Curcumin A

rPsychoneuroendocrinology (2011) 36, 15701581

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jou rn a l home pag e : ww w. el sepaindepression dyad: Behavioural, biochemical,neurochemical and molecular evidences

V. Arora, A. Kuhad, V. Tiwari, K. Chopra *

Pharmacology Research Laboratory, University Institute of Pharmaceutical Sciences, UGC Centre of Advanced Study,Panjab University, Chandigarh 160 014, India

Received 1 February 2011; received in revised form 21 April 2011; accepted 22 April 2011

1. Introduction

Several epidemiological studies demonstrate that pain anddepression frequently co-exist in up to 70% of chronic pain

cases (Bair et al., 2003; Arnow et al., 2006). Depression hasbeen shown to result in decreased pain threshold andincreased analgesic requirement (Jackson and Onge, 2003).It is estimated that the occurrence of depression in patientswith chronic pain is higher, ranging from 30% to 54%, than that(about 17%) in the general population (Ferrer-Garcia et al.,2006). In a World Health Organization Collaborative Study ofPsychological Disorders in Primary Care, International Classi-fication of Diseases-10, persistent somatoform pain disorder

KEYWORDSAllodynia;Biogenic amines;Paindepression dyad;Substance P

Summary An apparent clinical relationship between pain and depression has long beenrecognized. Depression and pain are often diagnosed in the same patients. The emerging conceptfor paindepression pathogenesis is the dysfunction of biogenic amine-mediated paindepres-sion control and the possible involvement of nitrodative stress-induced neurogenic inflammation.The present study was designed to investigate the effect of curcumin on reserpine-induced paindepression dyad in rats. Administration of reserpine (1 mg/kg subcutaneous daily for threeconsecutive days) led to a significant decrease in nociceptive threshold as evident from reducedpaw withdrawal threshold in Randall Sellitto and von-Frey hair test as well as significant increasein immobility time in forced swim test. This behavioural deficit was integrated with decrease inthe biogenic amine (dopamine, norepinephrine and serotonin) levels along with increasedsubstance P concentration, nitrodative stress, inflammatory cytokines, NF-kb and caspase-3levels in different brain regions (cortex and hippocampus) of the reserpinised rats. Curcumin(100, 200, 300 mg/kg; ip) dose dependently ameliorated the behavioural deficits associated withpain and depression by restoring behavioural, biochemical, neurochemical and molecularalterations against reserpine-induced paindepression dyad in rats.# 2011 Elsevier Ltd. All rights reserved.

* Corresponding author. Tel.: +91 172 2534105;fax: +91 172 2541142.

E-mail address: [email protected] (K. Chopra).

0306-4530/$ see front matter # 2011 Elsevier Ltd. All rights reserved.doi:10.1016/j.psyneuen.2011.04.012Curcumin ameliorates rese pine-induced

v ie r. com/ loca te /psyn eu en

Curcumin and Pain Depression Dyad 1571was found in 32.4% of patients with depression and in 8.6% ofprimary care patients without depression (Sartorius et al.,1993). Recently, Miller and Cano (2009) reported that preva-lence of chronic pain due to any cause was 21.9% and approxi-mately 35% of participants with chronic pain also had comorbiddepression. This relationship between pain and depressiongave rise to the theories that depression might increase painperception or that depression is a common consequence ofpain symptoms (Fishbain et al., 1997; Landi et al., 2005). Thiscomplex interaction is often labeled as DepressionPain syn-drome or PainDepression dyad (Lindsay and Wyckoff, 1981;Bair et al., 2004; Goldenberg, 2010), and implying that theconditions often coexist and respond to similar treatments,exacerbate one another, and share common biological path-ways. Although this intricate relationship between pain anddepression has attracted increasing attention in all areas ofresearch, but the mechanisms underlying the association ofdepression and pain are however not clear (Dersh et al., 2002).A combination of interactions between neurotransmitters(Russell et al., 1992; Elhwuegi, 2004), neuropeptides (Malm-berg and Yaksh, 1992; Kramer et al., 1998) nitrodative stress(Bagis et al., 2005; Maes et al., 2010) and cytokines (Wallace,2006; Dowlati et al., 2010) are thought to take part in patho-genesis of paindepression dyad.

The biochemical theory of depressionpain dyad posits aneurochemical imbalance or a functional deficiency of keyneurotransmitters, the monoamines: serotonin, norepinephr-ine, and dopamine (Fishbain et al., 1997; Stahl, 2002; Campbellet al., 2003). As with major depressive disorder (MDD) patients,studies in patients with chronic pain have consistently foundlower serotonin levels and/or reduced reuptake and lowerplasma and cerebrospinal fluid tryptophan levels comparedto controls (Moldofsky, 1982; Russell et al., 1992). Furthermore,both conditions involve increased plasma and cerebrospinalfluid substance P concentrations. Similarly, as in MDD, elevatedconcentrations of substance P in cerebrospinal fluid have beenfound in patients with chronic pain (Larson et al., 2000) andsubstance Pis also thought to play a role in the development andtreatment of MDD (Blier et al., 2004). Neurotransmission ofsubstance P is negatively modulated by efferent serotonergicneurons (Naranjo et al., 1989). It has also been observed thatincreased levels of substance P in brain increases 5-HT levels inspinal cord, while 5-HT decreases the release of substance Pinto the spinal cord (Moldofsky, 1982). Finally, since substance Pis involved in pain control as well as depression, there is a needto investigate the role of substance P along with the biogenicamines, particularly because empirical data suggest that sub-stance P and biogenic amines are involved in both the etio-pathogenesis and treatment response of pain and depression(Blier et al., 2004; Staud and Spaeth, 2008).

The second facet of the paindepression dyad is the invol-vement of nitrodative stress-induced neurogenic inflamma-tion. Human studies have reported a number of oxidativedisturbances in patients with major depression suggested bythe elevated lipid peroxidation products (Bilici et al., 2001;Khanzode et al., 2003; Sarandol et al., 2007), findings ofaltered antioxidant enzyme with reduced levels of superoxidedismutase (Herken et al., 2007). Moreover, a significant posi-tive correlation was found between oxidative stress index andthe Hamilton depression rating scale (Yanik et al., 2004).Similarly higher serum levels of pentosidine and malondialde-hyde along with reduced serum superoxide dismutase (amarker of antioxidant capacity) were found in patients withchronic pain compared with normal controls (Hein and Franke,2002; Bagis et al., 2005). Cordero et al. (2009) found higherlevels of ROS production in mononuclear cells from fibromyal-gia patients again suggesting enhanced oxidative stress. Thisenhanced oxidative stress can stimulate the production of NF-kb and can lead to increase in the levels of pro-inflammatorycytokines TNF-a, IL-1b, IL-6, IL-8, IFN-g (Pall, 2007).Depressed patients and patients with pain disorders oftendisplay enhanced cytokine levels including interleukin-6 (IL-6), C-reactive protein, interleukin-1-beta (IL-1b), and tumornecrosis factor alpha (TNF-a) (Raison et al., 2006; Omoigui,2007; Dowlati et al., 2010) Recently, Kadetoff et al. (2009)found higher mRNA levels of TNF-a in patients of fibromyalgia.Thus, enhanced ROS along with generation of pro-inflamma-tory cytokines (TNF-a and IL-1b) may modulate NF-kb signal-ling and caspase-3 pathway which may be responsible for thedevelopment and perpetuation of pain in depression or depres-sion in pain (Joseph and Levine, 2004).

Thus, an animal model of pain depression dyad ideallyshould include widespread pain as well as the depression.Previously published study from our lab (Kulkarni and Robert,1982) and recently published reserpine-mediated animalmodel of fibromyalgia implicate reserpine-induced dysfunc-tion of biogenic amines in mediation of CNS pain control(Nagakura et al., 2009). Reserpine is a monoamine depletorthat exerts a blockade on the vesicular monoamine trans-porter for neuronal transmission or storage, promoting dopa-mine-autoxidation and oxidative catabolism by monoamineoxidase may result in oxidative stress (Lohr et al., 2003). Thisdual action of reserpine (monoamine depletion and oxidativestress) makes it pronominal to address both facets involved inthe pathophysiology of the pain depression dyad.

Curcumin, a polyphenol found in turmeric, is a yellow curryspice with a long history of use in traditional Indian diets andherbal medicine. Curcumin (diferuloyl methane) has manypharmacological activities including replenishment of themonoamines (Xu et al., 2005) anti-inflammatory properties(Jain et al., 2009) and powerful antioxidant activity, studieshave shown that curcumin is a powerful scavenger of thesuperoxide anion, the hydroxyl radical, and nitrogen dioxide(Unnikrishnan and Rao, 1995). Curcumin exerts anti-inflam-matory and growth inhibitory effects in TNF-a treated HaCaTcells through inhibition of NF-kb and mitogen activated proteinkinase pathways (Cho et al., 2007). Curcumin is also known toexhibit anti-hyperalgesic (Sharma et al., 2006) and antidepres-sant effects in wide variety of animal models (Xu et al., 2005).

Thus, the aim of the present study was two-fold, first toinvestigate the protective effects of curcumin on the reser-pine-induced pain, tactile allodynia and accompanieddepression in rats and second to investigate the protectivepotential of curcumin against the reserpine induced biogenicamine depletion and nitrodative stress mediated inflamma-tory cascade and apoptotic signalling pathway.

2. Materials and methods

2.1. Animals

Adult male Wistar rats (200220 g) bred in Central AnimalHouse facility of Panjab University were used. The animals

were housed under standard laboratory conditions, main-tained on a 12:12 h light: dark cycle and had free access tofood (Ashirwad Industries, Mohali, India) and water. Animalswere acclimatized to laboratory conditions before the tests.All experiments were carried out between 09:00 and 17:00 h.The experimental protocols were approved by the Institu-tional Animal Ethics Committee of Panjab University andperformed in accordance with the guidelines of Committeefor Control and Supervision of Experimentation on Animals,Government of India on animal experimentation.

2.2. Drugs

Reserpine and curcumin were purchased from Sigma (St.Louis, MO, USA). TNF-a, IL-1b, substance P ELISA kit waspurchased from R&D Systems (USA). While NF-kb and cas-pase-3 ELISA kits were procured from Imegenex, San Diego,USA and Biovision, USA respectively. All other chemicals usedfor biochemical estimations were of analytical grade.

2.3. Experimental design

Pain and depression were induced by administration of reser-

the last reserpine injection. After behavioural assessment,rats were sacrificed under deep anaesthesia and differentbrain regions were isolated and stored at 80 8C for bio-chemical estimations. Reserpine was dissolved in glacialacetic acid, diluted to a final concentration of 0.5% aceticacid with distilled water. Curcumin was injected intraper-itoneally as absorption seems to be higher by this route thanafter oral administration, as gavage results in very low levelsinto the blood. Curcumin was prepared by using a conven-tional pharmaceutically acceptable carrier using a mixture of1% sodium carboxy methylcellulose and Tween 80 as theseagents possess absorption enhancing capacity in formula-tions. The animals were sacrificed under deep anaesthesiaon the fifth day immediately after behavioural assessmentand brain samples were rapidly removed and placed on dryice for isolation of cerebral cortex and hippocampus. Brainsamples were incubated with 1 ml of ice cold 1 hypotonicbuffer supplemented with 1 mM dithiothreitol and 1% deter-gent solution for 30 min on ice. After incubation, the sampleswere centrifuged for 10 min at 10,000 rpm at 4 8C. Thesupernatant (Cytoplasmic Fraction) was transferred into aseparate tube and stored at 4 8C. The nuclear pellet was re-suspended in 100 ml nuclear lysis buffer by pipetting up and

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1572 V. Arora et al.pine (1 mg/kg subcutaneous daily) for three consecutive days(Nagakura et al., 2009). The animals were randomly dividedinto six experimental groups with 8 animals in each. Group Icomprised control animals receiving 1 ml/kg vehicle subcu-taneously; Group II animals were administered reserpine(1 mg/kg; subcutaneously) for three consecutive days (i.e.day 1, 2 and 3); Group III, IV, V consisted of reserpinised ratsreceiving curcumin (100, 200 and 300 mg/kg; ip) for two daysafter reserpine (i.e. day 4 and 5); Group VI consisted ofcontrol animals receiving curcumin (300 mg/kg; intraperito-neally) (Fig. 1). Dose of curcumin was selected on the basis ofprevious studies stating CNS effects of curcumin (Dohareet al., 2008; Mehla et al., 2010) and from the study doneby Mittal et al. (2009) who shows the antinociceptive effectof curcumin at the selected dose range. Hyperalgesia (ther-mal and mechanical) and allodynia were assessed 48 h after

Figure 1 Pain and depression was induced by administration offollowed by intraperritoneal administration of curcumin 100, 200 behaviour experiments were conducted 30 min after curcumin admeasurement of the behaviour paradigms and the samples weredown. The samples were vortexed vigorously and suspensionwas incubated at 4 8C for 30 min. The suspension was vor-texed again for 30 s and centrifuged at 14,000 rpm for 10 minat 4 8C in a microcentrifuge. The supernatant was transfered(Nuclear Fraction) into a pre-chilled microcentrifuge tube.The cytoplasmic fractions were separated from the brainhomogenate for the biochemical estimations and for quanti-fication of TNF-a, IL-1b and caspase-3 while nuclear fractionwas used only for estimation of NF-kb levels.

2.4. Behavioural tests

Thermal hyperalgesia was assessed in a water bath main-tained at 42 8C (a temperature that is normally innocuous innaive rats until tail withdrawal or signs of struggle wereobserved (cut-off time: 15 s) (Chopra et al., 2010). Mechanical

erpine (1 mg/kg subcutaneous daily) for three consecutive days 300 mg/kg for two days after the last reserpine injection and allnistration. Brain samples were harvested immediately after thered at 80 8C for biochemical estimations on next day.

Curcumin and Pain Depression Dyad 1573hyperalgesia: the nociceptive flexion reflex was quantifiedusing the Randall Selitto paw pressure device (IITC, WoodlandHills, USA), which applies a linearly increasing mechanicalforce (in g) to the dorsum of the rats hindpaw (Chopraet al., 2010). Mechanical allodynia: rats were placed indivi-dually on an elevated mesh (1 cm2 perforations) in a clearplastic cage and adapted to the testing environment for atleast 15 min. von-Frey hairs (IITC, Woodland Hills, USA) withcalibrated bending forces (in g) of different intensities wereused to deliver punctuated mechanical stimuli of varyingintensity. Starting with the lowest filament force, von-Freyhairs were applied from below the mesh floor to the plantarsurface of the hindpaw, with sufficient force to cause slightbending against the paw, and held for 1 s (Chopra et al., 2010).Each stimulation was applied 5 times with an inter-stimulusinterval of 45 s. Care was taken to stimulate random loca-tions on the plantar surface. A positive response was noted ifthe paw was robustly and immediately withdrawn. Paw-with-drawal threshold was defined as the minimum pressurerequired to elicit a withdrawal reflex of the paw, at leastone time on the five trials. Voluntary movement associatedwith locomotion was not considered as a withdrawal response.Mechanical allodynia was defined as a significant decrease inwithdrawal thresholds to von-Frey hair application. All thepain measurement studies were done by the experimenter whowas blind to the drug treatment

Immobility period: the forced swim test was performedbased on the original method described by Porsolt et al.(1977). One day prior to the test, a rat was placed forconditioning in a clear plastic tank (45 cm 35 cm 60 cm)cm) containing 30 cm of water (24 0.5 8C) for 15 min (pre-test session). Twenty-four hours later (test session), the totalduration of immobility within a 5-min session was recorded asimmobility scores (in s). A rat was judged to be immobilewhen its hind legs were no longer moving and the rat washunched forward (a floating position). The immobility timewas recorded manually by an observer who was blind to thedrug treatment.

2.5. Biochemical estimations

2.5.1. Estimation of lipid peroxidationThe malondialdehyde content, a measure of lipid peroxida-tion, was assayed in the form of thiobarbituric acid-reactivesubstances by the method of Wills (1965). Briefly, 0.5 ml ofcytosolic fraction of both brain regions and 0.5 ml of TrisHClwere incubated at 37 8C for 2 h. After incubation 1 ml of 10%trichloroacetic acid was added and centrifuged at 1000 gfor 10 min. Then 1 ml of 0.67% thiobarbituric acid was addedto 1 ml of supernatant and the tubes were kept in boilingwater for 10 min. After cooling, 1 ml double-distilled waterwas added and absorbance was measured at 532 nm. Thio-barbituric acid-reactive substances were quantified using anextinction coefficient of 1.56 105 M1 cm1 and expressedas nmol of malondialdehyde per mg protein. Tissue proteinwas estimated using the Biuret method and the malondial-dehyde content expressed as nmol/mg protein.

2.5.2. Estimation of non protein thiolsNon protein thiols were assayed by the method of Jollowet al. (1974). Briefly, 1.0 ml of cytosolic fraction of both brainregions was precipitated with 1.0 ml of sulphosalicylic acid(4%). The samples were kept at 4 8C for at least 1 h and thensubjected to centrifugation at 1200 g for 15 min at 4 8C.The assay mixture contained 0.1 ml supernatant, 2.7 mlphosphate buffer (0.1 M, pH 7.4) and 0.2 ml 5,5-dithiobis-(2-nitrobenzoic acid) (Ellmans reagent, 0.1 mM, pH 8.0) in atotal volume of 3.0 ml. The yellow color developed was readimmediately at 412 nm and the reduced glutathione levelswere expressed as mmol/mg protein.

2.5.3. Estimation of superoxide dismutaseSuperoxide dismutase activity was assayed by the method ofKono (1978). The assay system consisted of 0.1 mM EDTA,50 mM sodium carbonate and 96 mM of nitro-blue tetrazolium(NBT). In a cuvette, 2 ml of the above mixture was taken and0.05 ml of cytosolic fraction of both brain regions and 0.05 mlof hydroxylamine hydrochloride (adjusted to pH 6.0 withNaOH) were added to it. The auto-oxidation of hydroxyla-mine was observed by measuring the change in opticaldensity at 560 nm for 2 min at 30-/60-s intervals.

2.5.4. Estimation of catalaseCatalase activity was assayed by the method of Claiborne(1985). Briefly, the assay mixture consisted of 1.95 ml phos-phate buffer (0.05 M, pH 7.0), 1.0 ml hydrogen peroxide(0.019 M) and 0.05 ml cytosolic fraction of both brain regionsin a final volume of 3.0 ml. Changes in absorbance wererecorded at 240 nm. Catalase activity was calculated interms of k min1 and expressed as mean S.E.M.

2.5.5. Nitrite estimationNitrite was estimated in the cytosolic fraction of differentbrain regions using the Greiss reagent and served as anindicator of nitric oxide production. A measure of 500 mlof Greiss reagent (1:1 solution of 1% sulphanilamide in 5%phosphoric acid and 0.1% napthaylamine diamine dihydro-chloric acid in water) was added to 100 ml of post-mitochon-drial supernatant and absorbance was measured at 546 nm(Green et al., 1982). Nitrite concentration was calculatedusing a standard curve for sodium nitrite and nitrite levelswere expressed as mg/ml. Although, the Griess spectropho-tometric assay is not a leading methodology for the quanti-fication of nitric oxide, it employs an indirect measure ofnitric oxide content.

2.6. Neurotransmitters estimation

Biogenic amines (dopamine, serotonin and norepinephrine)were estimated by HPLC with electrochemical detector.Waters standard system consisting of a high pressure isocraticpump, a 20 ml sample injector valve, C18 reverse phasecolumn and electrochemical detector were used. Data wasrecorded and analyzed with the help of empower software.Mobile phase consisting of sodium citrate buffer (pH 4.5)acetonitrile (87:13, v/v). Sodium citrate buffer consist of10 mM citric acid, 25 mM NaH2HPO4, 25 mM EDTA, and 2 mMof 1-heptane sulphonic acid (Patel et al., 2005). Electroche-mical conditions for the experiment were +0.75 V, sensitivityranges from 5 to 50 nA. Separation was carried out at a flowrate of 0.8 ml/min. Samples (20 ml) were injected manually.On the day of experiment frozen brain samples were thawed

and they were homogenized in homogenizing solution con-taining 0.2 M perchloric acid. After that samples were cen-trifuged at 12000 g for 5 min. The supernatant was furtherfiltered through 0.22 mm nylon filters before injecting in theHPLC injection pump. Data was recorded and analyzed withthe help of empower software.

2.7. TNF-a, IL-1b and substance P ELISA

The quantifications of TNF-a, IL-1b and substance P weredone with the help and instructions provided by R&D SystemsQuantikine Rat TNF-a, IL-1b and substance P immunoassaykit.

2.8. Quantification of NF-kb p65 unit

The nuclear levels of p65 may correlate positively with theactivation of NF-kb pathway. The NF-kb/p65 ActivELISA(Imgenex, San Diego, USA) kit was used to measure NF-kbfree p65 in the nuclear lysate. The NF-kb ActivELISA is asandwich ELISA. Free p65 was captured by anti-p65 antibodycoated plates and the amount of bound p65 was detected by

reaction. The enzymatic reaction for caspase activity wascarried out using R&D systems caspase-3 colorimetric kit.

2.10. Statistical analysis

Results were expressed as means S.E.M. The intergroupvariation was measured by one-way analysis of variance(ANOVA) followed by Tukeys test. Statistical significancewas considered at p < 0.05. The statistical analysis was doneusing the SPSS Statistical Software version 16 (SPSS Inc. 233South Wacker Drive, 11th Floor Chicago, IL 60606-6412).

3. Results

3.1. Effect of curcumin on behaviouralparadigms

3.1.1. Modulation of thermal hyperalgesiaReserpine produced a significant decrease in tail flick latency(4.40 0.34 s, p < 0.05) as compared to control group(6.73 0.88 s). Curcumin (100, 200 and 300 mg/kg) signifi-

fferl,

1574 V. Arora et al.adding a second anti-p65 antibody followed by alkalinephosphatase (AKP)-conjugated secondary antibody using col-orimetric detection in an ELISA plate reader at 405 nm.

2.9. Caspase-3 colorimetric assay

Caspase-3, also known as CPP-32, Yama or Apopain, is anintracellular cysteine protease that exists as a pro-enzyme,becoming activated during the cascade of events associatedwith apoptosis. The tissue lysates/homogenates can then betested for protease activity by the addition of a caspase-specific peptide that is conjugated to the color reportermolecule p-nitroaniline (pNA). The cleavage of the peptideby the caspase releases the chromophore pNA, which can bequantitated spectrophotometrically at a wavelength of405 nm. The level of caspase enzymatic activity in the celllysate/homogenate is directly proportional to the color

Figure 2 Data are expressed as mean S.E.M. *( p < 0.05) diadministered group; $( p < 0.05) different from one another. Ctcurcumin (200 mg/kg); C3, curcumin (300 mg/kg).cantly and dose-dependently increased the shortened tailflick latency in reserpinised rats (4.87 0.39, 5.93 0.24and 6.53 0.25) respectively [F(6,29) = 6.179 ( p < 0.001)].However, there was no significant change in the mean tailflick latency in per se group.

3.1.2. Modulation of mechanical hyperalgesiaReserpine produced a significant decrease in paw-withdrawalthreshold (60.77 3.99 g, p < 0.05) as compared to controlgroup (136.17 3.63 g) (Fig. 2). Curcumin (100, 200 and300 mg/kg), significantly and dose-dependently increasedthe paw-withdrawal threshold [F(6,29) = 84.68 ( p < 0.01)]in reserpine-treated rats. However, there was no significantchange in the mean paw-withdrawal threshold in per se group

3.1.3. Effect on mechanical allodyniaIn von-Frey hair test, reserpinised rats showed significantincrease in pain sensitivity to non-noxious stimulus

rent from control group; #( p < 0.05) different from reserpine-control; R, reserpine (1 mg/kg) C1, curcumin (100 mg/kg); C2,

was no significant change in the mean immobility time inper se group.

3.3. Effect of curcumin on neurotransmitterlevels

Chronic administration of reserpine resulted into decreasedlevels of dopamine, norepinephrine and serotonin in bothcortex and hippocampal region (Table 1) which was dosedependently replenished by curcumin (100, 200 and300 mg/kg). Curcumin 300 mg/kg produced a significantincrease in the NE [2.26 fold], DA [2.62-fold] and 5-HT[2.86-fold] in the cortex region and similar increase in thelevels of NE [1.84-fold], DA [1.87-fold] and 5-HT [3.72-fold] inthe hippocampus of reserpine administered rats. Curcumin(300 mg/kg) per se did not cause any significant change indopamine, norepinephrine and serotonin concentration ascompared to control.

3.4. Effect of curcumin on substance P levels

There was significant increase in substance P levels in the

Figure 3 Data are expressed as mean S.E.M. *( p < 0.05)different from control group; #( p < 0.05) different from reser-pine-administered group; $( p < 0.05) different from one anoth-er. Ctrl, control; R, reserpine (1 mg/kg) C1, curcumin (100 mg/kg); C2, curcumin (200 mg/kg); C3, curcumin (300 mg/kg).

Curcumin and Pain Depression Dyad 1575(23.67 2.70 g, p < 0.05) as compared to control rats(63.00 0.85 g, p < 0.05) (Fig. 2). Curcumin (100, 200 and300 mg/kg) produced significant and dose-dependentincrease in paw-withdrawal threshold in response to von-Frey hair stimulation [F(6,29) = 88.70 ( p < 0.01)]. However,there was no significant change in the mean paw-withdrawalthreshold in per se group.

3.2. Effect on immobility period in Forced swimtest

The mean immobility period (Fig. 3) of reserpine treated rats(86.60 4.73 s, p < 0.05) was significantly increased as com-pared to control group (66.60 5.43 s, p < 0.05). Treatmentwith curcumin (100, 200 and 300 mg/kg) significantly anddose-dependently decreased immobility time in reserpine-treated rats [F(6,29) = 29.06 ( p < 0.01)]. However, thereTable 1 Effect of curcumin (C) on neurotransmitter levels norepexpressed as mean S.E.M.Treatment NE (pg/mg tissue

CtrlR Cerebral cortex 2.772 0.011 Hippocampus 4.420 0.020 Cerebral cortex 0.814 0.020 *Hippocampus 1.690 0.020 *

R + C1 Cerebral cortex 1.290 0.015#,$Hippocampus 2.210 0.030#,$



C3 Cerebral cortex 2.783 0.019 Hippocampus 4.400 0.040

Ctrl, control; R, reserpine (1 mg/kg), C1, curcumin (100 mg/kg); C2, c* Different from control group ( p < 0.05).# Different from reserpine-administered group ( p < 0.05).$ Different from one another ( p < 0.05).cerebral cortex [1.87-fold] and hippocampus [2.02-fold](Fig. 4) of reserpine administered rats respectively. Curcumin(100, 200 and 300 mg/kg) produced a significant reduction insubstance P levels in a dose-dependent manner in bothcerebral cortex [F(6,29) = 2223 ( p < 0.001)] and hippocam-pus [F(6,29) = 741.1 ( p < 0.001)] of reserpine administeredrats.

3.5. Effect of curcumin on biochemical indices

3.5.1. Effect of curcumin on reserpine-inducedchanges in lipid peroxidationLipid peroxide levels were increased significantly in the cere-bral cortex and hippocampus of reserpine administered rats ascompared to control group (Table 2). Treatment with curcumin(100, 200 and 300 mg/kg) produced a significant reduction inlipid peroxide levels in the cerebral cortex [F(6,29) = 67.23

inephrine (NE), dopamine (DA) and serotonin (5-HT). Data are

) DA (pg/mg tissue) 5-HT (pg/mg tissue)

0.61 0.01 3.06 0.012.12 0.01 8.18 0.040.13 0.00 * 0.58 0.00 *0.61 0.01 * 1.15 0.02 *0.19 0.00#,$ 0.78 0.01#,$0.77 0.02#,$ 1.63 0.02#,$0.24 0.00#,$ 1.05 0.01#,$0.97 0.00#,$ 2.50 0.02#,$0.34 0.01#,$ 1.65 0.01#,$1.14 0.01#,$ 4.26 0.020#,$0.63 0.01 3.08 0.032.10 0.02 8.27 0.03

urcumin (200 mg/kg); C3, curcumin (300 mg/kg).

Figure 4 Data are expressed as mean S.E.M. *( p < 0.05) different from control group; #( p < 0.05) different from reserpine-administered group; $( p < 0.05) different from one another. Ctrl, control; R, reserpine (1 mg/kg) C1, curcumin (100 mg/kg); C2,curcumin (200 mg/kg); C3, curcumin (300 mg/kg).

1576 V. Arora et al.( p < 0.01)] and hippocampus [F(6,29) = 143.6 ( p < 0.01)] ofreserpine administered rats.

3.5.2. Effect of curcumin on reserpine-inducedchanges in the anti-oxidant profileThe non-protein thiols and enzymatic activity of superoxidedismutase and catalase significantly decreased in the cere-bral cortex and hippocampus of reserpine administered ratsas compared to control group (Table 2). This reduction wassignificantly and dose dependently restored with differentdoses of curcumin in the cerebral cortex and hippocampus ofreserpine administered rats.

3.5.3. Effect of curcumin on reserpine-inducednitrosative stressTotal nitric oxide was significantly elevated in cerebral cortex(2-fold) and hippocampus (2-fold) of reserpine administeredTable 2 Effect of curcumin (C) on lipid peroxide (LPO), reducednitrite levels. Data are expressed as mean S.E.M.Treatment LPO (nmol/mg pr) GSH (nmol/m

Ctrl Cerebral cortex 1.65 0.20 0.68 0.03 Hippocampus 0.88 0.03 0.42 0.02

R Cerebral cortex 11.21 1.01 * 0.19 0.02 *Hippocampus 4.14 0.21 * 0.12 0.03 *

R + C1 Cerebral cortex 7.23 0.25#,$ 0.26 0.03#,$Hippocampus 2.17 0.09#,$ 0.18 0.01#,$



C3 Cerebral cortex 1.75 0.18# 0.55 0.02#Hippocampus 0.87 0.05# 0.39 0.03#

Ctrl, control; R, reserpine (1 mg/kg), C1, curcumin (100 mg/kg); C2, c* Different from control group ( p < 0.05).# Different from reserpine-administered group ( p < 0.05).$ Different from one another ( p < 0.05).animals (Table 2). Curcumin (100, 200 and 300 mg/kg) treat-ment significantly inhibited this increase in nitrite levels inthe cerebral cortex [F(6,29) = 81.89 ( p < 0.01)] and hippo-campus [F(6,29) = 123.3 ( p < 0.01)] of reserpine adminis-tered rats.

3.6. Effect of treatment on TNF-a and IL-1blevels

3.6.1. Effect of curcumin on brain TNF-a levelThere was 2 fold and 3 fold increase in TNF-a level in thecerebral cortex and hippocampus (Table 3) of reserpineadministered rats respectively. Treatment with curcumin(100, 200 and 300 mg/kg) produced a significant reductionin TNF-a levels in a dose-dependent manner in cerebralcortex [F(6,29) = 36.71 ( p < 0.001)] and hippocampus[F(6,29) = 36.17 ( p < 0.001)] of reserpine administered rats. glutathione (GSH), superoxide dismutase (SOD), catalase and

g pr) SOD (U/mg pr) Catalase (U/mg pr) Nitrite (mg/ml)

1.49 0.07 0.88 0.02 5.35 0.101.34 0.09 0.87 0.01 4.55 0.300.36 0.03 * 0.20 0.03 * 11.55 0.19 *0.29 0.02 * 0.16 0.00a * 9.12 0.30 *0.48 0.03$ 0.35 0.02#,$ 8.42 0.27#,$0.49 0.04$ 0.38 0.01#,$ 6.92 0.30#,$0.68 0.05#,$ 0.58 0.02#,$ 7.05 0.40#,$0.73 0.01#,$ 0.58 0.01#,$ 5.05 0.30#,$0.89 0.03#,$ 0.81 0.03#,$ 4.35 0.50#,$1.02 0.04#,$ 0.83 0.01#,$ 3.92 0.30#,$1.42 0.05# 0.89 0.01# 4.15 0.251.35 0.11# 0.88 0.03# 4.58 0.30

urcumin (200 mg/kg); C3, curcumin (300 mg/kg).

b a

-1b

.21

.30

.93

.29

.61

.88

.14

.57

.59

.71

.61

.96

2, c

Curcumin and Pain Depression Dyad 15773.6.2. Effect of curcumin on brain IL-1b levelsThere was significant increase in the IL-1b level in the cortexand hippocampus (Table 3) of reserpine administered rats ascompared to control group. Curcumin (200 and 300 mg/kg)treatment significantly decreased IL-1b levels in the cerebralcortex [F(6,29) = 8.104 ( p < 0.001)] and hippocampus[F(6,29) = 24.87 ( p < 0.001)] of reserpinised rats.

3.7. Effect of curcumin on nuclear factor kappabeta (NF-kb)

NF-kb p56 subunit was significantly elevated in cerebralcortex [2.09-fold] and hippocampus [3.6-fold] (Table 3) ofreserpine administered rats as compared to control group.Curcumin treatment significantly ( p < 0.05) and dose depen-dently prevented NF-kb p56 subunit levels in the nuclearfraction of cortex [F(6,29) = 29.15 ( p < 0.001)] and the hip-pocampus [F(6,29) = 77.22 ( p < 0.01)] of reserpinised rats.

Table 3 Effect of curcumin (C) on TNF-a, IL-1b, p-65 of NFk

Treatment TNF-a (pg/ml) IL

Ctrl Cerebral cortex 89.20 6.10 24Hippocampus 29.56 2.27 12

R Cerebral cortex 180.73 6.70 * 51Hippocampus 92.70 8.17 * 35

R + C1 Cerebral cortex 151.43 7.08#,$ 43Hippocampus 88.42 6.06#,$ 21



C3 Cerebral cortex 99.70 2.29 29Hippocampus 23.90 2.19 12

Ctrl, control; R, reserpine (1 mg/kg), C1, curcumin (100 mg/kg); C* Different from control group ( p < 0.05).# Different from reserpine-administered group ( p < 0.05).$ Different from one another ( p < 0.05).3.8. Effect of curcumin on caspase-3 activity

Caspase-3 levels were significantly elevated in cerebral cor-tex [3.26-fold] and hippocampus [4.36-fold] (Table 3) ofreserpine administered rats as compared to control group.Treatment with curcumin significantly ( p < 0.05) inhibitedcaspase 3 activity in cortex [F(6,29) = 307.6 ( p < 0.01)] andhippocampus [F(6,29) = 601.2 ( p < 0.01)] of reserpinisedrats in a dose-dependent manner.

4. Discussion

Clinical depression is a multifactorial and multisymptomaticdisease, and apparently so is depression is associated withpain, thats why we aimed to investigate pain perception inrats with depressive-like behaviour in comparison to non-depressed controls. In the present study, reserpinised ratsexhibited increased pain sensitivity in tail flick latency (ther-mal hyperalgesia) and decreased paw-withdrawal threshold inRandall-sellito test (mechanical hyperalgesia) and von-Freyhair test (mechanical allodynia). These findings corroborateprevious reports published from our lab, Kulkarni and Robert(1982) and from Nagakura et al. (2009) who found a time-dependent decrease in nociceptive threshold as observed intail immersion test in reserpinised rats suggesting reserpine-induced hyperalgesia. Curcumin increased the pain thresholdin reserpinized rats in all the behavioural paradigms of painwhich is in line with evidence from previous studies done in ourlaboratory where curcumin attenuated the diabetic neuro-pathic pain (Sharma et al., 2006).

The enhanced pain sensitivity in reserpinised rats was alsocoupled with depression as indicated by increased immobilitytime in forced swim test which is in line with the resultsreported by Zeng et al. (2008) stating that the presence ofdepression-like behaviour in rats exacerbated mechanicalallodynia under the condition of chronic neuropathic pain.Curcumin significantly and dose-dependently decreased theimmobility time in forced swim test in reserpinised rats whichis in conformity with the antidepressant activity of curcumin

nd caspase-3 levels. Data are expressed as mean S.E.M. (pg/ml) p-65 of NFkb

(ng/mg of protein)Caspase-3 (%Control)

2.25 18.95 2.93 100.00 1.08 1.25 10.93 0.46 100.00 0.81 4.70 * 39.76 1.81 * 326.91 2.18 * 2.42 * 39.58 2.35 * 436.36 10.20 * 5.30#,$ 31.46 0.92#,$ 281.27 6.20#,$ 2.32#,$ 25.25 0.98#,$ 314.44 8.81#,$ 3.67#,$ 21.59 1.54#,$ 230.56 10.94#,$ 1.31#,$ 18.02 1.20#,$ 205.35 2.04#,$ 3.97#,$ 17.42 0.87#,$ 193.37 2.54#,$ 1.97#,$ 12.15 1.21#,$ 109.09 2.36#,$ 3.02 20.19 0.39 98.82 0.48 2.13 10.22 0.73 99.47 0.95urcumin (200 mg/kg); C3, curcumin (300 mg/kg).in various rodent models of depression (Xu et al., 2005).Analyses of cerebrospinal fluid and serum from patients

with chronic pain have suggested a decrease in biogenicamines, i.e., dopamine (DA), norepinephrine (NE), and 5-hydroxytryptamine (5-HT) (Russell et al., 1992). Basic neu-robiological research as well as clinical studies has alsorevealed that the monoamines (5-HT, DA, NE) have a crucialrole in the development of the depression syndrome (Elh-wuegi, 2004). Serotonin and norepinephrine are both impor-tant modulators in pain perception and depression in normalsubjects, thus it is reasonable to suspect that disturbances inthese functions may be the consequences of abnormalities inserotonin and norepinephrine metabolism and transmission(Kundermann et al., 2009). In the present study, curcuminrestored 5-HT, norepinephrine and dopamine levels in dif-ferent brain regions of reserpinised rats and the results are inaccordance with the previous findings from our laboratory(Kulkarni et al., 2008). Xu et al. (2005) also found increasedlevels of serotonin, dopamine and norepinephrine in both thefrontal cortex and hippocampus of mice treated with curcu-min (10 mg/kg) and this effect was attributed to monoamine

1578 V. Arora et al.oxidase inhibiting activity of curcumin. These findings sug-gest that the neuroprotective effects of curcumin mayinvolve the modulation of central monoaminergic neuro-transmitter systems.

Substance P is an active neuropeptide in the CNS and thereare studies which show the role of substance P, as it lowerpain thresholds (Malmberg and Yaksh, 1992) and causesdepression (Kramer et al., 1998). Substance P shows thestrong negative correlation between serum concentrationsof the primary serotonin metabolite, 5-hydroxyindoleaceticacid (Schwarz et al., 1999) and secondly NE may inhibitsubstance P, thus low NE could indirectly cause more noci-ception (Gureje et al., 1998). In the present study repeatedadministration of reserpine showed a significant increase inthe substance P levels in both cortex and hippocampusregions of the rat brain and treatment with curcumin sig-nificantly relegated the increased levels of the Substance P.To the best of our knowledge, this is the first study whichstates the inhibitory effect of curcumin on substance P.

The second facet of our hypothesis involves nitrodativestress-induced neurogenic inflammation which may beresponsible for the development and perpetuation of painin depression. Reserpine is a monoamine depletor that exertsa blockade on the vesicular monoamine transporter forneuronal transmission or storage, promoting dopamine-auto-xidation and oxidative catabolism by monoamine oxidase(Lohr et al., 2003). This accelerated mechanism leads tothe formation of dopamine-quinones and hydrogen peroxide,related to the oxidative stress process (Bilska et al., 2007).Bagis et al. (2005) demonstrated significantly higher serumlevels of pentosidine and malondialdehyde, together withserum superoxide dismutase reduction in patients withchronic pain as compared with normal controls. This genera-tion of advanced glycation end products that results from theincreased nitrodative stress activates transcription factorNF-kb, leading to pro-inflammatory gene expression (Pall,2007). It includes expression of cytokines and growth factorsby macrophages and mesangial cells (IL-1b, IGF-1, TNF-a). Inthe present study, lipid peroxidation was significantlyincreased whereas the levels of nonprotein thiols, superoxidedismutase and catalase were significantly decreased in thecerebral cortex and hippocampus of reserpinised rats. Cur-cumin inhibited lipid peroxidation and restored endogenousantioxidant profile in a dose-dependent manner signifying itsanti-oxidant potential and this is in addition to the powerfulscavenger activity of curcumin for the superoxide anion, thehydroxyl radical and peroxynitrite (Unnikrishnan and Rao,1995). In this study, nitrite levels were also significantlyincreased in cerebral cortex and hippocampus regions ofreserpine administered rats suggesting that NO is an impor-tant messenger molecule in signal transduction pathwaysthat enhance nociceptive transmission in the central nervoussystem (Wu et al., 2001). Curcumin decreased nitrite levels inreserpinised rats.

Nitrodative stress is also linked to the generation ofinflammatory cytokines and NF-kb. We also found increasedlevels of IL-1b and TNF-a in the reserpinised rats and ourfindings are in concurrence with the Huang et al. (2004), whofound increased IL-1b levels in brains of reserpinised rats.Szelenyi et al. (2000) reported dramatically increased TNF-alevels in lipopolysaccharide treated mice on treatment withreserpine. Recently, Uceyler et al. (2007) had also reportedthat the patients with the complex regional pain haveincreased mRNA and protein levels for TNF-a. In our study,curcumin significantly reduced TNF-a and IL-1b levels incortex and hippocampus of reserpinised rats which is attrib-uted to the potent anti-inflammatory properties of curcumin(Jain et al., 2009). The current findings are further supportedby results from Cho et al. (2007) who found a significantdecrease in pro-inflammatory cytokines (TNF-a, IL-1b, IL-8)on treatment with curcumin (Cho et al., 2007).

We also observed a significant increase in levels of NF-kb and caspase-3 in the cerebral cortex and hippocampusof reserpine administered rats suggesting a possible role ofapoptotic pathway in reserpine-induced pain depressiondyad. Our findings are supported by observations fromRuster et al. (2005) who found activated NF-kb and higherNe-carboxymethyllysine levels in the serum of patientswith chronic pain (Ruster et al., 2005). Ne-carboxymethyl-lysine is the major advanced glycation end product inhuman tissues and a marker for cumulative oxidativestress. Increased formation of advanced glycation endproducts was observed in patients with chronic pain,and a relation to NF-kb activation was suspected (Heinand Franke, 2002). Kislinger et al. (1999) clearly demon-strated that Ne-carboxymethyllysine adducts are ligands ofthe receptor for advanced glycated endproducts (RAGE).RAGE has been demonstrated to convert short-lastingredox-dependent signals to a sustained cellular responseby perpetuated activation of NF-kb (Bierhaus et al., 2001).In the present study, treatment with curcumin significantlyinhibited both NF-kb and caspase-3 in cerebral cortex andhippocampus of reserpine treated rats. These results are inaccordance with the studies done by Bharti et al. (2003)who suggested curcumin as a potent inhibitor of nucleartranscription factor kb in several cell types (Bharti et al.,2003).

4.1. Possible mechanism of curcumins action

The results of the present study raised the possibility thatcurcumin showed multiple effects by virtue of its strong anti-inflammatory and antioxidant properties. Moreover, curcu-min mediated increase in monoamine transmission may be astep in a potentially complex cascade of events that ulti-mately results in antidepressant and anti-nociceptive activ-ities (Bhutani et al., 2009).

Conclusively, the findings from the current study sug-gested that reserpine-induced neurochemical alterationsand nitrodative-inflammatory cascade-induced apoptoticsignalling may be responsible for inducing pain symptomsand associated depression in rats. Curcumin being a multi-targeted compound and thereby blocking various steps of thiscascade has a potential to attenuate paindepression syn-drome in rats. However, further studies are needed to clarifythe mechanism of curcumin action in the reserpinized animaland to establish the clinical effectiveness of curcumin inpatients suffering from paindepression dyad.

Contributors

All authors were involved in drafting the article or revising itcritically for important intellectual content, and all authors

Curcumin and Pain Depression Dyad 1579approved the final version to be published. Dr Kanwaljit hadfull access to all of the data in the study and takes respon-sibility for the integrity of the data and the accuracy of thedata analysis.

Study conception and design: Vipin Arora, Anurag Kuhad,Kanwaljit Chopra.

Acquisition of data: Vipin Arora, Vinod Tiwari.Analysis and interpretation of data: Vipin Arora.

Role of the funding source

UGC provided research fellowship for meritorious students toMr Vipin Arora and contingency to procure chemicals, kits andanimals.

Conflict of interest

Authors have no conflict of interest.

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Curcumin and Pain Depression Dyad 1581

Curcumin ameliorates reserpine-inducedpain-depression dyad: Behavioural, biochemical, neurochemical and molecular evidencesIntroductionMaterials and methodsAnimalsDrugsExperimental designBehavioural testsBiochemical estimationsEstimation of lipid peroxidationEstimation of non protein thiolsEstimation of superoxide dismutaseEstimation of catalaseNitrite estimation

Neurotransmitters estimationTNF-, IL-1 and substance P ELISAQuantification of NF- p65 unitCaspase-3 colorimetric assayStatistical analysis

ResultsEffect of curcumin on behavioural paradigmsModulation of thermal hyperalgesiaModulation of mechanical hyperalgesiaEffect on mechanical allodynia

Effect on immobility period in Forced swim testEffect of curcumin on neurotransmitter levelsEffect of curcumin on substance P levelsEffect of curcumin on biochemical indicesEffect of curcumin on reserpine-induced changes in lipid peroxidationEffect of curcumin on reserpine-induced changes in the anti-oxidant profileEffect of curcumin on reserpine-induced nitrosative stress

Effect of treatment on TNF- and IL-1 levelsEffect of curcumin on brain TNF- levelEffect of curcumin on brain IL-1 levels

Effect of curcumin on nuclear factor kappa beta (NF-)Effect of curcumin on caspase-3 activity

DiscussionPossible mechanism of curcumin's action

ContributorsRole of the funding sourceConflict of interestReferences

Curcumin A

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