Crocin - 6-hydroxydopamine - oxidative stress - nitric oxide - aversive memory - medial
forebrain bundle
Crocin - 6-hidroxidopamina - estresse oxidativo - óxido nítrico - feixe medial do
cérebro anterior
Parkinson’s disease (PD) is a progressive neurodegenerative disorder that is characterized
by the degeneration of dopaminergic nigrostriatal neurons, which leads to motor symptoms
of bradykinesia, rigidity, rest tremor, and postural imbalance[1]. Parkinson’s disease also causes impairments in cognitive performance, and the progression
of these deficits can lead to dementia[2]. The hippocampus, which is involved in cognitive processes such as learning and
memory, is implicated in memory deficits observed in PD since both structural and
functional changes of the hippocampus have been observed in PD patients[3],[4]. Several MRI studies have also clearly revealed that the reduction of hippocampal
volume was accompanied by cognitive deficits in PD patients[5].
Numerous studies have suggested that oxidative stress plays a major role in the pathogenesis
of PD[6]. Free radicals and other reactive oxygen species (ROS) resulted from dopamine auto-oxidation
and metabolism, lipid peroxidation, impaired mitochondrial function, and deficiencies
in endogenous antioxidant systems that may all contribute to a progressive loss of
dopaminergic neurons[7],[8]. There is also some evidence showing that the neurotoxicity of 6-hydroxydopamine
(6-OHDA), for the modeling of PD, is due to its oxidation and the formation of various
oxidants and free radicals and the depletion of reduced glutathione[9]. This leads to lipid peroxidation, protein damage and ultimately degeneration of
the nigrostriatal dopaminergic system. 6-OHDA has also been reported to produce reactive
nitrogen species (RNS) such as nitric oxide (NO) by elevated expression of inducible
nitric oxide synthase (iNOS), especially in neurons[10]. Reactive oxygen species can rapidly interact with NO and subsequently produce more
powerful oxidant peroxynitrite. Peroxynitrite is known to structurally and functionally
modify critical cellular macromolecules and cause oxidative damages, which finally
leads to apoptotic cell death[11].
Recently, the major focus of many preclinical studies is the identification of drugs
or approaches that might prevent or inhibit the neurodegenerative process. In this
context, it has been suggested that antioxidant molecules (carotenoids and polyphenols)
and compounds interfering with production of reactive oxygen species and nitric oxide
might be protective.
Crocin is a water- soluble carotenoid and an active constituent of saffron (Crocus sativus L.). It has been reported that crocin possesses multiple pharmacological properties,
including antioxidative activity[12],[13], anti-inflammatory[14], protection against cardiovascular diseases, inhibition of tumor cell proliferation,
neuroprotection and protection of hepatocytes[12]. It has also been shown that the spice saffron, which contains powerful antioxidants
such as crocin, protects nigral and retinal dopaminergic cells in an acute MPTP mouse
model of Parkinson’s disease[15]. The antioxidant and radical scavenging activity of crocins have also been shown
in several in vitro models[12],[16]. Based on the role of oxidative and nitrosative stress on the pathophysiology of
PD and considering the antioxidant and anti-inflammatory effects of crocin, the present
study was designed to evaluate whether it protects against 6-OHDA-induced oxidative
damage and memory deficits in rats.
METHODS
Animals
Adult male Wistar rats, weighing 250–300g were housed in an air conditioned colony
room at 22°C ± 2°C on a standard pellet diet and tap water ad libitum. The Ethics Committee for Animal Experiments at Isfahan University of Medical Sciences
approved the study and all experiments were conducted in accordance with the National
Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications
Nº 8023, revised 1996).
Chemicals
Crocin, 6-OHDA, and apomorphine hydrochloride were purchased from Sigma Aldrich Co.
2,2´ Dinitro-5,5´-dithiodibenzoic acid (DTNB), trichloro acetic acid (TCA), 2-thiobarbituric
acid (TBA), Tris-EDTA, chloral hydrate and hydrochloric acid (HCL) were obtained from
Merck.
Experimental design
The animals were randomly divided into four groups, with eight rats in each group,
as follows: normal saline sham-operated group, normal saline-treated lesioned group
(6-OHDA), and the crocin-treated lesioned groups (crocin 30 and 60 mg/kg/day). Crocin
was dissolved in normal saline and injected at doses of 30 and 60 mg/kg/day intraperitoneally,
three days before the surgery for six weeks. The surgical procedures were carried
out under general (chloral hydrate, 450 mg/kg, ip) and local anaesthesia (lidocaine
2%). The rats were placed in a stereotaxic apparatus and the lesion was induced by
injection of 6-OHDA (16 μg/4μl 0.2% ascorbate saline) into the left medial forebrain
bundle through microinjection pump (Kd Scientific, USA) according to the coordinates:
AP: -4.5 mm; ML: -1.7 mm; DV: -8.2 mm[17]. The rats of the sham-operated group also received an identical volume of the ascorbate
saline as the vehicle. The injection rate was 1 μl/min and the needle was kept in
place for an additional five minutes before slowly being retracted. At the end of
the experiment, the animals were sacrificed and the hippocampus and cortex were dissected
out, washed immediately in ice-cold saline, and homogenized in the NaCl solution by
a homogenizer. The hippocampus and cortex were obtained bilaterally, and the data
are measures from combined bilateral tissues.
Lipid peroxidation levels
The lipid peroxidation level of the hippocampus and cortex was measured as malondialdehyde,
which is the end product of lipid peroxidation. The malondialdehyde reacts with TBA
as a thiobarbituric acid reactive substance (TBARS) and produces a red colored complex
that has a peak absorbance (A) at 535 nm. Briefly, a mixture of TCA, TBA, and HCl
were added to 1mL of homogenate, and the mixture was heated for 45 min in a boiling
water bath. After cooling, the samples were centrifuged at 1000× g for 10 min and
the absorbance was measured at 535 nm. The level of TBARS was calculated according
to follow equation[13]. Concentration (Molar) = Absorbance /1.65 x 105.
Total thiol concentration
Total sulfhydryl (SH) groups were measured using DTNB as the reagent. This reagent
reacts with the SH groups to produce a yellow colored complex that has a peak absorbance
at 412 nm. Briefly, 1mL Tris-EDTA buffer (pH = 8.6) was added to 50 μL homogenate
in 2 mL cuvettes and the sample absorbance was read at 412 nm against the Tris-EDTA
buffer alone (A1). Then, 20 μL of the DTNB reagent (10 mM in methanol) was added to
the mixture and after 15 min, the sample absorbance was read again (A2). The absorbance
of the DTNB reagent was also read as a blank (B). The total thiol concentration (mM)
was calculated by: The total thiol concentration (mM) = (A2-A1-B) × 1.07/0.05 × 13.6[13].
Glutathion peroxidase assay
GSH peroxidase concentration was measured with the GSH peroxidase kit (Randox Labs,
Crumlin, UK).
Nitrite levels
The level of nitrite was measured using a colorimetric assay kit (Promega Corporation,
USA) that involved the Griess reaction. Briefly, after adding sulphanilamide solution
and incubation, N-(1-naphthyl) ethylenediamine solution was added. Then, the sample
absorbance was measured by a microreader in the wavelength of 492 nm. The nitrite
concentration of samples was determined by comparing comparison against with the nitrite
standard reference curve.
Passive avoidance learning
The passive avoidance test was used in the current study, because it is a suitable
model for evaluating hippocampal-dependent memory deficits in experimental animals.
Passive avoidance learning was assessed by shuttle box at the end of week 6. The apparatus
consisted of a light compartment (25×25×20 cm) and a dark compartment (50×25×20 cm)
with a grid floor and wooden walls. The two compartments were separated by a sliding
guillotine door. On the day before training, each rat was placed into the apparatus
and allowed to move around 5 min for five minutes for habituation. In the training
session, animals were placed individually in the light compartment for one minute.
After opening the door and entrance of the rat entering into the dark chamber, the
door was closed and a 1mA foot electric shock was delivered for 3s through the grid
floor. The current intensity for foot shock was selected based on previous studies
in our laboratory. In the test session, each rat was again placed into the light compartment.
The step-through latency to enter the dark compartment was measured as a positive
index of memory performance, with a 300 s cut-off time[18].
Histology
The animals were sacrificed by a high dose of the anaesthetic. Then, the brains were
removed and stored in 10% formalin for 72 h. The brains were sectioned coronally at
40 μm by a freezing microtome (Leica, Germany). Sections were mounted on gelatin-coated
slides and studied using a light microscope. The track of the needle and injection
site of 6-OHDA ([Figure 1]) was determined by reference to a rat brain atlas[17].
Figure 1 Photograph of the coronal section of rat brain representing injection site of 6-OHDA
in the medial forebrain bundle (open circle).mt: mammilothalamic tract, F: fornix.
Statistical analysis
The data were expressed as mean ± SEM. Statistical analysis was carried out using
one-way ANOVA followed by the LSD post hoc test. A statistical p-value < 0.05 was
considered significant.
RESULTS
Effects of crocin on lipid peroxidation levels
As shown in [Figure 2], a significant increase in the levels of TBARS, an index of lipid peroxidation,
was found in the hippocampus of 6-OHDA-lesioned rats (p = 0.01) as compared with the
sham group. Moreover, treatment of lesioned rats with crocin at a dose of 60 mg/kg
reduced the TBARS levels in the hippocampus at the end of week 6 (p < 0.05, [Figure 2]). Meanwhile, 6-OHDA increased the TBARS levels in the cortex of lesioned rats, although
the changes were not significant.
Figure 2 Lipid peroxidation levels (thiobarbituric acid reactive substance [TBARS]) in the
hippocampus and cortex of the sham, 6-OHDA-lesioned rats and lesioned rats treated
with crocin at doses of 30 and 60 mg/kg at the end of week 6. Data are mean ± SEM
for eight animals in each group. *p = 0.01 vs sham group, +p < 0.05 vs 6-OHDA-lesioned
group.
Effects of crocin on nitrite levels
[Figure 3] shows the nitrite levels in the hippocampus and cortex of the sham and experimental
groups. A significant increase in the nitrite levels in the hippocampus (p < 0.05)
of 6-OHDA-lesioned rats was observed as when compared with sham group rats. Treatment
of 6-OHDA-lesioned rats with crocin at a dose of 30 mg/kg significantly decreased
the nitrite levels in the hippocampus as when compared to with the lesioned group
(p < 0.05, [Figure 3]). Meanwhile, 6-OHDA did not change the nitrite levels in the cortex of lesioned
rats compared with sham group rats ([Figure 3]).
Figure 3 Nitrite levels in the hippocampus and cortex of the sham, 6-OHDA-lesioned rats and
lesioned rats treated with crocin at doses of 30 and 60 mg/kg at the end of week 6.
Data are mean ± SEM for eight animals in each group. *p < 0.05 vs sham group, +p <
0.05 vs 6-OHDA-lesioned group.
Effects of crocin on total thiol concentration
[Figure 4] shows the total thiol concentration in the hippocampus and cortex of the sham and
experimental groups. There was no significant change in total thiol concentrations
in the hippocampus and cortex of sham and experimental groups ([Figure 4]).
Figure 4 Total thiol concentrations in the hippocampus and cortex of the sham, 6-OHDA-lesioned
rats and lesioned rats treated with crocin at doses of 30 and 60 mg/kg at the end
of week 6. Data are mean ± SEM for eight animals in each group.
Effects of crocin on glutathione peroxidase levels
As shown in [Figure 5], there was no significant difference in glutathione peroxidase concentration in
the hippocampus and cortex of sham, 6-OHDA-lesioned rats and lesioned groups treated
with crocin at 30 and 60 mg/kg at the end of week 6.
Figure 5 Glutathione peroxidase concentrations in the hippocampus and cortex of the sham,
6-OHDA-lesioned rats and lesioned rats treated with crocin at doses of 30 and 60 mg/kg
at the end of week 6. Data are mean ± SEM for eight animals in each group.
Effects of crocin on passive avoidance learning
As shown in [Figure 6], the step-through latency of 6-OHDA-lesioned rats was shorter than the sham group
rats at the end of week 6 (p < 0.05, [Figure 6]). Moreover, treatment of lesioned rats with crocin at a dose of 30 mg/kg increased
the latency as compared with lesioned rats (p < 0.05).
Figure 6 Step-through latencies in the passive avoidance test in the sham, 6-OHDA-lesioned
rats and lesioned rats treated with crocin at doses of 30 and 60 mg/kg at the end
of week 6. Data are mean ± SEM for eight animals in each group. *p < 0.05 vs sham
group, +p < 0.05 vs 6-OHDA-lesioned group.
DISCUSSION
In the present study, we evaluated the effect of crocin on biochemical and behavioral
parameters using the 6-OHDA rat model, because this model imitates the pathological
and biochemical features of PD, such as oxidative stress, mitochondrial dysfunction
and apoptosis[19]. Oxidative stress, imbalance of free radicals and antioxidants, plays a critical
role in the pathogenesis of PD. Dopaminergic neurons are particularly sensitive to
oxidative stress because of their low antioxidant capacity, as evidenced by low intracellular
glutathione[20]. A number of studies have also demonstrated that the neurotoxicity of 6-OHDA for
the modeling of PD is due to its oxidation, the formation of various oxidants and
free radicals and the depletion of reduced glutathione, which leads to lipid peroxidation,
protein damage and ultimately degeneration of the neurons[9]. In the present study, we observed an elevated levels of TBARS in the hippocampus
of PD rat brains, which was not accompanied by a depleted glutathione peroxidase level.
This was, in part, in agreement with previous observations[21]. Our findings also revealed that long-term treatment with crocin reduced the TBARS
levels in the hippocampus of parkinsonian rats. This is in agreement with previous
studies which that report the antioxidant effects of crocin in other models. For example,
Rajaei et al.[13] reported that crocin attenuated hepatorenal oxidative damage in streptozotocin-induced
diabetic rats, as indicated by a significant decrease in TBARS levels and an elevation
in total thiol concentrations. Furthermore, the radical-scavenging and neuroprotective
effects of crocins have also been shown in several in vitro models[12],[16]. For instance, Zhang et al.[16] have recently shown that crocin protects PC12 cells against 1-methyl-4-phenylpyridinium-induced
injury. Collectively, it seems that the antioxidant activity of crocin in the hippocampus
in a 6-OHDA model of PD is related to its antioxidant and radical scavenging activity.
A large body of evidence also supports the involvement of inflammation in the pathogenesis
of PD. Microglial activation is considered as a rapid cellular response to inflammation.
Activation of microglia induces cytotoxic mediators such as NO and inflammatory cytokines,
which may contribute to the PD progression[22]. NO production resulting from induced NOS gene expression and subsequent iNOS enzyme
activation is a primary contributor to the inflammatory response[22],[23]. Examination of postmortem PD brains has revealed robust microgliosis and the presence
of high levels of iNOS expression in the substantia nigra (SN) compared with control
brains[24]. It has also been reported that unilateral injection of 6-OHDA into the SNc increased
NOS expression in the SN and striatum of PD rats[25]. Along with this, the present findings showed that the 6-OHDA increased the levels
of the nitrite, stable NO metabolite, in the hippocampus of parkinsonian rats. The
results also showed that treatment with crocin attenuated the nitrite levels in this
tissue. To our the best of knowledge, this is the first study reporting the anti-inflammatory
effects of crocin through reducing NO levels in a 6-OHDA model of Parkinson’s disease,
which is in agreement with its anti-inflammatory effects that have previously been
reported[14]. In this context, it has been reported that crocin inhibited the lipopolysaccaride-induced
NO release from cultured rat brain microglial cells[14]. Moreover, treatment with crocin decreased NO levels and NOS activity in cortical
microvascular homogenates in an ischemic model in rat[26].
In the present study, 6-OHDA injections also produced memory deficit, which acts by
increasing oxidative stress within the brain of rats. Previous studies have also demonstrated
that 6-OHDA could produce cognitive impairments in animals, and oxidative stress has
been shown to play an important role in memory impairment[27]. Reactive oxygen species induced by 6-OHDA can react with biological target molecules
and contribute to increased neuronal damage and death through protein oxidation, DNA
damage, and peroxidation of membrane lipids. In our study, the passive avoidance test
was used to examine whether crocin could improve memory of parkinsonian rats. This
task is based on the motivation of passive avoidance from the fear of foot shock.
Crocin at a dose of 30 mg/kg increased the step-through latency during the testing
session as when compared to parkinsonian rats, in the other words, it produced the
amelioration of retention memory in parkinsonian rats. Consistent with this, memory-enhancing
effects of crocin have previously been previously reported in other models, such as
cerebral ischemia.
However, it looks like that 60 mg/kg crocin is more efficient in decreaseing TBARS
in the hippocampus compared to the 30 mg/kg dose, but 60 mg/kg crocin did not significantly
affect the behavior impairment. This discrepancy could be related to the fact that
several other factors, rather other than reactive oxygen species and oxidative stress,
are also involved in the development of cognitive impairments in PD. These factors
include the imbalance in NO production and increased nitrosative stress[28], dysfunction of the cholinergic system[29], neuroinflammation and apoptosis[30]. For instance, Kuhad and Chopra[28] have reported a significant increase in nitrite levels in the cortex and hippocampus
of diabetic rats having with cognitive deficits. In our study, nitrite levels were
also significantly increased in the hippocampus of parkinsonian rats. Excessive production
of NO by increased iNOS expression leads to the formation of an extremely potent oxidizing
agent, peroxynitrite (ONOO–), which causes neuronal death[10]. Peroxynitrite, which is formed by reaction between superoxide and NO, reacts with
many biological target molecules and damages the neurons by oxidizing or nitrating
proteins, lipids, and DNA[11]. In fact, the cell membrane has no significant barrier against diffusion of peroxynitrite
into cells, thus allowing peroxynitrite to induce DNA damage and to mediate the activation
of apoptosis pathways[10]. Taken together, the data presented here suggest that the improvement of memory
by 30 mg/kg crocin might be mediated, at least in part, by decreased production of
NO and inhibition of nitrosative stress in the hippocampus.
In conclusion, the present study demonstrated that the crocin acts as an antioxidant
and anti-inflammatory agent in the hippocampus of parkinsonian rats and could improve
aversive memory, at least in part, through reducing nitrite levels in the brain. Considering
the safety of crocin in clinical trials, it might be used as an adjunct therapy for
improvement of memory impairments in PD in the future.
