Neuroprotective and Antiapoptotic Potential of Trigonelline in a Striatal 6-Hydroxydopamine Rat Model of Parkinson’s Disease
Considering neuroprotective and antioxidant effects of trigonelline, our study was undertaken to evaluate its protective effect in a 6-hydroxydopamine-induced model of Parkinson’s disease (PD) in rats. Unilateral intrastriatal 6-OHDA-lesioned rats were pretreated with trigonelline at doses of 50...
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Інститут фізіології ім. О.О. Богомольця НАН України
2016
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irk-123456789-1483132019-02-19T01:28:19Z Neuroprotective and Antiapoptotic Potential of Trigonelline in a Striatal 6-Hydroxydopamine Rat Model of Parkinson’s Disease Mirzaie, M. Khalili, M. Kiasalari, Z. Roghani, M. Considering neuroprotective and antioxidant effects of trigonelline, our study was undertaken to evaluate its protective effect in a 6-hydroxydopamine-induced model of Parkinson’s disease (PD) in rats. Unilateral intrastriatal 6-OHDA-lesioned rats were pretreated with trigonelline at doses of 50 and 100 mg/kg. Significant rotational behavior, a significant reduction in the number of Nissl-stained neurons on the left side of substantia nigra pars compacta (SNC), increased apoptosis, enhanced levels of malondialdehyde (MDA) and nitrite, and a lower level of glutathione (GSH) were observed in 6-OHDA-lesioned rats. Trigonelline at a dose of 100 mg/kg significantly reduced rotations, prevented reduction of SNC neurons, prevented apoptosis, and restored the MDA level. These results suggest that pre-lesion trigonelline treatment exerts dose-dependent neuroprotective and antiapoptotic effects under conditions of 6-OHDA toxicity and may be, henceforth, advantageous for the management of early PD. Беручи до уваги нейропротекторний та антиоксидантний вплив тригонелліну, ми дослідили його захисну дію в моделі хвороби Паркінсона у щурів, індукованій стереотаксичним уведенням 6-гідроксидофаміну (6-OHDA). Щурам, котрим робили унілатеральні ін’єкції 6-OHDA в стріатум, попередньо щоденно тричі вводили тригонеллін у дозах 50 і 100 мг/кг. В умовах використаної 6-OHDA-моделі істотно посилювалась обертальна моторна поведінка, викликана ін’єкцією апоморфіну, значно знижувалася кількість забарвлених, за Ніслем, нейронів у лівій половині компактної частини чорної субстанції, посилювався процес апоптозу нейронів, зростали рівні малональдегіду та нітриту та знижувався рівень відновленого глутатіону. Тригонеллін у щоденних дозах 100 мг/кг вірогідно знижував кількість обертальних рухів, протидіяв зменшенню числа нейронів у чорній субстанції та розвитку апоптозу, а також нормалізував рівень малональдегіду. Отримані результати свідчать про те, що уведення тригонелліну перед ін’єкціями 6-OHDA забезпечує дозозалежні нейропротекторний та антиапоптотичні ефекти в умовах токсичної дії 6-OHDA. Цей агент та його аналоги можуть бути тестовані як допоміжні засоби при лікуванні хвороби Паркінсона на ранніх стадіях. 2016 Article Neuroprotective and Antiapoptotic Potential of Trigonelline in a Striatal 6-Hydroxydopamine Rat Model of Parkinson’s Disease / M. Mirzaie, M. Khalili, Z. Kiasalari, M. Roghani // Нейрофизиология. — 2016. — Т. 48, № 3. — С. 197-204. — Бібліогр.: 35 назв. — англ. 0028-2561 http://dspace.nbuv.gov.ua/handle/123456789/148313 616.858:611.813.2:615.322 en Нейрофизиология Інститут фізіології ім. О.О. Богомольця НАН України |
| institution |
Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| collection |
DSpace DC |
| language |
English |
| description |
Considering neuroprotective and antioxidant effects of trigonelline, our study was undertaken
to evaluate its protective effect in a 6-hydroxydopamine-induced model of Parkinson’s disease
(PD) in rats. Unilateral intrastriatal 6-OHDA-lesioned rats were pretreated with trigonelline
at doses of 50 and 100 mg/kg. Significant rotational behavior, a significant reduction in the
number of Nissl-stained neurons on the left side of substantia nigra pars compacta (SNC),
increased apoptosis, enhanced levels of malondialdehyde (MDA) and nitrite, and a lower
level of glutathione (GSH) were observed in 6-OHDA-lesioned rats. Trigonelline at a dose of
100 mg/kg significantly reduced rotations, prevented reduction of SNC neurons, prevented
apoptosis, and restored the MDA level. These results suggest that pre-lesion trigonelline
treatment exerts dose-dependent neuroprotective and antiapoptotic effects under conditions
of 6-OHDA toxicity and may be, henceforth, advantageous for the management of early PD. |
| format |
Article |
| author |
Mirzaie, M. Khalili, M. Kiasalari, Z. Roghani, M. |
| spellingShingle |
Mirzaie, M. Khalili, M. Kiasalari, Z. Roghani, M. Neuroprotective and Antiapoptotic Potential of Trigonelline in a Striatal 6-Hydroxydopamine Rat Model of Parkinson’s Disease Нейрофизиология |
| author_facet |
Mirzaie, M. Khalili, M. Kiasalari, Z. Roghani, M. |
| author_sort |
Mirzaie, M. |
| title |
Neuroprotective and Antiapoptotic Potential of Trigonelline in a Striatal 6-Hydroxydopamine Rat Model of Parkinson’s Disease |
| title_short |
Neuroprotective and Antiapoptotic Potential of Trigonelline in a Striatal 6-Hydroxydopamine Rat Model of Parkinson’s Disease |
| title_full |
Neuroprotective and Antiapoptotic Potential of Trigonelline in a Striatal 6-Hydroxydopamine Rat Model of Parkinson’s Disease |
| title_fullStr |
Neuroprotective and Antiapoptotic Potential of Trigonelline in a Striatal 6-Hydroxydopamine Rat Model of Parkinson’s Disease |
| title_full_unstemmed |
Neuroprotective and Antiapoptotic Potential of Trigonelline in a Striatal 6-Hydroxydopamine Rat Model of Parkinson’s Disease |
| title_sort |
neuroprotective and antiapoptotic potential of trigonelline in a striatal 6-hydroxydopamine rat model of parkinson’s disease |
| publisher |
Інститут фізіології ім. О.О. Богомольця НАН України |
| publishDate |
2016 |
| url |
http://dspace.nbuv.gov.ua/handle/123456789/148313 |
| citation_txt |
Neuroprotective and Antiapoptotic Potential of Trigonelline in a Striatal 6-Hydroxydopamine Rat Model of Parkinson’s Disease / M. Mirzaie, M. Khalili, Z. Kiasalari, M. Roghani // Нейрофизиология. — 2016. — Т. 48, № 3. — С. 197-204. — Бібліогр.: 35 назв. — англ. |
| series |
Нейрофизиология |
| work_keys_str_mv |
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| first_indexed |
2025-07-12T19:07:34Z |
| last_indexed |
2025-07-12T19:07:34Z |
| _version_ |
1837469282332573696 |
| fulltext |
NEUROPHYSIOLOGY / НЕЙРОФИЗИОЛОГИЯ.—2016.—T. 48, № 3 197
UDC 616.858:611.813.2:615.322
M. MIRZAIE,1,2 M. KHALILI,1 Z. KIASALARI,1 and M. ROGHANI1
NEUROPROTECTIVE AND ANTIAPOPTOTIC POTENTIAL OF TRIGONELLINE
IN A STRIATAL 6-HYDROXYDOPAMINE RAT MODEL OF PARKINSON’S DISEASE
Received July 28, 2014
Considering neuroprotective and antioxidant effects of trigonelline, our study was undertaken
to evaluate its protective effect in a 6-hydroxydopamine-induced model of Parkinson’s disease
(PD) in rats. Unilateral intrastriatal 6-OHDA-lesioned rats were pretreated with trigonelline
at doses of 50 and 100 mg/kg. Significant rotational behavior, a significant reduction in the
number of Nissl-stained neurons on the left side of substantia nigra pars compacta (SNC),
increased apoptosis, enhanced levels of malondialdehyde (MDA) and nitrite, and a lower
level of glutathione (GSH) were observed in 6-OHDA-lesioned rats. Trigonelline at a dose of
100 mg/kg significantly reduced rotations, prevented reduction of SNC neurons, prevented
apoptosis, and restored the MDA level. These results suggest that pre-lesion trigonelline
treatment exerts dose-dependent neuroprotective and antiapoptotic effects under conditions
of 6-OHDA toxicity and may be, henceforth, advantageous for the management of early PD.
Keywords: Parkinson’s disease, trigonelline, 6-hydroxydopamine, apoptosis, oxidative
stress.
INTRODUCTION
Parkinson’s disease (PD) is a rather widespread
neurodegenerative disorder with such cardinal clinical
symptoms as bradykinesia, rigidity, resting-state
tremor, postural imbalance, and gait impairment [1].
These motor disabilities inevitably lead to a lower
quality of life [2]. The global occurrence of PD is
about 0.3% in the general population and 1–2% in
subjects elder than 60–65 years [2]. Behavioral and
cognitive problems, including dementia, depression,
anxiety, and sleep disturbances, also appear within
late stages of PD [3, 4]. At present, treatment for PD
includes mainly symptomatics and usually starts with
Levodopa and/or dopamine agonists [5]. Nevertheless,
after some time, most PD patients experience side
effects, including motor and nonmotor fluctuations
and dyskinesia [6].
Oxidative stress-mediated neuronal death plays a
key role in the pathogenesis of PD [7]. A neurotoxin,
6-hydroxydopamine (6-OHDA), is routinely used to
damage mesencephalic dopaminergic neurons and to
yield a widely used animal model of PD [8]. Because
environmental factors are engaged in the majority of
PD cases, it is important to assess the effectiveness
of natural products in neuroprotective interventions
for PD [9]. Meanwhile, patients with PD often turn to
complementary and alternative medicine [10].
The alkaloid trigonelline (N-methylnicotinic acid)
is a metabolite of nicotinic acid. It is mainly found
in fenugreek seeds and coffee beans. Trigonelline
demonstrates a potent antioxidant property and strong
free-radical scavenging activity [11, 12]. This agent
was shown to attenuate diabetes-induced auditory
neuropathy [13] and was, in general, reported to
induce neurite outgrowth and reconstruction of
neuronal networks in the damaged brain, which is
exclusively important for the therapeutic treatment
of neurodegenerative diseases [14]. In addition, a
standardized extract of fenugreek seeds, which contain
high amounts of trigonelline, was capable of reversing
motor dysfunction in rats with 6-OHDA-induced
unilateral cerebral lesion [15]. For these reasons,
trigonelline may be regarded as a prospective effective
mean against PD. Our study was undertaken to assess
the possible neuroprotective potential of pre-lesion
trigonelline treatment in the 6-OHDA-induced early
model of PD in rats.
1,2 Shahed University, Tehran, Iran
(1Neurophysiology Research Center; 2Student Research Committee).
Correspondence should be addressed to M. Roghani
(mehjour@yahoo.com).
NEUROPHYSIOLOGY / НЕЙРОФИЗИОЛОГИЯ.—2016.—T. 48, № 3198
M. MIRZAIE, M. KHALILI, Z. KIASALARI, and M. ROGHANI
METHODS
Chemicals and Reagents. Trigonelline, 6-OHDA,
apomorphine, Cresyl violet, and oxidative stress kits
were obtained from Sigma-Aldrich (Germany). All
other chemicals were procured by Merck (Germany).
Animals. Adult male Wistar rats (180–210 g, n = 60)
were provided by the Pasteur’s Institute (Tehran, Iran).
The animals were housed in a temperature-controlled
colony room under 12/12 h light/dark cycle with free
access to food and water and held in the above room for
at least one week before being tested. Only rats showing
no rotational behavior (net rotations less frequent than
30 h–1) were subjected to i.p. injections of apomorphine
hydrochloride (2 mg/kg) in our study. The animals
were randomly divided into five groups: (i) sham-
operated; (ii) sham-operated + trigonelline(100); (iii)
6-OHDA-lesioned; (iv) lesioned + trigonelline(50), and
(v) lesioned + trigonelline(100; see below). Stereotaxic
unilateral intrastriatal 6-OHDA injections (left side) were
performed using a 5 ml Hamilton syringe into anesthetized
rats (ketamine 80 mg/kg and xylazine 10 mg/kg, i.p.).
A stereotaxic apparatus (Stoelting, USA) was used
(coordinates: L –3 mm, AP +9.2 mm, and V +5 mm from
the center of the interaural line, according to the atlas of
Paxinos and Watson [16]). After injection, the needle was
left in place for an additional 5 min and then withdrawn
at a rate of 1 mm/min. The lesioned groups received
single injections of 2.5 mg/ml of 6-hydroxydopamine-
HCl in 5 ml of 0.9% saline containing 0.2% ascorbic acid
(w/v) at a rate of 1 ml/min. The sham group received an
identical volume of the ascorbate-saline solution. The
6-OHDA+trigonelline(50) and 6-OHDA+trigonelline
(100) groups received the neurotoxin in addition to
trigonelline i.p. dissolved in normal saline at doses of 50
and 100 mg/kg. Trigonelline was daily administered three
times before the surgery, with the last injection made
1 h pre-surgery. The doses of trigonelline were chosen
according to our preliminary study.
Behavioral Evaluation. The animals (n = 9 in each
group) were tested for apomorphine hydrochloride
(2 mg/kg, i.p.)-induced rotational behavior one week
before surgery (baseline) and at 1st week post-surgery.
The rotations were measured according to a method
described previously [8]. Briefly, the animals were
allowed to habituate for 10 min; then, 1 min after the
injection, full rotations were counted in a cylindrical
container (diameter 33 cm and height 35 cm) within
10-min-long intervals for 60 min (in a dimly-lighted
and quiet room). Net number of rotations was defined
as the positive score minus the negative score.
Assessment of Oxidative Stress. Midbrain
samples (n = 5 for each group) were dissected from
the rat brains and washed-off in cold normal saline.
Their anterior one-thirds were blotted dry, weighed,
homogenized (5% of tissue) in ice-cold normal saline,
and centrifuged at 4°C. The obtained supernatant was
aliquotted and stored at –70°C until being assayed.
Estimation of the MDA Content in the Midbrain.
The malondia ldehyde (MDA) concent ra t ion
(thiobarbituric acid reactive substances, TBARS) in
the supernatant was measured as described before [8].
Briefly, trichloroacetic acid and a TBARS reagent were
added to the supernatant, then mixed, and incubated
at boiling water for 90 min. After cooling on ice, the
samples were centrifuged at 1000g for 10 min, and the
absorbance of the supernatant was read at 532 nm. The
TBARS results were expressed as MDA equivalents
using tetraethoxypropane as the standard.
Midbrain Nitrite Assay. The nitrite content in the
supernatant was assayed by the Griess method accord
ing to previous studies [17]. Because NO is a com
pound with a short half-life and is rapidly convert
ed into stable end products, nitrate (NO3
–) and nitrite
(NO2
–), the principle of the assay is the conversion of
nitrate into nitrite by cadmium. This is followed by
color development with the Griess reagent (contain
ing sulfanilamide and N-naphthyl ethylenediamine)
in an acidic medium. The total nitrite was measured
by Griess reaction. The absorbance was measured at
540 nm with a spectrophotometer.
Catalase Activity Asssay. For this purpose, the
Claiborne’s method was used [18]. Briefly, H2O2 was
added to a mixture of 50 mM potassium phosphate
buffer (pH 7.0) and supernatant, and the rate of
H2O2 decomposition was assessed by measuring the
absorbance changes at 240 nm for 2 min. One unit of
the catalase activity was defined as 1.0 μmol of H2O2
decomposed within 1 min.
Measurement of the Reduced Glutathione
(GSH) Level. The GSH level was measured
spectrophotometrically as described before [19, 20].
Briefly, the supernatant was centrifuged with 5%
trichloroacetic acid, to centrifuge out the proteins.
Two milliliters of phosphate buffer (pH 8.4), 0.5 ml
of 5’5 dithiobis (2-nitrobenzoic acid, DTNB), and
0.4 ml of bidistilled water were added to 0.1 ml of
the homogenate. The mixture was vortexed, and the
absorbance was read at 412 nm within 15 min.
Protein Assay. The protein content in the
supernatant was measured with the Bradford method
using bovine serum albumin as the standard [21].
NEUROPHYSIOLOGY / НЕЙРОФИЗИОЛОГИЯ.—2016.—T. 48, № 3 199
NEUROPROTECTIVE AND ANTIAPOPTOTIC POTENTIAL
450
A
B
* +
4
360
2
270
0
180
–2
90
1
5
3
4 5
–4
0
–6
F i g. 1: Numbers of apomorphine-induced rotations (within a
1-h-long observation period) in different groups (A) and decrement/
increment of this index in 6-OHDA-lesioned groups (B). 1–5 are
indications of the experimental groups (see Methods). *P < 0.05
in comparison with the control group; + P < 0.05 in comparison of
groups 4 and 5.
Р и с. 1. Інтенсивність викликаної апоморфіном обертальної
моторної активності в моделі хвороби Паркінсона, індукованій
6-OHDA.
Estimation of DNA Fragmentation (Apoptosis).
In this experiment, three rats from each group were
euthanized one day after intrastriatal 6-OHDA injection,
and the midbrain tissue supernatant was prepared
as above-mentioned. The determination of histone-
associated DNA fragments was performed using the
Cell Death Detection ELISA kit (Roche Diagnostics,
Germany) as an indicator of apoptosis according
to the protocol from the company and the procedure
as described before [22]. The assay is based on a
quantitative sandwich-enzyme-immunoassay principle
using mouse monoclonal antibodies directed against
DNA and histones, respectively. This allows the specific
determination of mono- and oligonucleosomes (histone-
associated DNA fragments) in the fraction of tissue
lysates. The amount of nucleosomes demonstrating
DNA degradation was quantified by peroxidase retained
in the immunocomplex. The peroxidase content was
determined photometrically at 405 nm with 2,2’-azino-
bis(3-ethylbenzothiazoline-6-sulphonic acid) as a
substrate by a microplate reader (BioTek, USA) after
15 min of the substrate reaction time. The results were
expressed as the optical density (OD).
Histochemistry. At the end of the study, the rats
(n = 4 for each group) were deeply anesthetized with
a high dose of ketamine (150 mg/kg) and perfused
through the ascending aorta with 50–100 ml of
0.9% saline followed by 100–150 ml of the fixative
solution containing 4% paraformaldehyde in 0.1 M
phosphate buffer (PB, pH 7.4) followed by 100 ml of
0.1 M PB. After perfusion, the brains were removed
from the skull; blocks of the forebrain and brainstem
were prepared, and, after final steps of preparation
(immersion in 30% sucrose solution for 1–2 days),
30-mm-thick sections were cut on a freezing
microtome and collected in PB (0.1 M). Every second
section was Nissl-stained with 0.1% cresyl violet.
Histology. For each animal, mesencephalic sections
(interaural 2.9-4.2 mm) were examined by a method
described previously [7]. Nissl-stained neurons in the
SNC were manually counted (light microscopy, ×400)
using a superimposed grid to facilitate the procedure.
At least two sections representative of each of four
Paxinos-Watson planes (4.2, 3.7, 3.2, 2.97, interaural)
were examined by scanning the entire extent. Counting
was done blind to the treatments received.
Statistical Analysis. All data are expressed as
means ± s.e.m. For statistical evaluation of the data,
the parametric one-way ANOVA followed by the
Tukey’s posthoc test was used. In all analyses, the
null hypothesis was rejected at P < 0.05.
RESULTS
The beneficial effect of trigonelline at doses of 50 and
100 mg/kg was evaluated according to apomorphine-
induced rotations for a period of 1 h (Fig. 1). There
were no significant differences among the groups
at baseline (before surgery). Statistical analysis of
the total net number of rotations at the first week
post-surgery showed that apomorphine injections
caused a very significant contralateral turning in
the rats of the 6-OHDA-lesioned group (P < 0.001)
and induced significant but less intense rotations
in the 6-OHDA+trigonelline(50) (P < 0.005) and
6-OHDA+trigonelline(100) groups (P < 0.01) in
comparison with the sham group. Moreover, the group
6-OHDA+trigonelline(100) showed a significantly
smaller number of rotations (P < 0.05) when compared
to 6-OHDA-lesioned rats.
NEUROPHYSIOLOGY / НЕЙРОФИЗИОЛОГИЯ.—2016.—T. 48, № 3200
M. MIRZAIE, M. KHALILI, Z. KIASALARI, and M. ROGHANI
240 A
B
C
210
140
70
0
180
120
60
2.9
1
1 2 3 4 5 SNC SNR
2
3
4 5
** **
*
*
#
#
#
#
##
3.2 3.7 4.2
0
F i g. 2: Total numbers of Nissl-stained neurons on the left side of the substantia nigra within different frontal planes (A), averaged numbers
of such units in all planes (B), and photomicrographs of the substantia nigra (C) one week after 6-ODHA introduction. SNC and SNR are
substantia nigra pars compacta and substantia nigra pars reticulate, respectively. *P < 0.05, **P < 0.01 in comparisons with the sham
group; # P < 0.05, ## P < 0.01 in comparison with the 6-OHDA group with no trigonelline. Designations of the groups are similar to those in
Fig. 1.
Р и с. 2. Кількості забарвлених, за Ніслем, нейронів у лівій частині компактної зони чорної субстанції (SNC) у тварин різних
експериментальних груп.
Findings of Nissl staining (Fig. 2) showed that there
was no significant difference between the sham and
sham+trigonelline(100) groups regarding the number
of Nissl-stained neurons within the left SNC side. In
addition, a significant reduction was noticed in the
6-OHDA-lesioned group (P < 0.05). There were no
significant reductions in the 6-OHDA+trigonelline(50)
and 6-OHDA+trigonelline(100) groups when compared
to the sham group. In this respect, the number of
Nissl-stained neurons on the left side of SNC was
significantly higher in the 6-OHDA+trigonelline(100)
group vs. the 6-OHDA-lesioned group (P < 0.05).
With respect to biochemical markers of oxidative
stress (Fig. 3), 6-OHDA-lesioned rats demonstrated a
significant elevation of the MDA content (P < 0.05)
and nitrite amount (P < 0.05), as well as reduction of
the GSH level (P < 0.05). At the same time, there was
no significant reduction of the activity of the defensive
enzyme (catalase) vs. the respective index in the sham
group. The treatment of lesioned rats with trigonelline
at a dose of 100 mg/kg significantly lowered only the
MDA content (P < 0.05). Meanwhile, there were no
significant changes in the trigonelline(100)-treated
sham group relatively to sham animals regarding
these parameters. Furthermore, the measurement
of chromosomal breakdown of DNA (which is a
100 μm
NEUROPHYSIOLOGY / НЕЙРОФИЗИОЛОГИЯ.—2016.—T. 48, № 3 201
NEUROPROTECTIVE AND ANTIAPOPTOTIC POTENTIAL
MDA
Catalase
Nitrite
Glutathione
1
1 1
2
2 2
3
3 3
4
4 4
5
5 5
reliable indicator of apoptosis) showed a significant
increase of DNA fragmentation in 6-OHDA-lesioned
rats (P < 0.005) vs. the sham group. The trigonelline
pretreatment at a dose of 100 mg/kg significantly
reduced this index in comparison to that in the
6-OHDA group (P < 0.05), as is shown in Fig. 4.
DISCUSSION
In our study, we confirmed that trigonelline pretreat
ment in the 100 mg/kg daily dose significantly reduces
apomorphine-induced rotations in the respective mo-
del, increases the viability of SNC neurons, prevents
their apoptosis, and restores the MDA level in 6-OHDA-
lesioned rats.
Selective degeneration of dopaminergic neurons
in patients with PD results from a genetically
and/or environmentally-induced process of the
neurodegeneration (mostly in the SNC) [23]. 6-OHDA,
the agent generally used for PD induction in rodent
models, is assumed to cause selective degeneration
of dopaminergic neurons in this structure [24]. The
unilateral damage of the nigro-striatal dopaminergic
system, induced by intrastriatal injection of 6-OHDA,
is followed by a reduction in the striatal dopamine
level and upregulation of dopaminergic postsynaptic
receptors at the same side. These changes produce a
prominent functional and motor asymmetry that can
be evaluated by direct-acting dopaminergic agonists,
apomorphine in particular [25]. These rotations
are considered reliable indicators of nigro-striatal
dopamine depletion [26]. In our study, significant
attenuation of apomorphine-induced rotational
60 40
10
3.0
2.5
2.0
1.5
1.0
0.5
0
15
40
20
6 9
30
8 12
20
10
4 6
2 3
0 0
0 0
*
#
*
*
F i g. 3. Effects of trigonelline on midbrain oxidative stress markers. *P < 0.05 in comparisons with the sham group; # P < 0.05 in comparison
with the 6-OHDA group with no trigonelline. Designations of the groups are similar to those in Fig. 1.
Р и с. 3. Маркери оксидативного стресу в середньому мозку тварин різних експериментальних груп.
F i g. 4. The effect of trigonelline on DNA fragmentation in 6-OHDA-
lesioned rat. *P < 0.05 in comparisons with the sham group;
# P < 0.05 in comparison with the 6-OHDA group with no trigonelline.
Designations of the groups are similar to those in Fig. 1.
Р и с. 4. Вплив попередніх уведень тригонелліну на
інтенсивність фрагментації ДНК у моделі хвороби Паркінсона,
індукованій 6-OHDA.
**
* #* #
NEUROPHYSIOLOGY / НЕЙРОФИЗИОЛОГИЯ.—2016.—T. 48, № 3202
M. MIRZAIE, M. KHALILI, Z. KIASALARI, and M. ROGHANI
behavior was observed in trigonelline(100)-pretreated
6-OHDA-lesioned rats. The observed attenuation
of rotational behavior in the mentioned group could
be attributed to a clear neuroprotective effect of
trigonelline against SNC neurodegeneration and
maintenance of striatal dopamine at a level that was
not accompanied with a marked rotational behavior.
In other words, nigro-striatal neurons within a SNC
were mainly preserved in the presence of trigonelline
against neurodegenerative effects induced by the
neurotoxin 6-OHDA. In agreement with our earlier
observations, a standardized hydroalcoholic extract
of Trigonella foenumgraecum seeds, which contains
considerable amounts of trigonelline, was capable
of significantly lowering the intensity of induced
rotations in unilaterally 6-OHDA-lesioned rats and
to reverse motor dysfunctions, including enhanced
spontaneous motor activity in 4-phenyl-1,2,3,6-
tetrahydropyridine-induced lesioned mice. These
effects have been attributed to the neuroprotective
potential of this mean [15].
In addition, an overproduction of free radicals,
especially of reactive oxygen species, is also
cons iderab ly invo lved in 6 -OHDA-induced
neurodegeneration [26]. Oxidative stress is among
most important factors affecting the survival of
dopaminergic neurons in PD. Neurons crucially
depend on adequate energy supply produced by
mitochondria and are simultaneously faced with
high levels of reactive oxygen species, as well with
increased levels of free iron. The latter phenomenon
can promote hydroxyl production [27]. Overloading by
the free radicals inevitably leads to intensification of
the process of cell death. In addition, autooxidation of
dopamine may produce dopamine quinine [28]. The
formation of such species (semiquinones and other
free radicals) can especially intensely damage nucleic
acids, proteins, and membrane lipid components [29].
Therefore, the adequate therapeutic approaches should
be aimed at attenuation of oxidative stress. Free
radical scavengers may also be helpful in prolonging
the survival time of dopaminergic neurons [30]. In
this respect, trigonelline has been demonstrated to
attenuate neuronal damage and to strongly counteract
oxidative stress. The measurement of oxidative stress
markers in our study also showed that trigonelline
is able to attenuate this stress and to augment the
antioxidant defensive system in the midbrain. In this
way, this compound can significantly decrease the
toxicity of 6-OHDA. This finding is consistent with
literature data on the antioxidant ability of this mean
via regulating the antioxidant defense system, as well
as via suppression of free radical generation [11].
The inflammatory process initiated in the brain
structures is also an important causative factor for
PD [31, 32]. Pro-inflammatory cytokines released
from glial cells can considerably stimulate nitric
oxide production and exert a deleterious effect on
dopaminergic neurons by activating receptors that
contain intra-cytoplasmic death domains involved
in the apoptotic pathway [33]. It has been shown
that coffee (a food product that also contains high
amounts of trigonelline) demonstrates noticeable
anti-inflammatory activity [11]. Thus, it is possible
that trigonelline may also lowered the level of the
respective “PD-related” inflammatory mediators
within the brain, and this also contributes in some a
way to neuroprotection in 6-OHDA-induced PD model
in rats, as was observed in our study. In addition,
apoptosis is another factor that plays a critical role
when cells are exposed to neurotoxins, including
6-OHDA [34]. In our study, trigonelline was able
to prevent apoptosis, as evidenced by a lower DNA
fragmentation. The anti-apoptotic effect of trigonelline
has also been reported before [35]. Nevertheless,
some aspects of the respective issue need further
investigation.
Taken together, our results demonstrated that pre-
lesion treatment with trigonelline in the animal
model of PD exert, in a dose-dependent manner, clear
neuroprotective and antiapoptotic effects against
6-OHDA toxicity. Henceforth, the above agent may be
included in a set of therapeutic means advantageous
for the management of early PD.
Acknowledgements. This research study was the result of
MD thesis project approved and financially supported by the
Shahed University in 2013.
The used procedures for animals and their care were in
accordance with the existing international ethic norms for
experimental work mentioned in the NIH guidelines.
The authors, M. Mirzaie, M. Khalili, Z. Kiasalari, and
M. Roghani, confirm that they have no conflicts with respect to
any kind related to commercial or financial problems, relations
with organizations or persons, which could in any way be
associated with the investigation, and with the relationship of
the co-authors of the article.
NEUROPHYSIOLOGY / НЕЙРОФИЗИОЛОГИЯ.—2016.—T. 48, № 3 203
NEUROPROTECTIVE AND ANTIAPOPTOTIC POTENTIAL
М. Мірзайє1,2, М. Халілі1, З. Кіасаларі1, М. Рогані1
НЕЙРОПРОТЕКТОРНИЙ ТА АНТИАПОПТОТИЧ
НИЙ ВПЛИВИ ТРИГОНЕЛЛІНУ В СТРІАТНІЙ МОДЕЛІ
ХВОРОБИ ПАРКІНСОНА У ЩУРІВ, ІНДУКОВАНІЙ
6-ГІДРОКСИДОФАМІНОМ
1,2 Нейрофізіологічний науково-дослідницький центр
Університету Шахед, Тегеран (Іран).
2 Студентський дослідницький комітет Університету Ша
хед, Тегеран (Іран).
Р е з ю м е
Беручи до уваги нейропротекторний та антиоксидантний
вплив тригонелліну, ми дослідили його захисну дію в мо
делі хвороби Паркінсона у щурів, індукованій стереотак
сичним уведенням 6-гідроксидофаміну (6-OHDA). Щурам,
котрим робили унілатеральні ін’єкції 6-OHDA в стріатум,
попередньо щоденно тричі вводили тригонеллін у дозах 50
і 100 мг/кг. В умовах використаної 6-OHDA-моделі істот
но посилювалась обертальна моторна поведінка, виклика
на ін’єкцією апоморфіну, значно знижувалася кількість за
барвлених, за Ніслем, нейронів у лівій половині компактної
частини чорної субстанції, посилювався процес апопто
зу нейронів, зростали рівні малональдегіду та нітриту та
знижувався рівень відновленого глутатіону. Тригонеллін
у щоденних дозах 100 мг/кг вірогідно знижував кількість
обертальних рухів, протидіяв зменшенню числа нейронів у
чорній субстанції та розвитку апоптозу, а також нормалі
зував рівень малональдегіду. Отримані результати свідчать
про те, що уведення тригонелліну перед ін’єкціями 6-OHDA
забезпечує дозозалежні нейропротекторний та антиапопто
тичні ефекти в умовах токсичної дії 6-OHDA. Цей агент та
його аналоги можуть бути тестовані як допоміжні засоби
при лікуванні хвороби Паркінсона на ранніх стадіях.
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