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JAC Advance Access originally published online on January 29, 2008
Journal of Antimicrobial Chemotherapy 2008 61(3):679-688; doi:10.1093/jac/dkm503
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© The Author 2008. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Original research

Novel strategy for treatment of Japanese encephalitis using arctigenin, a plant lignan

Vivek Swarup{dagger}, Joydeep Ghosh{dagger}, Manoj Kumar Mishra and Anirban Basu*

National Brain Research Centre, Manesar, Gurgaon, Haryana 122050, India

* Corresponding author. Tel: +91-124-2338921 ext. 225; Fax: +91-124-2338910/28; E-mail: anirban{at}nbrc.res.in

Received 11 September 2007; returned 27 November 2007; revised 15 October 2007; accepted 29 November 2007


   Abstract
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 Abstract
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 Materials and methods
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 Discussion
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Objectives: To evaluate therapeutic efficacy of arctigenin in an experimental model of Japanese encephalitis (JE).

Methods: Four- to 5-week-old BALB/c mice of either sex were infected intravenously with lethal dose of 3 x 105 pfu of Japanese encephalitis virus (JEV). By the 9th day post-infection, all untreated animals succumbed to the infection. Arctigenin was dissolved in DMSO at a concentration of 0.5 mg/mL and stored at 4°C. After one day following virus inoculation, animals were given arctigenin intraperitoneally, twice daily (10 mg/kg of body weight) for next 7 days.

Results: Treatment with arctigenin provided complete protection against experimental JE. Arctigenin’s neuroprotective effect was associated with marked decreases in: (i) viral load; (ii) active caspase-3 activity; (iii) reactive oxygen species and reactive nitrogen species; (iv) microgliosis and proinflammatory cytokines; (v) levels of stress-associated signalling molecules; and (vi) neuronal death. Furthermore, treatment with arctigenin also improves the behavioural outcome following JE.

Conclusions: In conclusion, our findings provide a novel mechanistic insight into the actions of arctigenin in JE. Results from our in vivo and in vitro experiments clearly indicate that arctigenin reduced (i) viral load and viral replication within the brain, (ii) neuronal death and (iii) secondary inflammation and oxidative stress resulting from microglial activation, thereby suggesting its potential for treating JE. The antiviral, neuroprotective, anti-inflammatory and antioxidative effects of arctigenin successfully reduced the severity of disease induced by JEV.

Keywords: caspase-3 , microglia , apoptosis , cytokine , reactive oxygen species


   Introduction
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 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Funding
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 References
 
Flaviviruses are important human pathogens causing a variety of diseases ranging from mild febrile illness to severe encephalitis and haemorrhagic fever. Among them, Japanese encephalitis virus (JEV), a neurotropic virus, commonly affects children and is a major cause of acute encephalopathy.1 JEV is active over a vast geographic area that includes India, China, Japan and virtually all of South-East Asia. Approximately 3 billion people live in the JEV endemic area covering much of Asia, with nearly 50 000 cases of JE reported each year. Of these, ~10 000 cases result in death and a high proportion of survivors have serious neurological and psychiatric sequelae.2 Therapy for JE is supportive and no clearly effective antiviral agents exist. Therefore, the search is on for compounds which are cheap, easily available and with no or tolerable side effects combined with a protective potential when administered several hours after infection. Recently, we have identified the antiviral and anti-inflammatory effect of rosmarinic acid in an experimental murine model of JE.3

Arctigenin (AR), naturally occurring in Bardanae fructus, Saussurea medusa, Arctium lappa, Torreya nucifera, Ipomea cairica and Forsythia intermedia, is a phenylpropanoid dibenzylbutyrolactone lignan with antioxidant, anti-inflammatory and antiviral activities.4–6 Arctigenin has been reported to be a biologically active lignan with potent anti-HIV activity.7,8 The lignan also showed anti-influenza virus effect in vivo.9 It has been demonstrated earlier that arctigenin protects cultured cortical neurons from glutamate-induced neurodegeneration by binding to the kainite receptor.10 Arctigenin also showed cytostatic activity against the human leukaemic cell line, HL-60,11 anti-promotional12 and immunomodulatory effects.13

The studies presented here recommend arctigenin as a strong candidate for further consideration as a therapeutic measure to reduce the neurological and inflammation-related complications observed in JE.


   Materials and methods
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 Abstract
 Introduction
 Materials and methods
Results
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AR administration in an animal model of JE

Four- to 5-week-old BALB/c mice of either sex were infected intravenously (via the tail vein) with a lethal dose of 3 x 105 pfu of JE virus (GP78 strain). From the 5th day post-infection, animals started to show symptoms of JE including limb paralysis, poor pain response, restriction of movements, pilo-erection, body stiffening and whole body tremor. By the 8th day post-infection, all untreated animals succumbed to the infection. AR (Tocris Bioscience, UK) was dissolved in DMSO at a concentration of 0.5 mg/mL and stored at 4°C. One day after virus inoculation, animals started receiving AR intraperitoneally, twice daily (10 mg/kg of body weight) for the next 7 days (Figure 1). Observation of animal survival experiments was performed in a blinded manner to avoid bias towards any one group of animals. All experiments were performed according to the protocol approved by the Institutional Animal Ethics Committee of National Brain Research Centre (NBRC).


Figure 1
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  		Figure 1. Treatment regimen of arctigenin in an experimental model of JE. C, control; AR, AR-treated alone (drug control); JEV, JEV-infected; JEV + AR, JEV-infected/AR-treated. Schematic representation depicts AR therapy following JEV infection. On the 8th day post-infection, animals from four different groups were sacrificed for further analysis. Animals found dead due to JEV infection (without AR treatment) were excluded from any analysis. Only infected animals that were terminally ill on the 8th day (at the last stage of survival) were included in the analysis.

 
Plaque assay

For analysis of viral load in CNS of infected mice, brains were recovered after extensive cardiac perfusion with PBS, dissected, cooled on ice, homogenized and titrated for virus by plaque formation on Porcine stable (PS) kidney cell monolayers. PS cells were seeded in 35 mm dishes and formed semi-confluent monolayers after ~18 h. Monolayers were inoculated with 10-fold dilutions of virus sample made in minimal essential medium (MEM) containing 1% FBS and incubated for 1 h at 37°C with occasional shaking. The inoculum was removed by aspiration and the monolayers were overlaid with MEM containing 4% FBS, 1% low-melting-point agarose and a cocktail of antibiotic/antimycotic solution (Gibco, Invitrogen Corporation, NY, USA) containing penicillin, streptomycin and amphotericin B. Plates were incubated at 37°C for 4 days until plaques became visible. The cells were fixed with 10% formaldehyde, stained with crystal violet and the plaques were counted.14

To determine the effect of AR on viral titre in vitro, mouse neuroblastoma Neuro2a cells (N2a) were plated in 6-well plates at a density of 5 x 105 cells/well in 3 mL of medium and were cultured for 18 h. After 18 h in Dulbecco’s modified Eagle's medium (DMEM) with 10% serum, the cells were switched to serum-free media for 12 h. N2a cells were then infected with JEV [multiplicity of infection (moi) = 5] for 1 h. The virus was removed and the plates were then washed with 1x PBS to remove the unbound virus. The plates were further incubated with AR (dissolved in DMSO, final concentration 0.1%)-containing serum-free medium at 37°C. Cells were collected at various time points (6, 12 and 24 h), harvested and the virus titre was determined by plaque assay.15

Immunohistochemistry

On the 8th day post-infection, animals from the JEV-infected, JEV-infected/AR-treated and control (PBS-injected) groups were perfused with PBS containing 7 U/mL heparin, followed by a fixative containing 2.5% PFA in PBS. The brains were then processed for cryostat sectioning. Coronal sections of cerebral cortex were taken. The sections were stained with Iba-1 (1:200, Wako Chemical, Japan), a marker for activated microglia, antibody against the Nakayama strain of JEV (1:200, Chemicon, CA, USA) and active caspase-3 (1:100, BD Biosciences, NJ, USA). The sections were developed using corresponding biotinylated secondary antibody and developed with 3,3'-diaminobenzidine (DAB). The sections were then mounted with distyrene plasticizer xylene (DPX) and were observed under a Leica 4000DB light microscope (Leica Microsystems, Germany) (40x magnification). Cryostat sections from control, JEV-infected and JEV-infected/AR-treated animals were also processed for thionin staining.16

Immunoblot analysis

Western blot analysis was performed with protein isolated from brain tissues of all the four groups of animals [control (PBS-injected), JEV-infected, JEV-infected/AR-treated and AR-treated only (drug control)], on the 8th day post-infection (Figure 1).3 Briefly, 10 µg of each sample was electrophoresed and transferred onto a nitrocellulose membrane. The membranes were then blocked and probed with several primary antibodies that include: anti-JEV Nakayama strain and iNOS (1:1000, Chemicon), ERK-1/2, phospho-ERK-1/2, Akt, phospho-Akt, phospho-c-Jun and phospho-p38 MAPK (1:1000, Cell Signaling Tech., MA, USA), SOD-1 and HSP-70 (1:1000, Santa Cruz, CA, USA) and β-tubulin (1:2500, Sigma, MO, USA). Appropriate HRP-conjugated secondary antibodies were used for all of them. Using chemiluminescence reagent, blots were developed and images were captured and analysed using the Chemigenius Bioimaging System (Syngene, UK).

Measurement of reactive oxygen and nitrogen species (ROS/RNS)

To monitor the level of ROS produced within cells, the cell permeable, non-polar, H2O2-sensitive probe DCFDA (Sigma, USA) was used. To detect ROS in whole brain lysate, supernatant of fresh brain homogenate sample was taken and incubated with DCFDA (5 µM) for 30 min at room temperature.17

To detect RNS, nitric oxide (NO) levels in whole brain lysate were measured. Equal volumes of supernatant of fresh brain homogenate sample and Griess reagent were taken and incubated for 10 min. The absorbance was measured at 540 nm as described previously.18

Cytokine bead array

The BD mouse cytokine bead array kit (mouse inflammation CBA kit; BD Biosciences) was used to quantitatively measure cytokine expression levels in mouse brain tissue lysates. Fifty microlitres of mouse inflammation standard and sample dilutions were used and the assay was performed according to the manufacturer’s instructions and analysed on the FACS Calibur (Becton–Dickinson, NJ, USA). FACS analysis using special CBA software (BD Biosciences) allows the calculation of concentrations in the unknown lysates.19

Infection of Neuro2a cells with JE virus, MTT assay and cytochrome c assay

Mouse neuroblastoma Neuro2a cells (N2a) were plated in 12-well plates at a density of 3 x 105 cells/well in 1 mL of medium and were cultured for 18 h. After 18 h in DMEM with 10% serum, the cells were switched to serum-free media for 12 h. N2a cell line was then infected with JEV (moi = 5) for 1 h. The virus was removed and the plates were then washed with 1x PBS to remove the unbound virus. The plates were further incubated with AR (dissolved in DMSO, final concentration 0.1%)-containing serum-free medium at 37°C for next 24 h. Upon infection and AR treatment, viability of N2a cells was documented using the Trypan blue exclusion test. The viability was also evaluated by MTT assay as described elsewhere.10

To study the release of cytochrome c, N2a cells were mock-infected, JEV-infected (moi 1:5), AR-treated (drug control) or JEV-infected/AR-treated. The mitochondrial fraction was isolated and the cytochrome c assay (Sigma) was performed according to the manufacturer’s instructions.20 The absorbance was measured at 550 nm in a Biorad Microplate reader (Biorad, CA, USA).

Intracellular staining by flow cytometry for JEV-specific antigen

Mouse neuroblastoma Neuro2a cells (N2a) were plated in 6-well plates at a density of 5 x 105 cells/well in 3 mL of medium and were cultured for 18 h. After 18 h in DMEM with 10% serum, the cells were switched to serum-free medium for 12 h. N2a cell line was then infected with JEV (moi = 5) for 1 h. The virus was removed and the plates were then washed with 1x PBS to remove the unbound virus. The plates were further incubated with AR (dissolved in DMSO, final concentration 0.1%)-containing serum-free media at 37°C. Cells were collected at various time points (6, 12 and 24 h). After two washes with 1x PBS, cells were first fixed with BD cytofix solution (BD Biosciences) for 15 min. Then the cells were permeabilized by resuspension in permeabilization buffer (BD Cytoperm plus, BD Biosciences) and incubated at room temperature for 10 min. Cells were washed twice in wash buffer (PBS containing 1% BSA), then resuspended in wash buffer at 1 x 106 cells per 100 µL. Primary antibody (JEV Nakayama strain, Chemicon) were added in 1:100 dilutions and incubated for 30 min at room temperature. The cells were washed five times with wash buffer, pelleted and then incubated with FITC-conjugated secondary antibody for 30 min. After three washes with wash buffer, finally, the samples were resuspended in 400 µL FACS buffer and analysed. Samples were analysed on a FACS Calibur. Fold change was calculated by dividing the median fluorescence intensity of the infected and AR-treated sample by that of the control sample.21 The percentage of population was calculated after gating the populations on Dot plot in Cell Quest Software.

Statistics

All comparisons between groups were performed using one-way ANOVA, with the Bonferroni method for post hoc pairwise multiple comparisons to detect P values <0.05 between individual group means.


   Results
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 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Funding
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 References
 
AR confers complete protection to animal from JE

AR treatment following JEV infection completely prevented mortality (15/15 animals survived following AR treatment in JEV-infected group) (Figure 2). While all infected animals that did not receive any AR treatment succumbed to infection, AR alone (drug control) had no effect on the behavioural outcome of animals (data not shown). Infection with JEV was accompanied with distinct symptoms and treatment with AR following virus infection significantly reduced morbidity.


Figure 2
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  		Figure 2. AR treatment confers complete protection from JEV infection. Animals were infected with 3 x 105 pfu of JEV and either treated with AR or left untreated (n = 15 for each group). Treatment of uninfected animals with AR alone had no effect. Observation of animal survival experiments was performed in a blinded manner to avoid bias.

 
AR act as a potent antiviral agent

AR acts as a potent antiviral agent against JEV infection. We evaluated the antiviral properties of AR by using immunoblot analysis and immunohistochemistry. Brain sections from infected animals had significantly higher number of cells positive for JEV-specific proteins (Figure 3b) when compared with brain sections from control animals (Figure 3a). In contrast, brain sections obtained from JEV-infected/AR-treated animals showed reduced numbers of cells positive for JEV-specific proteins (Figure 3c). This observation was further confirmed by immunoblot for JEV-specific proteins. There was a significant induction of JEV-specific proteins (84 and 17 kDa) in the infected group (Figure 3d); AR treatment completely abolished the expression of viral proteins.


Figure 3
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  		Figure 3. AR acts as a potent antiviral agent. C, control; AR, AR-treated alone (drug control); JEV, JEV-infected; and JEV + AR, JEV-infected/AR-treated. (a–c) Processed brain sections from control, JEV-infected and JEV-infected/AR-treated mice were subjected to immunohistochemistry using anti-JEV Nakayama antibody. JEV-infected/AR-treated samples (c) had significantly reduced levels of JEV proteins when compared with JEV infected samples (b; arrow indicates cells which are positive for JE virus specific protein). Brain sections from control animals (a) were subjected to immunohistochemistry using same antibody. Scale bar is 25 µm. (d) AR treatment in JEV-infected mice completely reduced the levels of viral proteins. Protein isolated from control, only AR-treated, JEV-infected, JEV-infected/AR-treated BALB/c mice was analysed by immunoblot. A significant decrease in the levels of JEV-specific proteins (84 and 17 kDa) in AR-treated samples compared with infected ones were observed. (e) Protein isolated from control, JEV-infected and JEV-infected/AR-treated BALB/c mice was subjected to plaque assay to determine the viral load. The viral titre obtained after infection with JEV was 2 x 105 pfu and AR treatment significantly decreased the viral titre to 3 x 103 pfu (*P < 0.001). All the tissue samples were collected on the 8th day post-infection. Data shown are representative of three independent experiments with six individual animals from each group. Protein isolation and immunoblot analysis to determine viral load were performed in a blinded manner to avoid bias towards any group of animals.

 
AR treatment reduces the viral titre in vivo

To assess whether AR has any effect on the viral load in brain, virus isolated from JEV-infected and JEV-infected/AR-treated animal brains was titrated by plaque formation on PS monolayers. Treatment with AR significantly reduced the virus titres in JEV-infected animals (2 x 105 pfu/mL in JEV-infected mice), and after treatment it reduced to 3 x 103 pfu/mL (Figure 3e). There was a significant and dramatic decrease in JEV titre in brain after AR treatment (P < 0.001).

In vivo neuroprotective effect of AR

To further characterize the effects of AR on neuronal death following JEV infection, brain sections from control, JEV-infected and JEV-infected/AR-treated animals were stained with thionin dye to assess the extent of neuronal death. Numerous healthy cells were observed in sections obtained from a control brain (Figure 4a) when compared with a JEV-infected one (Figure 4b). AR treatment following JEV infection significantly rescued the integrity of neurons as well as reduces the enhanced gliosis observed following JE (Figure 4c).


Figure 4
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  		Figure 4. AR treatment reduces neuronal death. C, control; AR, AR-treated alone (drug control); JEV, JEV-infected; JEV + AR, JEV-infected/AR-treated. (a–c) Brain sections from control, JEV-infected and JEV-infected/AR-treated mice were processed and stained with thionin dye to assess the extent of neuronal death. Numerous healthy cells were observed in sections obtained from control brain (a) when compared with JEV-infected one (b; arrows indicate pyknotic cells). AR treatment following JEV infection dramatically rescued the loss of neuronal health and reduced the extent of gliosis observed in JEV-infected animals (c). (d–f) JEV-infected samples had a significantly increased number of active caspase-3-positive cells (e; arrows) when compared with control (d). In contrast, JEV-infected/AR-treated samples had significantly reduced levels of caspase-3-positive cells (f). Scale bar is 25 µm. (g) Protein expression levels of active caspase-3 were analysed. There was a significant induction of active caspase-3 proteins (17 and 12 kDa) in infected group. AR treatment completely inhibited the expression of active caspase-3. All tissue samples were collected on the 8th day post-infection. Data shown are representative of three independent experiments with six individual animals from each group.

 
To elucidate the molecular basis of JEV-induced neuronal death, brain sections from control, JEV-infected and JEV-infected/AR-treated mice were analysed for the presence of activated caspase-3. Immunohistochemistry results revealed that JEV-infected animals had a significantly increased number of active caspase-3-positive cells (Figure 4e) when compared with controls (Figure 4d). In contrast, JEV-infected/AR-treated samples had significantly reduced levels of activated caspase-3-positive cells (Figure 4f). We also evaluated the levels of active caspase-3 using immunoblot. There was a significant induction of active caspase-3 proteins (17 and 12 kDa) in infected group (Figure 4g), AR treatment completely down-regulated the expression of active caspase-3.

AR reduces neuronal death by its antioxidative properties

Since AR reduces neuronal death, we evaluated the antioxidative properties of AR in JEV-infected mice. We measured the ROS level in control, JEV-infected, JEV-infected/AR-treated and only AR-treated mouse brain lysates. We found that there was an 8-fold increase in ROS level (P < 0.01) in JEV-infected samples when compared with control ones. On the other hand, JEV-infected/AR-treated samples had significantly reduced (8-fold decrease over JEV-infected samples, P < 0.01) levels of ROS (Figure 5a). Since a major endogenous antioxidant in mammalian cells is the enzyme superoxide dismutase (SOD), which catalyses the conversion of the superoxide anion (O2–) into hydrogen peroxide (H2O2) and molecular oxygen (O2), we evaluated the expression levels of SOD1 in protein isolated from control, JEV-infected, JEV-infected/AR-treated and only AR-treated mouse brain samples. SOD1 levels were significantly down-regulated in JEV-infected samples (2-fold decrease when compared with control samples, P < 0.05). In contrast, JEV-infected/AR-treated samples had increased levels of SOD1 (1.8-fold when compared with only JEV-infected samples, P < 0.05) (Figure 5b).


Figure 5
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  		Figure 5. AR acts as a potent antioxidant. (a) Protein extracted from control, AR-treated alone (drug control), JEV-infected and JEV-infected/AR-treated mice was evaluated for ROS levels. While there was a significant increase (P < 0.01) in ROS levels in JEV-infected samples when compared with controls, JEV-infected/AR-treated samples had significantly reduced ROS levels. Data represent mean ± SEM of six animals from each group from three independent experiments. Asterisks indicate differences that were statistically significant (*P < 0.01, **P < 0.001). (b) SOD1 levels were significantly down-regulated in JEV-infected samples. In contrast, JEV-infected/AR-treated samples had increased levels of SOD1. Data shown are a representative blot of six individual animals from each group from three independent experiments. (c) NO levels were measured in control, AR-treated alone (drug control), JEV-infected and JEV-infected/AR-treated mice brain samples. A significant increase in NO levels was observed in JEV-infected mice (P < 0.001) when compared with controls. On the other hand, NO levels in JEV-infected/AR-treated mice were significantly reduced when compared with JEV-infected mice. Data represent mean ± SEM of six animals from each group from three independent experiments. Asterisks indicate differences that were statistically significant (*P < 0.01, **P < 0.001). (d) Expression levels of iNOS in control, JEV-infected and JEV-infected/AR-treated mice brain samples. We found that iNOS expression levels were significantly up-regulated in JEV-infected samples when compared with control samples. In JEV-infected/AR-treated mice brain samples, iNOS levels were significantly down-regulated when compared with JEV-infected samples. All the tissue samples were collected on the 8th day post-infection. Data shown are representative of three independent experiments with six individual animals from each group.

 
We also measured NO levels in control, JEV-infected and JEV-infected/AR-treated mouse brain samples. A 15-fold increase in NO levels was observed in JEV-infected mice (P < 0.001) when compared with controls. On the other hand, NO levels in JEV-infected/AR-treated mice were significantly reduced (12-fold decrease over JEV-infected animals, P < 0.001; Figure 5c). We also compared inducible nitric oxide synthase (iNOS) protein expression levels in control, JEV-infected and JEV-infected/AR-treated mouse brain samples. We found that iNOS expression levels were significantly up-regulated in JEV-infected samples when compared with control samples (3-fold increase, P < 0.01). In JEV-infected/AR-treated mouse brain samples, iNOS levels were significantly down-regulated when compared with JEV-infected samples (2.8-fold decrease, P < 0.01; Figure 5d).

AR abrogates microglial activation and induction of proinflammatory cytokines

Immunohistochemistry revealed both qualitative and quantitative differences in microglial activation in infected animals treated with AR when compared with those infected without treatment. In the JEV-infected brain sections, the number of star-shaped ‘activated’ microglia (Figure 6b) appeared to be more frequent (more than 30-fold, data not shown) than in controls (Figure 6a) or JEV-infected/AR-treated group (Figure 6c).


Figure 6
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  		Figure 6. AR reduces microglial activation and induction of proinflammatory cytokines. (a–c) Cryostat sections from control (a), JEV-infected (b) and JEV-infected/AR-treated (c) mouse brains were processed for Iba-1. While the control sections exhibited only resting microglia (a; arrows), the infected brains showed the presence of activated state of microglia (b; arrowheads). JEV-infected/AR-treated mice had mostly resting microglia (c; arrows). The scale bar represent 25 µm. (d) The protein expression levels of various proinflammatory cytokines in control, JEV infected and JEV-infected/AR-treated mice brain lysates were measured. AR dramatically reduced the level of proinflammatory cytokines and chemokine. A significant (P < 0.001) decrease in the levels of TNF-{alpha}, IFN-{gamma}, MCP-1 and IL-6, respectively, was observed in infected animals treated with AR when compared with those infected but not treated (n = 6 for each group). All the tissue samples were collected on the 8th day post-infection. Data shown are representative of three independent experiments.

 
We also measured the expression levels of various proinflammatory cytokines in control, JEV-infected and JEV-infected/AR-treated mouse brain lysates. As shown in Figure 6(d), AR dramatically reduced the level of proinflammatory cytokines and chemokines. A significant (P < 0.001) 5-, 1800-, 5000- and 16-fold decrease in the level of TNF-{alpha}, IFN-{gamma}, MCP-1 and IL-6, respectively, was observed in JEV-infected animals treated with AR when compared with infected animals without treatment. With AR treatment alone, no significant changes were observed in the level of cytokines.

AR modulates the expression pattern of several stress-associated proteins

JEV infection up-regulates the induction of several stress-associated proteins, and treatment with AR significantly reduced the level of those proteins in infected animals (Figure 7). In protein samples isolated from JEV-infected/AR-treated animals, there was a significant decrease in the expression of phospho-p38 MAPK (2-fold decrease when compared with JEV infected, P < 0.01), phospho-c-Jun (2.5-fold decrease when compared with JEV-infected, P < 0.05), Hsp70 (3-fold decrease when compared with JEV-infected, P < 0.01) and phospho-ERK-1/2 (3-fold decrease when compared with JEV-infected, P < 0.01). Interestingly, AR treatment in JEV-infected mice dramatically increased the levels of phopsho-Akt (2.5-fold increase when compared with JEV-infected mice without AR treatment, P < 0.05). With AR treatment alone, no significant changes were observed in the level of different stress-associated proteins.


Figure 7
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  		Figure 7. AR regulates the expression pattern of several key proteins associated with stress. C, control; AR, AR-treated only; JEV, JEV-infected; JEV + AR, JEV-infected/AR-treated. In JEV-infected/AR-treated brain samples, there was a significant decrease in the expression of phospho-p38 MAPK (P < 0.01), phospho-c-Jun (P < 0.05), Hsp70 (P < 0.01) and phospho-ERK-1/2 (P < 0.01). Interestingly, AR treatment in JEV-infected mice dramatically increased the levels of phopsho-Akt (P < 0.05). The protein isolated from AR-treated drug control mice had various expression levels of different signalling molecules comparable to controls. All the tissue samples were collected on 8th day post-infection. Data shown are representative of three independent experiments with six individual animals from each group.

 
AR prevents JEV-induced neurotoxicity in mouse neuroblastoma cells

Since we have shown that AR acts as a potent antiviral and anti-inflammatory agent in an experimental murine model of JE, we corroborated our results in mouse neuroblastoma cells (N2a). We performed survival studies using the Trypan blue and MTT assay. We found that there was a significant decrease in the percentage of dead cells in JEV-infected/AR-treated cells (4.5-fold decrease over JEV-infected cells, P < 0.001) (Figure 8a). Significant increases in cell viability of JEV-infected/AR-treated cells were also observed in MTT assay (6.5-fold increase over JEV-infected cells, P < 0.001) (Figure 8b).


Figure 8
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  		Figure 8. AR prevents JEV-induced cell death and decreases viral load in mouse neuroblastoma cells (N2a). (a–c) N2a cell line was infected with JEV (moi = 5) for 1 h. The virus was removed and the plates were then washed with PBS to remove the unbound virus. The plates were further incubated in AR-containing serum-free media at 37°C for next 24 h. (a) Survival studies were performed using the Trypan blue exclusion test. There was a significant decrease in the percentage of dead cells in JEV-infected/AR-treated cells. (b) Significant increases in cell viability of JEV-infected/AR-treated cells were also observed in MTT assay. (c) There was a significant decrease in the cytochrome c activity levels in JEV-infected/AR-treated cells. (d) N2a cells were incubated with JE virus for 1 h. The plates were then washed with 1x PBS to remove the unbound viruses. The plates were further incubated in AR-containing serum-free media at 37°C for 6, 12 and 24 h. Cells were harvested and plaque assay was performed to determine the viral titre. There was a significant decrease in viral titre in JEV-infected/AR-treated samples with respect to JEV-infected samples at each time point. Values reflect the mean ± SEM from three independent experiments. Asterisks indicate differences that were statistically significant (*P < 0.01, **P < 0.001). (e–g) N2a cells were incubated with JE virus for 1 h. The plates were then washed with 1x PBS to remove the unbound viruses. The plates were further incubated in AR-containing serum-free media at 37°C for 6, 12 and 24 h. AR treatment significantly reduces the JEV-specific viral antigen in N2a cells (P < 0.001). Histograms from FACS analysis represent results from one experiment that is representative of three independent experiments. (h) Values obtained from intracellular staining by FACS are depicted as a line graph.

 
As JEV induces apoptosis via a mitochondrion-dependent mechanism,20 we evaluated the cytochrome c levels in N2a cells. We found that there was a significant decrease in the cytochrome c activity levels in JEV-infected/AR-treated cells (4-fold decrease over JEV-infected cells, P < 0.001) (Figure 8c).

AR treatment reduced viral titre in vitro and decreased the intracellular viral load

We performed plaque assays to determine the effect of AR treatment on viral titre in JEV-infected N2a cells at different time points (6, 12 and 24 h post-infection). Treatment with AR significantly reduces the viral titre in N2a cells at each time point (P < 0.001) (Figure 8d). After 6, 12 and 24 h of infection with JEV, the viral titres in AR-untreated N2a cells were 1.2 x 106, 5.7 x 106 and 7 x 106, respectively. Treatment with AR significantly reduced the viral titre from 10-fold (at 6 h) to ~100-fold (at 12 and 24 h). To assess whether AR could decrease intracellular viral load, we co-cultured JEV with N2a cell-lines. Then intracellular staining of JEV was measured by flow cytometry in N2a cells at various time points after infection. We found that after 6, 12 and 24 h, the JEV-infected cells formed 32.0%, 59.38% and 68.22% of the population, respectively, whereas after AR treatment, it came down to 25.1%, 45.41% and 32.0% of the total cell population (P < 0.001) (Figure 8e–h).


   Discussion
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The lignan arctigenin has been shown to have neuroprotective and antiviral properties in diverse experimental models.22,23 However, this had not been tested in experimental models of CNS infection. The major finding in this study is that treatment with arctigenin provides complete protection against experimental JE. Arctigenin’s neuroprotective effect is associated with marked decreases in (i) viral load, (ii) active caspase-3 activity, (iii) ROS/RNS, (iv) microgliosis and proinflammatory cytokines, (v) levels of stress-associated signalling molecules and (vi) neuronal death. Furthermore, treatment with arctigenin also improves the behavioural outcome following JE (data not shown).

We showed previously that the increased microglial activation following JEV infection influences the outcome of viral pathogenesis and it is likely that the increased microglial activation triggers bystander damage, as the animals eventually succumb to infection.19 Inhibition of chronic neuroinflammation, particularly of microglial activation, has been suggested to be a practical strategy in the treatment of neurodegenerative diseases. We show here that treatment with arctigenin following JE reduces the number of activated microglia as well as the level of proinflammatory cytokines TNF-{alpha}, IFN-{gamma}, IL-6 and chemokine MCP-1. These data further support that microglial activation and subsequent inflammation is critical in determining the outcome of viral pathogenesis observed in JE.

We have recently reported that JEV infection is also accompanied by profound neuronal apoptosis.17,20 In addition to its direct actions on microglia, arctigenin has also been shown to exert a neuroprotective effect.10,22 These actions likely contributed to our in vivo findings of reduced caspase-3 activity as well as our in vitro findings with the Neuro2a cell line showing reduced apoptosis. These data suggest that arctigenin is also working as an anti-apoptotic molecule in this model of infection. In vivo microglial activation could be a response to neuronal damage with the subsequent inflammation resulting in negative consequences. Henceforth, early inhibition of neuronal apoptosis compounded by a decrease in the subsequent release of proinflammatory mediators by activated microglia would attenuate the severity of disease observed in JE. Because arctigenin’s anti-inflammatory and anti-apoptotic effects will be beneficial in reducing the severity of diseases induced by JEV, our findings provide compelling evidence to further consider arctigenin as a therapeutic measure for JE.

Generation of ROS with the accumulation of oxidative damage has been implicated in neurodegenerative diseases and in the degradation of nervous system function. We have earlier reported that JEV infection induce the level of ROS both in vivo and in vitro,17,24 and here we report that arctigenin’s protective function is associated with reduced levels of ROS, which reduces JEV-mediated neuronal death. We have earlier reported that JEV infection induces the level of RNS both in vivo and in vitro and here we report that arctigenin’s beneficial effect is also associated with reduced level of RNS,19 which reduces JEV-mediated neuronal death. Based on our findings, it is tempting to speculate that antioxidant property of arctigenin is one of the factors that are responsible in conferring neuroprotection following JEV infection.

Arctigenin has been shown to display in vitro and in vivo antiviral activity against several viruses including HIV.8,9,23 Arctigenin acts as an inhibitor of HIV-1 integrase.8 Here in the present communication, we have showed that arctigenin also inhibits the replication of JEV, although a more detailed study is needed to determine the exact antiviral mechanism of arctigenin for JEV.

It was reported earlier that arctigenin inhibits activation of MAP kinases including ERK1/2, p38 kinase and JNK through the inhibition of MKK activities.25 Recently, we have reported that JEV infection in vivo increases the level of p38 and p-JNK;20 here in the present communication, we have shown that treatment with arctigenin significantly down-regulated the level of phospho-p38, phospho-ERK1/2 and phospho-c-Jun. The MAP kinase cascade plays a central role in regulating inflammation following infection; hence pharmacological intervention of this pathway might be beneficial in targeting neurological complications observed in JE. Recently, it was reported that arctigenin also acts as a novel suppressor of the heat shock response in mammalian cells.26 We have found that in vivo JEV infection in animals up-regulates the level of Hsp70 protein, and treatment with arctigenin reversed this. Previously, it was reported that flaviviruses such as JEV and dengue not only induce cell apoptotic signalling in vitro but also activate survival signalling involving the phosphotidyl-inisitol-3 kinase (PI3K/Akt pathway).27 Contrary to that report, we have found that, at least in vivo, JEV infection decreased the level of phospho-Akt and treatment with arctigenin significantly up-regulated it. It is possible that following infection, induction of phospho-Akt changes in a time-dependent fashion.

In conclusion, our findings provide a novel mechanistic insight into the actions of arctigenin in JE. Results from our in vivo and in vitro experiments clearly indicate that arctigenin reduces (i) viral replication within the brain, (ii) neuronal death and (iii) secondary inflammation and oxidative stress resulting from microglial activation, thereby suggesting its potential for treating JE. The antiviral, neuroprotective, anti-inflammatory, antioxidative effects of arctigenin are essential for reducing the severity of diseases induced by JEV. To the best of our knowledge, this is the first study to have identified an agent with the potential to be an antiviral, neuroprotective and anti-inflammatory agent in JE. The studies presented here recommend arctigenin as a strong candidate for further consideration as a therapeutic measure to reduce the neurological and inflammation-related complications observed in JE.


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This work was supported by grants awarded to AB from the Department of Biotechnology (DBT), Award # BT/PR/5799/MED/14/698/2005. This work is also supported by a core grant from DBT to NBRC. M. K. M is a recipient of Senior Research Fellowship from University Grants Commission.


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None to declare.


   Footnotes
 
{dagger} These authors contributed equally to this work. Back


   Acknowledgements
 
We thank Kanhaiya Lal Kumawat for his help in this study. We are grateful to Dr. Nihar Ranjan Jana of our institute for providing the Hsp70 antibody. We thank Prof. Vijayalakshmi Ravindranath, Director, NBRC, for her continuous support and encouragement.


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