INTRODUCTION:
The International Association
for the Study of Pain (IASP) defines neuropathic pain (NP) as "pain resulting
from disease or damage of the peripheral or central nervous systems, and from dysfunction
of the nervous system" characterized by hyperalgesia and allodynia1.
The prevalence of neuropathy in india was reported as 78.2 %2. Acrylamide is one of the neurotoxic agents,
and it is used in various industries such as dye manufacturing, fiber processing
unit, polymer production, gel electrophoresis application, and water treatment process3.
Furthermore, it is widely distributed in ground water due to industrial waste products.
It is known to cause the potential distal multifocal axonal degeneration that
leads to ‘‘dying back’’ (i.e. progressive axonal loss) neuropathy4.
The neurotoxic effects of acrylamide involving
both the peripheral and central nervous system have been documented in both humans
and animals5. Acrylamide-induced neuropathic pain model is one of the
major methods of induction of neuropathic pain in experimental animals due to its
specific pathological mechanism4,5.
Pain after injury to the nervous system (neuropathic pain)
is a major chronic condition that remains difficult to treat. Tricyclic anti-depressants
and anticonvulsants have also been reported to produce antiallodynic effects in
neuropathy but they exhibit a wide spectrum of adverse effects6. Hence,
there is an urgent need to find an alternative medicine which can attenuate neuropathic
pain effectively with few side effects. This led researchers to find a drug from
natural sources7. Isolated
plant compounds such as α-β- Amyrin8, β- Caryophyllene9,
Cannabidiol10, Genistein11, Hesperitin12, Kaempferol13,
Lappaconitine14, Linalool15, Liquiritigenin16,
Luteolin17, Mangiferin18, Naringin19, Rutin20,
papain21, Quercetin22, Silibinin23, Oxymatrine24,
Triptolide25, Verbacoside26, Tormentic acid27 were
scientifically reported their effectiveness against neuropathic pain through mitigation
of various pathways such as inflammation, oxidative stress, inhibiting sodium channels,
inhibiting calcium channels, inhibiting glutaminergic system.
Sinapic
acid is widely distributed in the plant kingdom and is obtained from various sources
such as rye, fruits and vegetables28. Sinapic acid has potent antioxidant,
anxiolytic, anti-inflammatory, peroxynitrite scavenging and neuroprotective effects29.
This is the first study attempting to explore protective effect of sinapic acid
on toxic effects of acrylamide.
MATERIALS AND METHODS:
Materials:
Sinapic acid was purchased from Sigma Chemical Co. (St
Louis, MO, USA). Acrylamide, reduced glutathione (GSH) were obtained from Merck.
All other chemicals used were of analytical grade.
Cell culture:
U87MG
were obtained from American type culture collection (ATCC, USA). Cells were maintained
in a humidified atmosphere of 5% CO2 at 370C. Cells were grown
in Dulbecco’s Modified Eagles medium (Gibco Invitrogen, Paisley, UK) supplemented
with 10% fetal bovine serum, 1% non-essential amino acids, 1% penicillin (1000U/mL),
1% streptomycin (1000μg/mL) and 1% amphotericin (250U/mL). The cells were passaged
enzymatically with 0.25% trypsin- 1mM EDTA and sub-cultured on 75 cm2
plastic flasks at a density of 2.2 x104 cells/cm2. Culture medium was
replaced every 2 days. Cell confluence (80%) was confirmed by microscopic observance.
Treatment was performed 12 hours post-seeding to prevent cell differentiation.
Cell viability:
The
viability of cultured cells was determined by assaying the reduction of 3-(4, 5-dimethyl
thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) to formazan. Briefly, U87MG
cells were cultured in a 96- well microtiter plate at a density of 105 cells/well.
After pretreatment with sinapic acid (5, 50, 100, 150, 200, 250, 300µg/ml) for 24
h, ACR at a concentrarion of 5.46mM was added to each well. The cells were then
incubated for 48 and 72hrs and then treated with MTT solution (0.5 mg/ml PBS) at
370C until intracellular purple formazan crystals are visible under microscope.
100µL of the dimethyl sulfoxide (DMSO) was added to each well and mixed gently on
an orbital shaker for one hour at room temperature. The volume of DMSO was adjusted
depending on the volume of cell culture. Absorbance was measured at 570nm for each
well on an absorbance plate reader. The entire experiment was repeated thrice.
Experimental
animals:
Male Wistar rats, 230 to 250g were housed in rooms with
12/12 h light/dark cycle at 21±20C and fed with standard laboratory chow
(Hindustan lever limited, Mumbai) and water ad libitum. Animals were acclimatized
to the laboratory conditions prior to experimentation. The experimental protocol
was approved by Institutional Animal Ethics Committee and care of the animals was
carried out as per the guidelines of Committee for the Purpose of Control and Supervision
of Experiments on Animals (CPCSEA), Ministry of Environment and Forest, Government
of India (Reg. No. 1529/PO/Re/11/CPCSEA./CHIPS/ IAEC5/PRO-5/2016-17).
Experimental design:
Rats were randomly divided into 5 groups (n=6 in each group).
Group I rats served as control and received the vehicle
normal saline only. Group II, III and IV rats were administered with ACR (50mg/kg
bw, i.p. thrice a week) for 4 weeks. After administration of ACR Groups III, IV
rats received the SA (15 and 30mg/kg orally) and Group V rats received 30mg/kg of
SA orally. Rats of all the groups were monitored for the manifestation of neuropathy.
Daily food intake and weekly body weights were recorded throughout the experimental
period of 4 weeks. Rats were subjected to behavioral tests each week. Finally, after
assessing behavior rats were sacrificed under light ether anesthesia and the excised
SN was processed for biochemical analysis.
The
behavioral index (neurological scores) examination:
At the end of the treatment, the neurological scores were
examined. Rats were placed in a clear plexi glass box and were observed for 3 min,
and a neurological score, from 1 to 4, was assigned; where 1= a normal, unaffected
gait; 2= a slightly affected gait (foot splay, slight hind limb weakness and spread);
3= a moderately affected gait (foot splay, moderate hind limb weakness, moderate
limb spread during ambulation,); and 4= a severely affected gait (foot splay, severe
hind limb weakness, dragging hind limbs, inability to rear).
Motor coordination:
Motor coordination was evaluated by Rota-Rod device as
described by Jones and Roberts
(1968). 
Rats were placed for 2 min
on the rotating rod, the time taken for the falling from the rotating rod, was recorded30.
Narrow beam test:
Narrow beam test was employed to measure hind-limb impairments
according to a previously described method with minor modifications31.
Animals were trained to traverse a 150cm long wooden beam, divided into three segments
(1, 2 and 3) of 50cm each, from a platform at one end to the animal’s home cage
at the other, placed horizontally 60cm above the floor. The number of foot slips
onto an under-hanging ledge was recorded. Each rat was tested three times. The scoring
was between 0 and 4; a score of 0 for a rat that traverses the beam without falling;
a score of 1 if the animal fell off in the third segment; a score of 2 if it fell
in the second segment; a score of 3 if it fell in first segment and a score of 4
if the rat failed to even balance/sit on the beam. The average of scores for three
trials per rat were taken and mean value was calculated.
Cold-Allodynia Test (Acetone Drop Test):
Cold-allodynia of the hind paw was assessed using acetone
drop method as described by Choi et al. (1994), with slight modification
(De la Calle et al., 2002) for assessing the reactivity to non-noxious cold
chemical stimuli. The rats were placed on the top of a wire mesh grid, which allowed access to the hind paws. Acetone
0.1 ml (100μl) was sprayed on the plantar
surface of the hind paw of rat. Cold sensitive reaction with respect to either paw
licking, shaking or rubbing the hind paw was observed and recorded for 20 s test
period27.
Biochemical Analysis in SN:
The bilateral SN was excised (about 1 inch length) from
L4 segment of the spinal cord at the mid thigh level till it’s branching. It was
washed in ice-cold saline, freed from the adherent blood vessels and connective
tissue, blotted and weighed. SN was minced and homogenized in 0.1 M tris HCl buffer
of pH 7.4. The cytosolic fraction was obtained by centrifuging the samples.
Estimation of superoxide dismutase (SOD):
SOD activity was estimated according to the method of Misra
and Fridovich (1972) at room temperature. The sciatic nerve was homogenized in ice
cold 50mM phosphate buffer (pH-7) containing 0.1mM EDTA to give 5% w/v
homogenate. The homogenate was centrifuged at 10,000 rpm for the enzyme assay. 100
µl of tissue extract was added to 880 µl (0.05M, PH-10.2, containing
0.1mM EDTA) carbonate buffer, and 20 µl of 30mM epinephrine (in 0.05 % acetic acid)
was added to the mixture and the optical density values were measured at 480nm for
4 min on a UV-Vis Spectrophotometer. Activity is expressed as the amount of enzyme
that inhibits the oxidation of epinephrine by 50 % is equal to 1 unit32.
Assessment of Lipid Peroxidation (LPO):
LPO was assessed by measuring the formation of thiobarbituric
acid reactive substances (TBARS). Briefly, the reaction mixture contained 0.2ml
of SN homogenate, 1.5ml of acetic acid (pH 3.5, 20 %), 1.5ml of 0.8 % thiobarbituric
acid (0.8 % w/v) and 0.2ml SDS (8 % w/v). The mixture was heated to boiling for
45 min and TBARS adducts were extracted into 3ml of 1-butanol and its absorbance
was measured at 532nm and quantified as malondialdehyde (MDA) equivalents using
1,1,3,3-tetramethoxypropane as the standard33.
Reduced Glutathione (GSH):
Reduced glutathione was measured according to
the method of Ellman (1959). Equal quantity of sciatic nerve homogenate was mixed
with 10% trichloroacetic acid and centrifuged to separate proteins. To 0.01ml of
supernatant, 2ml of phosphate buffer (pH 8.4), 0.5ml of 5, 5_-dithio, bis (2-nitrobenzoic acid) and 0.4 ml double-distilled water was
added. Mixture was vortexed and the absorbance was read at 412nm within 15 min.
The concentration of reduced glutathione was expressed as n mol/mg of protein34.
Estimation
of total calcium:
To 4.5ml of deproteinated buffer
in a glass centrifuge tube, 0.5ml of the sample was added and was placed in water
bath for 3 minutes. Tubes were centrifuged while they were still hot, 0.5ml of each
supernatant was transferred into clean test tubes. For the reagent blank, 0.5ml
of blank solution was prepared by mixing 9 volumes of deproteination buffer with
one volume of water. 5ml of working colouring reagent was added to each tube, mixed
well and then read at 570nm35.
Statistical
analysis:
Data
were expressed as mean±S.E.M (n=6) and was analyzed using one way analysis of variance
for antioxidant parameters and two-way analysis of variance (ANOVA) followed by
Dunnet’s T test for behavioral tests using Graph pad prism 8.0. A value of P
< 0.05 was considered to be statistically significant.
RESULTS:
Effect of SA on ACR induced cytotoxicity in U87MG cells:
SA treatment decreased the ACR induced cytotoxicity significantly
(p<0.001) in comparision to acrylamide group (Figure 1).
Effect of SA on ACR induced alterations in neurological
score:
Exposure to ACR (50mg/kg, i.p) for 4 weeks led to progressive
gait abnormalities in rats as shown in Figure 2a. In animals treated with SA (15
and 30mg/kg) showed dose dependent reversal of ACR induced neurological deficits
(P<0.01) as compared to control group. At the end of 4 weeks SA treatment
caused a significant reduction in neurological scores compared to untreated ACR
administered rats indicating its protective effect.
Effect of SA on Motor coordination test:
Administration of SA significantly attenuated ACR induced decrease
in motor performance in a dose-dependent manner as assessed by time spent on rota
rod. Rats treated with SA (15 and 30mg/kg) showed improvement in motor performance
on 3rd and 4th week respectively (P<0.001) when compared
to control group (Figure 2b).
Narrow Beam Test:
ACR administered rats showed significant and progressive
motor dysfunction in the narrow beam test from week 3 onwards. The protective effect
of SA was evident only from week 3 onwards. The intensity of motor dysfunction and
inability to balance on the beam among ACR rats was very high at week 4. SA treatment
significantly (P<0.001) reversed ACR induced decrease in motor function
(Figure 3a).
Effect of SA on Acetone Drop Test:
ACR administration produced a significant decrease in the
nociceptive threshold for cold chemical pain sensation. From 2nd week
after the ACR administration and lasted throughout the experimental period. Treatment
with SA 15 and 30mg/kg p.o. attenuated ACR induced decrease in the non-noxious
nociceptive threshold in a dose dependent manner. Similar results were observed
in SA 30mg/kg; p.o. only treated rats (Figure 3b).
Effect of SA on Oxidative stress markers:
ACR treatment significantly decreased SOD (P<0.001)
and GSH (P<0.01) levels in SN. SA
dose dependently and significantly (P<0.01), (P<0.001) restored
the levels of SOD and GSH
compared to ACR treated rats. Treatment with 30mg/kg of SA alone had no change in
GSH as compared to control, indicating SA does not have any effect in healthy condition.
Lipid peroxidation in the SN was determined by measuring MDA content. ACR treated
rats showed a significant (P<0.001) increase in the level of MDA and Calcium levels when compared to
control rats. Treatment with SA at doses of 15 and 30 mg/kg significantly reversed
ACR induced increase in MDA and Calcium
levels in SN (Figure 4).
Fig.
1 Effect of Acrylamide and Sinapic acid on U87 MG cells; *** (P<0.001)
Vs control cells and ### (P<0.001) Vs acrylamide treated cells.
DISCUSSION:
In the present study, the neuroprotective effect of sinapic
acid on ACR induced cytoxicity in U87MG cells and ACR induced neurotoxicity in rats
was evaluated. Our results showed that ACR decreased the cell viability in U87MG
cells which was suppressed by the administration of sinapic acid. The results of
the present study also indicate that administration of ACR induced a significant
degree of painful neuropathy in rats, manifested as alterations in neurological
score, motor coordination, cold allodynia, narrow beam test and total calcium levels
and decreased superoxide dismutase, reduced glutathione levels. SA (15 and 30mg/kg)
attenuated ACR induced neuropathy. Exposure to ACR could damage both
the central and the peripheral nervous systems, manifesting as weight loss, ataxia,
and skeletal muscle weakness37,3.
Several mechanisms are involved in ACR-induced neurotoxicity,
including the induction of oxidative stress38,39 the apoptosis pathway40,41
and modulation of neurotransmitter release.
Previous studies have shown that ACR exposure caused significantly
decreased cell viability and increased ROS production in neutrally associated cell
lines such as PC12, SHSY5Y, U-1240MG, U-87MG and U-251MG cells42,43,44,45.
ACR induces apoptosis in neurons and astrocytes in a time- and dose-dependent manner
in PC12 cells41,42,43 and through activation of caspase 3 in human
neuroblastoma cells (SHSY5Y)40. In our study, ACR (5.46 mM) decreased
cell viability in U-87MG cells and pretreatment with sinapic acid (5, 50, 100, 150,
200, 250 and 300 μg/ml) increased the cell viability.
Antioxidant enzymes, such as SOD, CAT, GSH, GR & GPX
are considered to be the first line of cellular defence against oxidative injury46.
SOD acts as a primary defence against superoxide anion and prevents further generation
of free radicals47. Reduction in SOD activity in ACR exposed animals
reflects enhanced production of superoxide radical anions3. Results showed
that exposure to ACR markedly declined GSH contents while enhanced MDA levels in
SN. SA administration dose dependently reduced lipid peroxidation through enhancement
of GSH content in SN. GSH plays an important role in maintaining cell integrity
against exogenous and endogenous derived ROS48. Previous studies indicated
that ACR is detoxified by conjugation with GSH. Gycidamide, as a metabolite of ACR,
could also conjugate with GSH38. Our study clearly exhibited that exposure
to ACR markedly decreased GSH content in SN. Massive intracellular calcium accumulation
has been implicated to play a pivotal role in neuronal and tissue injury49,50.
Moreover, it has also been documented as a key player in various types of neuropathic
disorders such as post-trauma, axotomy, anti-HIV drugs, tibial and sural nerve transection,
chronic constriction injury, ischemia–reperfusion injury and vincristine induced
neuropathy51. ACR treatment in our study produced a rise in the levels
of calcium and superoxide anion concentrations, these observations support the contention
that enhanced Ca2+ ions and free radicals together
play a key role in ACR induced painful neuropathy. Recent evidence has implicated Ca2+ dysregulation and
excessive production of oxidative stress and the role of TRP channels in the development
of neuropathic pain in diabetes39. Although speculative, similar mechanism/s
may be operative in the ACR-model of neuropathy. Since treatment with SA significantly
restored the ACR-induced Ca2+
levels in SN, we hypothesize that this
specific effect may be largely responsible for the observed improvement in behavioral
tests. An imbalance between the production and removal of free radicals and reactive
oxygen species (ROS) increases oxidative stress that is implicated in pathogenesis
of some neurodegenerative diseases52,53. According to recent studies,
the potential role of oxidative stress has been proven in the ACR induced pathogenesis
in both in- vitro and in -vivo models40,52.
Because of crucial role of oxidative stress in ACR induced
cytotoxicity, it is suggested that antioxidants could be considered as an alternative
approach in modulation of ACR toxicity40. Numerous researches indicated
the antioxidant effect of SA in different conditions. SA attenuated lipid peroxidation
in liver and laryngeal carcinoma cell lines54 in apoptosis induced oxidative
damage in rats. Sinapic acid
mitigates gentamicin-induced nephrotoxicity and associated oxidative/nitrosative
stress, apoptosis, and inflammation in rats by down regulation of NF-kB55.
It has protective role in arsenic induced toxicity56. SA significantly
attenuated kainic acid induced hippocampal cell death through its GABAA receptor
activation induced anticonvulsive and free radical scavenging activities57.
It also possess neuroprotective activity in a mouse model of amyloid β (Aβ)1-42
protein induced Alzheimers disease58. In support of this, in the present
study, SOD, GSH levels were decreased and MDA, calcium levels were increased after
ACR in rats which were significantly attenuated with SA treatment indicating the
antioxidant role of SA in alleviation of ACR induced neuropathic pain. It
can be concluded that SA exhibits potential antioxidant action in sciatic nerve
and has a significant scavenging action towards biologically generated free radicals
which would contribute to the anti-hyperalgesic action of SA, and may contribute
towards the therapeutic benefit in the management neuropathic pain.
ACKNOWLEDGMENTS:
The
authors are grateful to the management of Chebrolu Hanumaiah Institute of Pharmaceutical
Sciences for extending their support and providing facilities to complete this work.
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