Glial activation involvement in neuronal death by Japanese encephalitis virus infection

Japanese encephalitis is characterized by profound neuronal destruction/dysfunction and concomitant microgliosis/astrogliosis. Although substantial activation of glia is observed in Japanese encephalitis virus (JEV)-induced Japanese encephalitis, the inflammatory responses and consequences of astrocytes and microglial activation after JEV infection are not fully understood. In this study, infection of cultured neurons/glia with JEV caused neuronal death and glial activation, as evidenced by morphological transformation, increased cell proliferation and elevated tumour necrosis factor (TNF)-alpha, interleukin (IL)-1 beta, IL-6 and RANTES (regulated upon activation, normal T-cell expressed and secreted) production. Replication-competent JEV caused all glial responses and neurotoxicity. However, replication-incompetent JEV lost these abilities, except for the ability to change microglial morphology. The bystander damage caused by activated glia also contributed to JEV-associated neurotoxicity. Microglia underwent morphological changes, increased cell proliferation and elevated TNF-alpha, IL-1 beta, IL-6 and RANTES expression in response to JEV infection. In contrast, IL-6 and RANTES expression, but no apparent morphological changes, proliferation or TNF-alpha/IL-1 beta expression, was demonstrated in JEV-infected astrocytes. Supernatants of JEV-infected microglia, but not JEV-infected astrocytes, induced glial activation and triggered neuronal death. Antibody neutralization studies revealed that TNF-alpha and IL-1 beta, but not RANTES or IL-6, released by activated microglia appeared to play roles in JEV-associated neurotoxicity. In conclusion, following JEV infection, neuronal death was accompanied by concomitant microgliosis and astrogliosis, and neurotoxic mediators released by JEV-activated microglia, rather than by JEV-activated astrocytes, had the ability to amplify the microglial response and cause neuronal death

Tyrosine kinase inhibitors attenuate Japanese encephalitis virus-induced neurotoxicity

The cellular signaling molecules that underlie Japanese encephalitis virus (JEV)-induced inflammation and neurotoxicity are not well understood. We examined whether protein tyrosine kinase (PTK) inhibitors play roles in JEV replication and cytopathic effect in neuron/glia cultures. JEV infection caused significant neuronal injury. PTK inhibitors. genistein, herbimycin A, and PP2, attenuated JEV-induced neurotoxicity but failed to affect JEV replication. Infection of neuron/glia cultures with JEV produced elevated levels of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta). PTK inhibitors suppressed JEV-induced TNF-alpha and lL-1beta production at the transcriptional level. Neutralizing antibodies against TNF-alpha and IL-1beta partially supressed JEV-induced neurotoxicity. JEV infection modulated tyrosine phosphorylation events within the course of infection. Currently, the nature of the affected phosphorylated proteins was not characterized. Our results suggest that PTKs, especially Src-related PTK. play roles in the production of TNF-alpha and IL-1beta during JEV infection and in the induction of neuronal death in neuron/glia cultures. (C) 2004 Elsevier Inc. All rights reserved

Japanese encephalitis virus infection stimulates Src tyrosine kinase in neuron/glia

Japanese encephalitis virus (JEV) is a neurotropic virus. The clinically manifestation of JEV-induced encephalitis is characterized by the brain inflammation and neuronal dysfunction and/or destruction. Currently, the cellular signaling molecules that underlie JEV-induced cerebral inflammation and cellular alterations are not well understood. Protein tyrosine phosphorylation events are key regulators of cellular signaling processes, including inflammation. We investigated whether Src protein tyrosine kinase (PTK) function in JEV-induced cellular chances in neuron/glia cultures. JEV infection modulated tyrosine phosphorylation events. Src PTK was hyperphosphorylated at the early stage of infection. Biochemical studies demonstrated that both inhibitors of the Src family PTK and Ras attenuated JEV-induced extracellular signal-regulated kinase (ERK) activation. Our results further revealed that PTK, Ras, and ERK inhibitors effectively suppressed JEV-induced pro-inflammatory cytokine expression and neurotoxicity. Pharmacological studies suggested that microglia secreted pro-inflammatory cytokine via Src/Ras/ERK pathway in responding to JEV infection. Another interesting observation was that nonstructural protein 3 (NS3) was able to interact with Src and showed tyrosine phosphorylation. However, the biological consequences of their interaction and exact control of NS3 tyrosine phosphorylation required further investigation. Our results suggest that the Src/Ras/ERK signaling cascade is involved in JEV-induced pro-inflammatory cytokine expression and neurotoxicity. (C) 2007 Elsevier Ireland Ltd. All rights reserved

Inhibition of nitric oxide production by quercetin in endotoxin/cytokine-stimulated microglia

Aims: Flavonoids possess several biological and pharmacological activities. Quercetin, a naturally occurring flavonoid, has been shown to down-regulate inflammatory responses and provide neuroprotection. However, the mechanisms underlying the anti-inflammatory properties of quercetin are poorly understood. In the present study, we investigated the modulatory effect of quercetin against neuroinflammation. Main methods: We herein describe a potential regulatory mechanism by which quercetin suppresses nitric oxide (NO) production by lipopolysaccharide (LPS)/interferon-gamma (IFN-gamma)-stimulated BV-2 microglial cells. The underlying regulatory cascades were approached by biochemical and pharmacological strategies. Key findings: Quercetin produced an inhibitory effect on inducible nitric oxide synthase (iNOS) expression and NO production. Biochemical studies revealed that the anti-inflammatory effect of quercetin was accompanied by the down-regulation of extracellular signal-regulated kinase, c-Jun N-terminal kinase, p38, Akt, Src, Janus kinase-1, Tyk2, signal transducer and activator of transcription-1, and NF-kappa B. In addition, quercetin scavenged free radicals and produced inhibitory effects on serine/threonine and tyrosine phosphatase activities. Intriguingly, the accumulation of lipid rafts, which is the critical step for signaling, was disrupted by quercetin. Significance: The data indicate that the anti-inflammatory action of quercetin may be attributable to its raft disrupting and anti-oxidant effects. These distinct mechanisms work in synergy to down-regulate iNOS expression and NO production. (C) 2010 Elsevier Inc. All rights reserved

Treatment of Atlantoaxial Rotatory Fixation With Botulinum Toxin Muscle Block and Manipulation

Slippage after reduction of atlantoaxial rotatory fixation (AARF) is usually treated with repeated cervical traction and brace immobilization. To date, no data have been published on the management of muscle spasm during treatment. Here, we describe the case of a 7-year-old girl with AARF for 1 month who visited our hospital for treatment. During physical examination, spasm of the sternocleidomastoid muscle was noted. The patient was treated with manipulative reduction, and slippage after reduction was managed with botulinum spasticity block of the sternocleidomastoid and splenius capitis muscles, and repeated manipulation. Cervical orthosis immobilization with a rehabilitation program of isometric contract-relax exercise for the neck was conducted for 3 months. The subject had full recovery from AARF at 1-year follow-up. This report demonstrates that, in selected cases of slippage after reduction from AARF, conservative management with manipulation under anesthesia is a good method, and the muscle components may play a crucial role in AARF. [J Chin Med Assoc 2010;73(4):222-224

Indomethacin induces apoptosis in 786-O renal cell carcinoma cells by activating mitogen-activated protein kinases and AKT

(NSAIDs) involves cyclooxygenase (COX)-dependent and COX-independent mechanisms. Evidence suggests that mitogen-activated protein kinases (MAPKs) may mediate apoptotic signaling induced by anti-neoplastic agents. While many reports have revealed the existence of MAPK activation in apoptosis induced by various stimuli, the signaling transduction pathways used by NSAIDs to trigger apoptosis in human renal cell carcinoma (RCC) remain largely unknown. Treatment of RCC 786-O cells with indomethacin resulted in growth regression and apoptosis. Caspase-dependent apoptosis was evidenced by the detection of enzymatic activities of caspase-3, caspase-6, and caspase-9 and suppression of toxicity using a caspase inhibitor. Indomethacin treatment was associated with increased expression of glucose-regulated protein 78 (GRP78) and C/EBP homologus protein (CHOP) and activation of ATF-6, characteristics of endoplasmic reticulum stress. In addition, the concomitant induction of peroxisome proliferator-activated receptor (PPAR), especially PPAR-beta, was apparent in treated cells. Western blotting revealed the activation of extracellular signal-regulated kinase (ERK), p38 MAPK, and c-Jun N-terminal kinase (JNK) with indomethacin treatment. Selective inhibitors of ERK, p38 MAPK, and JNK suppressed the induction of GRP78, CHOP, and PPAR-beta, attenuated indomethacin-induced cytotoxicity and reduced increased caspase activity. LY294002, a phosphomositide-3 kinase (PI3K)/AKT inhibitor, and Trolox, an antioxidant, suppressed indomethacin-induced cytotoxicity and caspase activation. Furthermore, Trolox attenuated indomethacin-induced increased phosphorylation in ERK, p38 MAPK, JNK, and AKT. In conclusion, our findings establish a mechanistic link between the oxidative stress, PI3K/AKT pathway, MAPK pathway and indomethacin-induced cellular alterations and apoptosis in 786-O cells. (c) 2007 Elsevier B.V. All rights reserved

Stearic acid attenuates cholestasis-induced liver injury

Inflammation is involved in cholestasis-induced hepatic damage. Stearic acid has been shown to possess anti-inflammatory potential. We assessed whether stearic acid has protective effects against cholestasis-related liver damage. Cholestasis was produced by bile duct ligation (BDL) in male Sprague-Dawley rats for 3 weeks. Daily administration of stearic acid was started 2 weeks before injury and lasted for 5 weeks. In comparison with the control group, the BDL group showed hepatic damage as evidenced by elevation in serum biochemicals, ductular reaction, fibrosis, and inflammation. These pathophysiological changes were attenuated by chronic stearic acid Supplementation. The anti-fibrotic effect of stearic acid was accompanied by reductions in alpha-smooth muscle actin-positive matrix-producing cells and critical fibrogenic cytokine transforming growth factor beta-1 production. Stearic acid also attenuated BDL-induced leukocyte accumulation and NF-kappa B activation. The data indicate that Stearic acid attenuates BDL-induced cholestatic liver injury. The hepatoprotective effect of stearic acid is associated with anti-inflammatory potential. (c) 2009 Elsevier Inc. All rights reserved

Antiviral effect of dehydroepiandrosterone on Japanese encephalitis virus infection

Japanese encephalitis virus (JEV), which causes neurological disorders, completes its life cycle and triggers apoptotic cell death in infected cells. Dehydroepiandrosterone (DHEA), an adrenal-derived steroid, has been implicated in protection against neurotoxicity and protection of animals from viral-induced encephalitis, resulting in an increased survival rate of the animals. Currently, the mechanisms underlying the beneficial effects of DHEA against the virus are largely unknown. In this study, DHEA suppression of JEV replication and virus-induced apoptosis in murine neuroblastoma (N18) cells was investigated. It was found that DHEA suppressed JEV-induced cytopathic effects, JEV-induced apoptotic; cell death and JEV propagation in a concentration-dependent manner. Antiviral activity was more efficient in cultures treated with DHEA immediately after viral adsorption compared with that in cultures receiving delayed administration after adsorption or transient exposure before adsorption. JEV-induced cytotoxicity was accompanied by the inactivation of extracellular signal-regulated protein kinase (ERK). Inactivation of ERK by JEV infection was reversed by DHEA. When cells were treated with the ERK inhibitor U0126, DHEA lost its antiviral effect. Activation of ERK by anisomycin mimicked the action of DHEA in suppressing JEV-induced cytotoxicity. DHEA-related compounds, such as its sulfate ester (DHEAS) and pregnenolone, were unable to suppress JEV-induced cytotoxicity and ERK inactivation. The hormone-receptor antagonists ICI 182780 and flutamide failed to abrogate the antiviral effect of DHEA. These findings suggest that the antiviral effect of DHEA is not linked directly to the genomic steroid-receptor pathways and suggest that the signalling pathways of ERK play a role in the antiviral action of DHEA

Indomethacin causes renal epithelial cell injury involving Mcl-1 down-regulation

Nonsteroidal anti-inflammatory drugs (NSAIDs) exert anti-tumor action in a variety of cancer cells. However, several treatment side effects such is gastrointestinal injury, cardiovascular toxicity, and acute renal failure limit their clinical use. We found that indomethacin caused renal epithelial cell injury independently of cyclooxygenase inhibition. Indomethacin treatment was associated with the disruption Of mitochondrial transmembrane potential, release of cytochrome c, down-regulation of Bcl-2 and Mcl-1, up-regulation of Bax, and elevation of caspases activity, Enhanced Mcl-1 but not Bcl-2 expression alleviated indomethacin-increased caspase-3 activity. Down-regulation of Akt-related and signal transducer and activator of transcription (STAT-3)-related Pathways was found in indomethacin-treated cells. Pharmacological and genetic studies revealed a potential mechanistic link between Akt/Mcl-1 and STAT-3/Mcl-1 signaling pathways and indomethacin-induced cytotoxicity. Mcl-1 is a determinant molecule for the induction of epithelial cell injury caused by indomethacin. Therefore, the maintenance of Mcl-1 levels is important for prevention of renal epithelial cell injury and apoptosis. (C) 2009 Elsevier Inc. All rights reserved

Tetramethylpyrazine reduces ischemic brain injury in rats

Tetramethylpyrazine (TMP), which is widely used in the treatment of ischemic stroke by Chinese herbalists, is one of the most important active ingredients of the traditional Chinese herbal medicine, Ligusticum wallichii Franchat (Chung Xiong). However, the mechanism by which TMP protects the brain is still not clear. We examined neuroprotective effects of TMP after transient focal cerebral ischemia using common carotid artery and middle cerebral artery occlusion model in rats and evaluated the involvement of anti-inflammation. TMP administrated intraperitoneally significantly protected the brain against ischemic insult as evidenced by the reduction in infarction volume, preservation of neurons, and decrease in brain edema. TMP markedly reduced cerebral ischemia/reperfusion-induced inflammatory cell activation and proinflammatory mediator production. Moreover, TMP suppressed lipopolysaccharide/interferon-gamma-induced inflammation and prostaglandin E-2 production in cultured glial cells. Our findings suggest that one of neuroprotective effects of TMP against ischemic brain injury might involve its anti-inflammatory potential. (C) 2004 Elsevier Ireland Ltd. All rights reserved