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Wipawan angnipon, Ph.D.*, Sukonthar Ngampramuan, Ph.D.*, Nopparat Suthprasertporn, Ph.D.*, Chanati

Jantrachotechatchawan, Ph.D.**, Patoomratana Tuchinda, Ph.D.***, Saksit Nobsathian, Ph.D.****

*Research Center for Neuroscience, Institute of Molecular Biosciences, Mahidol University, Salaya, Nakhonpathom 73170, ailand, **Wolfson Centre

for Age-Related Diseases, Institute of Psychiatry, Psychology & Neuroscience, King’s College London, United Kingdom, ***Department of Chemistry,

Faculty of Science, Mahidol University, Bangkok 10700, ailand, ****Nakhon Sawan Campus, Mahidol University, Phayuhakiri, Nakhon Sawan

60130, ailand.

Protective Roles of N-trans-feruloyltyramine Against

Scopolamine-Induced Cholinergic Dysfunction on

Cortex and Hippocampus of Rat Brains

ABSTRACT

Objective: To study the protective eects of N-trans-feruloyltyramine (NTF) on scopolamine-induced cholinergic

dysfunction, apoptosis, and inammation in rat brains.

Materials and Methods: Treatments were administered intraperitoneally (i.p.). Wistar rats (8-week-old) were

allocated into 4 groups (n = 3) as follows: scopolamine-only, NTF-only, NTF + scopolamine and control. Spatial

cognition was evaluated by Morris water maze. ROS assay and Western blot analyses were conducted in 3 brain

regions: the frontal cortex, hippocampus, and temporal cortex.

Results: NTF treatment inhibited scopolamine-induced memory impairment and signicantly attenuated scopolamine-

induced changes in the three brain regions. Investigated scopolamine-associated changes were as follows: increases

in ROS production and BACE1 level, decrease in ChAT level, increases in inammatory and apoptotic markers,

and activation of signaling pathway kinases related to inammation and apoptosis.

Conclusion: With its in vivo antioxidant, cholinergic-promoting, anti-apoptosis, and anti-inammatory biological

activities, NTF is a promising candidate to be further investigated as a potential treatment for Alzheimer’s-associated

neurodegeneration.

Keywords: Acetylcholine; Alzheimer’s disease; antioxidant; Morris water maze; N-trans-feruloyltyramine (Siriraj

Med J 2021; 73: 413-422)

Corresponding author: Wipawan angnipon

E-mail: wipawan.tha@mahidol.ac.th

Received 5 February 2021 Revised 20 April 2021 Accepted 22 April 2021

ORCID ID: http://orcid.org/0000-0001-7889-2801

http://dx.doi.org/10.33192/Smj.2021.55

INTRODUCTION

Alzheimer’s disease (AD) causes progressive and

irreversible deterioration of cognitive functions especially

memory.

e major pathological characteristics of the

human AD brain are extracellular aggregates of amyloid-β

(Aβ) and intracellular aggregates of hyperphosphorylated

tau, namely the senile plaques and the neurobrillary

tangles respectively.

The underlying mechanism of

sporadic AD involves cholinergic dysfunction including

degeneration of basal forebrain cholinergic neurons

and loss of hippocampal cholinergic bers.

Impaired

cholinergic transmission aects learning and memory,

cortical and hippocampal information processing, and

ultimately behaviors.

Scopolamine, an antagonist of a

muscarinic acetylcholine receptor,

induces cholinergic

dysfunction and cognitive impairment through oxidative

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414

stress and neuroinflammation in animal models.

Intraperitoneal injection of scopolamine blocks cholinergic

neurotransmission, dysregulates cholinergic system, and

consequently impairs cognition in rodents.

N-trans-feruloyltyramine (NTF) was puriﬁed from

Polyalthia suberosa, a shrubby tree found throughout

southeast Asia and south China whose parts are used

in ai traditional medicine.

NTF has potent radical-

scavenging antioxidant property, especially from phenolic

hydroxyls. NTF and the other puried compound studied

by our group, N-benzylcinnamide (PT-3), have been

demonstrated to be eective in protecting rat cultured

cortical neurons against Aβ-induced cytotoxicity by

inhibiting ROS production, suppressing apoptotic caspase-3

and Bax, and in turn elevating anti-apoptotic Bcl-2.

7-8

As PT-3 that has elicited protective eects on primary

cortical neurons also showed promising outcome in

aged rat brain in vivo,

neuroprotective eects of NTF

in vivo had become our next primary study target.

In this research study, the animals were used in the

experiment, and they were calculated for the sample size

in accordance with the ethical guideline to protect the

unnecessary wastage of resources. Consistently, there are

several studies that have demonstrated the statistically

signicant results with a small sample size (n = 3).

9-11

Meanwhile, our preliminary results showed the protective

eects of NTF treatment against scopolamine-induced

cholinergic dysfunction, including, ROS production,

apoptosis, inammation, and associated signaling pathways

in several rat brain regions, specically the frontal cortex,

hippocampus and temporal cortex.

e frontal cortex integrity is correlated with the

higher reservation of cognitive performance in aging

populations.

e hippocampus and especially its associated

cholinergic signaling pathway play a crucial role in memory

formation.

e medial temporal lobe cortex or, in short,

the temporal cortex is reciprocally interconnected to the

hippocampus and it greatly involved in the hippocampus-

associated cognitive processes and cognitive decline such

as AD in humans

and ischemia-associated dementia in

rats.

Furthermore, ChAT activity in the temporal cortex

is positive correlated with cognitive preservation, but it is

negatively correlated with AD pathologies.

Importantly,

the frontal cortex, hippocampus, and temporal cortex are

three of the regions heavily aected by scopolamine

17,18

and aging

in the rat brain.

Herein, we studied the neuroprotective eects of

NTF treatment on scopolamine-associated cholinergic

dysfunction, namely, ROS production, apoptosis,

inammation, and relevant signaling pathways in the

rat frontal and temporal cortices and hippocampus.

MATERIALS AND METHODS

Reagents

Scopolamine (Sigma-Aldrich) and NTF were dissolved

in a vehicle solution of (v/v) 40% dimethylsulfoxide,

59% phosphate-buered saline (PBS), and 1% ethanol

and diluted in PBS for animal administration. NTF was

isolated from the acetone extract of Polyalthia suberosa

stems as previously described.

e test drug NTF was

in its form of pure compound. From 997.2 mg of the

semi-solid fraction, 88.1 mg (8.83 %) of NTF could be

puried by preparative layer chromatography.

Animal experiments

All experimental procedures were approved by

the Institute of Molecular Biosciences Animal Care and

Use Committee (MB-ACUC) (COA.NO.MB-ACUC

2016/002).

Twelve 8-week male Wistar rats (250-300 g) from the

National Laboratory Animal Center, Mahidol University,

were individually cared for in cages under 12 h light/

dark cycle, 22±1 °C temperature, 45-55 % humidity, and

ad libitum water and diet. e 12 rats were separated

into 4 groups, each with 3 animals, namely, scopolamine

treatment only, NTF treatment only, scopolamine plus

NTF treatment, and control. Following habituation for 5

days, animals were once per day injected intraperitoneally

(i.p.) for 14 days.

In the control group, animals were

injected with vehicle 1 h before the water maze test for 14

days. In the scopolamine-only group, animals were rst

injected for 7 days with vehicle followed by scopolamine

treatment on day 8 to day 14 (3.0 mg/kg BW) 1 h before

the water maze test. In the NTF treatment only group,

animals were injected with 1 ml aliquot of NTF (1.5

mg/kg BW) for 14 days 1 h before the water maze test.

In the scopolamine plus NTF treatment group, animals

were administered with NTF as described above for 7

days and then together with n day 8 to day 14 (3.0 mg/

kg BW) 1 h before the water maze test.

Rats were treated with NTF for 7 days prior to

MWM training because we would like to investigate the

neuroprotective eects of NTF against scopolamine. e

NTF eects as a memory enhancer can still remain during

the MWM training.

e dose of NTF was selected in

accordance with our previous ndings on the protective

roles of N-benzylcinnamide (PT-3).

In animal studies the

model of scopolamine has been performed using doses

between 0.5,1 or 3 mg/kg IP). e eect of scopolamine

to induce impairment of learning and memory is dose

dependent.

19-20

e highest dose of scopolamine exhibits

the greater severity of memory dysfunction. Scopolamine

was administered via the injection 1 hr before MWM

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training because the half-life of scopolamine is 2.4±1.4

h for its elimination.

Central pharmacodynamic eects

of scopolamine peak between 1 and 3 h and disappear

aer 5–6 h.

Morris water maze test

Water maze tests were conducted to evaluate the

eects of NTF and scopolamine on the acquisition and

retention of spatial memory.

Visible platform trial

was performed on the day before the training trial.

Animals have to associate the relative location of the

visible platform with the provided visual cues. On the

following training trials, the platform was submerged

and invisible. e training trials were conducted over a

period of 7 days as follows: day 1, visible platform test;

days 2-6, hidden platform tests; and on the same day 6

aer hidden platform, probe trial test. ese consecutive 6

days correspond to the days 8-13 of the treatment period.

Rats in each group were put in the water facing dierent

quadrants, which were altered for each experiment,

and the escape latency dened as time spent to nd the

submerged platform was recorded. In the probe trial,

time spent in the target quadrant with the platform was

recorded.

e experimental schedule was designed to investigate

the protective eect of NTF as a memory enhancer in

rats. Rats underwent habituation for 5 days. Aer that,

NTF (1 ml, 1.5 mg/kg. i.p.) or vehicle was administrated

once a day for 7 days. en, 1 h prior to the rst trial

session, rats were administrated of veh, NTF, scopolamine

(3.0 mg/kg) or NTF+Scop (i.p) every MWM training

for 6 days, in accordance with the previously described

protocol.

ROS production assay

After animals were decapitated without prior

anesthesia, the 3 regions of the rat brains – the frontal

cortex, hippocampus, temporal cortex – were dissected

and frozen at -80°C. For the frontal cortex, the frontal

part of the brain was cut straight down coronally and

attached subcortical brain regions were removed. For

the hippocampus, the bilateral hippocampal tissues with

all subsectors were collected. For the temporal cortex, all

cortical regions encompassing the hippocampus or the

medial temporal lobe were dissected. Reactive oxygen

species (ROS) production was measured according to the

protocol of our previous work.

In brief, 2.4-3.0 mg of

brain tissue from each dissected region were homogenized

in 80-100 µl of ice-cold Locke’s buer, then 10 µl of

homogenate was adjusted to a concentration of 3 mg/ml

tissue, incubated with 10 µM 2',7'-dichlorouorescin

diacetate (DCFH-DA) (stock solution in 100% ethanol)

for 45 min at 37°C. Fluorescence of the oxidized product

2',7'-dichlorouorescein (DCF) (485 nm excitation and

535 nm emission) was measured every 10 minutes for

4 times in total at 37°C in a DTX 880 multimode plate

reader.

Western blot analysis

Rat brains were dissected into three regions: frontal

cortex, hippocampus and temporal cortex).

e brain

protein contents were extracted with the lysis buer,

centrifuged at 11,000 ×g for 15 min, and collected as

a supernatant. e protein samples were loaded onto

polyacrylamide gel. e proteins were separated by gel

electrophoresis and transferred onto (PVDF) membranes

where they were reacted to specic antibodies. On the

PVDF membrane, proteins of interest were identied using

a detection reagent. e protein bands were quantied

and analyzed with the same loading sample groups of

its own gel. PVDF membrane was incubated overnight

at 4°C with primary rabbit anti-Bax, -Bcl-2, -activated

caspase-3, -BACE1, -phospho-p38, -p38, -phospho-JNK,

-JNK, -phospho-ERK1/2, -ERK1/2, -phospho-Akt, -Akt,

-phospho-NF-κB p65 (Ser536), -NF-κB antibodies (1:1000

dilution) (Cell signaling), -IL-1β (1:1000) (Santa Cruz);

and primary mouse anti-ChAT (1:1000) (Millipore) and

-IL-6 (1:1000) (Santa Cruz); and rabbit anti-actin (1:2500)

(Cell Signaling) for normalization of protein loading.

Immunoreactive proteins were visualized by incubating

PVDF membranes with secondary horseradish peroxidase-

conjugated antibodies (1:1000) (Cell Signaling) at room

temperature for 1 h and subsequently with enhanced

chemical luminescence (ECL) reagents (Bio-Rad) before

signal exposure to Hyperlm

(GE Healthcare). Intensities

of the visualized protein bands were measured by scanning

densitometry (Image J soware). e optical density

(OD) of protein bands on Hyperlm was determined

and normalized to the OD of β-actin and total phospho

proteins. e results were expressed as the percentage

of OD values by using the ImageJ soware.

Each gel contains four sample groups (Control,

Sco, NTF+sco, NTF). Each protein sample of three rats

on dierent brain regions, including frontal cortex (3),

hippocampus (3), and temporal cortex (3) was loaded

separately on dierent polyacrylamide gel. Four sample

groups of each region in each rat were underwent Western

blotting. Each blot was repeated for at least 3 times from

dierent batches of protein samples. e representative

blots were shown in gures.

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Statistical analysis

Statistical analyses were performed using Prism

6.0 (GraphPad). Dierences in the escape latency from

the training/hidden platform trials were analyzed using

two-way analysis of variance (ANOVA). Dierences

between means of the 4 groups from all other experiments

were determined using one-way ANOVA with pair-

wise p-values corrected by Tukey’s posthoc test. Results

are deemed signicant at p < 0.05. In all Western blot

analyses, non-phospho proteins (ChAT, Bcl-2, Bax,

Activated caspase-3, IL-6, and IL-1β) were quantied and

normalized with β-Actin. Meanwhile, phosphoproteins

(p-NFκB, p-ERK, p-JNK, p-p38, p-Akt) were quantied

and normalized with the total protein counterparts.

RESULTS

Learning and memory in a Morris water maze test of

rats administered scopolamine with and without NTF

Control rats became proficient at locating the

submerged platform by day 5 of training compared to

day 1 (p < 0.05) in a hidden platform test (Fig 1A). On day

5 of training scopolamine-treated rats perform signicantly

less well than controls (p < 0.01), but, interestingly, rats

treated with scopolamine together NTF have prociencies

superior to those of controls, whereas NTF treatment

alone was no more eective than non-treated control.

In the probe trial tests, as expected, compared to control

rats, those in the scopolamine plus NTF treatment group

stay signicantly longer in the platform quadrant. ose

in the NTF only treatment group spent the same time.

Lastly as expected, those in the scopolamine-only group

stayed for a signicantly shorter duration (Fig 1B).

ROS production in brain tissues of rats administered

scopolamine with and without NTF

Scopolamine treatment for 7 days elevated ROS

level 2 folds in all three types of brain tissues (frontal

cortex, hippocampus, and temporal cortex) over control

levels (Fig 2). ROS levels were restored to control levels

by NTF administration (7 days prior and during the

scopolamine treatment period), while NTF alone (for

7 days) had no eect on ROS levels in all three types of

brain tissues.

Fig 1. NTF (1.5 mg/kg) was injected i.p. daily to rats 7 days before and together with scopolamine (3.0 mg/kg BW i.p.) 1 h before the 7-day

Morris water maze test; probe trial was conducted on day 7. Each data point (n=3) is expressed as mean ± SEM. A NTF prevented scopolamine

from elevating escape latency. *p < 0.05 scopolamine-treated with NTF versus (vs) the scopolamine group on day 3, **p < 0.01 scopolamine-

treated with NTF vs the scopolamine group on day 5. B NTF restored scopolamine-induced decrease in retention measured as time spent

in the target quadrant. *p < 0.05 scopolamine vs NTF, **p < 0.01 scopolamine vs scopolamine with NTF treated group.

Fig 2. Eects of NTF treatment in scopolamine-treated rats

on ROS. Production of ROS in brain homogenates were

measured by uorometric agent DCFH-DA. Each data bar

(n=3) is expressed as mean ± SEM. *p < 0.05, **p < 0.01 vs

control; #p < 0.05, ##p < 0.01 vs scopolamine-treated group.

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Levels of cholinergic, apoptosis, and inammatory

proteins in brain tissues of rats administered scopolamine

with and without NTF

Administration of scopolamine to rats signicantly

decreases ChAT and increases BACE1 levels in the 3 brain

regions compared to control tissues (Fig 3A-C), and NTF

cotreatment was able to ameliorate these alterations.

However, NTF treatment alone had no eect on basal

levels of the two proteins.

Exposure to scopolamine causes a signicant increase

in apoptotic proteins, Bax and activated caspase-3, and

a corresponding decrease in anti-apoptotic Bcl-2 in the

three types of brain tissues compared to controls with

NTF treatment reversing these changes (Fig 4A-C).

However, these relative changes in apoptosis-related

proteins levels in the brain tissues of rats treated with

NTF alone varied with tissue types and thus impacting the

ability of NTF to restore the normal levels of apoptosis-

related proteins in the scopolamine-treated animals.

Scopolamine significantly increased levels of

cytokines – interleukin (IL)-6 and IL-1β – and of activated

transcription factor – phosphorylated NF-κB – which

were reversed by NTF. e extent of these phenomena

relative to control levels also depended on brain tissue

types (Fig 5A-C).

Activated cell signaling pathways in brain tissues of

rats administered scopolamine with and without NTF

We investigated the activation indicated by the

proportion of phosphorylated proteins of JNK, p38, ERK1/2,

and Akt. In the frontal cortex (Fig 6A), scopolamine

increased p-JNK and p-p38, decreased p-ERK1/2, and

increased p-Akt. NTF downregulated p-ERK1/2 but

upregulated p-Akt without aecting p-JNK or p-p38.

A combination of scopolamine and NTF could only

restore p-p38 to control level. In the hippocampus (Fig

6B), scopolamine elevated levels of p-JNK and p-p38,

depressed p-ERK1/2 and had no eect on p-Akt. NTF had

no eect on levels of p-JNK, increased p-p38, decreased

p-ERK1/2, and had no eect on p-Akt. A combination

of scopolamine and NTF could only restore p-ERK1/2

to control level and had no eect on the p-Akt level. In

the temporal cortex (Fig 6C), scopolamine raised levels

p-JNK and p-p38, lowered p-ERK1/2, and had no eect

on p-Akt. NTF had no eect on levels of p-JNK, p-p38,

or p-ERK1/2 but decreased p-Akt. A combination of

scopolamine and NTF restored levels of p-JNK, p-p38

and p-ERK1/2, and had no eect on p-Akt. Table 1

shows the quantitative protein values as the percentage

of change in expression relative to control groups in

Western blot analysis.

Fig 3. Eects of NTF on scopolamine-induced cholinergic dysfunction in rats on the levels of ChAT and BACE1 in: A frontal cortex, B

hippocampus, and C temporal cortex. Each data bar (n=3) is expressed as mean ± SEM. *p < 0.05, **p < 0.01 vs control group; ##p < 0.01,

###p < 0.001 vs scopolamine-treated group

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Fig 4. Eects of NTF on scopolamine-induced apoptosis represented by the levels of Bax, Bcl-2, and activated caspase-3 in: A frontal cortex,

B hippocampus, and C temporal cortex. Each data bar (n=3) is expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 vs control

group; #p < 0.05, ###p < 0.001 vs scopolamine-treated group.

Fig 5. Eects of NTF on scopolamine-induced inammatory responses indicated by the levels of p-NF-κB, IL-6, and IL-1β in: A frontal

cortex, B hippocampus, and C temporal cortex. Each data bar (n=3) is expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 vs

control group; #p < 0.05, ##p < 0.01, ###p < 0.001 vs scopolamine-treated group.

DISCUSSION

Scopolamine has been demonstrated to increase

oxidative stress including ROS production in the mouse

cortex and hippocampus, which could be reversed by

pretreatment with sodium tanshinone IIA sulfonate,

a radical-scavenging antioxidant.

e current study

shows that administration of NTF was able to prevent

scopolamine-augmented ROS production in the frontal

and temporal cortices and hippocampus.

Oxidative stress contributes to the mechanisms of

multiple neurodegenerative disorders including AD.

Both oxidative damage and cholinergic dysfunction

have been implicated in cognitive impairment in rat

dementia models. Treatment with antioxidant Terminalia

chebula extract attenuated scopolamine-induced ROS

generation and reduction in ChAT and ACh levels in

mouse hippocampal tissue.

BACE1 is an enzyme necessary

for the production of Aβ42 and Aβ40 peptides in AD.

Consistent with our nding, an antioxidant ipriavone

decreased BACE1 expression in the hippocampus of a

scopolamine-treated rat.

Furthermore, NTF treatment alone

signicantly decreased apoptosis through downregulation

of activated caspase-3 and Bax and upregulation of

Bcl-2 in the hippocampus. Similarly, treatment with

an antioxidant sodium tanshinone IIA sulfonate in a

scopolamine-injected mouse signicantly decreased

Bax:Bcl-2 expression ratio and downregulated expression

of activated caspase-3 in the hippocampus and cortex.

Scopolamine also induces cytokines and NF-κB

phosphorylation;

all of these changes were restored

to normal levels in the rat cortex and hippocampus by

NTF as described in the current study. Pretreatment

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with a water-soluble ginseng oligosaccharide extract

decreased scopolamine-induced expression of IL-1β and

IL-6 mRNAs in the murine hippocampus and signicantly

prevented scopolamine from inducing spatial memory

impairment during the water maze test.

In addition,

antioxidant isoavones from soybean-fermented food

tempeh restored ACh level and suppressed scopolamine-

induced IL-1β expression in the rat brains.

The effects of scopolamine treatment on phosphorylation

of the four signaling proteins (JNK, p38, ERK1/2, and Akt)

were consistent across the three brain regions. Whether

NTF could restore these changes could not be predicted

based on the eects of NTF treatment alone in each brain

tissue type. However, where scopolamine or NTF alone

resulted in similar eects to a cell signaling protein, e.g.

p-Akt (stimulated in the frontal cortex but unaected

in the temporal cortex and hippocampus), the results

of their combination were as expected. In scopolamine-

treated rats, NTF inhibited phosphorylation of JNK

and p38 in these three brain regions. Previous studies

have reported that JNK inhibitor SP600125 increased

the number of surviving cells aer ischemia in the rat

hippocampus

and that SB202190 p38 inhibitor provided

neuroprotection against cell apoptosis in mice.

Scopolamine-decreased p-ERK1/2 levels in the three

brain regions were accompanied by elevated BACE1

levels. Congruently, the expression and activity of BACE1

are negatively regulated by the ERK pathway.

Similar

to our study, treatment of an antioxidant ipriavone

restored ERK1/2 phosphorylation and decreased BACE-1

expression and subsequent Aβ pathologies in a scopolamine-

administered rat.

ERK1/2 inhibitor U0126 also blocked

alleviating eects of amyrin, a triterpene with antioxidant

activity, on scopolamine-induced memory decits.

CONCLUSION

is preliminary result demonstrates that NTF

was able to attenuate scopolamine-induced impairment

of the spatial cognitive performance, oxidative stress,

inﬂammation, and apoptosis in the brain cortex and

hippocampus, but its eects on cell signaling pathways

in these brain tissues remained ambiguous. e utility of

NTF as a potential preventive and therapeutic candidate

for AD requires further experimentation. In a future study,

Fig 6. Eects of NTF on scopolamine-induced dysfunction in signaling pathways indicated by the levels of p-JNK, p-p38, p-ERK1/2 and

p-Akt in: A frontal cortex, B hippocampus, and C temporal cortex. Each data bar (n=3) is expressed as mean ± SEM. *p < 0.05, **p < 0.01,

***p < 0.001 vs control group; ##p < 0.01, ###p < 0.001 vs scopolamine-treated group.

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TABLE 1. Eects of NTF treatment on western blot protein expression in the frontal, hippocampus and temporal cortex in scopolamine-treated rats. Expression of target

proteins is represented as the percentage of control. Results are expressed as mean ± SEM of three independent experiments.

Frontal cortex Hippocampus Temporal cortex

NTF (1.5 mg/kg) - + + - + + - + +

Sco (3 mg/kg) + + - + + - + + -

ChAT 76.9 ± 0.90* 125.9 ± 4.70

###

101.1 ± 4.51 57.7 ± 1.41** 137.4 ± 0.73

###

141.6 ± 12.38** 52.9 ± 3.78** 119.4 ± 10.98

###

104.1 ± 4.11

BACE1 147.8 ± 4.45** 99.3 ± 2.43

114.3 ± 11.36 145.0 ± 1.23** 68.6 ± 9.74

###

80.7 ± 0.60 137.0 ± 6.40** 95.1 ± 8.43

81.8 ± 2.13

Bax 137.7 ± 0.67*** 112.33 ± 5.82

81.1 ± 2.90*** 188.5 ± 15.4*** 105.6 ± 7.41

###

42.3 ± 0.50** 143.0 ± 6.87*** 104.2 ± 0.19

###

76.2 ± 0.61*

Bcl-2 68.4 ± 4.11** 109.8 ± 6.23

98.9 ± 5.14 58.2 ± 4.01*** 189.7 ± 3.88

###

143.5 ± 5.11*** 54.0 ± 1.22*** 96.6 ± 1.94

###

119.8 ± 0.86***

Act. casp 3 211.2 ± 17.09*** 163.5 ± 1.85

97.2 ± 3.42 186.8 ± 2.32*** 112.2 ± 0.92

###

42.7 ± 0.83*** 148.1 ± 8.3** 83.4 ± 5.29

###

69.6 ± 5.92*

p-NFkB 224.8 ± 10.48*** 170.2 ± 9.14

105.3 ± 4.77 204.2 ± 7.93*** 115.1 ± 12.8

###

110.9 ± 3.90 130.1 ± 2.59* 91.1 ± 10.58

92.2 ± 5.04

IL-6 163.5 ± 4.31*** 133.7 ± 3.71

104.8 ± 4.28 153.6 ± 7.63*** 80.1 ± 2.50

###

53.9 ± 6.29** 160.2 ± 8.74*** 103.7 ± 6.28

###

60.6 ± 4.00**

 IL-1β 190.5±5.52*** 118.7±15.4

117.2 ± 0.79 137.5 ± 2.36** 62.2 ± 3.73

###

40.7 ± 10.02*** 126.2 ± 9.26* 67.6 ± 5.94

###

63.4 ± 1.57**

p-JNK 120.2 ± 3.86** 77.8 ± 3.84

###

98.5 ± 3.10 133.6 ± 9.39* 70.8 ± 4.67

###

94.8 ± 7.94 159.7 ± 6.84*** 91.3 ± 2.56

###

85.6 ± 6.68

p38 171.2 ± 9.98** 91.9 ± 20.2

96.1 ± 3.33 145.2 ± 2.44*** 123.2 ± 2.26

107.9 ± 4.02 162.0 ± 4.81*** 109.0 ± 3.58

###

107.1 ± 3.53

p-ERK 36.9 ± 2.68* 124.7 ± 20.27

66.8 ± 14.29 55.1 ± 5.05** 93.4 ± 0.88

52.3 ± 11.29** 58.8 ± 4.43* 116.9 ± 3.28

101.7 ± 13.36

p-Akt 125.1 ± 16.05 134.0 ± 9.97 123.2 ± 9.56 90.3 ± 1.05 104.3 ± 3.20 103.2 ± 12.61 92.9 ± 4.52 55.5 ± 0.75

###

54.4 ± 2.02***

*p < 0.05, **p < 0.01, *** p < 0.001 compared to control;

p < 0.05,

p < 0.01,

###

p < 0.01 compared to scopolamine-treated group.

Thangnipon et al.

Volume 73, No.6: 2021 Siriraj Medical Journal

https://he02.tci-thaijo.org/index.php/sirirajmedj/index

421

Original Article

SMJ

the eect of NTF on the altered activities of dierent cell

signaling pathways and their mechanisms in rat brain

tissues (frontal cortex, hippocampus, and temporal

cortex) are needed to be investigated and the sample size

should be expanded in order to acquire more conclusive

results.

ACKNOWLEDGEMENTS

We thanked Professor Prapon Wilairat for critical

and helpful suggestions on the manuscript. is project

was partly sponsored by a joint grant IRG5780009 between

Mahidol University and the ailand Research Fund

(TRF).

Conicts of Interest: All authors conrmed the absence

of potential conicts of interest as declared in the ICMJE

form.

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