Pine needle extract prevents hippocampal memory impairment in acute restraint stress mouse model

Author’s Accepted Manuscript Pine needle extract prevents hippocampal memory impairment in acute restraint stress mouse model Jin-Seok Lee, Hyeong-Geug Kim, Hye-Won Lee, Won-Yong Kim, Yo-Chan Ahn, Chang-Gue Son www.elsevier.com/locate/jep

PII: DOI: Reference:

S0378-8741(16)31948-1 http://dx.doi.org/10.1016/j.jep.2017.06.024 JEP10902

To appear in: Journal of Ethnopharmacology Received date: 17 November 2016 Revised date: 1 June 2017 Accepted date: 18 June 2017 Cite this article as: Jin-Seok Lee, Hyeong-Geug Kim, Hye-Won Lee, Won-Yong Kim, Yo-Chan Ahn and Chang-Gue Son, Pine needle extract prevents hippocampal memory impairment in acute restraint stress mouse model, Journal of Ethnopharmacology, http://dx.doi.org/10.1016/j.jep.2017.06.024 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Pine needle extract prevents hippocampal memory impairment in acute restraint stress mouse model Jin-Seok Leea, Hyeong-Geug Kima, Hye-Won Leeb, Won-Yong Kima, Yo-Chan AhncChang-Gue Sona* a

Liver and Immunology Research Center, Oriental Medical College of Daejeon University, 22-5

Daehung-dong, Jung-gu, Daejeon, 301-724, Republic of Korea b

TKM-based Herbal Drug Research Group, Korea Institute of Oriental Medicine, Daejeon 305-811,

Republic of Korea c

Department of Health Service Management, Daejeon University, 96-3 Yongun-dong, Dong-gu,

Daejeon, 300-716, Republic of Korea

*

Correspondence to: Chang-Gue Son, O.M.D., Ph.D. Professor, Liver and Immunology Research

Center, Daejeon Oriental Hospital of Oriental Medical Collage of Daejeon University, 22-5 Daehungdong, Jung-gu, Daejeon, 301-724, Republic of Korea. Tel.: +82 42 257 6397; fax: +82 42 257 6398. [email protected]

 

Abstract Ethnopharmacological relevance The Pinus densiflora leaf has been traditionally used to treat mental health disorders as a traditional Chinese medicine. Here we examined the ethnopharmacological relevance of pine needle on memory impairment caused by stress.

Aim of the study To elucidate the possible modulatory actions of 30% ethanolic pine needle extract (PNE) on stressinduced hippocampal excitotoxicity, we adopted an acute restraint stress mouse model.

Materials and methods Mice were orally administered with PNE (25, 50, or 100 mg/kg) or ascorbic acid (100 mg/kg) for 9 days, and were then subjected to restraint stress (6h/day) for 3 days (from experimental day 7 to 9). To evaluate spatial cognitive and memory function, the Morris water maze was performed during experimental days 5 to 9.

Results Restraint stress induced the memory impairment (the prolonged escape latency and cumulative pathlength, and reduced time spent in the target quadrant), and these effects were significantly prevented by PNE treatment. The levels of corticosterone and its receptor in the sera/hippocampus were increased by restraint stress, which was normalized by PNE treatment. Restraint stress elicited the hippocampal excitotoxicity, the inflammatory response and oxidative injury as demonstrated by the increased glutamate levels, altered levels of tumor necrosis factor (TNF)-α and imbalanced oxidantantioxidant balance biomarkers. Two immunohistochemistry activities against glial fibrillary acidic protein (GFAP)-positive astrocytes and neuronal nuclei (NeuN)-positive neurons supported the finding of excitotoxicity especially in the cornu ammonis (CA)3 region of the hippocampus. Those alterations were notably attenuated by administration of PNE.

Conclusions

 

The above findings showed that PNE has pharmacological properties that modulate the hippocampal excitotoxicity-derived memory impairment under severe stress conditions.

Graphical abstract

1. Introduction Stress responses are necessary processes for adaptation to constantly changing environments in life. A stress status could have beneficial or detrimental effects depending on the conditions, and then severe stress may cause the development of neurological disorders including schizophrenia, cerebrovascular accident, and neurodegenerative diseases (Esch et al., 2002; Cohen et al., 2007). Among functional regions of brain tissues, the hippocampus plays a crucial role for negative feedback in the stress response. Uncontrolled stress can elicit dendritic atrophy of hippocampal neurons via alterations in neuronal homeostasis, such as neuroendocrine, neurotransmitters and normal neuroinflammatory responses (Chen et al., 2010b; Mondelli et al., 2011). Excessive stress activates the hypothalamic-pituitary-adrenal (HPA) axis and sequentially induces over-secretion of corticosterone (Dallman, 1993). In particular hippocampus is known to be vulnerable to glucocorticoids (Kim and Diamond, 2002), therefore the stress-induced high concentration of corticosterone could impair the hippocampal main functions; learning and memorizing (McEwen and Magarinos, 2001; Garcia-Bueno et al., 2008). The stress-induced corticosterone is also known to elevate the extracellular glutamate level in synaptic clefts, which one of the main causes in stress-derived neurodegeneration, as form of neuronal excitotoxicity (Popoli et al., 2012; Mehta et al., 2013). In addition, the reactive oxygen/nitrogen species (ROS/RNS) are major contributors to pathological processes of neurodegenerative disorders; furthermore, brain tissue is susceptible to oxidative injury due to relatively high concentrations of irons, unsaturated fatty acids, and blood-brain barrier (BBB) sensitivity (Halliwell, 1992; Pun et al., 2009). Under uncontrolled stress status, over-generation of free radicals by glial cells is well recognized (Gleichmann and  

Mattson, 2011). On the other hand, recently natural products have been pharmaceutical recourse in drug developments for neurodegenerative disorders (Essa et al., 2012; Gao et al., 2013). The pine needle (Pinus densiflora Sieb & Zucc.) has been traditionally used to treat neurological disorders in East Asian countries, which is well documented in the traditional Chinese medical book called Bencao Gangmu (膩誣笾聺) (Li and Luo, 2003). Moreover, Pinus densiflora is recorded as a one of the herbs frequently cited for treating memory disorders (May et al., 2013). Our previous study also found notable anti-amnesic actions in a scopolamine-induced memory deficits model (Lee et al., 2015). In order to measure the potential of pine needle extract (PNE) as a neuroprotective resource, we verified it activity under on acute restraint stress-induced hippocampal memory impairment, and investigate its underlying mechanisms.

 

2. Materials and methods 2.1. Preparation of PNE Pine needles (Pinus densiflora Sieb & Zucc.) were obtained from Guryong Mountain in August, 2016 (Chungbuk province, South Korea). Soak chopped pine needles in the 30% ethanol for 72 h to extract it, and supernatant liquid was filtered using a Whatman filter paper (Advantec®, Toyo Roshi Kaisha, Tokyo, Japan). Filtrate was concentrated and lyophilized to powder, and its final yield was 10.46% w/w.

2.2. Fingerprinting analysis of PNE The PNE and four reference compounds including catechin, astragalin, quercetin dihydrate, and kaempferol (250 μg/ml) were dissolved in 50% methanol. These solutions were filtered using an Acrodisc® LC 13 mm syringe filter (0.45 μm, Ann Arbor, MI, USA), and then filtrates were subjected to the high performance liquid chromatography (HPLC). To carry out quantitative analysis the main compound of PNE, catechin and astragalin were diluted into six concentrations with methanol. Calibration curves were obtained from estimating the peak areas for six concentrations in the range of the 0.31 to 500 μg/ml. The linearity of the peak area (y) versus the concentration (x, μg/ml) curve for each component was used to calculate the contents in the PNE. A quantitative analysis was performed under the simultaneous conditions using an 1100 HPLC (Agilent Technologies, Santa Clara, CA, USA) equipped with an autosampler (G11313A), column oven (GA1316A), binary pump (G1312), diodearray-detector (DAD), and degasser (GA1379A). The analytical column was a Gemini C18 (Phenomenex, Torrance, CA, USA) and was kept at 30°C during performance. The data were analyzed by Chemstation software (Agilent Technologies, Santa Clara, CA, USA). The mobile phase conditions contained 10% acetonitrile, 0.05% formic acid and 90% acetonitrile in water. The gradient flow was as follows; 0 - 10 min, 5-10 % B; 10-15 min, 10-20 % B; 15-30 min, 20-25 % B; 30-40 min, 25-60 % B; 40-50 min, 60-100 %. The injection volume was 10 μL, and the analysis was carried out at a flow rate of 1.0 ml/min with detection at 280 nm.  

For confirmation of whether or not PNE contains a two terpenoids compounds including α-pinene and β-pinene, analysis was performed using Agilent 7890A gas chromatography (GC; Agilent Technologies, Santa Clara, CA, USA) equipment with capillary column (30 m × 250 µm × 0.25 µm). PNE was prepared in diethyl ether, and it was injected in to a GC/mass selective detector (MSD). The injector, detector and interface temperatures were maintained at 250°C. GC/mass spectrometry (MS) was analyzed using an Agilent 5975C GC/MS with a column. Electron impact mass spectra were recorded at 70 eV, and each peak was identified using a NIST 5.0 mass spectra library.

2.3. Chemicals and reagents The following reagents were obtained from Sigma (St. Louis, MO, USA); 2,2-azino-bis (3ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 4-amino-3-hydrazino-5-mercapto1,2,4-triazole (Purpald), 1-chloro-2,4-dinitrobenzene (CDNB), N,N-diethyl-p-phenylendiamine (DEPPD), 2,4-dinitrophenylhydrazine (DNPH), 5,5-dithio-bis-2-nitrobenzoic acid (DTNB), ferrous sulfate, potassium phosphate, reduced glutathione (GSH), myoglobin, glutathione reductase (GSHRd), L-glutathione oxidized disodium salt (GSSG), guanidine hydrochloride, potassium phosphate reduced form of β-nicotinamide adenine dinucleotide phosphate (β-NADPH), trichloroacetic acid (TCA), 1,1,3,3-tetraethoxypropane (TEP), and tert-butyl hydroperoxide. The other reagents were obtained from other vendors: Western blotting antibodies 4-hydroxynonenal (4HNE), NADPH oxidase 2 (NOX2), NF-E2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), β-actin, and horseradish peroxidase (HRP)-conjugated horseradish peroxidase secondary antibody (Abcam, Cambridge, MA; Thermo Fisher Scientific, Rockford, IL; and Santa Cruz Biotechnology, Santa Cruz, CA, USA), immunohistochemical staining antibodies glial fibrillary acidic protein (GFAP), neuronal nuclei (NeuN), and universal Pan-specific antibody (Dako, Hamburg, Germany; Millipore, MA, USA; Vector Lab, CA, USA), thiobarbituric acid (TBA; Lancaster Co.; Lancashire, England), H2O2, (Junsei Chemical Co., Ltd.; Tokyo, Japan), n-butanol (J.T. Baker; Mexico City, Mexico), 1 M Tris-HCl solution (pH 7.4) and 500 mM ethylene diaminetetraacetic acid (EDTA) solution (pH 8.0; Bioneer;

 

Daejeon, Republic of Korea). 

2.4. Animals and experimental design Sixty SPF C57BL/6N mice (12 weeks old; male, 24–27 g) were obtained from Koatech (Gyeonggido, Korea). Mice were allowed free access to the food (Cargill Agri Furina; Gyeonggido, Korea) and water, and were housed at a temperature of 23 ± 2°C and 60% relative humidity under a 12 h light/dark cycles. After adaptation for 1 week, the mice were randomly divided into six groups (n = 10 in each group): naïve, control, the PNE (25, 50 or 100 mg/kg) and ascorbic acid (100 mg/kg, as a positive control). The PNE and ascorbic acid were dissolved in distilled water, and they were administered once daily by gavage to mice for 9 days. Administration volume was a 10 µL/g (v/w). After 7th administration (on experimental day 7; 30 min after each drug administration) of drug, all mice (except the naïve group) were subjected to restraint stress through placement inside a 50 ml conical tube (6 h per day) for 3 days. The restraint stress model was described previously (Buynitsky and Mostofsky, 2009), and procedure was performed between 10:00 and 16:00 daily. Restraint tube (3-cm diameter × 8-cm long) comprises a 0.5 cm air hole for breathing (Fig. 1C). The dosage of PNE was based on the prescreening results including in vivo and vitro assay. The protocol was approved by the Institutional Animal Care and Use Committee of Daejeon University (DJUARB 2014-001) and was conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health (NIH).

2.5. Morris water maze task After 5th administration (on experimental day 5) of the drug (distilled water, PNE, or ascorbic acid), all mice were subjected to the Morris water maze for 5 days to evaluate spatial learning and memory function. Morris water maze task was performed in a circular pool (100 cm × 50 cm) with a circular acrylic platform (10 cm × 35 cm). To discriminate the location of platform, stable distal cues  

can be either provided using a curtain around the pool. The pool was filled with water and milk at 22 ± 1°C and divided into equal quadrants. A platform was placed in one of the quadrants ~1 cm below the surface, as described previously (Morris, 1984). To learn the escape from pool, mice were placed on the platform for 10 s and then removed from the pool. The animals were guided into the water facing the pool wall at defined starting positions. Mice were given acquisition trial for 4 days (from experimental day 5 to 8), and the probe trial was performed for 60 s on the last experimental day (9th day). Spatial learning and memory were assessed by detecting parameters including escape latency and path-length (during each acquisition trial) and time spent in the target quadrant (probe trail). Morris water maze task was performed one trial per day. Data were recorded using a video camera connected to the corresponding software (Smart Junior, Panlab SL; Barcelona, Spain).

2.6. Sample preparation Mice were sacrificed under ether anesthesia following the behavior test on last experimental day. Blood and the brain were collected under standard conditions following IACUC of Deajeon University, and the hippocampal region was immediately isolated from the whole brain. Serum was separated by centrifugation at 3000 × g for 15 min at 4°C. Seven hippocampal tissues and sera were stored at -80°C for biochemical analysis or in RNAlater (Ambion, TX, USA) for gene analysis. Three mouse

brains

from

each

group

were

stored

in

4%

paraformaldehyde

solution

for

immunohistochemical staining.

2.7. Determination of hippocampal glutamate, TNF-α levels and serum corticosterone level A 10% (w/v) hippocampal tissue was homogenized on ice using radioimmunoprecipitation assay (RIPA) buffer. The glutamate level in hippocampal tissue was determined using an available glutamate colorimetric assay kit (Biovision; Milpitas, CA, USA) according to manufacturer’s protocol. Briefly, homogenates was incubated with reaction mix (assay buffer, glutamate developer, and glutamate enzyme mix) for 30 min at 37°C under avoid from light. Absorbance at 450 nm was  

measured using a UV spectrophotometer (Molecular Devices; Sunnyvale, CA, USA). Tumor necrosis factor (TNF)-α level in hippocampal tissue was determined using a commercially available enzyme immunoassay (EIA) kit (BD Biosciences, San Diego, CA, USA). Briefly, homogenates was incubated for 2 h at room temperature (RT) in the TNF- α antibody coated plate, and then incubated with working detector (detection antibody and biotin/streptavidin-HRP conjugate) for 1 h at RT. The TMB substrate was added and incubated for 30 min at RT in dark. After add a stop solution, absorbance at 450 and 570 nm was measured using a UV spectrophotometer. The corticosterone level in serum was measured using a commercially available EIA kit (Oxford Biomedical Research, Rochester Hills, MI, USA). Briefly, homogenates were prepared following extraction methods, and then equal volume of samples and corticosterone-HRP conjugate added. After incubation for 1 h at RT, the TMB substrate was added and incubated for 30 min at RT. Then, absorbance at 650 nm was measured using a UV spectrophotometer.

2.8. Determination of ROS levels in serum and hippocampus Hippocampal and serum ROS levels were determined as described previously (Hayashi et al., 2007). Hippocampal homogenates or sera were prepared with sodium acetate buffer (0.1 M, pH 4.8). After incubation at 37°C for 5 min, the DEPPD (10 mM)/ferrous sulfate solution (4.37 μM) mixture (1:25) was added. Absorbance was measured at 505 nm using a UV spectrophotometer, and results were calculated using a standard curve of H2O2.

2.9. Determination of NO and MDA level in hippocampus Hippocampal nitric oxide (NO) level was determined using the Griess reagent method (Green et al., 1982). Hippocampal homogenates were mixed with Griess reagent (1% sulfanilamide, 0.1% N-(1naphthyl) ethylenediamine hydrochloride, 2.5% phosphoric acid). After incubation at 37°C for 20 min, absorbance was measured at 540 nm using a UV spectrophotometer. Hippocampal malondialdehyde (MDA) level was determined using the thiobarbituric acid  

reactive substance (TBARS) method (Mihara and Uchiyama, 1978). The hippocampal tissue was homogenized on ice cold 1.15% KCl buffer. Homogenates mixed with 1% phosphoric acid and 0.67% of TBA. After incubation at 100°C for 45 min, samples were placed on the ice for 5 min. Then, nbutanol was added and centrifuged at 3,000 × g for 15 min at 4°C. Absorbance at 535 and 520 nm was measured using a UV spectrophotometer. The concentration of TBARS was calculated using a standard curve of TEP.

2.10. Determination of TAC in hippocampus Hippocampal total antioxidant capacity (TAC) was determined as described previously (Kambayashi et al., 2009). Hippocampal homogenates were gradually mixed with phosphate-buffered saline (10 mM, pH 7.2), myoglobin solution (18 μM), and ABTS solution (3 mM). After incubation at 25°C for 3 min, the H2O2 was added and incubated for 5 min. Absorbance at 600 nm was measured using a UV spectrophotometer, and results were calculated using a standard curve of gallic acid equivalent antioxidant capacity.

2.11. Determination of total GSH content in hippocampus Hippocampal glutathione (GSH) content was determined as described previously (Ellman, 1959). Hippocampal homogenates were combined with NADPH (0.3 mM)/DTNB (4 mM) mixture (7:1), and then GSH-Rd solution (0.06 units) was added. Absorbance at 405 nm was measured using a UV spectrophotometer, and GSH contents were calculated using standard curve of glutathione reduced.

2.12. Determination of SOD and catalase activities in hippocampus Hippocampal superoxide dismutase (SOD) activity was determined using a SOD assay kit (Dojindo Laboratories; Kumamoto, Japan). Absorbance was measured at 450 nm using a UV spectrophotometer, and results were calculated using dilutions of bovine erythrocyte SOD (Sigma) ranging from 0.01–50 unit/ml.  

Hippocampal catalase activity was determined as described previously (Wheeler et al., 1990). Hippocampal homogenates were methodically mixed with phosphate buffer (250 mM, pH 7.0), methanol (12 mM), and H2O2 (44 mM). After incubation at room temperature for 20 min, reaction was stopped by Purpald solution (22.8 mM Purpald in 2 N potassium hydroxide), followed by incubation at 25°C for 20 min. Finally, potassium periodate (65.2 mM in 0.5 N potassium hydrate) was added. Absorbance at 550 nm was measured using a UV spectrophotometer, and results were calculated using standard curve of catalase.

2.13. Immunohistochemical staining Immunohistochemical staining was analyzed by identifying the expression of neurons with neuronal nuclei (NeuN) and astrocytes with glial fibrillary acidic protein (GFAP) in hippocampus. Brain tissue was immersed in the 4% paraformaldehyde solution for 4 h. To cryoprotect from freezing damage, the brain was immersed for 24 h in 10 to 30% sucrose respectively. Brain tissues were embedded in tissue-freezing medium with liquid nitrogen, and cut into coronal frozen sections (35 μm) using a Leica CM3050 cryostat. Frozen sections of brain were stored in the anti-freeze buffer. To block endogenous peroxidase activity, the free-floating sections were immersed in 1% H2O2. Sections were subjected to blocking buffer (5% normal chicken serum and 0.3% Triton X-100 in PBS) for 1 h, and then incubated with primary NeuN (1:500, MAB377, Millipore) and GFAP (1:200, Z0334, Dako) antibodies overnight at 4°C. After 4 times washing with PBS, sections were subjected to biotinylated pan-specific universal secondary antibody (BA-1300, Vector Lab.) for 2 h, and exposed to an avidin-biotin peroxidase complex (PK-6200, VECTASTAIN Elite ABC kit, Vector Lab.) for 1 h. The

peroxidase

activity

was

color-developed

with

stable

3,

3’-diaminobenzidine,

and

immunoreactions were observed using an Axio-phot microscope (Carl Zeiss, Germany). Quantification analysis of staining results were used the ImageJ 1.46 version (NIH, Bethesda, MD, USA).

 

2.14. Western blot analysis The hippocampal protein expression of 4-hydroxylnonenal (4HNE), NADPH oxidase 2 (NOX2), NF-E2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), and β-actin was analyzed by Western blot. The proteins from hippocampal homogenates were separated by 10% polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes. To minimize the nonspecific response, membranes were blocked with 5% skim milk for 1 h. Then, membranes were subjected to primary antibodies (4HNE, NOX2, Nrf2, HO-1 and β-actin) overnight at 4°C. After 3 times wash, membranes were incubated with HRP-conjugated anti-rabbit or anti-mouse antibody for 2 h. Western blots were visualized using an enhanced chemiluminescence (ECL) advanced kit.

2.15. Gene expression analysis by quantitative real-time PCR The hippocampal mRNA expression of glucocorticoid receptor (GR), N-methyl-D-aspartate receptor subtype 1 (NMDAR1), glutamate decarboxylase 65 (GAD65), and inducible nitric oxide synthase (iNOS) were analyzed by quantitative real-time PCR. Total RNA was isolated from hippocampal tissue using an RNeasy Mini Kit (QIAGEN; Valencia, CA, USA), and cDNA was synthesized using a High-Capacity cDNA reverse transcription kit (Ambion; Austin, TX, USA). Realtime PCR was performed using SYBRGreen PCR Master Mix (Applied Biosystems; Foster City, CA, USA) and PCR amplification was performed using a standard protocol with the IQ5 PCR Thermal Cycler (Bio-Rad; Hercules, CA, USA). Information regarding the primer sequences, product sizes and annealing temperatures is summarized in Table 1.

2.16. Statistical analysis Data are expressed as the mean ± standard deviation (SD) and behavioral results are expressed as the mean ± standard error of mean (SEM). The statistical significance differences between the groups were evaluated by one-way analysis of variance (ANOVA) followed by post hoc multiple comparisons with Tukey’s HSD t-test using the IBM SPSS statistics software, ver. 20.0 (SPSS Inc.; Chicago, IL,  

USA). Differences at P < 0.05, P < 0.01, or P < 0.001 were considered statistically significant.

3. Results 3.1. Compounds present in the 30% ethanolic PNE The 30% ethanolic PNE and four reference compounds (i.e., catechin, astragalin, quercetin dihydrate and kampferol) were subjected to HPLC analysis. Among them, catechin and astragalin were detected at retention times of 19.00 and 31.34 min (Fig. 1A), and the concentration of catechin and astragalin following quantitative analysis were 0.41 ± 0.04 and 2.65 ± 0.03 μg/mg, respectively (Fig. 1B). GC-MS data confirmed that terpenoids of α-pinene or β-pinene were not present in the PNE (data not shown).

3.2. Morris water maze task Restraint stress significantly delayed the escape latency time (P < 0.001), and cumulative pathlength was significantly extended compared with the naïve group on experimental day 8 during acquisition trial (P < 0.001). Administration of PNE significantly ameliorated the increase in escape latency (P < 0.05 for 50 and 100 mg/kg) and path-length (P < 0.01 for 50 and 100 mg/kg; Fig. 2A and B) compared with control group. As a probe trial, the time spent in target quadrant on experimental day 9 was significantly shortened by restraint stress compared with the naïve group (P < 0.001), whereas administration of PNE significantly reversed the alteration compared with the control group (P < 0.05 for 25 and 100 mg/kg, P < 0.01 for 50 mg/kg; Fig. 2C). Ascorbic acid had effects similar to those of PNE treatment.

3.3. Effects on corticosterone level in the serum Serum corticosterone level was significantly elevated by restraint stress compared with the naïve group (P < 0.001). Administration of PNE significantly attenuated the increase in corticosterone level compared with the naïve group (P < 0.01 for 50 and 100 mg/kg; Fig. 3A).  

3.4. Effect on glutamate and TNF-α levels in the hippocampus The glutamate and TNF-α levels in hippocampal tissue were significantly increased by restraint stress compared with naïve group (P < 0.001 and P < 0.01, respectively), whereas administration of PNE significantly attenuated these alterations compared with the control group (P < 0.05 for 25 mg/kg, P < 0.001 for 50 and 100 mg/kg in glutamate; P < 0.05 for 25 mg/kg, P < 0.01 for 50 and 100 mg/kg; Fig. 3B and C). Ascorbic acid had an effect similar to that of PNE.

3.5. Effects on ROS levels in serum and hippocampus Restraint stress significantly increased the ROS levels in both serum and the hippocampus compared with the naïve group (P < 0.001 in both). Administration of PNE significantly attenuated those elevations in both serum (P < 0.05 for 25 and 50 mg/kg, P < 0.01 for 100 mg/kg) and hippocampal tissue (P < 0.05 for 25 and 50 mg/kg, P < 0.001 for 100 mg/kg) compared with the control group (Table 2). Ascorbic acid had an effect on both serum and hippocampal ROS level similar to that of PNE.

3.6. Effects on NO and MDA level in hippocampus NO level in hippocampal tissue were significantly increased by restraint stress compared with the naive group (P < 0.001), whereas administration of PNE significantly attenuated the increase in NO level compared with the control group (P < 0.01 for 25 and 50 mg/kg, P < 0.001 for 100 mg/kg). MDA level in hippocampal tissue was increased significantly by extreme restraint stress compared with the naive group (P < 0.01), whereas administration of PNE significantly attenuated the elevation of MDA level compared with the control group (P < 0.05 for 25 m/kg, P < 0.01 for 50 and 100 mg/kg; Table 2). A similar effect of ascorbic acid was observed only with MDA level.

3.7. Effects on antioxidant biomarker profiles in the hippocampus  

Restraint stress significantly depleted the antioxidant capacity, including TAC, GSH content, activities of SOD and catalase in hippocampal tissue, compared with the naïve group (P < 0.01 in TAC, P < 0.05 in catalase, P < 0.001 for GSH and SOD). These depletions were almost completely reversed by administration of PNE for all biomarkers; administration of PNE showed a higher antioxidant capacity than the naïve group in both TAC and catalase (P < 0.05 or P < 0.01 or P < 0.001 for all groups). These effects were also seen with ascorbic acid in the hippocampus (Table 2).

3.8. Effect on mRNA expression in the hippocampus The GR, NMDAR1, GAD65 and iNOS (P < 0.05 for all) mRNA levels in hippocampal tissue were significantly altered by restraint stress compared with naïve group. These alterations of mRNA levels were significantly ameliorated by administration of PNE compared with the control group (P < 0.05 or P < 0.01 for all groups; Fig. 3D). Moreover, restraint stress significantly down-regulated the mRNA level of GAD65 in hippocampal tissue compared with the naïve group (P < 0.05), whereas administration of PNE significantly normalized the down-regulation of GAD65 compared with the control group (P < 0.05 for 100 mg/kg, Fig 3D). Similar effect of ascorbic acid was observed in the GAD65 mRNA levels.

3.9. Western blot analysis in the hippocampus Restraint stress significantly elevated the 4HNE (P < 0.01), NOX2 (P < 0.01) and HO-1 (P < 0.05) protein levels in hippocampus compared with naïve group, whereas administration of PNE attenuated the increase of 4HNE and NOX2 levels compared with the control group (P < 0.05 or P < 0.01 or P < 0.001 for all groups). The protein level of HO-1 was significantly augmented by administration of PNE more than the control group (P < 0.05 for 50 mg/kg). In contrast, restraint stress significantly reduced the Nrf2 protein level in the hippocampus compared with the naïve group (P < 0.01). Administration of PNE significantly normalized the reduction of Nrf2 level (P < 0.01 for 50 mg/kg). Ascorbic acid had an effect on the expression of 4HNE and NOX2 similar to that of PNE (Fig. 4 A  

and B).

3.10. Immunohistochemical analysis in the hippocampus Restraint stress significantly activated astrocytes in hippocampal regions including the dentate gyrus (DG), cornu ammonis 1 (CA1) and CA3 regions compared with the naïve group (P < 0.05 or P < 0.01), as evidenced by GFAP-positive cell staining. Administration of PNE completely attenuated the numerical increase in astrocytes in hippocampus, as well as their over-activation (P < 0.05 or P < 0.01 for all groups; Fig. 5A and B). The NeuN-labeled number of neurons was not changed by restraint stress in the hippocampal DG and CA1, whereas restraint stress significantly reduced the neuronal cells in CA3, compared with the naïve group (P < 0.05). Administration of PNE significantly increased the neurons in hippocampal CA1 (P < 0.05 for 100 mg/kg) and DG (P < 0.05 for 25 mg/kg). In particular, the reduction in hippocampal CA3 pyramidal neurons was significantly ameliorated by administration of PNE with the NeuN-positive cells (P < 0.05 for 50 and 100 mg/kg; Fig. 6A and B). In contrast, ascorbic acid ameliorated these alterations only in the GFAP staining result.

 

4. Discussion Although moderate stress can help mental health and brain activity, uncontrolled severe stress stimuli impairs brain functions, especially memory function (Wolf, 2009). Memory impairment involves pathophysiological alterations in the brain such as cholinergic abnormalities, neuroendocrine dysfunction, and excitotoxicity (Terry and Buccafusco, 2003; Dong et al., 2009). Our previous study demonstrated the anti-amnesic property of PNE in a cholinergic system-blocked condition (Lee et al., 2015). Under an excessive stress status, excitotoxicity is especially responsible for the neuronal cell death and brain structural atrophy, leading to brain dysfunction including memory impairment (Brown et al., 1999; Mattson, 2000; Hynd et al., 2004). We herein adapted an acute restraint stress mouse model, with purpose of verifying the neuroprotective effects of PNE under stress condition. Both acute and chronic stress was shown to cause hippocampal synaptic loss and suppression of neurogenesis, which caused cognitive and memory impairment (McEwen, 1999). To investigate the spatial learning and memory, the Morris water maze has been commonly used in the animal study. Our results showed that PNE treatment (particularly 50 and 100 mg/kg) remarkably protected the amnesic behavior in the Morris water maze task (Fig. 2A-C). Intriguingly, PNE treatment also showed the tendency to reduce the escape latency even no stress condition (especially 6th day), but without statistical significance (Fig. 2A). We examined the effects of PNE on major modulator of the stress-related hormone system. The HPA axis is a central route to adaptation to psycho-physiological stress. Hyperactivation of HPA axis promotes the synthesis of glucocorticoid hormone from the adrenal cortex under stress conditions (Smith and Vale, 2006). Accumulating evidence has suggested that high level of glucocorticoids impairs hippocampal memory (de Quervain et al., 1998) whileas a GR antagonist inhibits both the stress-induced activation of microglial cell and neuroinflammatory reactions (Frank et al., 2012). The hippocampus is a key brain region for episodic memory and spatial navigation, but this region is particularly sensitive to stress due to the presence of abundant GR (Hoschl and Hajek, 2001). As expected, the PNE treatment attenuated the increase of serum corticosterone level and up-regulation  

of GR hippocampal gene expression (Fig. 3A and D). These results can predict the enhancement of memory function by PNE, and it correlates with positive results on amnesic behavior. Restraint stress has been commonly applied to psychological stress animal model, which elevate the glucocorticoid level and, glutamate release which in turn induce neuronal excitotoxicity and hippocampal atrophy (McEwen, 1999). Glutamate is an essential excitatory neurotransmitter that plays a crucial role especially in hippocampal memory; however, a glut of the extracellular glutamate leads to neuronal excitotoxicity and cell death via promotion of the excessive influx of Ca2+ into neurons (Flores-Soto et al., 2012). Under a stress status, glucocorticoids trigger the elevation of extracellular glutamate level (Stein-Behrens et al., 1994, Wang and Wang, 2009). Severe stress reduced conversion of glutamate into gamma-aminobutyric acid (GABA), a major inhibitory neurotransmitter (Maguire, 2014). Our results supposed them by the elevation of glutamate level, upregulation of NMDA receptor and down-regulation of GAD 65 gene expressions in hippocampus, and then those gene expression alterations were significantly normalized by administration of PNE (Fig. 3B and D). Glutamate and NMDA receptor are currently investigated as potential targets for treatment of patients with moderate to severe Alzheimer’s disease (Reisberg et al., 2003; Kamat et al., 2014). Our findings suggest that PNE ameliorated the hippocampal excitotoxicity via modulating glutamate levels and its-linked genes. We confirmed that PNE treatment completely alleviated the activation of astrocytes as evidenced by measuring GFAP-positive signal in brain tissues (Fig. 5A and B). As our results, restraint stress induces astrogliosis in the hippocampal regions, due to their ability to uptake glutamate and affects ions buffering (Pekny and Nilsson, 2005). In a state of excitotoxicity, reactive glial cells are deeply associated with neuroinflammatory response and oxidative stress (Agostinho et al., 2010) via releasing the inflammatory cytokines, such as TNF-α and interleukin 1β, and producing free radicals such as ROS (Reynolds et al., 2007; Takahashi et al., 2003). From our results, PNE treatment significantly attenuated the elevation of hippocampal TNF-α, ROS and NO levels including iNOS gene expression as well as the products of lipid peroxidation (MDA and 4HNE) in the hippocampus and/or sera under restraint stress (Fig. 3C and D, Fig. 4, Table 2). These results were supported by  

measurements of NOX2 activation (a key molecule of ROS), Nrf2 and HO-1 protein levels, and antioxidant capacity (TAC, GSH, SOD, and catalase) in the hippocampus (Fig. 4, Table 2). Interestingly, HO-1 protein level was notably elevated by restraint stress, which might be a temporal compensatory reaction for the production of antioxidant elements. Our restraints stress model induced excitotoxicity, as shown by various alterations including hyperactivation of the HPA axis, neuroinflammation, and oxidative stress. Those alterations obviously contributed to neuronal degenerations of the hippocampus (Fig. 6A) while administration of PNE significantly protected neurons, in mainly CA3 region, against apoptotic change (Fig. 6B). Several studies have shown that the CA3 region of the hippocampus is especially most vulnerable to oxidative injury, along with dendritic atrophy, under stress conditions (Wood et al., 2004; Chen et al., 2010a). These histopathological changes have been characterized in neurodegenerative diseases, especially Alzheimer's disease (Scheff et al., 2006). As a multi-target natural product, PNE has shown to ameliorate diverse neuropathologies in this animal model, and cholinergic benefits were already demonstrated by our previous study. Our scientific evidences support ethnopharmacological relevance of pine needle, and thus PNE could be prescribed for mild cognitive disorder and neurodegenerative diseases. Japanese Red Pine (Pinus densiflora) is distributed in East Asian countries, which has been widely used in folk remedies or supplementary foods or tea for the health. Pine needle was described in the wooden part of traditional Chinese medical book (Bencao Gangmu) as treating cerebral palsy (Li and Luo, 2003). Japanese herbal medicine, Sho-ju-sen including kumazasa leaves, ginseng radix and pine needle, has been prescribed to patients with mental disorders (Ichikawa et al., 1998). The P. densiflora leaf is composed of various compounds such as polyphenols, terpenoids and flavonoids. Recent studies suggested that pine needle has an antioxidant and anti-apoptotic effects in high cholesterol-fed rat model (Seo et al., 2014), and its polyphenols ameliorated cognitive impairment in a D-galactose-induced mouse model (Wang et al., 2014). Moreover, we speculate that PNE’s neuroprotective effect on hippocampal dysfunction might result from astragalin, as evidenced by mulberry leaves-derived flavonol glycosides inhibiting hippocampal neuroexcitotoxicity in HT22 cell  

(Rebai et al., 2017). Meanwhile, terpenoids as isocupressic acid from Ponderosa pines leaf have abortifacient effects on beef cattle (Gardner et al., 1994; Stegelmeier et al., 1996). Free amino acid such as GABA and alanine of P. densiflora leaf are expected to exert an impact on pharmaceutical activity (Arai et al., 1987). Our previous study demonstrated that PNE has potent anti-amnesic effects against memory deficits by cholinergic dysfunction (Lee et al., 2015). Ascorbic acid (100 mg/kg), a positive control agent in our study, revealed the potential actions against memory impairment (Shahidi et al., 2008; Harrison et al., 2009). Possible mechanism of ascorbic acid involved in the antioxidative activity via pass to BBB in endoplasmic reticulum of the brain (Kumar et al., 2009). In our results, ascorbic acid has shown the positive effects on especially hippocampal oxidative injury; however it couldn’t regulate glucocorticoid-related parameters. It was suggested that pharmacological potentiality of PNE on stress-derived memory impairment superior than ascorbic acid. The present study has limitations including the not therapeutics but preventionfocused design, no identification of active compounds, and unknown fact regarding pass of brain blood barrier of CNS. Further studies are necessary to clarify them in the future.

 

Conclusion Our findings provide insights into the preventive property of PNE on memory impairment under stress condition. The underlying mechanisms are supposed to engage the regulation of stress hormones, hippocampal excitotoxicity and oxidative injury. This study suggested the possibility of P. densiflora leaf as a natural material for stress-derived neurodegenerative disorders.

 

Conflicts of interest The authors declare no conflicts of interest. All of the authors have approved the final article.

Acknowledgements This research was supported by the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (grant number: 2015003195).

Author contribution statement Jin-Seok Lee ([email protected]) wrote the main manuscript text, and conducted experiments. Hyeong-Geug Kim ([email protected]) prepared the figure 4, and extracted the pine needle. Hye-Won Lee ([email protected]) prepared the figure 1A and B (HPLC analysis). Won-Yong Kim ([email protected]) supported the behavioral test for Morris water maze and immunohistochemical staining. Yo-Chan Ahn ([email protected]) performed a statistical analysis. ChangGue Son ([email protected]) supervised the manuscript, and directed final version of all contents. All authors reviewed and approved this manuscript.

This manuscript has been checked by at least two native speakers of English in scientific and technical editing service company (Nature publishing group language editing). For a certificate, please see: languageediting.nature.com/certificate. Reference number: B714-FB0E-4821-38D9-E81B

 

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Figure Legends Figure 1. HPLC chromatograms of 30% ethanolic PNE. (A) PNE and four reference compounds were subjected to HPLC analysis. (B) Retention time and quantification of main two compounds. (C) Experimental scheme of the present study.

Figure 2. Behavioral test for spatial learning and memory. (A) Escape latency time during the acquisition trial of 4 days. (B) Cumulative path-length of the acquisition trial on 4th day. (C) Time spent in the target quadrant of the probe trial on 5th day. Data are expressed as the mean ± SEM (n = 7). ###P < 0.001 compared with the naïve group; *P < 0.05, **P < 0.01 compared with the control group.

Figure 3. Hormone, neurotransmitter, cytokine and their-related gene expressions. (A) Corticosterone serum level. (B) Levels of glutamate. (C) TNF-α in the hippocampus. (D) NMDAR1, GAD65, GR and iNOS gene expressions in the hippocampus, determined by real-time PCR. Gene expression was normalized to that of β-actin. Data are expressed as the mean ± SD (n = 7). #P < 0.05, ###

P < 0.001 compared with the naïve group; *P < 0.05, **P < 0.01,

##

P < 0.01,

***

P < 0.001 compared with the

control group.

Figure 4. Oxidative stress and antioxidant protein levels. (A) 4HNE, NOX2, Nrf2 and HO-1 protein levels in the hippocampus were determined by Western blotting and (B) were quantified. Data are expressed as the mean ± SD (n = 7). #P < 0.05, ##P < 0.01 compared with the naïve group; *P < 0.05, **

P < 0.01, ***P < 0.001 compared with the control group.

Figure 5. GFAP-positive astrocytes were apparent in the hippocampus including DG, CA1 and CA3. (A) Astrocyte activation was confirmed by GFAP immunohistochemical analysis. (B) Quantification of GFAP-positive hippocampal astrocytes. Representative photomicrographs were taken at magnifications of 100 and 400™. Data are expressed as the mean ± SD (n = 3). #P < 0.05, ##P < 0.01  

compared with the naïve group; *P < 0.05, **P < 0.01 compared with the control group.

Figure 6. NeuN-positive neurons were apparent in the hippocampus including DG, CA1 and CA3. (A) Neuronal cells were confirmed by NeuN immunohistochemical analysis. (B) Quantification of NeuNpositive hippocampal neurons. `ative photomicrographs were taken at magnifications of 100™. Data are expressed as the mean ± SD (n = 3). #P < 0.05 compared with the naïve group; *P < 0.05 compared with the control group. 



 

Table 1. Sequence of the primers used in real-time PCR analysis. Annealing

Gene (number)

Primer sequencing (Forward and Reverse)

Product size (base pair)

temperature (Ȕ)

GR (NM_008173)

5′-GGA AGC GTG ATG GAC TTG TAT AAA A-3′ 5′-TGG AAT CTG CCT GAG AAG CA-3′

100

59

GAD65 (NM_008078)

5'-TCG ATT TCC ATT ACC CCA ATG-3' 5'-GTT GTT TGG CAA TGC GTC AA-3’

100

60

NMDAR 1 (NM_001177656)

5'-AGC GTG AGT CCA AGA GTA AAA AAA G-3' 5'-GGG TCA AAC TGC AGC ACC TT-3'

103

60

iNOS (NM_010927)

5′-GGC AGC CTG TGA GAC CTT TG-3′ 5′-TGC ATT GGA AGT GAA GCG TTT-3′

120

60

β-actin (NM_007393)

5'-GGC ACC ACA CCT TCT ACA ATG A-3' 5'-ATC TTT TCA CGG TTG GCC TTA G-3'

100

59

GR; glucocorticoid receptor, NMDAR; N-methyl-D-aspartate receptor, GAD; glutamate decarboxylase, iNOS; inducible nitric oxide synthase, β-actin as a housekeeping gene.

 

Table 2. Effects on serum ROS level and biomarkers of oxidative stress/antioxidant in hippocampus Ascorbic acid (mg/kg)

PNE (mg/kg) Treatment

Naive

Control 25

50

100

100

Serum ROS (U/ml)

24.24 ± 5.51

61.82 ± 15.14###

38.17 ± 12.19*

31.85 ± 15.52*

27.99 ± 13.42**

31.72 ± 11.00*

Hippocampal ROS (U/mg protein)

54.62 ± 10.65

193.28 ± 48.67###

129.36 ± 13.23*

134.71 ± 25.70*

111.33 ± 32.39***

145.26 ± 51.70**

NO (μM/mg protein)

64.37 ± 7.23

97.47 ± 10.73###

72.29 ± 5.15**

74.79 ± 5.93**

59.21 ± 8.05***

87.58 ± 14.91

MDA (μM/mg protein)

11.38 ± 2.43

18.80 ± 3.80##

12.78 ± 1.77*

10.94 ± 2.58**

10.63 ± 2.62**

12.60 ± 5.20*

TAC (μM/mg protein)

19.82 ± 3.45

11.83 ± 2.39##

21.15 ± 4.05**

22.52 ± 3.80***

24.94 ± 5.04***

20.41 ± 3.16**

GSH (μM/mg protein)

38.54 ± 9.70

18.18 ± 4.84###

28.28 ± 5.87*

22.29 ± 7.35

26.55 ± 5.54*

28.89 ± 6.12*

SOD (U/mg protein)

51.10 ± 6.59

32.25 ± 5.32###

42.63 ± 4.48

48. 52 ± 9.33**

50.96 ± 8.31***

46.51 ± 2.36**

Catalase (U/mg protein)

569.17 ± 39.47

558.31 ± 193.03

650.56 ± 88.62**

586.99 ± 78.40*

382.39 ± 70.41#

669.05 ± 144.19**

Data are expressed as the mean ± SD (n = 7). ##P < 0.01, ###P < 0.001 compared with the naïve group; *P < 0.05, **

P < 0.01, ***P < 0.001 compared with the control group.

GSH, glutathione; MDA, malondialdehyde; NO, nitric oxide; ROS, reactive oxygen species; SOD, superoxide dismutase; TAC, total antioxidant capacity.

 

   

 

PNE

Standard

Fig. 1

Fig. 2

Fig. 3

Fig. 4

Fig. 5

Fig. 6