toxicity in PC12

455

cells through inhibiting ROS production and Ca2+ influx. The different protective

456

effects of PAYCS and CVGSY might be partly related to the differences in the amino

457

acid compositions and sequences. The AChE inhibitory peptides, PAYCS and

458

CVGSY may be potential neuroprotective compounds for protecting against apoptosis

459

induced by glutamate cytotoxicity. Further study is needed to elucidate its apoptosis

460

cascade signaling and the underlying molecular mechanisms as well as the absorption

461

and transport mechanisms.

462

Conflicts of interest


Page 20 of 34

Page 21 of 34


463

The authors declare that there are no conflicts of interest.

464

Acknowledgements

465

The authors gratefully acknowledge the Strategic Emerging Industry Key

466

Scientific and Technological Program of Guangdong Province (No. 2012A020800002

467

and No. 2012A080800014), the Science and Technology Program of Guangzhou,

468

China (2012Y2-00012) and State Key Research and Development Plans (Project No.

469

2017YFD0400200 and 2017YFD0400100) for their financial supports.

470 471

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Cancer 2002, 2, 647-656.

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603

Figure captions:

604

Fig. 1 (A) Chromatography of APH separated by gel filtration on a Sephadex G-25 column (at 220

605

nm). (B) AChE inhibitory activity of each fraction；(C) RP-HPLC purification chromatography of

606

Fr.4; (D) AChE inhibitory activity of each subfraction (Fr.4-1~10); (E) Mass spectrum of

607

Pro-Ala-Tyr-Cys-Ser (PAYCS, m/z 540.1936 Da); (F) Mass spectrum of Cys-Val-Gly-Ser-Tyr

608

(CVGSY, m/z 527.1955 Da); (G) AChE inhibitory capacities of PAYCS and CVGSY; The sample

609

concentration for AChE inhibitory capacity detection was 10 mg/mL. Superscript letters (a, b, c)

610

represent the statistical significances between samples, p < 0.05.

611

Fig. 2 Effects of PAYCS and CVGSY on glutamate-induced oxidative stress in PC12 cells. (A)

612

Cell viability after treatment with PAYCS and CVGSY (0.5 mg/mL); (B) Cell viability after

613

treatment with PAYCS and CVGSY (0.5 mg/mL) and 32.5 mM glutamate for 24 h; (C) LDH

614

leakage in PC12 cells. △: vs. control group, p < 0.05. *: vs. glutamate group, p < 0.05. #

615

represents the significant differences among sample groups, p < 0.05.

616

Fig. 3 Morphological analysis of nuclear chromatins in PC-12 cells as determined by Hoechst

617

33258 staining (100×).

618

Fig. 4 Effects of PAYCS and CVGSY (0.5 mg/mL) on the glutamate-induced (A) intracellular

619

ROS accumulation; (B) SOD activity down-regulation; (C) GSH-px activity down-regulation; (D)

620

MDA levels overproduction. △: vs. control group, p < 0.05. *: vs. glutamate group, p < 0.05. #

621

represents the significant differences among sample groups, p < 0.05.

622

Fig. 5 Effects of PAYCS and CVGSY (0.5 mg/mL) on the glutamate-induced intracellular Ca2+

623

influx (A) and MMP loss (B). [Ca2+]i was detected with a fluorescence spectrophotometer. PAYCS

624

and CVGSY (0.5 mg/mL) could inhibit the [Ca2+]i increment. JC-1 mmonomer and aggregates

625

were measured by fluorescence spectrophotometer. The ratio of aggregates intensity and monomer

626

intensity of JC-1 could be used to represent the change of MMP. △: vs. control group, p < 0.05. *:

627

vs. glutamate group, p < 0.05. # represents the significant differences among sample groups, p

Neuroprotective Effects of Acetylcholinesterase Inhibitory Peptides

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