Department of Education and Research, Taichung Veterans General Hospital, Taichung, Taiwan, Republic of China1
Institute of Preventive Medicine, National Defence Medical Center, Taipei, Taiwan, Republic of China2
Author for correspondence: Chun-Jung Chen. Fax +886 4 23592705. e-mail cjchen{at}vghtc.vghtc.gov.tw
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Non-steroidal anti-inflammatory drugs (NSAIDs) have long been used to treat fever and inflammatory diseases. Traditional NSAIDs like aspirin, ibuprofen and indomethacin can inhibit the activity of both cyclooxygenase (COX) isoforms, COX-1 and COX-2, thereby blocking the production of prostaglandins (Vane, 1971 ). Aspirin is a pro-drug form of salicylate, which is rapidly hydrolysed to salicylate in vivo (Gilman et al., 1990
). Salicylate, however, inhibits the synthesis of prostaglandins in vivo, but has little effect on COX1 and COX2 activities in vitro (Gilman et al., 1990
). Salicylate must therefore modulate the synthesis of prostaglandins through an alternate mechanism not involving direct effects on COXs. In addition to the anti-inflammatory action of salicylate, increasing evidence indicates its ability to protect against cancer, cardiovascular disease and viral infection (Huang & Dietsch, 1988
; Primache et al., 1998
; Speir et al., 1998
; Chen et al., 2000
; Patrignani, 2000
). This suggests that some of the biochemical effects of salicylate are independent of effects on COX activity.
The antiviral effects of NSAIDs have been shown to exert an influence on the influenza virus, cytomegalovirus and varicella-zoster virus (Huang & Dietsch, 1988 ; Primache et al., 1998
; Speir et al., 1998
). Inhibition of COX activity, scavenging of free radicals and down-regulation of transcription factors may be attributed to the antiviral action of NSAIDs. Despite extensive studies of NSAIDs, little is known about their effects on viral infection in the CNS. The propagation of vesicular stomatitis virus (VSV) in mouse CNS is suppressed by NSAIDs leading to a decrease in encephalitis (Chen et al., 2000
). Moreover, arachidonic acid and its metabolites are involved in the death of neuroblastoma cells induced by Dengue virus, one of the flaviviruses (Jan et al., 2000
). In the present study, we explored the effects of NSAIDs on JEV infection. We found that salicylate suppressed JEV replication and its cytotoxicity in neuroblastoma cells. The mechanism of its antiviral action was shown to be by modulation of the mitogen-activated protein kinase (MAPK) signalling pathway, not by inhibition of COX activity.
![]() |
Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Virus infection and titre determination.
For infection of cells with JEV, monolayers of each cell type were first adsorbed with JEV at an m.o.i. of 5 for 1 h at 37 °C. After adsorption, unbound virus was removed by gentle washing with PBS, pH 7·4. Fresh medium was added to each plate for further incubation at 37 °C. To determine virus titres, culture media were harvested and used in plaque-forming assays. Briefly, various dilutions of the culture media were added to 80% confluent BHK21 cells and incubated at 37 °C for 1 h. After adsorption, the cells were washed and overlaid with 1% agarose (SeaPlaque; FMC BioProducts) in RPMI 1640 plus 2% FBS. After incubation for 4 days, the cells were fixed with 10% formaldehyde, stained with 0·5% crystal violet and examined for the presence of plaques.
Cytotoxicity assessment.
Cytotoxicity, as indicated by cell membrane integrity, was assessed by measuring the activity of lactate dehydrogenase (LDH) in the cultured media by the colorimetric detection of formazan, using an LDH diagnostic kit (Promega). After each experiment, the supernatants were transferred to a microtitre plate and incubated with reaction mixture at room temperature for 30 min for colour development. The OD492 was measured using a spectrophotometer (PowerWaveX 340, BIO-TEK Instruments).
MTT assay.
An MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay was used to measure mitochondrial function as an index of living cells. In brief, MTT (Sigma) was dissolved in 0·1 M TBS to make a 5 mg/ml solution. Fifty µl of MTT solution was added to each well containing tested cells in a 12-well plate and incubated at 37 °C for 4 h. After incubation, the MTT solution was removed and 500 µl isopropanol containing 0·04 M HCl was added to dissolve the dark-blue crystals precipitated in the wells. A 100 µl sample of the resulting solution was removed from each well and the absorbance at 540 nm was determined using a microplate reader (PowerWaveX 340, Bio-Tek Instruments).
Assessment of intracellular redox potential.
Levels of intracellular free radicals were analysed by the fluorescent signal produced after the oxidation of non-fluorescent 2',7'-dichlorofluorescein (Molecular Probe) by reactive oxygen species (ROS), as described previously (Zhu et al., 1994 ). 2',7'-Dichlorofluorescein was added to cultures to a concentration of 5 µM in a defined salt buffer (116 mM NaCl, 1·8 mM CaCl2, 0·8 mM MgSO4, 5·4 mM KCl, 1 mM NaH2PO4, 14·7 mM NaHCO3, 10 mM HEPES and 5·5 mM glucose) at 37 °C for 5 min, washed and then incubated with buffer for 60 min. The fluorescent signal resulting from oxidation of 2',7'-dichlorofluorescein was observed using a fluorescent microscope (Leica; excitation 488 nm and emission 510 nm) or measured using a fluorometer (Fluoroskan Ascent, Labsystems).
TdT-mediated dUTP nick end-labelling (TUNEL).
Apoptosis-induced DNA strand breaks were end-labelled with dUTP by use of terminal deoxynucleotidyltransferase (TdT) with a commercial kit (In Situ Cell Death Detection Kit, Boehringer Mannheim), according to the manufacturer's instructions. Briefly, the cells were fixed with 4% paraformaldehyde in PBS for 30 min at room temperature and permeabilized in 0·1% Triton X-1000·1% sodium citrate for 2 min on ice. The reaction was performed by labelling with fluorescein isothiocyanatedUTP at 37 °C for 60 min, then incubated with an alkaline phosphatase-conjugated secondary antibody. The colour was developed by incubation with NBT/BCIP substrate and observed under a light microscope.
Western blot.
Protein extracts (100 µg) were resolved by SDSpolyacrylamide gel electrophoresis and transferred on to a blotting membrane. The membrane was first incubated with 5% (w/v) skimmed milk in PBS for 30 min to reduce non-specific binding, then incubated with primary antibody overnight at 4 °C, followed by washing with 0·05% Tween 20 in PBS. After washing, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody. The signals were developed by chemiluminescent detection. The intensity of signals was determined by a computer image system (Alpha Innotech Corporation, IS1000).
Determination of prostaglandin E2 (PGE2) concentration.
PGE2 levels were detected by ELISA, following the instructions provided by the manufacturer (Cayman Chemical).
Northern blot.
Isolation of total cellular RNA and Northern blot analysis were performed as previously described (Chang et al., 1994 ). The probe used was the antigenomic strand of JEV RNA (nt 1039210976), synthesized in vitro with Sp6 RNA polymerase in the presence of Bio-11-UTP (Sigma). The signals were developed by chemiluminescent detection. The intensity of signals was determined by a computer image system (Alpha Innotech Corporation, IS1000). The 18S and 28S rRNA were detected by staining with ethidium bromide before probing.
Statistical analysis.
Data are expressed as mean±SEM. For comparisons, the statistical significance between means was determined using one-way analysis of variance (ANOVA) followed by Dunnetts t-test. A level of P<0·05 was considered statistically significant.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Phospholipids play roles in many biological processes, including the generation of proinflammatory lipid mediators, such as prostaglandins and leukotrienes, and the regulation of lipid metabolism (Glaser, 1995 ). The mobilization of AA from phospholipids requires the involvement of PLA2. AA serves as a substrate for COX enzymes, resulting in the production of prostaglandins. Prostaglandins activate cellular receptors, resulting in the subsequent initiation of signal cascades involving G-protein and cyclic AMP (Cirino, 1998
). These signalling cascades have an important role in cellular injury via their ability to mediate inflammatory responses. NSAIDs inhibit the activity of both COX isoforms, leading to decreased production of prostaglandins and the accumulation of AA. Therefore, the inflammatory action of prostaglandins and COX activity-associated free-radical generation are suppressed. In contrast, the biological action of AA is apparent.
NSAIDs such as aspirin, indomethacin and sodium salicylate suppressed JEV propagation in neuroblastoma cells, as indicated by the attenuation of cell death and the reduction of viral protein expression and virus yield (Figs 1 and 2
). Numerous studies have used PGE2 release as a marker for COX activity. JEV infection slightly increased the production of PGE2 by neuroblastoma cells (Fig. 4
). These NSAIDs suppressed mock-infection (data not shown) and JEV-induced PGE2 production, indicating the well-known pharmacological effects on COX enzymes. Previously, we reported that JEV infection caused generation of free radicals in neuroblastoma cells via the involvement of the flavon-containing enzymes (Raung et al., 2001
). The increased production of free radicals induced by JEV could be eliminated by NSAIDs (Fig. 3
). This result suggests that JEV-induced free-radical generation might be through the COX-associated pathway. However, it should be noted that the reduction of free radicals may be derived from the direct scavenging effect. Recently, Jan et al. (2000)
reported that AA, superoxide anion and nuclear factor kappa B (NF-
B) were sequentially involved in Dengue virus-triggered apoptotic pathways. However, exogenous AA and an inhibitor of PLA2 failed to mimick the action of NSAIDs in suppressing JEV-induced cell death (Fig. 5
). Although reduction of superoxide anion generation by superoxide dismutase protected Dengue virus-induced apoptosis (Jan et al., 2000
), antioxidants showed no protective actions against JEV-mediated cytotoxicity (Raung et al., 2001
). Moreover, the doses of NSAIDs used to eliminate free-radical generation (Fig. 3
) and PGE2 production (Fig. 4
) were lower than those required to suppress JEV propagation (Figs 1
4
). Therefore, these results suggest that the antiviral effect of NSAIDs was not through the conventional COX pathway.
Signal transduction pathways convey signals generated at the cell surface into the cell nucleus in order to initiate a programme of gene expression that is characteristic for particular stimuli. Among them, MAPKs, including ERK, p38 protein kinase and c-Jun N-terminal kinase (JNK), play a critical role in the regulation of cell proliferation and differentiation in response to mitogens and other extracellular stimuli (Karin, 1998 ). The activation of MAPKs can be modulated by salicylate (Schwenger et al., 1998
). We found that the antiviral effect of salicylate against JEV could be blocked by inhibitors of mitogen-activated protein kinase (MEK) (PD 98059) or p38 MAPK (SB 203580) (Fig. 6
). In addition, direct activation of p38 MAPK by anisomycin also markedly suppressed JEV-induced LDH efflux (Fig. 6
). The involvement of MAPK modulation on the antiviral effect of salicylate was observed not only in neuroblastoma cells but also in non-neuronal BHK21 cells (Fig. 7
). Recently, Liao et al. (2001)
have demonstrated that the antiflavivirus effect of salicylates was partially reversed by blocking p38 MAPK activation with SB 203580 in BHK21 cells. In this study, we have provided further evidence showing the involvement of p38 MAPK and ERK in JEV-mediated cytotoxicity as well as in the antiviral effect of salicylates. In general, the dynamic balance between branches of the MAPK family is believed to regulate neuronal decisions to live or die in response to stressors (Xia et al., 1995
). In particular, ERK activation may play a pivotal role. For example, many neuroprotective/neurotrophic factors activate receptor tyrosine kinases transmitting signals through the activation of ERK (Segal & Greenberg, 1996
). ERK activation appears to antagonize apoptotic pathways in some cell systems (Xia et al., 1995
). In contrast, several recent studies indicate that ERK activation may also play a pathologic role in neurons exposed to increased oxidative stress (Oh-hashi et al., 1999
; Stanciu et al., 2000
). Although the detailed mechanisms underlying the antiviral effect remain unclear, JEV infection-induced decreased phosphorylation of ERK could be reversed by salicylate (Fig. 6
). In addition, the activation of p38 MAPK also suppressed JEV-induced toxicity (Fig. 6
). Thus, the activation of MAPK plays a role in host-cell protection against JEV infection.
Oxidative stress has been suggested to be a mediator of apoptosis/necrosis induced by a variety of triggers, including virus infections (Schwarz, 1996 ; Peterhans, 1997
; Speir et al., 1998
; Schweizer & Peterhans, 1999
). Some antioxidants are known to inhibit cell death in virus systems (Cossarizza et al., 1995
; Verhaegen et al., 1995
; Lowy & Dimitrov, 1997
), although antioxidants do not protect cells against JEV-induced toxicity (Raung et al., 2001
). Free radicals are potent triggers for NF-
B activation. Accumulating evidence indicates the antiviral effects of NF-
B inhibition in cytomegalovirus, Dengue virus, respiratory syncytial virus and human immunodeficiency virus (Schwarz, 1996
; Bitko et al., 1997
; Peterhans, 1997
; Speir et al., 1998
; Jan et al., 2000
). In these viruses, antioxidant therapy is effective against virus infection (Staal et al., 1990
; Schwarz, 1996
; Bitko et al., 1997
; Peterhans, 1997
; Speir et al., 1998
; Jan et al., 2000
). NF-
B inhibition has been shown to be a potential mechanism of the neuroprotection effect exerted by aspirin and salicylate (Grilli et al., 1996
). However, both antioxidants (Raung et al., 2001
) and the inactivation of NF-
B (Liao et al., 2001
) failed to attenuate JEV propagation and JEV-mediated cytotoxicity. Therefore, the inactivation of NF-
B is not involved in the antiviral effect of salicylate against JEV.
The generation of ROS has been demonstrated to be critical in the rapid degradation of phagocytosed JE viral protein and nucleic acid (Srivastava et al., 1999 ). In addition, nitric oxide appears to be effective in restricting JE amplification (Lin et al., 1997
). Usually, oxidative stress is well associated with the activation of MAPK. In this study, we have demonstrated that MAPK signalling pathways are also involved in suppression of JEV propagation.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bitko, V., Velazquez, A., Yang, L., Yang, Y. C. & Barik, S. (1997). Transcriptional induction of multiple cytokines by human respiratory syncytial virus requires activation of NF-kappaB and is inhibited by sodium salicylate and aspirin. Virology 232, 369-378.[Medline]
Chambers, T. J., Hahn, C. S., Galler, R. & Rice, C. M. (1990). Flavivirus genome organization, expression, and replication. Annual Review of Microbiology 44, 649-688.[Medline]
Chang, M. F., Chen, C. J. & Chang, S. C. (1994). Mutational analysis of delta antigen: effect on assembly and replication of hepatitis delta virus. Journal of Virology 68, 646-653.[Abstract]
Chaturvedi, U. C., Mathur, A., Tandon, P., Natu, S. M., Rajvanshi, S. & Tandon, H. O. (1979). Variable effect on peripheral blood leucocytes during JE virus infection of man. Clinical and Experimental Immunology 38, 492-498.[Medline]
Chen, C. J., Kuo, M. D., Chien, L. J., Hsu, S. L., Wang, Y. M. & Lin, J. H. (1997). RNAprotein interactions: involvement of NS3, NS5, and 3 noncoding regions of Japanese encephalitis virus genomic RNA. Journal of Virology 71, 3466-3473.[Abstract]
Chen, N., Warner, J. L. & Reiss, C. S. (2000). NSAID treatment suppresses VSV propagation in mouse CNS. Virology 276, 44-51.[Medline]
Cirino, G. (1998). Multiple controls in inflammation, extracellular and intracellular phospholipase A2, inducible and constitutive cyclooxygenase, and inducible nitric oxide synthase. Biochemical Pharmacology 55, 105-111.[Medline]
Cossarizza, A., Franceschi, C., Monti, D., Salvioli, S., Bellesia, E., Rivabene, R., Biondo, L., Rainaldi, G., Tinari, A. & Malorni, W. (1995). Protective effect of N-acetylcysteine in tumor necrosis factor-alpha-induced apoptosis in U937 cells: the role of mitrochondria. Experimental Cell Research 220, 232-240.[Medline]
Gilman, A. G., Rall, T. W., Nies, A. S. & Taylor, P. (1990). The Pharmacological Basis of Therapeutics, 8th edn. Elmsford, NY: Pergamon Press.
Glaser, K. B. (1995). Regulation of phospholipase A2 enzymes: selective inhibitors and their pharmacological potential. Advances in Pharmacology 32, 31-66.[Medline]
Grilli, M., Pizzi, M., Memo, M. & Spano, P. (1996). Neuroprotection by aspirin and sodium salicylate through blockade of NF-kappaB activation. Science 274, 1383-1385.
Huang, R. T. & Dietsch, E. (1988). Anti-influenza viral activity of aspirin in cell culture. New England Journal of Medicine 319, 797.[Medline]
Jan, J. T., Chen, B. H., Ma, S. H., Liu, C. I., Tsai, H. P., Wu, H. C., Jiang, S. Y., Yang, K. D. & Shaio, M. F. (2000). Potential Dengue virus-triggered apoptotic pathway in human neuroblastoma cells: arachidonic acid, superoxide anion, and NF- B are sequentially involved. Journal of Virology 74, 8680-8691.
Kalgutkar, A. S., Crews, B. C., Rowlinson, S. W., Garner, C., Seibert, K. & Marnett, L. J. (1998). Aspirin-like molecules that covalently inactivate cyclooxygenase-21211. Science 280, 1268-1270.
Karin, M. (1998). Mitogen-activated protein kinase cascades as regulators of stress responses. Annals of the New York Academy of Sciences 851, 139-146.
Khanna, N., Agnihotri, M., Mathur, A. & Chaturvedi, U. C. (1991). Neutrophil chemotactic factor produced by Japanese encephalitis virus stimulated macrophages. Clinical and Experimental Immunology 86, 299-303.[Medline]
Kumar, R., Mathur, A., Kumar, A., Sethi, G. D., Sharma, S. & Chaturvedi, U. C. (1990). Virological investigations of acute encephalopathy in India. Archives of Disease in Childhood 65, 1227-1230.[Abstract]
Liao, C. L., Lin, Y. L., Wu, B. C., Tsao, C. H., Wang, M. C., Liu, C. I., Huang, Y. L., Chen, J. H., Wang, J. P. & Chen, L. K. (2001). Salicylates inhibit flavivirus replication independently of blocking nuclear factor kappa B activation. Journal of Virology 75, 7828-7839.
Lin, Y. L., Huang, Y. L., Ma, S. H., Yeh, C. T., Chiou, S. Y., Chen, L. K. & Liao, C. L. (1997). Inhibition of Japanese encephalitis virus infection by nitric oxide: antiviral effect of nitric oxide on RNA virus replication. Journal of Virology 71, 5227-5235.[Abstract]
Lowy, R. J. & Dimitrov, D. S. (1997). Characterization of influenza virus-induced death of J774.1 macrophages. Experimental Cell Research 234, 249-258.[Medline]
Mathur, A., Bharadwaj, M., Kulshreshtha, R., Rawat, S., Jain, A. & Chaturvedi, U. C. (1988). Immunopathological study of spleen during Japanese encephalitis virus infection in mice. British Journal of Experimental Pathology 69, 423-432.[Medline]
Oh-hashi, K., Maruyama, W., Yi, H., Takahashi, T., Naoi, M. & Isobe, K. (1999). Mitogen-activated protein kinase pathway mediates peroxynitrite-induced apoptosis in human dopaminergic neuroblastoma SH-SY5Y cells. Biochemical and Biophysical Research Communications 263, 504-509.[Medline]
Patrignani, P. (2000). Nonsteroidal anti-inflammatory drugs, COX-2 and colorectal cancer. Toxicology Letters 112/113, 493-498.
Peterhans, E. (1997). Reactive oxygen species and nitric oxide in viral diseases. Biological Trace Element Research 56, 107-116.[Medline]
Primache, V., Binda, S., De Benedittis, G. & Barbi, M. (1998). In vitro activity of acetylsalicylic acid on replication of varicella-zoster virus. New Microbiologica 21, 397-401.[Medline]
Raung, S. L., Kuo, M. D., Wang, Y. M. & Chen, C. J. (2001). Role of reactive oxygen intermediates in Japanese encephalitis virus infection in murine neuroblastoma cells. Neuroscience Letters 315, 9-12.[Medline]
Ravi, V., Parida, S., Desai, A., Chandramuki, A., Gourie-Devi, M. & Grau, G. E. (1997). Correlation of tumor necrosis factor levels in the serum and cerebrospinal fluid with clinical outcome in Japanese encephalitis patients. Journal of Medical Virology 51, 132-136.[Medline]
Sagone, A. L.Jr & Husney, R. M. (1987). Oxidation of salicylates by stimulated granulocytes: evidence that these drugs act as free radical scavengers in biological systems. Journal of Immunology 138, 2177-2183.
Schwarz, K. B. (1996). Oxidative stress during viral infection: a review. Free Radical Biology and Medicine 21, 641-649.[Medline]
Schweizer, M. & Peterhans, E. (1999). Oxidative stress in cells infected with bovine viral diarrhoea virus: a crucial step in the induction of apoptosis. Journal of General Virology 80, 1147-1155.[Abstract]
Schwenger, P., Alpert, D., Skolnik, E. Y. & Vilcek, J. (1998). Activation of p38 mitogen-activated protein kinase by sodium salicylate leads to inhibition of tumor necrosis factor-induced IkappaB alpha phosphorylation and degradation. Molecular and Cellular Biology 18, 78-84.
Segal, R. A. & Greenberg, M. E. (1996). Intracellular signaling pathways activated by neurotrophic factors. Annual Review of Neuroscience 19, 463-489.[Medline]
Singh, A., Kulshreshtha, R. & Mathur, A. (2000). Secretion of the chemokine interleukin-8 during Japanese encephalitis virus infection. Journal of Medical Virology 49, 607-612.
Speir, E., Yu, Z. X., Ferrans, V. J., Huang, E. S. & Epstein, S. E. (1998). Aspirin attenuates cytomegalovirus infectivity and gene expression mediated by cyclooxygenase-2 in coronary artery smooth muscle cells. Circulation Research 83, 210-216.
Srivastava, S., Khanna, N., Saxena, S. K., Singh, A., Mathur, A. & Dhole, T. N. (1999). Degradation of Japanese encephalitis virus by neutrophils. International Journal of Experimental Pathology 80, 17-24.[Medline]
Staal, J. T., Roederer, M., Herzenberg, L. A. & Herzenberg, L. A. (1990). Intracellular thiols regulate activation of nuclear factor B and transcription of human immunodeficiency virus. Proceedings of the National Academy of Sciences, USA 87, 9943-9947.[Abstract]
Stanciu, M., Wang, Y., Kentor, R., Burke, N., Watkins, S., Kress, G., Reynolds, I., Klann, E., Angiolieri, M., Johnson, J. & Defranco, D. B. (2000). Persistent activation of ERK contributes to glutamate-induced oxidative toxicity in a neuronal cell line and primary cortical neuron cultures. Journal of Biological Chemistry 275, 12200-12206.
Vane, J. R. (1971). Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs. Nature 231, 232-235.
Verhaegen, S., McGowan, A. J., Brophy, A. R., Fernandes, R. S. & Cotter, T. G. (1995). Inhibition of apoptosis by antioxidants in the human HL-60 leukemia cell line. Biochemical Pharmacology 50, 1021-1029.[Medline]
Weissmann, G. (1991). Aspirin. Scientific American 264, 84-90.
Xia, Z., Dickens, M., Raingeaud, J., Davis, R. J. & Greenberg, M. E. (1995). Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science 270, 1326-1331.[Abstract]
Zhu, H., Bannenberg, G. L., Moldeus, P. & Shertzer, H. G. (1994). Oxidation pathways for the intracellular probe 2',7'-dichlorofluorescein. Archives of Toxicology 68, 582-587.[Medline]
Received 4 January 2002;
accepted 22 March 2002.