Protective roles of N-benzylcinnamide on cortex and hippocampus of aged rat brains

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Archives of Pharmacal Research ISSN 0253-6269 Arch. Pharm. Res.DOI 10.1007/s12272-015-0593-8

Protective roles of N-benzylcinnamide oncortex and hippocampus of aged rat brains

Wipawan Thangnipon, Nirut Suwanna,Chanati Jantrachotechatchawan,Sukonthar Ngampramuan,Patoomratana Tuchinda & SaksitNobsathian

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RESEARCH ARTICLE

Protective roles of N-benzylcinnamide on cortex and hippocampusof aged rat brains

Wipawan Thangnipon1• Nirut Suwanna1,2

• Chanati Jantrachotechatchawan1•

Sukonthar Ngampramuan1• Patoomratana Tuchinda3

• Saksit Nobsathian4

Received: 30 September 2014 / Accepted: 30 March 2015

� The Pharmaceutical Society of Korea 2015

Abstract Brain aging has been associated with oxidative

stress leading to inflammation and apoptosis. The protec-

tive effects and underlying mechanisms of N-benzylcin-

namide (PT-3), purified from Piper submultinerve, on

brains of 90-week-old Wistar rats were investigated fol-

lowing daily intraperitoneal injection with 1.5 mg of PT-3/

kg of body weight for 15 days. PT-3 treatment improved

spatial learning and memory of aged rats and caused sig-

nificant changes in brain frontal cortex, hippocampus, and

temporal cortex in parameters associated with oxidative

stress (decreased reactive oxygen species production and

iNOS and nNOS levels), inflammation (reduced levels of

IL-1b and IL-6), apoptosis (reduced levels of Bax and

activated caspase-3, and elevated level of Bcl-2), and sig-

naling pathways related to inflammation and apoptosis

(decreased amounts of phospho-JNK and -p38, increased

phospho-Akt level and no change in phospho-ERK1/2

content) compared to controls. PT-3 treatment also inhib-

ited aged rat brain AChE activity. These results suggest

that PT-3 with its intrinsic antioxidant and AChE inhibitory

properties has therapeutic potential in ameliorating, in part,

age-associated damages to the brain.

Keywords Aging � Antioxidant � Apoptosis �N-benzylcinnamide � Morris water maze

Introduction

Brain aging is characterized by progressive neuronal loss

and functional deficits, some of which may stem from

oxidative stress, inflammatory responses and mitochondrial

dysfunction (Poon et al. 2004). Age-associated changes in

frontal and temporal cortices and in hippocampus of aged

murines are associated with altered performance in Morris

water maze performance, indicative of impairment to the

spatial reference memory capacity (Lu et al. 2010).

Moreover, aged human brains show hippocampal atrophy

(Jack et al. 1998) and tissue loss in the frontal and temporal

cortices (Colcombe et al. 2003).

In the aged brain, neuronal apoptotic pathways are also

activated by oxidative stress-induced alterations in cell

signaling and disruption of synaptic connectivity (Hu et al.

2006). The enhanced susceptibility to apoptosis in aged rat

hippocampus is indicated by elevation in levels of cytosolic

(pro-apoptosis) Bax and activated caspase-3 (Jin et al.

2008), and reduction of cytosolic (anti-apoptosis) Bcl-2

(Kaufmann et al. 2003). The expressions of inflammatory

cytokines IL-1b (Jin et al. 2008) and IL-6 (Ye and Johnson

1999) are increased in the hippocampus of aged rodents.

Age-altered regulation of nitric oxide synthase (NOS) may

play a significant role in the process of brain aging as NO

contributes to brain deterioration (Liu et al. 2005). Oxida-

tive or inflammatory condition causes expression of in-

ducible (i)NOS in aged rat cortex (Uttenthal et al. 1998) and

& Wipawan Thangnipon

[email protected]

1 Research Center for Neuroscience, Institute of Molecular

Biosciences, Mahidol University, Salaya, Nakhonpathom,

Thailand

2 Department of Companion Animal Clinical Sciences, Faculty

of Veterinary Medicine, Kasetsart University,

Kamphaeng Saen, Nakhonpathom, Thailand

3 Department of Chemistry, Faculty of Science, Mahidol

University, Bangkok, Thailand

4 Nakhonsawan Campus, Mahidol University,

Amphoe Phayuhakiri, Nakhonsawan, Thailand

123

Arch. Pharm. Res.

DOI 10.1007/s12272-015-0593-8

Author's personal copy

hippocampus (Gavilan et al. 2007). Elevation in neuronal

(n)NOS expression occurs in aged rat cortex (Uttenthal

et al. 1998) and in aged murine brains (Sharman et al. 2000).

Furthermore, elevated levels of IL-1b and reactive oxygen

species (ROS) are associated with up-regulation of stress-

activated p38 and JNK signaling pathways in hippocampus

of aged rat brains (Jin et al. 2008, Martin et al. 2002), which

result in down-regulation of growth-related ERK1/2 and

survival-related Akt/PI3 K pathways (Jin et al. 2008). Anti-

inflammatory and antioxidant sodium ferulate prevents age-

induced elevation of caspase-3 and IL-1b levels in rat

hippocampus and reverse age-related changes in JNK and

Akt signaling pathways (Jin et al. 2008).

Age-related reduction in the function of cholinergic

system is responsible for short-term memory deficit (Braida

et al. 2000). An effective indicator of the status of

cholinergic metabolism is acetylcholinesterase (AChE)

activity, which allows temporal control of synaptic acti-

vation by rapidly hydrolyzing acetylcholine, a neurotrans-

mitter in the central nervous system, to acetate and choline

(Das et al. 2001). AChE activity in the brain declines with

age (Das et al. 2001) and is accompanied by a decrease in

acetylcholine content (Wu et al. 1988). AChE inhibitors,

which suppress acetylcholine hydrolysis and enhance

cholinergic transmission, have been used in the treatment

of cognitive disorders, particularly in aging and in Alz-

heimer’s disease (Kuhl et al. 1999).

N-benzylcinnamide (PT-3) from Piper submultinerve

provides neuroprotective effects on amyloid-b (Ab)-in-

duced toxicity (Thangnipon et al. 2013). N-trans-feruloyl-

tyramine and PT-3 attenuate Ab-induced cell death in rat

cultured cortical neurons by inhibiting the generation of

ROS, reducing caspase-3 and Bax levels and upregulating

Bcl-2 expression (Thangnipon et al. 2012, 2013). PT-3 also

suppresses inflammatory cytokines, IL-1b and IL-6, and

deactivates toxicity-associated p38 and JNK (Thangnipon

et al. 2013).

In this study, we investigated the protective effects of

PT-3 treatment on ROS production, apoptosis, inflamma-

tion, and related pathways of aged rat brains, specifically

the frontal cortex, hippocampus, and temporal cortex. In

addition, we examined the ability of PT-3 treatment to

ameliorate age-related neuropathological processes asso-

ciated with cognitive deficits.

Materials and methods

Animal experiments

At 90 weeks of age, three Wistar rats (450–700 g), indi-

vidually housed, kept on a 12 h light reversal schedule and

provided with food and water ad libitum, were injected

intraperitoneally (i.p.) daily at 12 PM with 1.5 mg of PT-3

(Nobsathian et al. 2012)/kg of body weight (BW) (freshly

prepared) for 15 days, and three control rats received vehicle

(40 % (v/v) dimethylsulfoxide, 59 % (v/v) phosphate-buf-

fered saline and 1 % (v/v) ethanol). All experimental pro-

cedures were approved by the Ethical Review Committee on

Animal Experimentation, Mahidol University.

Morris water maze test

In order to determine the effects of age and of PT-3 treatment

on spatial learning and memory, water maze experiments

were performed as previously described (Hernandez et al.

2006). In brief, a black plastic circular pool (diameter,

180 cm; height, 76 cm) was filled with water to a depth of

35 cm (maintained at 25 ± 1 �C). The pool was located in a

large room with a number of extra-maze visual cues, namely

geometric images hung on the wall. The pool was divided

into 4 quadrants of equal area (northeast, northwest, south-

east and southwest). All data were recorded via a comput-

erized video tracking recording system (S-MART: PanLab

Co., Barcelona, Spain). On the day prior to the water maze

hidden platform test, a visible platform test was performed to

assure that the study animals were visually capable of per-

forming the test and that they demonstrated normal search/

escape behaviors. For the hidden platform tests, an invisible

(black) 10 9 10-cm square platform was submerged ap-

proximately 2.0 cm below the surface of the water and

placed in the center of the southwest quadrant. In order to

avoid any other external spatial cues apart from the maze, a

black curtain surrounded the pool.

Each rat was given three trials per day for five con-

secutive days to locate and climb onto the hidden platform.

A trial was initiated by placing the rat in the water with its

nose directly facing the pool wall in one of the three

quadrants other than the target quadrant. The daily order of

entry into individual quadrants was pseudo-randomized

such that all three quadrants were used once every training

day. Twenty-four hours following the last drug treatment, a

single probe trial was conducted for each test animal to

ensure spatial bias for the previous platform location by

removing the platform from the pool and measuring the

time spent in the previous platform quadrant location. The

whole procedure took six consecutive days.

ROS production assay

Following the water maze tests, rats were euthanized by

decapitation without anesthesia. Three brain regions

(frontal cortex, hippocampus, and temporal cortex) were

rapidly dissected out and immediately frozen at -80 �C.

ROS production was determined as previously described

(Hashimoto et al. 2013). In brief, 2.5 mg brain tissues were

W. Thangnipon et al.

123


homogenized in 50 ll of ice-cold 100 mM potassium

phosphate buffer (KPB) (pH 7.4) and 10 ll aliquot of fresh

homogenate was added to 970 ll of 100 mM KPB and

incubated with 5 lM 20,70-dichlorofluorescin diacetate in

methanol for 15 min at 37 �C, followed by centrifugation

at 12,5009g for 10 min at 4 �C. The pellet was resus-

pended in 250 ll of ice-cold 100 mM KPB and incubated

for 60 min at 37 �C. Fluorescence (485 nm excitation and

535 nm emission) was measured at 37 �C at 10 min in-

tervals for 30 min in DTX 880 multimode plate reader

(Beckman Coulter).

AChE activity assay

AChE activity was determined colorimetrically using

acetylthiocholine iodide as a substrate (Ellman et al. 1961).

In short, 5 mg rat brain was homogenized in 100 ll of

100 mM PBS containing 1 % Triton X-100 (Thangnipon

et al. 1995), centrifuged at 10,0009g for 5 min and 25 ll

aliquot of the supernatant was added to 75 ll of 100 mM

PBS and 25 ll of 10 mM 5,5-dithio-bis nitrobenzoic acid.

The reaction was initiated by addition of 25 ll of 75 mM

acetylthiocholine iodide and the mixture was incubated at

room temperature for 2 min. Absorbance at 412 nm was

recorded at 1 min intervals for 8 min in a microplate reader

spectrophotometer (Molecular Devices SpectraMax, CA).

AChE activity is expressed as nmol/min/mg protein. Pro-

tein concentration was measured using Bradford method

(Bio-Rad protein assay kit).

Western blot analysis

Rat brain tissue contents of proteins and phospho-proteins

of interest were assayed by western blotting employing the

following primary antibodies: rabbit anti-iNOS (1:500 di-

lution) (Abcam); mouse anti-Bcl-2, -Bax, and -IL-6

(1:500); 1:1000 rabbit anti-IL-1b (1:1000) (Santa Cruz);

rabbit anti-phospho-p38 and -p38 (1:500); rabbit anti-ac-

tivated caspase-3, -nNOS, -phospho-ERK1/2, -ERK1/2,

-phospho-JNK, -JNK, -phospho-Akt, and -Akt (1:1000);

and rabbit anti-actin (1:2500) (Cell Signaling), latter used

to normalize gel loading). Protein bands were visualized by

treating with appropriate secondary horseradish per-

oxidase-conjugated antibodies (1:1000) (Zymed) at room

temperature for 1 h and quantitated using enhanced

chemiluminescence (Bio-Rad and ImageJ program).

Statistical analysis

Statistical significance of differences in the escape latency

and swimming path length of Morris water maze test was

evaluated using two-way analysis of variance (ANOVA)

with repeated measures. Other data were analyzed using

unpaired Student’s t test with Welch’s correction. Results

are considered statistically significant at p \ 0.05. All data

are expressed as mean ± SEM of three independent ex-

periments conducted in triplicate.

Results

Effects of PT-3 on learning and memory ability

of aged rats in Morris water maze tests

In the Morris water maze tests, at day 5 of PT-3 treatment

aged rats showed improved spatial reference memory as

reflected by significant decreases in escape latency time

(p \ 0.001) and interaction time (p [ 0.05) (Fig. 1a), and

in swimming path length (p \ 0.05) (Fig. 1b) compared to

controls. This effect was not attributed to difference in

motor activity as the two rat groups exhibited similar

swimming speeds (Fig. 1c). Moreover, there are statisti-

cally significant effects of PT-3 treatment on performance

in probe trials as indicated by the greater percent total time

spent in the previous target quadrant by treated compared

to control aged rats (Fig. 1d).

Effects of PT-3 on ROS production in aged rat

brains

PT-3 treatment (1.5 mg/kg BW daily for 15 days) sig-

nificantly reduces ROS production in aged rat frontal cortex,

hippocampus and temporal cortex to 82 ± 2 % (p \ 0.05),

81 ± 2 % (p \ 0.01), and 78 ± 2 % (p \ 0.05), respec-

tively compared to controls (Fig. 2a).

Effects of PT-3 on AChE activity in aged rat brains

AChE activity in the frontal cortex, hippocampus and

temporal cortex of PT-3-treated aged rats is significantly

reduced compared to controls: 446 ± 24 versus 723 ± 39

(p \ 0.01), 529 ± 37 versus 853 ± 44 (p \ 0.01), and

571 ± 7 versus 905 ± 56 (p \ 0.01) nmol/min/mg, re-

spectively (Fig. 2b).

Effects of PT-3 on expressions of apoptotic

and inflammatory proteins in aged rat brains

Treatment with PT-3 significantly attenuates apoptosis in

the aged rat brains compared to controls as evidenced by

reductions in the levels of pro-apoptotic Bax (Fig. 3a) and

activated caspase-3 (Fig. 3c) in hippocampus and temporal

cortex, and elevation in anti-apoptotic Bcl-2 (Fig. 3b) in

hippocampus. Furthermore, PT-3 treatment is able to al-

leviate significantly age-associated inflammatory responses

compared to control as shown by the reduced levels of IL-

Protective roles of N-benzylcinnamide on cortex and hippocampus of aged rat brains

123


1b in the hippocampus (Fig. 4a), IL-6 in the hippocampus

and temporal cortex (Fig. 4b), iNOS in the frontal cortex

and hippocampus (Fig. 4c), and nNOS in the hippocampus

and temporal cortex (Fig. 4d).

b Fig. 1 Effects of PT-3 treatment of aged rats on performance in a

Morris water maze test. Wistar rats (90 weeks of age; (n = 3)) were

injected i.p. daily for 15 days with 1.5 mg of PT-3/kg BW. Controls

(n = 3) received vehicle. There were 3 trials/day over five con-

secutive days of testing of a mean latency time to find a hidden

platform, and b mean path length. c Motor function of aged rats was

determined by swimming speed. d Performance of water maze probe

trial is reported as mean percent total time spent in the previous target

quadrant. Results are expressed as mean ± SEM of three independent

experiments. *p \ 0.05 compared to control

Fig. 2 Effects of PT-3 treatment of aged rats on intracellular a ROS

production and b AChE activity. Animals were treated with PT-3 as

described in legend to Fig. 1. ROS production and AChE activity of

brain homogenates were assayed fluorometrically using 20,70-dichlo-

rofluorescin diacetate and by Ellman reaction respectively. Results are

expressed as mean ± SEM of three independent experiments.

*p \ 0.05, **p \ 0.01 compared to control. Front frontal cortex,

hippoc hippocampus, temp temporal cortex


123


Cell signaling pathways of PT-3-mediated

neuroprotection in aged rat brains

JNK phosphorylation in the hippocampus of aged rats is

significantly inhibited by PT-3 treatment compared to

controls (Fig. 5a), as well as p38 phosphorylation in the

hippocampus and temporal cortex (Fig. 5b). However, no

changes were observed in ERK1/2 phosphorylation

(Fig. 5c), whereas Akt phosphorylation in the frontal cor-

tex and hippocampus are significantly increased in PT-3

treated compared to control aged rats (Fig. 5d).

Discussion

Oxidative stress resulting from elevated ROS generation in

aged rat brain, specifically the frontal cortex and hip-

pocampus, is significantly higher than in young rat brain

(Driver et al. 2000). Treatment with silymarin, a fruit-

derived antioxidant, lowers ROS production more sig-

nificantly in the cortex of young than aged rats brains

(Galhardi et al. 2009). In this study, treatment of aged rats

with PT-3 for 15 days was effective in reducing ROS

production in three regions of the brain. Similarly, hu-

perzine A, isolated from a Chinese herbal medicine, re-

duces lipid peroxidation and enhances superoxide

dismutase activity in aged rat cerebral cortex and hip-

pocampus (Shang et al. 1999). However, it is worth noting

that administration of an antioxidant extracted from Cen-

tella asiatica does not cause any marked changes in lipid

peroxidation levels in various regions of young rat brains

including cortex and hippocampus (Subathra et al. 2005).

In aged rat, low cholinergic activity in the brain is im-

proved through inhibition of AChE by a number of com-

pounds, such as eptastigmine (in frontal cortex and striatum)

(Braida et al. 2000; Garrone et al. 1998), huperzine A (in

frontal cortex, hippocampus, striatum, and hypothalamus)

(Tang et al. 1994), and phenylcinnamide derivatives (Saeed

et al. 2014). All of these compounds share an amide group,

which is capable of interacting with a serine residue in AChE

active site (Imramovsky et al. 2012). In addition, i.p. in-

jection with cholinesterase inhibitor, donepezil or galan-

tamine, improves cognitive performances of aged rats in

both hidden platform task and probe trial in Morris water

maze test (Hernandez et al. 2006). PT-3 may restore spatial

memory partly via its capability of reducing AChE activity.

PT-3 treatment was able to dampen oxidants (ROS and

NO, inferred from the decreased expression of iNOS and

nNOS) and associated inflammation in hippocampus and

temporal cortex of aged rat brains. A number of antioxidants

Fig. 3 Effects of PT-3 treatment of aged rats on levels of a Bax in

hippocampus and temporal cortex, b Bcl-2 in hippocampus and

c activated caspase-3 in hippocampus and temporal cortex. Animals

were treated with PT-3 as described in legend to Fig. 1. Brain

homogenates were assayed by western blotting. Results are expressed

as mean ± SEM of three independent experiments. *p \ 0.05,

**p \ 0.01 compared to control


123


exert similar effects. For example, eicosapentanoic de-

creases age-related elevation in IL-1b expression in rat

hippocampus (Martin et al. 2002); AChE inhibitor ladostigil

reduces age-related up-regulation of iNOS and IL-6 ex-

pression in rat parietal cortex (Panarsky et al. 2012); and

melatonin abrogates age-associated elevation of nNOS level

in mouse cortex (Sharman et al. 2000).

Oxidative stress also is capable of initiating apoptosis

through activation of JNK and p38 phosphorylation

(Thangnipon et al. 2013). Apoptosis was ameliorated in

hippocampus and temporal cortex of aged rat brains by PT-

3 treatment. Age-related elevation of Bax in rat hip-

pocampus is decreased by the antioxidant N-tert-butyl-a-

phenylnitrone (Kaufmann et al. 2003), and the antioxidant

catechins from green tea extract prevent reduction of Bcl-2

level in aged murine hippocampus (Li et al. 2009). Sodium

ferulate reduces JNK phosphorylation and lowers activated

caspase-3 expression in aged rat hippocampus (Jin et al.

2008). The p38 inhibitor SB203580 abolishes IL-1b-in-

duced caspase-3 activation (Martin et al. 2002). Similarly,

JNK inhibitor D-JNKI1 has been employed against in-

flammatory-associated apoptosis in the aged rat brains

(Maher et al. 2005). Furthermore, as Bcl-2 degradation is

facilitated by caspase-3 (Choi et al. 2010), the increase in

total Bcl-2 expression by PT-3 may be acting through the

down-regulation of activated caspase-3 via inactivation of

JNK and p38. Age-associated inflammation and JNK ac-

tivation are accompanied by a reduction in survival-related

Akt/PI3 K signaling (Jin et al. 2008). PT-3 treatment

elevated Akt phosphorylation in the aged rat frontal cortex

and hippocampus. This is in agreement with a previous

finding that, in an aging mouse model, elevation of an-

tioxidant genes, suppression of caspase-3 cleavage and

JNK phosphorylation, and restoration of impaired water

Fig. 4 Effects of PT-3 treatment of aged rats on expression levels of

a IL-1b in hippocampus, b IL-6 in hippocampus and temporal cortex,

c iNOS in hippocampus and frontal cortex, and d nNOS in

hippocampus and temporal cortex. Animals were treated with PT-3

as described in legend to Fig. 1. Brain homogenates were assayed by

western blotting. Results are expressed as mean ± SEM of three

independent experiments. *p \ 0.05, **p \ 0.01 compared to control


123


maze test performances, including both hidden platform

task and probe trial, by antioxidant anthocyanins from

sweet purple potato are abolished by treatment with PI3 K

inhibitor LY294002 (Lu et al. 2010). This lends support to

the notion that the amelioration of spatial memory deficits

by PT-3 may be attributable to JNK inhibition and PI3 K/

Akt activation, which ultimately reduce oxidative stress

and apoptotic cell death in aged rat cortex and hippocam-

pus. On the other hand, ERK1/2 mediates cell survival and

proliferation, and PT-3 did not alter ERK1/2 phosphory-

lation status in the aged rat brain, consistent with that re-

ported in rat cultured cortical neurons (Thangnipon et al.

2013). PT-3 treatment induced different effects in different

regions of the brain on the expression of proteins involved

in age-related alterations. Interestingly, PT-3 significantly

impacted all age-related parameters in the hippocampus.

This is congruent with numerous findings that spatial

memory-ameliorating treatments also affects the following

age-associated characteristics in hippocampus: cholinergic

transmission (Hernandez et al. 2006), oxidative stress,

apoptosis, JNK activation, Akt inhibition (Lu et al. 2010),

and inflammation (Briones and Darwish 2012). Consistent

with the effects of PT-3 on the cortex, it has been reported

that caspase-3 activity is increased only in aged rat tem-

poral cortex and hippocampus (Cetin et al. 2013). In ad-

dition, our result is supported by a previous finding

(Campuzano et al. 2009) that aging elevates the level of IL-

6 but not IL-1b in rat cortex.

In conclusion, PT-3 treatment of aged rat for even a

short period of time (15 days) was able to abrogate such

age-related phenomena as impaired spatial cognitive per-

formance, low cholinergic activity, oxidative stress, in-

flammation and apoptosis. This may be related to its ability

to act both as an AChE inhibitor and an antioxidant.

Fig. 5 Effects of PT-3 treatment of aged rats on levels of a JNK

phosphorylation in hippocampus, b p38 phosphorylation in hip-

pocampus and temporal cortex, c ERK1/2 phosphorylation in all three

brain regions, and d Akt phosphorylation in frontal cortex and

hippocampus. Animals were treated with PT-3 as described in legend

to Fig. 1. Brain homogenates were assayed by western blotting.

Results are expressed as mean ± SEM of 3 independent experiments.

*p \ 0.05, **p \ 0.01 compared to control


123


Acknowledgments This research project was supported by The

Thailand Research Fund (TRF) through the Royal Golden Jubilee

Ph.D. Program (Grant no. PHD/0354/2550) and the Office of the

Higher Education Commission, Ministry of Education, and Mahidol

University, Thailand. We are grateful to Prof. Prapon Wilairat,

Mahidol University, for critical reading of the manuscript. We thank

Ms. Paksinee Klaimala, Ms. Siriporn Leungsuchonkul and Ms.

Paphatsara Khunlert, Impact of Pesticide Use subgroup, Pesticide

Research group, Agricultural Production Science Research and

Development office, Department of Agriculture, Thailand, for helping

in the experiments.

Conflict of interest The authors declare that there are no conflicts

of interest.

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Protective roles of N-benzylcinnamide on cortex and hippocampus of aged rat brains

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