ORIGINAL ARTICLE

Curcumin Protects Neuronal Cells from JapaneseEncephalitis Virus-Mediated Cell Death and also InhibitsInfective Viral Particle Formation by Dysregulationof Ubiquitin–Proteasome System

Kallol Dutta & Debapriya Ghosh & Anirban Basu

Received: 13 February 2009 /Accepted: 22 April 2009 /Published online: 12 May 2009# Springer Science + Business Media, LLC 2009

Abstract Japanese encephalitis (JE) is an arboviral diseasecommon in Southeast Asia encompassing a population of 3billion people. Periodic outbreak of JE takes hundreds oflives. Children are major victims of JE. About one third ofJE patients die, and many of the survivors suffer frompermanent neuropsychiatric sequel, owing to the lack ofspecific therapeutic measure. Curcumin is a naturallyoccurring phenolic compound extracted from Curcumalonga L. Previous studies have reported that curcuminpossesses strong antioxidant, anti-inflammatory, antiviralactivity. We used Neuro2a cell line and infected them withJE virus. The infected cells were treated with varying dosesof curcumin. Cell viability, reactive oxygen species (ROS)production within the cells, and change in cellular mem-brane integrity were studied. The changes in expression ofsome signaling and stress-related proteins were alsoassessed. We also studied the inhibitory role of curcuminon the production of infective viral particles by dysregula-tion of the ubiquitin–proteasome system. In this study, wefound that curcumin imparts neuroprotection in vitro,probably by decreasing cellular reactive oxygen specieslevel, restoration of cellular membrane integrity, decreasingpro-apoptotic signaling molecules, and modulating cellularlevels of stress-related proteins. We have also shown thatcurcumin, by inhibition of ubiquitin–proteasome systemcauses reduction in infective viral particle production frompreviously infected neuroblastoma cells.

Keywords Japanese encephalitis . curcumin . plaque assay .

neuroprotection . reactive oxygen speciesubiquitin–proteasome system

Introduction

Japanese encephalitis virus (JEV) is an acute zoonoticinfection that commonly affects children and is a majorcause of acute encephalopathy (Chen et al. 2002). JEV isactive over a vast geographic area that includes India,China, Japan, and virtually all of Southeast Asia. Approx-imately three billion people live in the JEV endemic areacovering much of Asia, with nearly 50,000 cases ofJapanese Encephalitis (JE) reported each year (Kaur andVrati 2003). JEV, like many other viruses, initiate oxidativestress in infected cells (Betteridge 2000). Higher concen-trations of ROS damage proteins, DNA, and lipids(Halliwell 2001; Lombard et al. 2005) and initiatesapoptotic pathways (Ciriolo 2005; Halliwell et al. 1992;Swarup et al. 2008). Although there are no clear reports ofthe involvement of ubiquitin–proteasome system (UPS) inthe life cycle of JEV, UPS, besides being a fundamentalmachinery in the cell, has been shown to be involved in thereplication, virion maturation, and budding of several otherviruses (Galinier et al. 2002; Taylor et al. 2007; Si et al.2008; Yu and Lai 2005).

Curcumin is a phenolic compound extracted from therhizome of Curcuma longa L. and is commonly used in theAsian continent (Calabrese et al. 2008). It has been reportedto have anti-inflammatory, antioxidant, and antiproliferativeproperties by modulating multiple cellular machineries. Itinhibits several intracellular signaling pathways, includingthe MAPKs, PI3K/PKB, and pNFκβ (Joe et al. 2004). Inaddition, recent evidence has demonstrated that exposure to

J Neuroimmune Pharmacol (2009) 4:328–337DOI 10.1007/s11481-009-9158-2

K. Dutta and D. Ghosh contributed equally to this work.

K. Dutta :D. Ghosh :A. Basu (*)National Brain Research Centre,Manesar,Haryana 122050, Indiae-mail: anirban@nbrc.ac.in

curcumin leads to dysregulation of the UPS (Jana et al.2004). This property has been utilized to demonstrate animportant antiviral effect of curcumin (Si et al. 2007, 2008).

Till date, therapy for JE is entirely supportive, and noeffective antiviral agents exist. Therefore, the search is onfor compounds that are cheap, easily available, and with noor tolerable side effects (Ghosh and Basu 2008). In thisstudy, we demonstrate that the antioxidative property ofcurcumin renders substantial cytoprotection in neuronal cellline. We also demonstrate that curcumin reduces formationof infective viral particles from previously infected cell,probably by dysregulation of UPS.

Materials and methods

Determination of cell viability

Cell viability was assessed using the [3-(4,5-dimethylth-iazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] (MTS; Promega, USA) assay asdescribed earlier (Rahmani et al. 2005). Mouse neuroblas-toma (N2a) cells were plated onto 96-well plates at adensity of 2 × 104 cells/well. Cells were divided into mock-infected (control), JEV-infected, and three JEV-infectedcurcumin-treated groups. Cells of all the groups, exceptcontrol, were inoculated with JEV (multiplicity of infection=5) and incubated for 1 h. Subsequently, the cells were washedfree of the virus with serum-free media (SFM), and the threegroups marked for curcumin treatment were treated with 1, 5,and 10μM doses of curcumin for 8 h. Cells were then againwashed with SFM and incubated for 24 h. Twenty microlitersof MTS solution was then added in each well. After 4-h incubation, the absorbance the reflecting reduction of MTSby viable cells was determined at 490 nm. Values wereexpressed as a percentage relative to those obtained incontrols.

Terminal deoxynucleotide transferase-mediated dUTPnick-end labeling (TUNEL) assay

N2a was plated at a density of 5×104 cells/well of eight-well chamber slides (Nunc, Denmark) and treated asdescribed for MTS assay for 5μM dose of curcumin.Apoptotic cells were identified in situ, using Cell DeathDetection Kit, TMR red (Roche, Germany) as describedpreviously (Mishra et al. 2009). Briefly, the cells were fixedwith 4% paraformaldehyde in phosphate-buffered saline(PBS) and blocked with 4% bovine serum albumincontaining 0.02% Triton-X-100. The fixed cells were thenincubated in TUNEL mix (terminal deoxynucleotidyltransferase in storage buffer and TMR red labeled-nucleotide mixture in reaction buffer) for 1 h at room

temperature. The slides were mounted with Vectashieldmounting media containing 4′,6-diamidino-2-phenylindole(Vector Laboratories, California).

Measurement of ROS

The level of ROS produced within cells of control and eachtreatment groups were measured by the cell permeable,non-polar, H2O2-sensitive probe 5(and 6)-chlromethyl-20,70-dichlorodihydrofluoresceindiacetate (CM-H2DCFDA; Sigma, USA) by the method described previously(Schreck and Baeuerle 1994). Briefly, N2a cells were platedin five 90-mm plates at a density of 5× 105 cells permilliliter (after triplicate counts with a haemocytometer)and were treated as described above. Before lysing the cellswith lysis buffer, the cells were treated with 5μM solutionof CM-H2DCFDA and incubated at room temperature for45 min. The cells were then washed with ice-cold PBS, andthe protein isolated was used to measure the relativefluorescence with the help of Varioskan Flash multimodereader at excitation 500 nm and emission 530 nm (ThermoElectron, Finland). The fluorescence intensity of intracellu-lar DCFDA is a linear indicator of the amount of H2O2 inthe cells.

Measurement of membrane fluorescence anisotropy

Membrane fluidity of N2a cells was determined using thefluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH) asdescribed previously (Mishra et al. 2009). Briefly, N2a cellswere cultured and treated as described above and thenincubated with 1μmol/L DPH in PBS for 2 h at 37°C,following which cells were washed and resuspended inPBS. Spectrofluorometer recorded the fluorescence emis-sion of the DPH probe bound to the cell membrane at430 nm after exciting the cell at 365 nm. The fluorescenceanisotropy (r) value was calculated using the equation r=[(I||−I┴)/(I||+2I┴)], where I|| and I┴ are the fluorescenceintensities oriented parallel and perpendicular, respectively,to the direction of polarization of the excited light. Themicroviscosity parameter [(ro/r)−1]−1 was calculated ineach case knowing the maximal limiting fluorescenceanisotropy ro=0.362 for DPH.

Assay of proteasome activity

After culturing, virus infection, and subsequent curcu-min treatment as stated above, N2a cells were isolatedand suspended in 100µl of proteasome assay buffercontaining 10 mM Tris, pH7.4, 1 mM EDTA, 5 mMATP, 5 mM dithiothreitol, and 20% (v/v) glycerol, lysedby sonication, and then centrifuged at 15,000×g for 15 minat 4°C. The supernatant (containing 25µg protein) was

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incubated in the proteasome activity assay buffer (50 mMTris, pH7.4, 0.5 mM EDTA, and 50µM of each protea-some substrate) for different time periods to obtainlinearity of the reaction. The substrates Suc-Leu-Leu-Val-Tyr-MCA and Z-Leu-Leu-Glu-MCA were used to deter-mine chymotrypsin and post-glutamyl peptidyl hydrolytic(PGPH)-like activity, respectively (Jana et al. 2004).Protease activities at a particular time point (30 min)within the linear range were used to calculate the data. Thefluorescence intensity was measured at 380-nm excitationand 460-nm emissions by using Varioskan Flash multi-mode reader.

Immunoblot

N2a cells were plated in five 90-mm plates at a density of5× 105 cells per milliliter (after triplicate counts with ahaemocytometer) and were treated as described above.Cells from each treatment groups were washed twice withice-cold 1× PBS, then lysed in buffer containing 1% Triton-X-100, 10 mM Tris–HCl (pH8.0), 150 mM NaCl, 0.5%Nonidet P (NP-40), 1 mM EDTA, 0.2% EGTA, 0.2%sodium orthovanadate, and protease inhibitor cocktail(Sigma). Protein levels were determined by Bradfordmethod. Twenty micrograms of each sample was electro-phoresed on polyacrylamide gel and transferred onto anitrocellulose membrane. After blocking with 7% skimmedmilk, the blots were incubated overnight at 4°C withprimary antibodies against phospho-ERK1/2, phospho-p38MAPK, phospho-JNK, phospho-NFκβ (Cell-Signaling,USA), HSP70, SOD-1 (Santa Cruz Biotechnology, Cal-ifornia, USA), TRX-1 [(Ab Frontiers, Korea), a kind giftfrom Dr Ellora Sen, National Brain Research Center(NBRC)], and ubiquitin (a kind gift from Dr. Nihar RanjanJana, NBRC). After extensive washes in PBS–Tween, blotswere incubated with appropriate secondary antibodiesconjugated with peroxidase (Vector Laboratories). Theblots were again washed in PBS–Tween and processed fordevelopment using chemiluminescence reagent (Millipore,USA). The images were captured and analyzed usingChemigenius, Bioimaging System (Syngene, Cambridge,UK; Swarup et al. 2008). The blots were stripped andreprobed with anti-β-actin (Sigma) to determine equivalentloading of samples.

Plaque assay

Inhibition of infective viral particle formation in curcumin-treated cells was determined by plaque assay. N2a wereplated in 90-mm plates at a density of 5×105 cells permilliliter (after triplicate counts with a haemocytometer).Cells were divided into mock-infected (control), JEV-infected, and two JEV-infected curcumin-treated groups.

Cells of all the groups, except control, were inoculated withJEV (multiplicity of infection=5) and incubated for 1 h.Subsequently, the cells were washed free of the virus withSFM, and the two groups marked for curcumin treatmentwere treated with 5 and 10μM doses of curcumin for 8 h.Cells were then again washed and incubated in equalvolume of SFM for 48 h.

Porcine stable (PS) kidney cells were seeded in six-well culture plates and grown to a monolayer. Fivehundred microliters of supernatants from the control,JEV, and JEV–curcumin-treated N2a cells were admin-istered on separate PS cultures and incubated for 1 h at37°C with occasional shaking. The inoculum wasremoved by aspiration, and the monolayers wereoverlaid with minimum essential media containing 4%fetal bovine serum, 1% low-melting-point agarose and acocktail of antibiotic/antimycotic solution (Gibco, Invi-trogen, New York, USA) containing penicillin, strepto-mycin, and amphotericin B. The plates were incubatedat 37°C for 3 days until plaques became visible. Thecells were fixed with 10% paraformaldehyde and stainedwith crystal violet, and the plaques were counted(Swarup et al. 2008).

Determination of reduction in production of infective JEVparticle in previously infected Neuro2A cells by curcumin

N2a cells were plated in 90-mm plates at a density of 5×105 cells per milliliter (after triplicate counts with ahaemocytometer). Cells were divided into mock-infected,JEV-infected, two JEV-infected curcumin-treated, andmock-infected–curcumin-treated groups. Except the mockgroups, all the cells were incubated with JEV for 1 h,washed, and then the curcumin-treated groups wereincubated with curcumin (5 and 10µM) for 8 h, followedby washing and incubation with equal volume of SFM for48 h. Cells were then scrapped and collected along with theincubating media and homogenized using an Ultra Turraxhomogenizer. The homogenates were then filtered bysyringe filter (MDI), and aliquots of the filtrates wereinjected in equal volume, intracerebrally, into 6-day-oldmice pups (BALB/c). The survivality of these animals werethen monitored. All experiments were performed accordingto the protocol approved by the Institutional Animal EthicsCommittee of NBRC.

Statistical analysis

Statistical analysis was performed using SIGMASTATsoftware (SPSS Inc., Chicago, IL, USA). Data werecompared between groups using one-way, model 1 analysisof variance Fisher’s post hoc test. Differences with P<0.05were considered significant.

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Results

Curcumin is effective in reducing JEV-induced cell death

To determine whether curcumin has any cytoprotective rolein JE, MTS assay was done on JEV-infected and JEV-infected and curcumin-treated cells (Fig. 1a). The cellviability was found to increase in curcumin-treated cells as

compared to only virus-infected cells, but the viabilitycorresponded maximally for a dose of 5μM of curcumin.

Because maximum viability was observed with the5-μM dose, we performed TUNEL assay to observe theapoptotic phenomena in JEV-infected and JEV-infected andcurcumin-treated cells. The results showed a markeddecrease in TUNEL-positive cells after curcumin treatment(5μM), as compared to only JEV-treated cells (Fig. 1b–e).

Fig. 1 Curcumin imparts cytoprotection following JEV infection. aViability of control, only JEV-infected and JEV-infected andcurcumin-treated N2a cells were determined by MTS assay. Thefigure clearly depicts that curcumin renders significant cytoprotectionto the JEV-treated N2a cells at 5 and 10μM doses. The graphrepresents the percentage viability of cells. Values represent mean+SDfrom three independent experiments. b–d Apoptotic cell death assayof N2a cells showing TUNEL-positive cells. A profound increase inthe number of TUNEL-positive cells was observed when N2a wascultured in the presence of JEV (c). N2a cells cultured in the presenceof JEV and treated with 5μM dose of curcumin showed a drastic

decrease in TUNEL-positive cells (d). Scale bars 25μm (magnifica-tion, ×20). The figures are representative of three independentexperiments, performed in duplicate. e Graphical representation ofpercentage of TUNEL-positive cells. JEV infection leads to 50%increase in TUNEL-positive cells compared to control N2a cells.Following treatment with 5μM curcumin, the percentage of TUNEL-positive cells was decreased by about 50% of JEV-infected cells.Values represent mean+SD from three independent experiments,performed in duplicate. (*P< 0.01 for JEV-infected relative to control;#P<0.01 for JEV-infected curcumin-treated relative to only JEV-infected)

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Curcumin alleviates oxidative stress in JEV-infected cells

Western blot analysis of proteins associated with themaintenance of the redox balance in cells showed increasedexpression of SOD-1and HSP-70 in JEV-infected andcurcumin-treated cells (Fig. 2a, b). In case of SOD-1, the5 μM dose showed maximal effect. The HSP-70 levels werefound to be significantly increased in 5 and 10μM doses.Furthermore, the level of TRX-1 was diminished followingcurcumin treatment of JEV-infected cells (Fig. 2b).

Because ROS have been suggested as a possible mediator ofcell damage in JEV infection, we examined ROS production inJEV-infected and JEV-infected curcumin-treated cells using thefluorescent probe DCFDA. More than twofold increase wasobserved in intracellular ROS level after JEV infection, but thislevel was significantly decreased in curcumin-treated cells

(Fig. 2c). As generation of ROS affects cell membraneintegrity, we next determined membrane fluidity of control,JEV-infected, and JEV-infected curcumin-treated cells usingDPH. A significant decrease was observed in the membranemicroviscosity of JEV-infected N2a cells, which was reversedafter curcumin treatment. It was observed that membranemicroviscosity was significantly increased by application of1μM curcumin (threefold increase as compared to only JEV-infected cells), but 5 and 10μM doses resulted in approxi-mately a twofold increase (Fig. 2d).

Curcumin modulates the expression pattern of pJNK,phospho-p38MAPK, pERK-1/2 and pNFκβ

Western blot analysis demonstrated a significant inhibitionin the expression of different signaling proteins upon

Fig. 2 Curcumin reduces JEV-induced oxidative stress. a Westernblot analysis demonstrates induction of SOD-1, TRX-1, and HSP-70in N2a cell following JEV infection. Curcumin treatment induceslevels of SOD-1 and HSP-70 significantly in particular doses.However, TRX-1 level is down-regulated after curcumin treatment.b Densitometric analysis of individual immunoblots for each antibodytested. The graph represents the fold change in the expression levels ofSOD-1, TRX-1, and HSP-70 in JEV-infected and JEV-infectedcurcumin-treated N2a cells over control. Values represent mean+SDfrom three independent experiments performed in duplicate. c Fromsample containing equal amounts of protein isolated from control andtreated N2a cells, ROS levels were measured using H2O2-sensitiveprobe DCFDA. ROS level were found to be dramatically increased inJEV-infected cells. Curcumin treatment of JEV-infected cells signif-

icantly reduced the ROS level. The graph indicates the fold change inthe level of ROS in JEV-infected and JEV-infected curcumin-treatedcells over control. Values represent mean+SD from three independentexperiments performed in duplicate. d Membrane microviscosity ofJEV-infected and JEV-infected curcumin-treated N2a cells werecompared with control. Fluorescence intensity of cells labeled withDPH was measured, and membrane microviscosity was determined.As microviscosity and fluidity are inversely correlated, these resultsindicated an increase in membrane fluidity of N2a cells infected withJEV that was reversed by curcumin treatment. Values representmean+SD from three independent experiments performed in duplicate(e) (*P<0.01 for JEV-infected relative to control; #P<0.01 for JEV-infected curcumin treated relative to only JEV infected)

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curcumin treatment, whose levels were elevated followingJEV infection (Fig. 3a, b). There was a significant increasein the expression of pJNK, phospho-p38 MAPK, phospho-ERK-1,2, and pNFκβ in JEV-infected mice when comparedwith control. However, curcumin treatment significantlyreduced their levels. The reduction was found to be dose-dependent in case of phospho ERK-1 and 2 (Fig. 3c). Incase of phospho-p38 MAPK, both 5 and 10μM doses wasfound to be quite effective. In case of pJNK, all three doseswere found to be equally effective with no significantdifference in reduction by them. For pNFκβ, the 5- and 10-μM doses was found to be most effective.

Inhibition of UPS by curcumin in JEV-infected cells

To explore the potential roles of the UPS in mediating theantiviral activities of curcumin, we examined the effect ofcurcumin on the UPS. As shown in Fig. 4a, curcumintreatment resulted in an increased accumulation of protein–ubiquitin conjugates, which was accompanied by a decreasein the free ubiquitin level. Because ubiquitinated proteins are

degraded by the proteasome, increased accumulation ofubiquitinated proteins would result in inhibition of protea-some activity. Thus, we measured chymotrypsin and PGPH-like protease activity of the proteasome and observed that theactivity of both enzymes was dramatically inhibited bycurcumin in a dose-dependent manner [Fig. 4b (i and ii)].

Curcumin inhibits production of infective JEV particlein previously infected Neuro2A cells

Plaque assay was performed to directly assess the role ofcurcumin in reducing the viral titer within neuroblastomacells. Treatment of PS cells with supernatant of JEV-infected and JEV-infected curcumin-treated N2a cellsshows that, in the latter case, the number of plaques formedon the monolayer bed of PS cells was much less. Theresults indicate that, at 5 and 10μM doses of curcumin,there was a significant reduction in virus production inpreviously infected N2a cells (Fig. 4c).

In vivo determination of infective JEV particle produc-tion in infected neuroblastoma cells following curcumin

Fig. 3 The effect of JEV infection and subsequent curcumin treatmenton the expression of pJNK, phospho-p38MAPK, pERK-1/2, andpNFκβ. a, b Western blot analysis demonstrates significant inductionof pJNK, phospho P38MAPK, pERK-1/2, and pNFκβ over control inN2a cell line following JEV infection. Curcumin treatment followingJEV treatment reduces levels of pJNK, phospho-p38MAPK, pERK-1/2, and pNFκβ significantly in particular doses. c Densitometric

analyses were performed on individual immunoblots for each antibodytested. The graph represents the fold change in the expression levels ofpJNK, pP38MAPK, ERK-1/2, and pNFκβ in JEV-infected and JEV-infected and curcumin-treated N2a cells over control. Values representmean+SD from three independent experiments performed in duplicate(*P<0.01 for JEV-infected relative to control; #P<0.01 for JEV-infected curcumin treated relative to only JEV infected)

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Fig. 4 Curcumin likely inhibits the production of infective JEVparticle from infected neuroblastoma cells through the dysregulationof the ubiquitin–proteasome system. a Curcumin treatment resulted inan increased accumulation of protein–ubiquitin conjugates, which wasaccompanied by a decrease in the free ubiquitin level. b Curcumininhibits 20S proteasome activities. The activities of 20 S proteasome,including (i) chymotrypsin-like and (ii) PGPH-like activities, wereestimated in N2a cell lysate of JEV-infected and JEV-infectedcurcumin-treated cells. There is no substantial difference in theproteasome activities in the JEV-infected cell from the control.Curcumin treatment of the JEV-infected N2a cells causes significantdecrease in the 20S proteasome (both chymotrypsin-like and PGPHlike) activities. Values represent mean+SD from three independentexperiments performed in duplicate. (#P< 0.01 for JEV-infectedcurcumin treated relative to only JEV-infected). c Curcumin reduced

the production of infective viral particles from N2a cells. There was asignificant decrease in viral titer following incubation with curcuminin the JEV-infected N2a cells, which is manifested by a decrease in thenumber of plaque forming units. Values represent mean±SD fromthree independent experiments performed in duplicate. (#P<0.01 forJEV-infected curcumin treated relative to only JEV infected; Curcurcumin). d N2a cells were infected with JEV and treated withcurcumin as described in “Materials and methods.” Cells werecollected along with the incubating media and homogenized. Thehomogenates were filtered, and equal volume of the filtrate wasinjected intracerebrally into mice pups, and their survival wasobserved. Data represented as mean+SD from three independentexperiments performed in duplicate; n=3. (*P<0.01 for JEV-infectedrelative to control; #P<0.01 for JEV-infected curcumin treated relativeto only JEV infected; M mock-infected)

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treatment showed that curcumin at 5 and 10μM dosesreduces production of infective JEV particle in N2a cells.The homogenates from only JEV-infected and JEV-infectedcurcumin treated N2a cells killed the pups at different timepoints, whereas the pups that were injected with mock-infected or mock-infected curcumin-treated cells showedcomplete survival. The result showed a significant increasein the survival time of the pups treated with homogenatesfrom 5 and 10μM curcumin-treated JEV-infected N2acells, when compared with the survival time of pupsreceiving homogenates from only JEV-infected N2a cells(Fig. 4d).

Discussion

The anti-inflammatory, antioxidant, and antiproliferativeproperties of curcumin have led to several investigationsregarding the efficacy of this natural compound for thetreatment of diverse diseases. The neuroprotective role ofcurcumin has also been under investigation (Bala et al. 2006;Cole et al. 2007). These studies have shown that curcuminhas an outstanding safety profile and a number of pleiotropicactions with substantial potential for neuroprotective effica-cy. Recently, the antiviral effects of curcumin have generatedinterest with studies reporting promising results in in vitromodels of coxsackievirus (Si et al. 2007) and herpes simplexvirus infection (Kutluay et al. 2008).

In this study, we report for the first time that curcumin,in controlled doses, (1) imparts protection to N2a cellsagainst JEV, (2) the cytoprotection rendered may be due torestoration of intracellular redox balance and membraneintegrity, (3) helps to reverse the elevated level of stressrelated signaling molecules, and (4) reduces formation ofinfective JEV particle from previously infected N2a cell,probably by dysregulation of UPS.

Curcumin was found to possess cytoprotective effects onneuroblastoma cell line. After infection with JEV, cellsshowed a significantly reduced viability, which wasreversed by curcumin treatment. JEV is known to mediateneuronal death by inducing apoptosis. In confirmation withthe previous studies, TUNEL assay revealed significantincrease in apoptotic cells following JEV treatment (Mishraet al. 2009). We found a significant decrease in apoptoticcells following curcumin treatment of JEV-infected cells.The dosage of curcumin seems to be an important factor inthe evaluation of cytoprotection by curcumin. Any dosehigher than 10μm of curcumin has been shown to reduceviability of neuroblastoma cells (data not shown). Thus, ourchosen doses were either less than or equal to 10μM. Wefound that 5μM concentration of curcumin was highlyeffective in preventing JEV-induced cell death, in ourexperimental model. ROS are multi-potent diffusible

molecules capable of carrying out several signal transduc-tion processes in response to several extracellular stimuli.ROS are multi-potent diffusible molecules capable ofcarrying out several signal transduction processes inresponse to several extracellular stimuli. It has beenreported earlier that JEV infections induce the level ofROS both in vivo and in vitro (Mishra et al. 2009; Raung etal. 2001). We show in this study that curcumin’s protectivefunction is probably because of its antioxidant property andis associated with reduction in the level of ROS bycurcumin in JEV-infected cells, which may in turn reduceJEV-mediated cell death. ROS production increases plasmamembrane fluidity (Sergent et al. 2005). Alterations inplasma membrane fluidity in cells is also associated withapoptosis and exposure to various apoptotic stimuli(Fujimoto et al. 1999; Gorria et al. 2006). In this study,curcumin’s antioxidative action can be responsible for therestoration of membrane microviscosity and reduction ofmembrane fluidity in the JEV-infected cells. Curcuminelevates level of stress-related proteins like HSP-70 andSOD-1 significantly. Previous reports suggests that HSP-70and SOD-1 helps to ameliorate the oxidative stressgenerated by wide array of causes and also protects thecells from stress-induced apoptosis. SOD-1 was also beenreported to have been studied as a therapeutic agent forpost-ischemic condition (Agee et al. 1991; Halliwell 1992).Reports also show alleviation of inflammation-relatedsignaling molecules by HSP-70 (Gabai et al. 1997; Endoet al. 2007). Thus, curcumin causes elevation of SOD-1 andHSP-70, which may help to ameliorate the oxidative stressin the JEV-infected cells. TRX-1 have antioxidant role, asreported earlier (Billiet and Rouis 2008), but in this study,we observed that curcumin treatment at 1 and 5μM dosesdid not cause any significant difference in TRX-1 level incomparison to only JEV-treated samples. Interestingly,curcumin at 10μM dose treatment causes down-regulationof TRX-1.

Most apoptotic stimuli, including JEV infection, areknown to activate stress kinase pathways, leading toactivation of ERK1/2, JNK, and p38 MAPK pathway(Mishra et al. 2009; Su et al. 2002; Swarup et al. 2007a). Inthe present communication, we show that curcumin mayprevent apoptosis in JEV-infected N2a cells by significantlydown-regulating the level of phospho-p38MAPK, phospho-ERK1/2, and phospho-JNK, following JEV infection.Moreover, activation of pNFκβ regulates apoptotic genes,especially the TRAF1 and TRAF2, and thereby checks theactivities of the caspases, which are central to mostapoptotic processes. JEV is known to activate pNFκβ viaa PI3K-dependent pathway in the brain of infected animals,which is associated with apoptosis (Swarup et al. 2007b).The protective effect of curcumin may also be due toreduction of pNFκβ level induced by JEV infection.

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The UPS is fundamental machinery in the cell and hasbeen shown to be involved in the replication, maturation,assembly, and budding of several viruses. Moreover,inhibition of proteasome activity and reduction of cellularpool of free ubiquitin by proteasome inhibitors like MG132inhibits production of infective viral particles (Yu and Lai2005; Kasparia et al. 2008; Zhadina et al. 2007). Inconfirmation of the data from the previous studies (Jana etal. 2004), we found that curcumin treatment significantlydecreases proteasome action by decreasing chymotrypsinand PGPH-like protease activity of the proteasome. Mostimportantly, we found that the production of infective virusparticle from previously infected N2a cells were signifi-cantly decreased following curcumin treatment (Fig. 4c, d).We harvested the virus (of same multiplicity of infection) inequal number of N2a cells and incubated with same volumeof SFM for equal time span. All other conditions remainingsame, the production of the infectious virus particles insidethe cells depended only on one variable—the curcumintreatment. UPS is important for infective viral particleproduction, and we have demonstrated that curcumin issignificantly inhibiting proteasomal function and reducingcellular-free ubiquitin pool. Thus, the reduced infectiveviral particle formation following curcumin treatment couldbe attributed to its inhibition of UPS, though other factorsmay be responsible.

Acknowledgements This work is funded by the grant from theDepartment of Biotechnology, Government of India to A. B. (award nos.BT/PR/5799/MED/14/698/2005 and BT/PR8682/Med/14/1273/2007). Weare grateful to Dr. Nihar Ranjan Jana for his critical feedback.We thankMs.Richa Tewari and Mr. Ranjan Maity for their help.

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