Original Paper Pharmacology 2016;97:151–160 DOI: 10.1159/000443896
Received: September 22, 2015 Accepted after revision: January 7, 2016 Published online: January 30, 2016
Neuroprotective Activity of Curcumin in Combination with Piperine against Quinolinic Acid Induced Neurodegeneration in Rats Shamsher Singh a, b Puneet Kumar a
b
Department of Pharmacology, I.S.F. College of Pharmacy, Ferozepur Road, Ghal Kalan, Moga, and Research Scholar, Punjab Technical University, Jalandhar, India
Key Words Quinolinic acid · Curcumin · Piperine · Huntington’s disease · Excitotoxicity · Oxidative stress
Abstract Aim: Quinolinic acid (QA) is an excitotoxin that induces Huntington’s-like symptoms in animals and humans. Curcumin (CMN) is a well-known antioxidant but the major problem is its bioavailability. Therefore, the present study was designed to investigate the effect of CMN in the presence of piperine against QA-induced excitotoxic cell death in rats. Material and Methods: QA was administered intrastriatally at a dose of 200 nmol/2 μl saline, bilaterally. CMN (25 and 50 mg/kg/ day, p.o.) and combination of CMN (25 mg/kg/day, p.o.) and with piperine (2.5 mg/kg/day, p.o.) was administered daily for the next 21 days. Body weight and behavioral parameters were observed on 1st, 7th, 14th and 21st day. On the 22nd day, animals were sacrificed and striatum was isolated for biochemical (LPO, nitrite and GSH), neuroinflammatory (interleukin (IL)-1β, IL-6 and TNF-α) and neurochemical (dopamine, norepinephrine, GABA, glutamate, 5-HT, 3,4-dihydroxyphenylacetic acid and homovanillic acid) estimation. Results: CMN treatment showed beneficial effect against QA-induced motor deficit, biochemical and neurochemical abnormalities in rats. Combination of piperine (2.5 mg/kg/ day, p.o.) with CMN (25 mg/kg/day, p.o.) significantly en-
© 2016 S. Karger AG, Basel 0031–7012/16/0974–0151$39.50/0 E-Mail [email protected] www.karger.com/pha
hanced its protective effect as compared to treatment with CMN alone. Conclusion: This study has revealed that the combination of CMN and piperine showed strong antioxidant and protective effect against QA-induced behavioral and neurological alteration in rats. © 2016 S. Karger AG, Basel
Introduction
Quinolinic acid (QA) is a neurotoxic metabolite of the kynurenine pathway that, by acting as a competitive agonist on NMDA receptors, increases Ca2+ influx, excitotoxicity and neuronal cell death [1]. However, in the pathological condition, QA exerts Ca2+-mediated excessive excitation leads to mitochondrial dysfunction, decreased ATP levels and causes selective loss of GABAergic and cholinergic neurons responsible for Huntington’s-like symptoms [2]. The mechanism of QA-induced toxicity is considered very complex. Moreover, QA decreases oxidative defence by targeting number of pathways, increase level of inflammatory markers and alterations in neurochemical level in the striatum, which are similar with Huntington’s patients. Huntington’s disease (HD) is an autosomal dominant progressive neurodegenerative disorder initiated with excessive loss of medium spiny neurons in the striatum, reDr. Puneet Kumar, MPharm, PDCR, PhD Associate Professor, Department of Pharmacology I.S.F. College of Pharmacy Moga, Punjab 142001 (India) E-Mail punnubansal79 @ gmail.com
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a
Table 1. Effect of CMN and its combination with piperine on QA induced alteration in rotarod activity (fall off time)
Treatment, mg/kg
1st day
7th day
14th day
21st day
Control QA QA + CMN (25) QA + CMN (50) QA + piperine (2.5) + CMN (25)
172.8±2.8 172.2±3.6 172.2±4.1 173.3±2.0 172.2±3.2
171.2±9.6 127.3±12.4a 109.3±7.8b 131.6±8.4b, c 145.6±7.5b–d
171.4±8.2 74.5±9.7a 108±15.4b 124.6±13.3b, c 143.5±16.4b–d
170.8±8.0 60.6±9.8a 87.8±9.3b 123.3±8.8b, c 139.5±14.4b–d
Data expressed as mean ± SEM. a p < 0.01 vs. control, b p < 0.05 vs. QA, c p < 0.05 vs. QA + CMN (25), d p < 0.05 vs. QA + CMN (25) and QA + CMN (50).
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mechanism of CMN in the presence of piperine against QA-induced behavioral, biochemical, neuroinflammatory and neurochemical alterations.
Results
Effect of CMN on QA-Induced Decrease in Body Weight of Rats Rats treated with QA showed significant decrease in body weight on the last day, that is, the 21st day (–25% age change in body weight), as compared to normal control group (20% age change in body weight). CMN (25 and 50 mg/kg/day, p.o.) significantly restored (–10%) the body weight as compared to the QA treated groups. However, co-administration of piperine (2.5 mg/kg/ day, p.o.) with CMN (25 mg/kg/day, p.o.) showed significant improvement (–5%) in the body weight when compared to treatment with CMN alone (data not shown). Effect of CMN on QA-Induced Changes in Locomotor Activity, Rotarod and Grip Strength Performance of Rats There was significant decrease in grip strength, locomotor activity, rotarod activity (day 7th, 14th and 21st) with consistent improvement in transfer latency and foot error on narrow beam runway (21st day) after QA administration compared to normal control group. CMN (25 and 50 mg/kg/day, p.o.) treatment significantly ameliorated the impairment in grip strength, locomotor activity, rotarod activity, transfer latency and foot error compared to QA treated group. However, co-administration of piperine (2.5 mg/kg/day, p.o.) with CMN (25 mg/kg/day, p.o.) synergistically attenuated the behavioral abnormalities compared to treatment with CMN alone (tables 1, 2 and fig. 1, 2a, b). Singh/Kumar
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sulting in progressive cognitive impairment, neuropsychiatric symptoms and involuntary choreiform movements [3]. The GABAergic medium spiny neurons in HD are more vulnerable and their degeneration tends to alter the level of dopamine (DA) responsible for hyperkinetic movement. QA act by decreasing GABAergic neuron activity and its influence over glutaminergic discharge, promote excitotoxicity, thereby increases formation of reactive oxygen species (ROS) [4]. The administration of QA to rodents and non-human primates provides a good correlation of the various behavioral, biochemical and cellular parameters that are altered significantly in HD patients. On the other hand, antioxidants are effective therapeutic agents that help in slowing disease progression in transgenic mouse models of HD and show promise in human clinical trials [5]. Curcumin (CMN) a well-known natural (antioxidant) phenolic curcuminoid present in turmeric with reported wide range of biological activities including antihyperglycemic, anti-inflammatory, antioxidant and neuroprotective both in animals and humans [6]. CMN is neuroprotective in multiple animal models and have great potential for the prevention or treatment of neurological disorders like HD and Parkinson’s disease [7]. The major drawback with CMN is its very less oral bioavailability, because on oral administration in rats, 75% is excreted in feces and only a minute concentration appears in blood [8, 9]. Recently, numerous approaches have been undertaken to improve the bioavailability of CMN including the use of a glucuronidase inhibitor (e.g., piperine). Piperine is a major alkaloid obtained from black pepper. It is a powerful inhibitor of hepatic and intestinal glucuronidation, which also induces enzymes and increases the bioavailability of many drugs like CMN [10]. Based on this background, the present study has been designed with the aim of elucidating the neuroprotective
Table 2. Effect of CMN and its combination with piperine on QA induced alteration in locomotor activity
Treatment, mg/kg
1st day
7th day
14th day
21st day
Control QA QA + CMN (25) QA + CMN (50) QA + piperine (2.5) + CMN (25)
102.8±5.9 103.3±9.6 103.8±7.8 101.8±9.6 103.6±8.3
103±12 78.2±10.9a 81.6±6.2 85.6±8.9 90±10.9b
101.7±17.2 55.5±8.1a 62.5±10.2 71.2±8.1b 88.5±14.4b, c
103.2±13.5 30.2±7.5a 51.7±8.7b 67.7±6.9b, c 93.7±11.4b–d
Data expressed as mean ± SEM. a p < 0.01 vs. control, b p < 0.05 vs. QA, c p < 0.05 vs. QA + CMN (25), d p < 0.05 vs. QA + CMN (25) and QA + CMN (50).
1.6
Control
QA (200 nmol/2 μl)
QA + CMN (25)
QA + CMN (50)
QA + PP (2.5) + CMN (25)
Grip strength (KgF)
1.2 b, c, d b, c b
0.8 a 0.4
Fig. 1. Effect of CMN on QA induced changes in grip strength in rats: data expressed as mean ± SEM. One-way ANOVA with a Tukey’s post hoc test. a p < 0.01 vs. control, b p < 0.05 vs. QA, c p < 0.05 vs. CMN 25, d p < 0.05 vs. CMN 50.
0
21 days
Narrow beam walking
Transfer latency
Control QA + CMN (25) QA + CMN (50)
a
QA + PP (2.5) + CMN (25) b
15
b, c
10
b, c, d
5
a
0
21 days
a Time taken to cross narrow beam (s)
Number of slips
20
25
QA (200 nmol/2 μl)
25
b
20
b b, c
15 b, c, d 10
5
0
21 days
Fig. 2. a, b Effect of CMN on QA induced changes in narrow beam walking in rats: data expressed as mean ±
Neuroprotective Activity of CMN with Piperine
Pharmacology 2016;97:151–160 DOI: 10.1159/000443896
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SEM. One-way ANOVA with a Tukey’s post hoc test. a p < 0.01 vs. control, b p < 0.05 vs. QA, c p < 0.05 vs. CMN 25, d p < 0.05 vs. CMN 50.
Table 3. Effect of CMN and its combination with piperine on QA induced alteration in lipid peroxidation, nitrite and reduced GSH
Treatment, mg/kg
MDA, nmol/mg protein (percentage of control)
Nitrite level, μg/ml protein
GSH, nmol GSH/μg protein
Control QA QA + CMN (25) QA + CMN (50) QA + piperine (2.5) + CMN (25)
100±0.46 192±4.61a 156±5.73b 132±6.21b, c 118±3.42b–d
130±12.5 285±14.7 230±15.8b 205±13.9b, c 180±12.4b–d
0.095±4.3 0.032±5.3a 0.048±3.87b 0.067±5.27b, c 0.084±6.17b–d
Data expressed as mean ± SEM. a p < 0.01 vs. control, b p < 0.05 vs. QA, c p < 0.05 vs. QA + CMN (25), d p < 0.05 vs. QA + CMN (25) and QA + CMN (50).
200
a
QA + CMN (50)
QA + PP (2.5) + CMN (25)
QA + CMN (25) a
b
b b, c
a b, c, d
b, c b
b, c, d b, c
100
b, c, d
50
0
Effect of CMN on TNF-α, IL-1β and IL-6 Levels in QA Treated Rats Tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β and IL-6 are important pro-inflammatory markers in HD and other neurodegenerative disorders. In the Pharmacology 2016;97:151–160 DOI: 10.1159/000443896
QA (200 nmol/2 μl)
150
Effect of CMN on Lipid Peroxidation, Nitrite and Glutathione Levels in Rats Administered with QA Systemic administration of QA significantly increased MDA, nitrite levels and decrease in glutathione level in the striatum compared to the normal control group. CMN pre-treatment (25 and 50 mg/kg/day, p.o.) significantly restored the altered levels of oxido-nitrosative stress in QA administered rats as compared to the QA treated group. In addition, co-administration of piperine (2.5 mg/kg/day, p.o.) with CMN (25 mg/kg/day, p.o.) has showed significant decrease in oxido-nitrosative stress compared to treatment with CMN alone (table 3).
154
Control
TNF-į
IL-1DŽ
IL-6
present study, systemic administration of QA significantly increased the TNF-α, IL-1β and IL-6 levels in brain striatum compared to normal control group. The elevated levels of TNF-α, IL-1β and IL-6 were significantly prevented with CMN (25 and 50 mg/kg/day, p.o.) compared to treatment with QA alone. In addition, co-administration of piperine (2.5 mg/kg/day, p.o.) with CMN (25 mg/ kg/day, p.o.) significantly decreased TNF-α, IL-1β and IL-6 levels compared to treatment with CMN alone (fig. 3). Effect of CMN on Catecholamines Levels in the Striatum in QA Treated Rats In the brain, DA is metabolized enzymatically by monoamine oxidase (MAO)-B with intermediate 3, 4-dihydroxyphenylacetic acid (DOPAC), which is further metabolized to homovanillic acid (HVA). The catecholamine levels (nor-epinephrine, DA and serotonin) in striSingh/Kumar
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Fig. 3. Effect of CMN on TNF-α and IL-1β levels in QA treated rats: data expressed as mean ± SEM. One-way ANOVA with a Tukey’s post hoc test. a p < 0.01 vs. control, b p < 0.05 vs. QA, c p < 0.05 vs. CMN 25, d p < 0.05 vs. CMN 50.
Neuroinflammatory markers (pg/ml)
250
Catecholamines level (striatum) 120
Fig. 4. Effect of CMN on brain catechol-
Tissue sample (ng/mg)
100
3-NP (10)
CMN (25) + 3-NP
CMN (50) + 3-NP
PP (2.5) + CMN (25) + 3-NP
80 b, c, d
60 b, c 40 20
amines levels: data expressed as mean ± SEM. One-way ANOVA with a Tukey’s post hoc test. a p < 0.01 vs. control, b p < 0.05 vs. QA, c p < 0.05 vs. CMN 25, d p < 0.05 vs. CMN 50.
Control
b
a
0
b, c
b, c, d
b a a
NE
Dopamine
b
b, c
b, c, d
Serotonin
Catecholamines metabolites (striatum) 25
Fig. 5. Effect of CMN on brain catecholamines metabolite level: data expressed as mean ± SEM. One-way ANOVA with a Tukey’s post hoc test. a p < 0.01 vs. control, b p < 0.05 vs. QA, c p < 0.05 vs. CMN 25, d p < 0.05 vs. CMN 50.
Control
QA (200 nmol/2 μl)
QA + CMN (50)
QA + PP (2.5) + CMN (25)
b
15
QA + CMN (25)
a b
b, c
b, c b, c, d
b, c, d
10
5
0
DOPAC
HVA
atum were found to be significantly reduced with QA treatment. Pre-treatment with CMN (25 and 50 mg/kg/ day, p.o.) and co-administration of piperine (2.5 mg/kg/ day, p.o.) with CMN (25 mg/kg/day, p.o.) significantly restored the catecholamine levels in striatum compared to treatment with CMN alone. Further treatment with QA results in significant increase in DOPAC and HVA levels in striatum. Rats pre-treated with CMN (25 and 50 mg/kg/day, p.o.) showed significant attenuation in the increased levels of DOPAC and HVA when compared to treatment with QA alone. However, co-administration of piperine (2.5 mg/kg/day, p.o.) and CMN (25 mg/kg/day,
p.o.) significantly prevented the increase in DOPAC and HVA levels in striatum compared to treatment with CMN alone (fig. 4 and 5).
Neuroprotective Activity of CMN with Piperine
Pharmacology 2016;97:151–160 DOI: 10.1159/000443896
Effect of CMN on Striatal GABA, Glutamate and Adenosine Levels The treatment with QA alone had significantly lower GABA, adenosine levels and high glutamate when compared to the normal control group. Pre-treatment with CMN (25 and 50 mg/kg/day, p.o.) significantly and dose dependently prevented the alteration in GABA, glutamate and adenosine level in striatum. Moreover, co-ad155
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Tissue sample (ng/mg)
20
a
Fig. 6. Effect of CMN on striatal GABA,
Tissue sample (ng/mg)
200
glutamate and adenosine levels in QA treated rats: data expressed as mean ± SEM. One-way ANOVA with a Tukey’s post hoc test. a p < 0.01 vs. control, b p < 0.05 vs. QA, c p < 0.05 vs. CMN 25, d p < 0.05 vs. CMN 50.
a
150
Control
QA (200 nmol/2 μl)
QA + CMN (25)
QA + CMN (50)
QA + PP (2.5) + CMN (25)
b b, c b, c, d
100
b, c, d
50
b, c
a
b
b, c
b, c, d
a 0
Glutamate
GABA
Adenosine
Intrastriatal administration of Quinolinic acid Excess Ca2+ discharge by glutamate receptor
Neuronal firing and death
Increase IL-1, IL-6, TNF-į 1)NJ%
Increase ROS and RNS
Curcumin
Mitochondrial dysfunction
flammatory mechanism prevent QA induced neurodegeneration.
ministration of piperine (2.5 mg/kg/day, p.o.) and CMN (25 mg/kg/day, p.o.) in combination significantly prevented fluctuation in GABA, glutamate and adenosine levels compared to treatment with CMN alone (fig. 6).
Discussion
The present study reflects strong evidences that QA through excess Ca2+ discharge induces excitotoxicity dependent neuronal and glial cell death [11]. Intrastriatal administration of QA causes loss of medium spiny neurons in the basal ganglia that are responsible for motor and cognitive abnormalities indicated by impairment in 156
Pharmacology 2016;97:151–160 DOI: 10.1159/000443896
Huntington disease
locomotor activity, rotarod performance, grip strength and narrow beam walk performance in rats [12]. QA exerts neuropathologic changes through lipid peroxidation by acting as a pro-oxidant in concordance with the present study. Also, QA administration depleted antioxidant enzyme levels (glutathione) and raised oxidants (lipid peroxidation, nitrite level) suggesting the role of ROS in neurodegeneration (fig. 7) [13]. Interruption of ROS/ RNS levels by QA further activates microglia/macrophages, which promote the COX-2 dependent synthesis of various pro-inflammatory cytokines like IL-1β, IL-6 and TNF-α resembles the postpartum reports of HD patients [14]. Similarly, increased ROS and lipid peroxidation promote DNA-dependent transcription of inflamSingh/Kumar
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Fig. 7. CMN by antioxidant and anti-in-
matory cytokines and activation of nuclear factor of kappa B (NF-ĸB). This further triggers caspase-dependent cleavage of HD proteins, accumulation of insoluble oligomeric fragment-dependent striatal neuron damage responsible for all HD-like complications in concordance with our study. In the present study, QA-dependent loss of dopaminergic neurons showed significant reduction in motor function (impaired locomotor, rotarod, grip strength and narrow beam performance), oxidative damage (as evidenced by increase LPO, nitrite and depletion of glutathione level), increased IL-1β, IL-6 and TNF-α compared to normal control group. However, treatment with CMN at low dose (25 mg/kg, p.o.) and high dose (50 mg/kg, p.o.) results in remarkable improvement in locomotor activity, rotarod performance, narrow beam walk performance, decrease oxidative defence and attenuated IL-1β, IL-6 and TNF-α levels as compared to QA treated animals. Furthermore, co-administration of piperine (2.5 mg/kg, p.o.) and CMN (25 mg/kg, p.o.) showed more significant results than treatment with CMN alone. The previous reports suggest that QA on intrastriatal administration causes excitotoxicity, oxidative stress, activation of NF-ĸB and p53-induced neurodegeneration. Moreover, the damage in striatal neuron leads to lowering of catecholamine levels (norepinephrine (NE), DA and serotonin), 5-HIAA and increased the level of DOPAC and HVA in the striatum nuclei [15]. Striatal area mainly receives GABAergic neuronal projections and is the most vulnerable in HD; that is why the dysfunction of these neurons is responsible for the development of chorea [16]. The decrease in catecholamine level is due to the severe degeneration of nigrostriatal neurons, causing motor impairment as supported by present behavioral studies. Adenosine is a purine nucleoside that behaves as a typical neuromodulator in all neurons by inhibiting neurodegeneration through A1 and A2A G-protein-coupled receptors. Protection against neuronal damage is a major objective of current research in areas such as HD. The facilitation of A1 and blockade of A2A receptor decrease Ca2+-dependent glutamate release, lower the excitability of spinal neurons and helps to recover motor abnormalities in animals and humans [17]. In the present study, QA administration significantly decreased the level of catecholamines (NE, DA and serotonin) and adenosine with increase in the levels of DOPAC and HVA in striatal nuclei. Moreover, pre-treatment with CMN at 25 and 50 mg/kg, postoperatively, dose dependently attenuated the level of catecholamines and adenosine. Furthermore, the co-administration of piperine (2.5 mg/kg, p.o.) in combination with CMN (25 mg/kg,
p.o.) had showed significant protection by restoring the catecholamine levels compared with treatment by CMN alone. CMN is a potent antioxidant with reported neuroprotective and anti-inflammatory activity due to its inhibitory effect over COX-2, LOX-5, TNF-α, NF-ĸB, iNOS and P13K/AKT [15]. But the major complication is its very low oral bioavailability; so when piperine (bioavailability enhancer) is co-administered with CMN, it promotes its therapeutic effect. It is well reported that CMN at the dose of 1.0 g/kg, postoperatively, shows low plasma levels of 0.13 μg/ml after 15 min, while a maximum plasma level of 0.22 μg/ml was obtained at 1 h [18]. However, co-administration of piperine with CMN also inhibits MAO-A and MAO-B, thus enhanced the level of biological amines in stratum [19]. Piperine is a powerful inhibitor of hepatic and intestinal glucuronidation and increases the bioavailability of CMN in humans and rats [20]. In humans, piperine co-administration with CMN increases bioavailability by 2,000% at 45 min [21]. The present study thus illustrates that the co-administration of piperine (2.5 mg/kg, p.o.) and CMN (25 mg/kg, p.o.) synergistically showed significant improvement in QA induced loss of motor deficit abnormalities and oxidative stress. The present observations are supported by results configured from the whole study and also by the previous finding that CMN by targeting multifunctional pathways acts as potent antioxidant, inhibits COX and decreases the levels of various inflammatory cytokines. However, by inhibiting xanthine oxidase and by slowing ROS production, CMN further prevents neurodegeneration along with improvement in the level of catecholamines and purines in striatum. In conclusion, QA induced HD symptoms are confirmed by behavioral, biochemical, neuroinflammatory and neurochemical studies. Due to its well-established antioxidant and anti-inflammatory properties, CMN has the ability to prevent neurodegenerative changes seen in HD. Based on these above-detailed findings, CMN could be a promising treatment for neurodegenerative disorders like HD. To the best of my knowledge, CMN and piperine may act as a useful combination for the treatment of HD and other neurological disorders.
Neuroprotective Activity of CMN with Piperine
Pharmacology 2016;97:151–160 DOI: 10.1159/000443896
Material and Methods
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Experimental Animals The experiment was carried out on adult Wistar rats aged 4–5 months (250–280 g) that were obtained from central animal house of I.S.F. College of Pharmacy, Moga, Punjab (India). The animals
Drugs and Chemicals QA and piperine (Sigma Aldrich, St. Louis, Mo., USA), CMN (Himedia, Vadhani Industrial Estate, L.B.S. Marg, Mumbai, India) and IL-1β, IL-6 and TNF-α ELISA kits (Krishgen BioSystem, Mumbai, India) and, unless stated, all other chemicals and biochemical reagents of highest analytical grade were used in the study. The experimental protocol was divided into 5 groups and each treatment group consisted of 9 animals (total no of animals, i.e., n = 45). Experimental Procedure and Drug Treatment Animals were anesthetized with intraperitoneal administration of ketamine (80 mg/kg) and diazepam (5 mg/kg), and the surface of the skull was exposed by making an incision on the scalp. A small hole (1–2 mm diameter) was made on both sides of the skull at anterior +1.7 mm, lateral ±2.7 mm, ventral –4.8 mm from bregma as described by Paxinos and Watson, 2007. QA (200 nmol/2 μl saline) was administered bilaterally over a period of 2 min in the striatum using digital stereotaxic apparatus (Stoelting, Wood Dale, and USA). The injection needle was left in place for another 2 min to prevent back diffusion of the injected drug solution. QA was dissolved in normal saline (pH 7.4) and administered intrastriatally (i.s.) once at a dose of 200 nmol/2 μl. CMN was dissolved in sodium carboxymethyl cellulose and administered at a dose of 25 and 50 mg/kg and also combination of CMN (25 mg/kg) with piperine (2.5 mg/kg) daily for 21 days starting from the 7th day after QA administration. All the behavioral parameters like grip strength, narrow beam, rotarod and locomotor activity were assessed on days 1, 7, 14 and 21. Terminally, on day 22, animals were sacrificed and striatum was separated, homogenized, centrifuged and clear supernatant was used to estimate biochemical parameters (LPO, nitrite, reduced GSH levels) and neurochemical analysis (DA, NE, serotonin, DOPAC, HVA, adenosine). The levels of proinflammatory cytokines (IL-1β, IL-6 and TNF-α) were estimated using ELISA kits. The experimental procedure is summarized in table 3. Behavioral Assessments Measurement of Body Weight The body weight of animals was measured on the first (1st day) and last day (21st day) of study. The percentage change in body weight was as follows: Body weight (1st day – 21st day)/1st day body weight × 100. Assessment of Gross Behavioral Parameters (Locomotor, Rotarod, Grip Strength and Narrow Beam Walk Performance) Open field test is used to monitor spontaneous locomotor activity (crossing field) using a wooden, rectangular, light brown colored open field apparatus measuring 100 × 100 × 40 cm [22]. The motor coordination and integrity with rotarod test for all animals
158
Pharmacology 2016;97:151–160 DOI: 10.1159/000443896
were evaluated using weekly intervals. The rotarod apparatus comprises having a diameter of 7 cm (speed 25 rpm). The cutoff time was 180 s and the average time of the fall was recorded [23]. On the grip strength apparatus, the fore limbs grip was measured using digital grip force meter (DFIS series, Chatillon, Greensboro, N.C., USA). The grip strength was recorded in kilogram force [23]. During narrow beam walk task, the animals are required to walk on across a narrow wooden beam, measuring its motor coordination ability. Number of slips and time taken to cross in each trial were recorded [23]. Dissection and Homogenization On the 22nd day, all the animals were randomly divided into 3 groups: first group for biochemical estimations, second for neuroinflammatory markers and third for neurochemicals. The animals were sacrificed by decapitation immediately, that is, 24 h after behavioral assessments. Brains were put on the ice; the cortex and striatum regions were separated and weighed. A 10% (w/v) tissue homogenate was prepared in 0.1 mol/l phosphate buffer (pH 7.4). Homogenate was centrifuged for 15 min at 15,000 rpm, and the supernatant was stored at 80 ° C for assessing the biochemical parameters.
Measurement of Oxidative Stress Parameters Measurement of Lipid Peroxidation, Nitrite, Reduced Glutathione and Proteins The quantitative measurement of lipid peroxidation in the brain striatum was performed according to the method of Wills [24], using nitrite by Green et al. [25], using reduced glutathione as described by Ellman [26] and protein by the Lowry method using Folin phenol reagent [27]. Estimation of TNF-α, IL-1β and IL-6 in Striatum The levels of IL-1β, IL-6 and TNF-α were quantified by using rat IL-1β, IL-6 and TNF-α immunoassay kit (Krishgen BioSystem, USA). Concentrations of TNF-α were calculated from the plotted standard curves. Neurochemical Analysis Estimation of Brain Catecholamines by HPLC-ECD The brain catecholamine levels were estimated using the method described by Patel with slight modifications [28]. Catecholamines (DA, serotonin and NE) and their metabolites (DOPAC, HVA) levels in striatum were estimated by HPLC using electrochemical detector and C18 reverse phase column. Mobile phase consisted of sodium citrate buffer (pH 4.5) – acetonitrile (87: 13, v/v). The sodium citrate buffer consisted of 10 mmol/l citric acid, 25 mmol/l NaH2HPO4, 25 mmol/l EDTA and 2 mmol/l of 1-heptane sulfonic acid. The electrochemical conditions for the experiment were +0.75 V, with sensitivity ranging from 5 to 50 nA. Separation was carried out at a flow rate of 0.8 ml/min. Samples (20 μl) were injected manually. On the day of the experiment, the brain samples were homogenized in solution containing 0.2 mol/l perchloric acid. Then the samples were centrifuged at 12,000 g for 5 min. The supernatant was filtered through 0.22 mm nylon filters before being injected into the HPLC sample injector. Data were recorded and analyzed with breeze software. The concentrations of the neurotransmitters and their metabolites were calculated from the standard curve generated by using standard with concentration 10–100 mg/ml.
Singh/Kumar
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were housed under standard laboratory conditions (room temperature 22 ± 1 ° C and relative humidity of 60%) with 12 h light/dark cycle. The food and water were made available ad libitum. All the behavioral assessments were carried out between 9:00 and 17:00 h. The experimental protocol was approved by the Institutional Animal Ethics Committee, and experiments were carried out in accordance with the guidelines of the Indian National Science Academy for the use and care of experimental animals.
Brain GABA and Glutamate Estimation by HPLC-ECD The GABA and glutamate levels were estimated by the method described by Lashley with slight modifications [29]. GABA and Glutamate were estimated by HPLC by using mobile phase comprised of 100 mmol/l disodium hydrogen phosphate anhydrous, 25 mmol/l EDTA and 22% methanol. Electrochemical conditions for the experiment were +0.65 V, sensitivity ranges from 5 to 50 nA. The separation for the GABA and glutamate and data were recorded by similar method as discus above in catecholamine by using standard in a concentration range of 10–100 ng/ml.
Statistical Analysis The data obtained are expressed as mean ± SEM. All the data were analysed using one-way ANOVA was followed by Tukey’s post hoc test. Values of p < 0.05 were considered statistically significant. A p value of 0.05 was considered statistically significant.
Pre-Column Derivatization Procedure Amino acids were measured as (OPA/β-ME) derivatives according to the method of Wang et al. [30].
Authors are thankful to Science and Engineering Board (SERB), Department of Science and Technology, Government of India, New Delhi for providing financial assistance under Fast Track Scheme (DST-SERB-FTYS) to Dr. P. Kumar.
Brain Purines Estimation by HPLC-Photodiode Array The purine (adenosine) level was estimated by HPLC using a PDA detector and C18 reversed phase column. The mobile phase consisted of water, methanol and acetonitrile (88:5:7, v/v). Separation and data recording were carried out similarly as discussed above in catecholamines by using standard in the concentration range of 10–100 ng/ml [31].
Acknowledgments
Disclosure Statement The authors declare that they have no conflict of interest.
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Neuroprotective Activity of CMN with Piperine
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14 Kalonia H, Mishra J, Kumar A: Targeting neuro-inflammatory cytokines and oxidative stress by minocycline attenuates quinolinicacid-induced Huntington’s disease-like symptoms in rats. Neurotox Res 2012;22:310–320. 15 Bishnoi M, Chopra K, Rongzhu L, Kulkarni SK: Protective effect of curcumin and its combination with piperine (bioavailability enhancer) against haloperidol-associated neurotoxicity: cellular and neurochemical evidence. Neurotox Res 2011; 20: 215–225. 16 Reilmann R: Pharmacological treatment of chorea in Huntington’s disease-good clinical practice versus evidence-based guideline. Mov Disord 2013;28:1030–1033. 17 Chen JF, Sonsalla PK, Pedata F, Melani A, Domenici MR, Popoli P, Geiger J, Lopes LV, de Mendonça A: Adenosine A2A receptors and brain injury: broad spectrum of neuroprotection, multifaceted actions and ‘fine tuning’ modulation. Prog Neurobiol 2007; 83: 310– 331. 18 Subhashini, Chauhan PS, Kumari S, Kumar JP, Chawla R, Dash D, Singh M, Singh R: Intranasal curcumin and its evaluation in murine model of asthma. Int Immunopharmacol 2013;17:733–743. 19 Kulkarni SK, Bhutani MK, Bishnoi M: Antidepressant activity of curcumin: involvement of serotonin and dopamine system. Psychopharmacology (Berl) 2008;201:435–442. 20 Rinwa P, Kumar A, Garg S: Suppression of neuroinflammatory and apoptotic signaling cascade by curcumin alone and in combination with piperine in rat model of olfactory bulbectomy induced depression. PLoS One 2013;8:e61052.
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1 Leipnitz G, Schumacher C, Scussiato K, Dalcin KB, Wannmacher CM, Wyse AT, DutraFilho CS, Wajner M, Latini A: Quinolinic acid reduces the antioxidant defenses in cerebral cortex of young rats. Int J Dev Neurosci 2005; 23:695–701. 2 Kalonia H, Kumar P, Kumar A, Nehru B: Effect of caffeic acid and rofecoxib and their combination against intrastriatal quinolinic acid induced oxidative damage, mitochondrial and histological alterations in rats. Inflammopharmacology 2009;17:211–219. 3 Kumar P, Kalonia H, Kumar A: Possible GABAergic mechanism in the neuroprotective effect of gabapentin and lamotrigine against 3-nitropropionic acid induced neurotoxicity. Eur J Pharmacol 2012;674:265–274. 4 He S, Bausch SB: Synaptic plasticity in glutamatergic and GABAergic neurotransmission following chronic memantine treatment in an in vitro model of limbic epileptogenesis. Neuropharmacology 2014;77:379–386. 5 Acuña AI, Esparza M, Kramm C, Beltrán FA, Parra AV, Cepeda C, Toro CA, Vidal RL, Hetz C, Concha II, Brauchi S, Levine MS, Castro MA: A failure in energy metabolism and antioxidant uptake precede symptoms of Huntington’s disease in mice. Nat Commun 2013; 4:2917. 6 Tapia E, Soto V, Ortiz-Vega KM, ZarcoMárquez G, Molina-Jijón E, Cristóbal-García M, Santamaría J, García-Niño WR, Correa F, Zazueta C, Pedraza-Chaverri J: Curcumin induces Nrf2 nuclear translocation and prevents glomerular hypertension, hyperfiltration, oxidant stress, and the decrease in antioxidant enzymes in 5/6 nephrecto-
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Received: September 22, 2015 Accepted after revision: January 7, 2016 Published online: January 30, 2016
Neuroprotective Activity of Curcumin in Combination with Piperine against Quinolinic Acid Induced Neurodegeneration in Rats Shamsher Singh a, b Puneet Kumar a
b
Department of Pharmacology, I.S.F. College of Pharmacy, Ferozepur Road, Ghal Kalan, Moga, and Research Scholar, Punjab Technical University, Jalandhar, India
Key Words Quinolinic acid · Curcumin · Piperine · Huntington’s disease · Excitotoxicity · Oxidative stress
Abstract Aim: Quinolinic acid (QA) is an excitotoxin that induces Huntington’s-like symptoms in animals and humans. Curcumin (CMN) is a well-known antioxidant but the major problem is its bioavailability. Therefore, the present study was designed to investigate the effect of CMN in the presence of piperine against QA-induced excitotoxic cell death in rats. Material and Methods: QA was administered intrastriatally at a dose of 200 nmol/2 μl saline, bilaterally. CMN (25 and 50 mg/kg/ day, p.o.) and combination of CMN (25 mg/kg/day, p.o.) and with piperine (2.5 mg/kg/day, p.o.) was administered daily for the next 21 days. Body weight and behavioral parameters were observed on 1st, 7th, 14th and 21st day. On the 22nd day, animals were sacrificed and striatum was isolated for biochemical (LPO, nitrite and GSH), neuroinflammatory (interleukin (IL)-1β, IL-6 and TNF-α) and neurochemical (dopamine, norepinephrine, GABA, glutamate, 5-HT, 3,4-dihydroxyphenylacetic acid and homovanillic acid) estimation. Results: CMN treatment showed beneficial effect against QA-induced motor deficit, biochemical and neurochemical abnormalities in rats. Combination of piperine (2.5 mg/kg/ day, p.o.) with CMN (25 mg/kg/day, p.o.) significantly en-
© 2016 S. Karger AG, Basel 0031–7012/16/0974–0151$39.50/0 E-Mail [email protected] www.karger.com/pha
hanced its protective effect as compared to treatment with CMN alone. Conclusion: This study has revealed that the combination of CMN and piperine showed strong antioxidant and protective effect against QA-induced behavioral and neurological alteration in rats. © 2016 S. Karger AG, Basel
Introduction
Quinolinic acid (QA) is a neurotoxic metabolite of the kynurenine pathway that, by acting as a competitive agonist on NMDA receptors, increases Ca2+ influx, excitotoxicity and neuronal cell death [1]. However, in the pathological condition, QA exerts Ca2+-mediated excessive excitation leads to mitochondrial dysfunction, decreased ATP levels and causes selective loss of GABAergic and cholinergic neurons responsible for Huntington’s-like symptoms [2]. The mechanism of QA-induced toxicity is considered very complex. Moreover, QA decreases oxidative defence by targeting number of pathways, increase level of inflammatory markers and alterations in neurochemical level in the striatum, which are similar with Huntington’s patients. Huntington’s disease (HD) is an autosomal dominant progressive neurodegenerative disorder initiated with excessive loss of medium spiny neurons in the striatum, reDr. Puneet Kumar, MPharm, PDCR, PhD Associate Professor, Department of Pharmacology I.S.F. College of Pharmacy Moga, Punjab 142001 (India) E-Mail punnubansal79 @ gmail.com
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a
Table 1. Effect of CMN and its combination with piperine on QA induced alteration in rotarod activity (fall off time)
Treatment, mg/kg
1st day
7th day
14th day
21st day
Control QA QA + CMN (25) QA + CMN (50) QA + piperine (2.5) + CMN (25)
172.8±2.8 172.2±3.6 172.2±4.1 173.3±2.0 172.2±3.2
171.2±9.6 127.3±12.4a 109.3±7.8b 131.6±8.4b, c 145.6±7.5b–d
171.4±8.2 74.5±9.7a 108±15.4b 124.6±13.3b, c 143.5±16.4b–d
170.8±8.0 60.6±9.8a 87.8±9.3b 123.3±8.8b, c 139.5±14.4b–d
Data expressed as mean ± SEM. a p < 0.01 vs. control, b p < 0.05 vs. QA, c p < 0.05 vs. QA + CMN (25), d p < 0.05 vs. QA + CMN (25) and QA + CMN (50).
152
Pharmacology 2016;97:151–160 DOI: 10.1159/000443896
mechanism of CMN in the presence of piperine against QA-induced behavioral, biochemical, neuroinflammatory and neurochemical alterations.
Results
Effect of CMN on QA-Induced Decrease in Body Weight of Rats Rats treated with QA showed significant decrease in body weight on the last day, that is, the 21st day (–25% age change in body weight), as compared to normal control group (20% age change in body weight). CMN (25 and 50 mg/kg/day, p.o.) significantly restored (–10%) the body weight as compared to the QA treated groups. However, co-administration of piperine (2.5 mg/kg/ day, p.o.) with CMN (25 mg/kg/day, p.o.) showed significant improvement (–5%) in the body weight when compared to treatment with CMN alone (data not shown). Effect of CMN on QA-Induced Changes in Locomotor Activity, Rotarod and Grip Strength Performance of Rats There was significant decrease in grip strength, locomotor activity, rotarod activity (day 7th, 14th and 21st) with consistent improvement in transfer latency and foot error on narrow beam runway (21st day) after QA administration compared to normal control group. CMN (25 and 50 mg/kg/day, p.o.) treatment significantly ameliorated the impairment in grip strength, locomotor activity, rotarod activity, transfer latency and foot error compared to QA treated group. However, co-administration of piperine (2.5 mg/kg/day, p.o.) with CMN (25 mg/kg/day, p.o.) synergistically attenuated the behavioral abnormalities compared to treatment with CMN alone (tables 1, 2 and fig. 1, 2a, b). Singh/Kumar
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sulting in progressive cognitive impairment, neuropsychiatric symptoms and involuntary choreiform movements [3]. The GABAergic medium spiny neurons in HD are more vulnerable and their degeneration tends to alter the level of dopamine (DA) responsible for hyperkinetic movement. QA act by decreasing GABAergic neuron activity and its influence over glutaminergic discharge, promote excitotoxicity, thereby increases formation of reactive oxygen species (ROS) [4]. The administration of QA to rodents and non-human primates provides a good correlation of the various behavioral, biochemical and cellular parameters that are altered significantly in HD patients. On the other hand, antioxidants are effective therapeutic agents that help in slowing disease progression in transgenic mouse models of HD and show promise in human clinical trials [5]. Curcumin (CMN) a well-known natural (antioxidant) phenolic curcuminoid present in turmeric with reported wide range of biological activities including antihyperglycemic, anti-inflammatory, antioxidant and neuroprotective both in animals and humans [6]. CMN is neuroprotective in multiple animal models and have great potential for the prevention or treatment of neurological disorders like HD and Parkinson’s disease [7]. The major drawback with CMN is its very less oral bioavailability, because on oral administration in rats, 75% is excreted in feces and only a minute concentration appears in blood [8, 9]. Recently, numerous approaches have been undertaken to improve the bioavailability of CMN including the use of a glucuronidase inhibitor (e.g., piperine). Piperine is a major alkaloid obtained from black pepper. It is a powerful inhibitor of hepatic and intestinal glucuronidation, which also induces enzymes and increases the bioavailability of many drugs like CMN [10]. Based on this background, the present study has been designed with the aim of elucidating the neuroprotective
Table 2. Effect of CMN and its combination with piperine on QA induced alteration in locomotor activity
Treatment, mg/kg
1st day
7th day
14th day
21st day
Control QA QA + CMN (25) QA + CMN (50) QA + piperine (2.5) + CMN (25)
102.8±5.9 103.3±9.6 103.8±7.8 101.8±9.6 103.6±8.3
103±12 78.2±10.9a 81.6±6.2 85.6±8.9 90±10.9b
101.7±17.2 55.5±8.1a 62.5±10.2 71.2±8.1b 88.5±14.4b, c
103.2±13.5 30.2±7.5a 51.7±8.7b 67.7±6.9b, c 93.7±11.4b–d
Data expressed as mean ± SEM. a p < 0.01 vs. control, b p < 0.05 vs. QA, c p < 0.05 vs. QA + CMN (25), d p < 0.05 vs. QA + CMN (25) and QA + CMN (50).
1.6
Control
QA (200 nmol/2 μl)
QA + CMN (25)
QA + CMN (50)
QA + PP (2.5) + CMN (25)
Grip strength (KgF)
1.2 b, c, d b, c b
0.8 a 0.4
Fig. 1. Effect of CMN on QA induced changes in grip strength in rats: data expressed as mean ± SEM. One-way ANOVA with a Tukey’s post hoc test. a p < 0.01 vs. control, b p < 0.05 vs. QA, c p < 0.05 vs. CMN 25, d p < 0.05 vs. CMN 50.
0
21 days
Narrow beam walking
Transfer latency
Control QA + CMN (25) QA + CMN (50)
a
QA + PP (2.5) + CMN (25) b
15
b, c
10
b, c, d
5
a
0
21 days
a Time taken to cross narrow beam (s)
Number of slips
20
25
QA (200 nmol/2 μl)
25
b
20
b b, c
15 b, c, d 10
5
0
21 days
Fig. 2. a, b Effect of CMN on QA induced changes in narrow beam walking in rats: data expressed as mean ±
Neuroprotective Activity of CMN with Piperine
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SEM. One-way ANOVA with a Tukey’s post hoc test. a p < 0.01 vs. control, b p < 0.05 vs. QA, c p < 0.05 vs. CMN 25, d p < 0.05 vs. CMN 50.
Table 3. Effect of CMN and its combination with piperine on QA induced alteration in lipid peroxidation, nitrite and reduced GSH
Treatment, mg/kg
MDA, nmol/mg protein (percentage of control)
Nitrite level, μg/ml protein
GSH, nmol GSH/μg protein
Control QA QA + CMN (25) QA + CMN (50) QA + piperine (2.5) + CMN (25)
100±0.46 192±4.61a 156±5.73b 132±6.21b, c 118±3.42b–d
130±12.5 285±14.7 230±15.8b 205±13.9b, c 180±12.4b–d
0.095±4.3 0.032±5.3a 0.048±3.87b 0.067±5.27b, c 0.084±6.17b–d
Data expressed as mean ± SEM. a p < 0.01 vs. control, b p < 0.05 vs. QA, c p < 0.05 vs. QA + CMN (25), d p < 0.05 vs. QA + CMN (25) and QA + CMN (50).
200
a
QA + CMN (50)
QA + PP (2.5) + CMN (25)
QA + CMN (25) a
b
b b, c
a b, c, d
b, c b
b, c, d b, c
100
b, c, d
50
0
Effect of CMN on TNF-α, IL-1β and IL-6 Levels in QA Treated Rats Tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β and IL-6 are important pro-inflammatory markers in HD and other neurodegenerative disorders. In the Pharmacology 2016;97:151–160 DOI: 10.1159/000443896
QA (200 nmol/2 μl)
150
Effect of CMN on Lipid Peroxidation, Nitrite and Glutathione Levels in Rats Administered with QA Systemic administration of QA significantly increased MDA, nitrite levels and decrease in glutathione level in the striatum compared to the normal control group. CMN pre-treatment (25 and 50 mg/kg/day, p.o.) significantly restored the altered levels of oxido-nitrosative stress in QA administered rats as compared to the QA treated group. In addition, co-administration of piperine (2.5 mg/kg/day, p.o.) with CMN (25 mg/kg/day, p.o.) has showed significant decrease in oxido-nitrosative stress compared to treatment with CMN alone (table 3).
154
Control
TNF-į
IL-1DŽ
IL-6
present study, systemic administration of QA significantly increased the TNF-α, IL-1β and IL-6 levels in brain striatum compared to normal control group. The elevated levels of TNF-α, IL-1β and IL-6 were significantly prevented with CMN (25 and 50 mg/kg/day, p.o.) compared to treatment with QA alone. In addition, co-administration of piperine (2.5 mg/kg/day, p.o.) with CMN (25 mg/ kg/day, p.o.) significantly decreased TNF-α, IL-1β and IL-6 levels compared to treatment with CMN alone (fig. 3). Effect of CMN on Catecholamines Levels in the Striatum in QA Treated Rats In the brain, DA is metabolized enzymatically by monoamine oxidase (MAO)-B with intermediate 3, 4-dihydroxyphenylacetic acid (DOPAC), which is further metabolized to homovanillic acid (HVA). The catecholamine levels (nor-epinephrine, DA and serotonin) in striSingh/Kumar
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Fig. 3. Effect of CMN on TNF-α and IL-1β levels in QA treated rats: data expressed as mean ± SEM. One-way ANOVA with a Tukey’s post hoc test. a p < 0.01 vs. control, b p < 0.05 vs. QA, c p < 0.05 vs. CMN 25, d p < 0.05 vs. CMN 50.
Neuroinflammatory markers (pg/ml)
250
Catecholamines level (striatum) 120
Fig. 4. Effect of CMN on brain catechol-
Tissue sample (ng/mg)
100
3-NP (10)
CMN (25) + 3-NP
CMN (50) + 3-NP
PP (2.5) + CMN (25) + 3-NP
80 b, c, d
60 b, c 40 20
amines levels: data expressed as mean ± SEM. One-way ANOVA with a Tukey’s post hoc test. a p < 0.01 vs. control, b p < 0.05 vs. QA, c p < 0.05 vs. CMN 25, d p < 0.05 vs. CMN 50.
Control
b
a
0
b, c
b, c, d
b a a
NE
Dopamine
b
b, c
b, c, d
Serotonin
Catecholamines metabolites (striatum) 25
Fig. 5. Effect of CMN on brain catecholamines metabolite level: data expressed as mean ± SEM. One-way ANOVA with a Tukey’s post hoc test. a p < 0.01 vs. control, b p < 0.05 vs. QA, c p < 0.05 vs. CMN 25, d p < 0.05 vs. CMN 50.
Control
QA (200 nmol/2 μl)
QA + CMN (50)
QA + PP (2.5) + CMN (25)
b
15
QA + CMN (25)
a b
b, c
b, c b, c, d
b, c, d
10
5
0
DOPAC
HVA
atum were found to be significantly reduced with QA treatment. Pre-treatment with CMN (25 and 50 mg/kg/ day, p.o.) and co-administration of piperine (2.5 mg/kg/ day, p.o.) with CMN (25 mg/kg/day, p.o.) significantly restored the catecholamine levels in striatum compared to treatment with CMN alone. Further treatment with QA results in significant increase in DOPAC and HVA levels in striatum. Rats pre-treated with CMN (25 and 50 mg/kg/day, p.o.) showed significant attenuation in the increased levels of DOPAC and HVA when compared to treatment with QA alone. However, co-administration of piperine (2.5 mg/kg/day, p.o.) and CMN (25 mg/kg/day,
p.o.) significantly prevented the increase in DOPAC and HVA levels in striatum compared to treatment with CMN alone (fig. 4 and 5).
Neuroprotective Activity of CMN with Piperine
Pharmacology 2016;97:151–160 DOI: 10.1159/000443896
Effect of CMN on Striatal GABA, Glutamate and Adenosine Levels The treatment with QA alone had significantly lower GABA, adenosine levels and high glutamate when compared to the normal control group. Pre-treatment with CMN (25 and 50 mg/kg/day, p.o.) significantly and dose dependently prevented the alteration in GABA, glutamate and adenosine level in striatum. Moreover, co-ad155
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Tissue sample (ng/mg)
20
a
Fig. 6. Effect of CMN on striatal GABA,
Tissue sample (ng/mg)
200
glutamate and adenosine levels in QA treated rats: data expressed as mean ± SEM. One-way ANOVA with a Tukey’s post hoc test. a p < 0.01 vs. control, b p < 0.05 vs. QA, c p < 0.05 vs. CMN 25, d p < 0.05 vs. CMN 50.
a
150
Control
QA (200 nmol/2 μl)
QA + CMN (25)
QA + CMN (50)
QA + PP (2.5) + CMN (25)
b b, c b, c, d
100
b, c, d
50
b, c
a
b
b, c
b, c, d
a 0
Glutamate
GABA
Adenosine
Intrastriatal administration of Quinolinic acid Excess Ca2+ discharge by glutamate receptor
Neuronal firing and death
Increase IL-1, IL-6, TNF-į 1)NJ%
Increase ROS and RNS
Curcumin
Mitochondrial dysfunction
flammatory mechanism prevent QA induced neurodegeneration.
ministration of piperine (2.5 mg/kg/day, p.o.) and CMN (25 mg/kg/day, p.o.) in combination significantly prevented fluctuation in GABA, glutamate and adenosine levels compared to treatment with CMN alone (fig. 6).
Discussion
The present study reflects strong evidences that QA through excess Ca2+ discharge induces excitotoxicity dependent neuronal and glial cell death [11]. Intrastriatal administration of QA causes loss of medium spiny neurons in the basal ganglia that are responsible for motor and cognitive abnormalities indicated by impairment in 156
Pharmacology 2016;97:151–160 DOI: 10.1159/000443896
Huntington disease
locomotor activity, rotarod performance, grip strength and narrow beam walk performance in rats [12]. QA exerts neuropathologic changes through lipid peroxidation by acting as a pro-oxidant in concordance with the present study. Also, QA administration depleted antioxidant enzyme levels (glutathione) and raised oxidants (lipid peroxidation, nitrite level) suggesting the role of ROS in neurodegeneration (fig. 7) [13]. Interruption of ROS/ RNS levels by QA further activates microglia/macrophages, which promote the COX-2 dependent synthesis of various pro-inflammatory cytokines like IL-1β, IL-6 and TNF-α resembles the postpartum reports of HD patients [14]. Similarly, increased ROS and lipid peroxidation promote DNA-dependent transcription of inflamSingh/Kumar
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Fig. 7. CMN by antioxidant and anti-in-
matory cytokines and activation of nuclear factor of kappa B (NF-ĸB). This further triggers caspase-dependent cleavage of HD proteins, accumulation of insoluble oligomeric fragment-dependent striatal neuron damage responsible for all HD-like complications in concordance with our study. In the present study, QA-dependent loss of dopaminergic neurons showed significant reduction in motor function (impaired locomotor, rotarod, grip strength and narrow beam performance), oxidative damage (as evidenced by increase LPO, nitrite and depletion of glutathione level), increased IL-1β, IL-6 and TNF-α compared to normal control group. However, treatment with CMN at low dose (25 mg/kg, p.o.) and high dose (50 mg/kg, p.o.) results in remarkable improvement in locomotor activity, rotarod performance, narrow beam walk performance, decrease oxidative defence and attenuated IL-1β, IL-6 and TNF-α levels as compared to QA treated animals. Furthermore, co-administration of piperine (2.5 mg/kg, p.o.) and CMN (25 mg/kg, p.o.) showed more significant results than treatment with CMN alone. The previous reports suggest that QA on intrastriatal administration causes excitotoxicity, oxidative stress, activation of NF-ĸB and p53-induced neurodegeneration. Moreover, the damage in striatal neuron leads to lowering of catecholamine levels (norepinephrine (NE), DA and serotonin), 5-HIAA and increased the level of DOPAC and HVA in the striatum nuclei [15]. Striatal area mainly receives GABAergic neuronal projections and is the most vulnerable in HD; that is why the dysfunction of these neurons is responsible for the development of chorea [16]. The decrease in catecholamine level is due to the severe degeneration of nigrostriatal neurons, causing motor impairment as supported by present behavioral studies. Adenosine is a purine nucleoside that behaves as a typical neuromodulator in all neurons by inhibiting neurodegeneration through A1 and A2A G-protein-coupled receptors. Protection against neuronal damage is a major objective of current research in areas such as HD. The facilitation of A1 and blockade of A2A receptor decrease Ca2+-dependent glutamate release, lower the excitability of spinal neurons and helps to recover motor abnormalities in animals and humans [17]. In the present study, QA administration significantly decreased the level of catecholamines (NE, DA and serotonin) and adenosine with increase in the levels of DOPAC and HVA in striatal nuclei. Moreover, pre-treatment with CMN at 25 and 50 mg/kg, postoperatively, dose dependently attenuated the level of catecholamines and adenosine. Furthermore, the co-administration of piperine (2.5 mg/kg, p.o.) in combination with CMN (25 mg/kg,
p.o.) had showed significant protection by restoring the catecholamine levels compared with treatment by CMN alone. CMN is a potent antioxidant with reported neuroprotective and anti-inflammatory activity due to its inhibitory effect over COX-2, LOX-5, TNF-α, NF-ĸB, iNOS and P13K/AKT [15]. But the major complication is its very low oral bioavailability; so when piperine (bioavailability enhancer) is co-administered with CMN, it promotes its therapeutic effect. It is well reported that CMN at the dose of 1.0 g/kg, postoperatively, shows low plasma levels of 0.13 μg/ml after 15 min, while a maximum plasma level of 0.22 μg/ml was obtained at 1 h [18]. However, co-administration of piperine with CMN also inhibits MAO-A and MAO-B, thus enhanced the level of biological amines in stratum [19]. Piperine is a powerful inhibitor of hepatic and intestinal glucuronidation and increases the bioavailability of CMN in humans and rats [20]. In humans, piperine co-administration with CMN increases bioavailability by 2,000% at 45 min [21]. The present study thus illustrates that the co-administration of piperine (2.5 mg/kg, p.o.) and CMN (25 mg/kg, p.o.) synergistically showed significant improvement in QA induced loss of motor deficit abnormalities and oxidative stress. The present observations are supported by results configured from the whole study and also by the previous finding that CMN by targeting multifunctional pathways acts as potent antioxidant, inhibits COX and decreases the levels of various inflammatory cytokines. However, by inhibiting xanthine oxidase and by slowing ROS production, CMN further prevents neurodegeneration along with improvement in the level of catecholamines and purines in striatum. In conclusion, QA induced HD symptoms are confirmed by behavioral, biochemical, neuroinflammatory and neurochemical studies. Due to its well-established antioxidant and anti-inflammatory properties, CMN has the ability to prevent neurodegenerative changes seen in HD. Based on these above-detailed findings, CMN could be a promising treatment for neurodegenerative disorders like HD. To the best of my knowledge, CMN and piperine may act as a useful combination for the treatment of HD and other neurological disorders.
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Material and Methods
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Experimental Animals The experiment was carried out on adult Wistar rats aged 4–5 months (250–280 g) that were obtained from central animal house of I.S.F. College of Pharmacy, Moga, Punjab (India). The animals
Drugs and Chemicals QA and piperine (Sigma Aldrich, St. Louis, Mo., USA), CMN (Himedia, Vadhani Industrial Estate, L.B.S. Marg, Mumbai, India) and IL-1β, IL-6 and TNF-α ELISA kits (Krishgen BioSystem, Mumbai, India) and, unless stated, all other chemicals and biochemical reagents of highest analytical grade were used in the study. The experimental protocol was divided into 5 groups and each treatment group consisted of 9 animals (total no of animals, i.e., n = 45). Experimental Procedure and Drug Treatment Animals were anesthetized with intraperitoneal administration of ketamine (80 mg/kg) and diazepam (5 mg/kg), and the surface of the skull was exposed by making an incision on the scalp. A small hole (1–2 mm diameter) was made on both sides of the skull at anterior +1.7 mm, lateral ±2.7 mm, ventral –4.8 mm from bregma as described by Paxinos and Watson, 2007. QA (200 nmol/2 μl saline) was administered bilaterally over a period of 2 min in the striatum using digital stereotaxic apparatus (Stoelting, Wood Dale, and USA). The injection needle was left in place for another 2 min to prevent back diffusion of the injected drug solution. QA was dissolved in normal saline (pH 7.4) and administered intrastriatally (i.s.) once at a dose of 200 nmol/2 μl. CMN was dissolved in sodium carboxymethyl cellulose and administered at a dose of 25 and 50 mg/kg and also combination of CMN (25 mg/kg) with piperine (2.5 mg/kg) daily for 21 days starting from the 7th day after QA administration. All the behavioral parameters like grip strength, narrow beam, rotarod and locomotor activity were assessed on days 1, 7, 14 and 21. Terminally, on day 22, animals were sacrificed and striatum was separated, homogenized, centrifuged and clear supernatant was used to estimate biochemical parameters (LPO, nitrite, reduced GSH levels) and neurochemical analysis (DA, NE, serotonin, DOPAC, HVA, adenosine). The levels of proinflammatory cytokines (IL-1β, IL-6 and TNF-α) were estimated using ELISA kits. The experimental procedure is summarized in table 3. Behavioral Assessments Measurement of Body Weight The body weight of animals was measured on the first (1st day) and last day (21st day) of study. The percentage change in body weight was as follows: Body weight (1st day – 21st day)/1st day body weight × 100. Assessment of Gross Behavioral Parameters (Locomotor, Rotarod, Grip Strength and Narrow Beam Walk Performance) Open field test is used to monitor spontaneous locomotor activity (crossing field) using a wooden, rectangular, light brown colored open field apparatus measuring 100 × 100 × 40 cm [22]. The motor coordination and integrity with rotarod test for all animals
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Pharmacology 2016;97:151–160 DOI: 10.1159/000443896
were evaluated using weekly intervals. The rotarod apparatus comprises having a diameter of 7 cm (speed 25 rpm). The cutoff time was 180 s and the average time of the fall was recorded [23]. On the grip strength apparatus, the fore limbs grip was measured using digital grip force meter (DFIS series, Chatillon, Greensboro, N.C., USA). The grip strength was recorded in kilogram force [23]. During narrow beam walk task, the animals are required to walk on across a narrow wooden beam, measuring its motor coordination ability. Number of slips and time taken to cross in each trial were recorded [23]. Dissection and Homogenization On the 22nd day, all the animals were randomly divided into 3 groups: first group for biochemical estimations, second for neuroinflammatory markers and third for neurochemicals. The animals were sacrificed by decapitation immediately, that is, 24 h after behavioral assessments. Brains were put on the ice; the cortex and striatum regions were separated and weighed. A 10% (w/v) tissue homogenate was prepared in 0.1 mol/l phosphate buffer (pH 7.4). Homogenate was centrifuged for 15 min at 15,000 rpm, and the supernatant was stored at 80 ° C for assessing the biochemical parameters.
Measurement of Oxidative Stress Parameters Measurement of Lipid Peroxidation, Nitrite, Reduced Glutathione and Proteins The quantitative measurement of lipid peroxidation in the brain striatum was performed according to the method of Wills [24], using nitrite by Green et al. [25], using reduced glutathione as described by Ellman [26] and protein by the Lowry method using Folin phenol reagent [27]. Estimation of TNF-α, IL-1β and IL-6 in Striatum The levels of IL-1β, IL-6 and TNF-α were quantified by using rat IL-1β, IL-6 and TNF-α immunoassay kit (Krishgen BioSystem, USA). Concentrations of TNF-α were calculated from the plotted standard curves. Neurochemical Analysis Estimation of Brain Catecholamines by HPLC-ECD The brain catecholamine levels were estimated using the method described by Patel with slight modifications [28]. Catecholamines (DA, serotonin and NE) and their metabolites (DOPAC, HVA) levels in striatum were estimated by HPLC using electrochemical detector and C18 reverse phase column. Mobile phase consisted of sodium citrate buffer (pH 4.5) – acetonitrile (87: 13, v/v). The sodium citrate buffer consisted of 10 mmol/l citric acid, 25 mmol/l NaH2HPO4, 25 mmol/l EDTA and 2 mmol/l of 1-heptane sulfonic acid. The electrochemical conditions for the experiment were +0.75 V, with sensitivity ranging from 5 to 50 nA. Separation was carried out at a flow rate of 0.8 ml/min. Samples (20 μl) were injected manually. On the day of the experiment, the brain samples were homogenized in solution containing 0.2 mol/l perchloric acid. Then the samples were centrifuged at 12,000 g for 5 min. The supernatant was filtered through 0.22 mm nylon filters before being injected into the HPLC sample injector. Data were recorded and analyzed with breeze software. The concentrations of the neurotransmitters and their metabolites were calculated from the standard curve generated by using standard with concentration 10–100 mg/ml.
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were housed under standard laboratory conditions (room temperature 22 ± 1 ° C and relative humidity of 60%) with 12 h light/dark cycle. The food and water were made available ad libitum. All the behavioral assessments were carried out between 9:00 and 17:00 h. The experimental protocol was approved by the Institutional Animal Ethics Committee, and experiments were carried out in accordance with the guidelines of the Indian National Science Academy for the use and care of experimental animals.
Brain GABA and Glutamate Estimation by HPLC-ECD The GABA and glutamate levels were estimated by the method described by Lashley with slight modifications [29]. GABA and Glutamate were estimated by HPLC by using mobile phase comprised of 100 mmol/l disodium hydrogen phosphate anhydrous, 25 mmol/l EDTA and 22% methanol. Electrochemical conditions for the experiment were +0.65 V, sensitivity ranges from 5 to 50 nA. The separation for the GABA and glutamate and data were recorded by similar method as discus above in catecholamine by using standard in a concentration range of 10–100 ng/ml.
Statistical Analysis The data obtained are expressed as mean ± SEM. All the data were analysed using one-way ANOVA was followed by Tukey’s post hoc test. Values of p < 0.05 were considered statistically significant. A p value of 0.05 was considered statistically significant.
Pre-Column Derivatization Procedure Amino acids were measured as (OPA/β-ME) derivatives according to the method of Wang et al. [30].
Authors are thankful to Science and Engineering Board (SERB), Department of Science and Technology, Government of India, New Delhi for providing financial assistance under Fast Track Scheme (DST-SERB-FTYS) to Dr. P. Kumar.
Brain Purines Estimation by HPLC-Photodiode Array The purine (adenosine) level was estimated by HPLC using a PDA detector and C18 reversed phase column. The mobile phase consisted of water, methanol and acetonitrile (88:5:7, v/v). Separation and data recording were carried out similarly as discussed above in catecholamines by using standard in the concentration range of 10–100 ng/ml [31].
Acknowledgments
Disclosure Statement The authors declare that they have no conflict of interest.
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