Treadmill exercise alleviated prenatal buprenorphine exposure-induced depression in rats.

Mounting evidence suggests that physical exercise shows health benefits in a range of diseases, including psychiatric disorders. Perinatal opioid exposure produces neurobehavioral abnormality, which includes depression symptoms, in patients and their offspring following chronic use of buprenorphine, a mixed agonist/antagonist with a high affinity to opioid receptors, for pain control. Previously, we demonstrated that prenatal buprenorphine exposure in pregnant Sprague-Dawley rats starting from gestation day 7 and lasting for 14 days caused the development of depression-like phenotypes in pups at postnatal day 21. Using the same prenatal buprenorphine exposure model, we further demonstrated that a 4-week course of moderate treadmill exercise conducted on pups starting from postnatal day 22 improved depression-like neurobehaviors. Prenatal buprenorphine exposure-induced neurobehavioral changes were accompanied by reductions of neuronal survival, neural stem cell-associated genes, plasma level of brain-derived neurotrophic factor (BDNF) and serotonin, phosphorylated tropomyosin-related kinase receptor type B (TrkB), phosphorylated extracellular signal-regulated kinase (ERK), PKA activity, phosphorylated cAMP response element-binding protein (CREB), and CREB DNA binding activity, as well as elevation of repressor element-1 silencing transcription factor (REST), oxidative stress, and inflammatory responses. Those changes in parameters of plasma and brain were improved by treadmill exercise. In conclusion, the findings of the current study suggest that a non-pharmacological option, i.e., moderate treadmill exercise, alleviated the development of depression-like neurobehaviors by resolving the oxidative and inflammatory burden as well as by enhancing neurochemical and neuroendocrine signaling.

Susceptibility of naïve and differentiated PC12 cells to Japanese encephalitis virus infection.

Japanese encephalitis is a mosquito-borne disease caused by Japanese encephalitis virus (JEV) infection. Although JEV infects and replicates in cells with multiple tissue origins, neurons are the preferential cells for JEV infection. Currently, the identities of JEV cell tropism are largely unclear. To gain better insight into the underlying identities of JEV cell tropism, this study was designed to compare the JEV cell tropism with naïve or differentiated PC12 cells. Through nerve growth factor-differentiated PC12 cells, we discovered that JEV efficiently replicated in differentiated PC12 cells rather than naïve cells. Mechanistic studies revealed that viral adsorption/attachment seemed not to be a crucial factor. Supporting data showed that antagonizing postreceptor intracellular signaling of interferons, along with the activation of suppressor of cytokine signaling-3 (SOCS3) expression and protein tyrosine phosphatase activity, were apparent in differentiated PC12 cells after JEV infection. Independent of differentiating inducing agents, the upregulation of SOCS3 expression and protein tyrosine phosphatase activity, as well as preferential JEV tropism, were common in JEV-infected differentiated PC12 cells. Using cultured primary neurons, JEV efficiently replicated in embryonic neurons rather than adult neurons, and the preference was accompanied by higher SOCS3 expression and protein tyrosine phosphatase activity. Given that both SOCS3 and protein tyrosine phosphatases have been implicated in the process of neuronal differentiation, JEV infection seems to not only create an antagonizing strategy to escape host's interferon antiviral response but also takes advantage of cellular machinery to favor its replication. Taken together, current findings imply that dynamic changes within cellular regulators of antiviral machinery could be accompanied by events of neuronal differentiation, thus concurrently playing roles in the control of JEV cell tropism and replication. © 2017 IUBMB Life, 69(2):79-87, 2017.

Enterovirus 71 infection caused neuronal cell death and cytokine expression in cultured rat neural cells.

Fatal enterovirus type-71 (EV71) cases are associated with central nervous system infection characterized by inflammatory cell infiltration and activation, cytokine overproduction, and neuronal cell death. Although EV71 antigen has been detected in neurons and glia, the molecular mechanisms underlying EV71-associated neuroinflammation and neuronal cell death are not fully understood. Using cultured rodent neural cell models, we found that EV71 infection preferentially caused cell death in neurons but not brain-resident immune cells astrocytes and microglia. Neurons, astrocytes, and microglia responded to EV71 infection by releasing distinct profiles of cytokines, including nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, regulated on activation normal T cell expressed and secreted (RANTES), and glutamate. EV71 infection-induced neuronal cell death correlated well with the elevated production of NO, TNF-α, IL-1ß, and glutamate as well as activation of microglia. Exogenous addition studies further demonstrated the neurotoxic potential of NO, TNF-α, IL-1ß, and glutamate. EV71 infection-induced cytokine expression was accompanied by activation of protein tyrosine phosphorylation, mitogen-activated protein kinases (MAPKs), and NF-κB. Intriguingly, EV71 susceptibility was accompanied by infection-elevated neuronal human scavenger receptor class B member 2 expression in cultured neural cells with age-dependent manner. Biochemical and pharmacological studies revealed that after EV71 infection, microglia and accompanied cytokines play an active role in triggering bystander damage to neurons involving the tyrosine kinase/MAPKs/NF-κB signaling cascade. These data suggest that bystander damage caused by activated glia particularly the microglia could be an alternative mechanism of EV71-associated neuronal cell death. However, its clinical importance and implication require further investigation.

Tetramethylpyrazine inhibits neutrophil activation following permanent cerebral ischemia in rats.

Experimental studies have demonstrated the beneficial effects of tetramethylpyrazine (TMP) against ischemic stroke and highlighted its crucial role in anti-inflammatory activity. This study provides evidence of an alternative target for TMP and sheds light on the mechanism of its anti-inflammatory action against ischemic brain injury. We report a global inhibitory effect of TMP on inflammatory cell intracerebral activation and infiltration in a rat model of permanent cerebral ischemia. The results of immunohistochemistry, enzymatic assay, flow cytometric analysis, and cytological analysis revealed that intraperitoneal TMP administration reduced neuronal loss, macrophage/microglia activation, brain parenchyma infiltrative neutrophils, and circulating neutrophils after cerebral ischemia. Biochemical studies of cultured neutrophils further demonstrated that TMP attenuated neutrophil migration, endothelium adhesion, spontaneous nitric oxide (NO) production, and stimuli-activated NO production after cerebral ischemia. In parallel with these anti-neutrophil phenomena, TMP also attenuated the activities of ischemia-induced inflammation-associated signaling molecules, including plasma high-mobility group box-1 protein (HMGB1) and neutrophil toll-like receptor-4 (TLR4), Akt, extracellular signal-regulated kinase (ERK), and inducible nitric oxide synthase. Another finding in this study was that the anti-neutrophil effect of TMP was accompanied by a further elevated expression of NF-E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) in neutrophils after cerebral ischemia. Taken together, our results suggest that both the promotion of endogenous anti-inflammatory defense capacity and the attenuation of pro-inflammatory responses via targeting of circulating neutrophils by elevating Nrf2/HO-1 expression and inhibiting HMGB1/TLR4, Akt, and ERK signaling might actively contribute to TMP-mediated neuroprotection against cerebral ischemia.

Luteolin sensitizes human 786-O renal cell carcinoma cells to TRAIL-induced apoptosis.

AIMS: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been considered to be one of the most promising candidates in research on treatments for cancer, including renal cell carcinoma (RCC). However, many cells are resistant to TRAIL-induced apoptosis which limits the potential application of TRAIL in cancer therapy. Luteolin, a naturally occurring flavonoid, has been identified as a potential therapeutic and preventive agent for cancer because of its potent cancer cell-killing activity. In this study, we investigated whether luteolin treatment could modulate TRAIL-induced apoptosis in RCC. MAIN METHODS: The effect of luteolin on TRAIL sensitivity was assessed in human RCC 786-O, ACHN, and A498 cells. The underlying regulatory cascades were approached by biochemical and pharmacological strategies. KEY FINDINGS: We found that nontoxic concentration of luteolin alone had no effect on the level of apoptosis, but a combination treatment of TRAIL and luteolin caused significant extrinsic and intrinsic apoptosis. The sensitization was accompanied by Bid cleavage, Mcl-1 and FLIP down-regulation, DR4/DR5 protein expression and cell surface presentation, and Akt and signal transducer and activator of transcription-3 (STAT3) inactivation. Among these phenomena, changes in FLIP, Akt, and, STAT3 are more prone to the effects of luteolin treatment. Studies have further demonstrated that inactivation of Akt or STAT3 alone was sufficient to down-regulate FLIP expression and sensitized 786-O cells to TRAIL-induced apoptosis. SIGNIFICANCE: Data from this study thus provide in vitro evidence supporting the notion that luteolin is a potential sensitizer of TRAIL in anticancer therapy against human RCC involving Akt and STAT3 inactivation.

Infection of pericytes in vitro by Japanese encephalitis virus disrupts the integrity of the endothelial barrier.

Though the compromised blood-brain barrier (BBB) is a pathological hallmark of Japanese encephalitis-associated neurological sequelae, the underlying mechanisms and the specific cell types involved are not understood. BBB characteristics are induced and maintained by cross talk between brain microvascular endothelial cells and neighboring elements of the neurovascular unit. In this study, we show a potential mechanism of disruption of endothelial barrier integrity during the course of Japanese encephalitis virus (JEV) infection through the activation of neighboring pericytes. We found that cultured brain pericytes were susceptible to JEV infection but were without signs of remarkable cytotoxicity. JEV-infected pericytes were found to release biologically active molecules which activated ubiquitin proteasome, degraded zonula occludens-1 (ZO-1), and disrupted endothelial barrier integrity in cultured brain microvascular endothelial cells. Infection of pericytes with JEV was found to elicit elevated production of interleukin-6 (IL-6), which contributed to the aforementioned endothelial changes. We further demonstrated that ubiquitin-protein ligase E3 component n-recognin-1 (Ubr 1) was a key upstream regulator which caused proteasomal degradation of ZO-1 downstream of IL-6 signaling. During JEV central nervous system trafficking, endothelial cells rather than pericytes are directly exposed to cell-free viruses in the peripheral bloodstream. Therefore, the results of this study suggest that subsequent to primary infection of endothelial cells, JEV infection of pericytes might contribute to the initiation and/or augmentation of Japanese encephalitis-associated BBB breakdown in concerted action with other unidentified barrier disrupting factors.

Docosahexaenoic acid reduces cellular inflammatory response following permanent focal cerebral ischemia in rats.

Cellular inflammatory response plays an important role in ischemic brain injury and anti-inflammatory treatments in stroke are beneficial. Dietary supplementation with docosahexaenoic acid (DHA) shows anti-inflammatory and neuroprotective effects against ischemic stroke. However, its effectiveness and its precise modes of neuroprotective action remain incompletely understood. This study provides evidence of an alternative target for DHA and sheds light on the mechanism of its physiological benefits. We report a global inhibitory effect of 3 consecutive days of DHA preadministration on circulating and intracerebral cellular inflammatory responses in a rat model of permanent cerebral ischemia. DHA exhibited a neuroprotective effect against ischemic deficits by reduction of behavioral disturbance, brain infarction, edema and blood-brain barrier disruption. The results of enzymatic assay, Western blot, real-time reverse transcriptase polymerase chain reaction and flow cytometric analysis revealed that DHA reduced central macrophages/microglia activation, leukocyte infiltration and pro-inflammatory cytokine expression and peripheral leukocyte activation after cerebral ischemia. In parallel with these immunosuppressive phenomena, DHA attenuated post-stroke oxidative stress, c-Jun N-terminal kinase (JNK) phosphorylation, c-Jun phosphorylation and activating protein-1 (AP-1) activation but further elevated ischemia-induced NF-E2-related factor-2 (Nrf2) and heme oxygenase-1 (HO-1) expression. DHA treatment also had an immunosuppressive effect in lipopolysaccharide/interferon-γ-stimulated glial cultures by attenuating JNK phosphorylation, c-Jun phosphorylation and AP-1 activation and augmenting Nrf2 and HO-1 expression. In summary, we have shown that DHA exhibited neuroprotective and anti-inflammatory effects against ischemic brain injury and these effects were accompanied by decreased oxidative stress and JNK/AP-1 signaling as well as enhanced Nrf2/HO-1 expression.

Induction of apoptosis by luteolin involving akt inactivation in human 786-o renal cell carcinoma cells.

There is a growing interest in the health-promoting effects of natural substances obtained from plants. Although luteolin has been identified as a potential therapeutic and preventive agent for cancer because of its potent cancer cell-killing activity, the molecular mechanisms have not been well elucidated. This study provides evidence of an alternative target for luteolin and sheds light on the mechanism of its physiological benefits. Treatment of 786-O renal cell carcinoma (RCC) cells (as well as A498 and ACHN) with luteolin caused cell apoptosis and death. This cytotoxicity was caused by the downregulation of Akt and resultant upregulation of apoptosis signal-regulating kinase-1 (Ask1), p38, and c-Jun N-terminal kinase (JNK) activities, probably via protein phosphatase 2A (PP2A) activation. In addition to being a concurrent substrate of caspases and event of cell death, heat shock protein-90 (HSP90) cleavage might also play a role in driving further cellular alterations and cell death, at least in part, involving an Akt-related mechanism. Due to the high expression of HSP90 and Akt-related molecules in RCC and other cancer cells, our findings suggest that PP2A activation might work in concert with HSP90 cleavage to inactivate Akt and lead to a vicious caspase-dependent apoptotic cycle in luteolin-treated 786-O cells.

Glutamate released by Japanese encephalitis virus-infected microglia involves TNF-α signaling and contributes to neuronal death.

The substantial activation of microglia in Japanese encephalitis virus (JEV)-induced Japanese encephalitis found in numerous studies demonstrates that the disease pathogenesis involves bystander damage caused by microglia-released mediators. Previously, we reported that microglia synthesized and secreted bioactive mediators with neurotoxic potential into the cultured supernatants in response to JEV infection. In this study, we found that the supernatants of JEV-infected microglia caused MK801-inhibitable neuronal damage in cultured neurons, indicating a potential excitotoxic mechanism. Infection with JEV was found to elicit the extracellular glutamate accumulation from microglia but not from neuron and astrocyte cultures. The glutaminase inhibitor 6-diazo-5-oxo-L-norleucine, cystine/glutamate antiporter inhibitor α-aminoadipic acid, and the gap junction inhibitor carbenoxolone reduced JEV infection-induced microglial glutamate release and neurotoxicity. We further demonstrated that tumor necrosis factor-alpha (TNF-α) was a key cytokine which stimulated extensive microglial glutamate release by up-regulating glutaminase expression via signals involving protein kinase C, cAMP responsive element-binding protein, and CAAT-enhancer-binding protein-beta. Although the elevated expression of excitatory amino acid transporter 1 and 2 was observed in JEV-infected cells, the glutamate uptake activity was significantly inhibited by TNF-α. The JEV infection-induced alterations, such as the extracellular glutamate release and glutamate-mediated excitoneurotoxicity, also occurred in neuron/glia cultures. Our findings support a potential link between neuroinflammation and the development of excitotoxic neuronal injury in Japanese encephalitis. The link between neuroinflammation and excitotoxic death may involve a mechanism in which TNF-α released by microglia plays a facilitory role in glutamate excitoneurotoxicity via up-regulation of glutamate synthesis and down-regulation of glutamate uptake.

Src signaling involvement in Japanese encephalitis virus-induced cytokine production in microglia.

Numerous studies have demonstrated that the disease pathogenesis of Japanese encephalitis involves cytokine-mediated bystander damage. The mechanisms involved in the regulation of Japanese encephalitis virus (JEV)-induced cytokine expression are not well defined but rely mainly on the tight regulation of transcription factor NF-κB. The Src-family tyrosine kinases participate in diversity of cellular signaling and have been demonstrated in JEV-infected cells. A direct link leading from Src activation to NF-κB activation in JEV-induced cytokine expression is incompletely understood. Here, we report that Src-related Ras/Raf/extracellular signal-regulated kinase (ERK) cascades participate in NF-κB activation and consequent tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1ß) expression in JEV-infected microglia. Central microglia were capable of producing TNF-α and IL-1ß after JEV infection. However, JEV infection had a negligible effect on triggering TNF-α and IL-1ß production by neurons and astrocytes. The expression of TNF-α and IL-1ß caused by JEV was accompanied by increased Src phosphorylation, Ras membrane association, Raf serine-338 as well as tyrosine-340 phosphorylation, ERK phosphorylation, NF-κB DNA binding activity, and decreased Raf serine-259 phosphorylation. Pharmacological studies revealed that the integrity of lipid raft and the activation of Src, Ras, Raf, ERK, and NF-κB all contributed to JEV-induced TNF-α and IL-1ß expression. Pharmacological and biochemical studies further suggested that Src, upon activation, might transmit signals to the Raf/ERK cascades via Ras-dependent and -independent mechanisms that in turn might lead to NF-κB activation. Overall, our results show that the lipid raft might play a role in mediating JEV-initiated Src/Ras/Raf/ERK/NF-κB signaling and TNF-α/IL-1ß expression in microglia.

Luteolin inhibits cytokine expression in endotoxin/cytokine-stimulated microglia.

Microglial activation plays a pivotal role in the pathogenesis of neurodegenerative disease by producing excessive proinflammatory cytokines and nitric oxide (NO). Luteolin, a naturally occurring polyphenolic flavonoid antioxidant, has potent anti-inflammatory and neuroprotective properties both in vitro and in vivo. However, the molecular mechanism of luteolin-mediated immune modulation in microglia is not fully understood. In the present study, we report the inhibitory effect of luteolin on lipopolysaccharide (LPS)/interferon γ (IFN-γ)-induced NO and proinflammatory cytokine production in rat primary microglia and BV-2 microglial cells. Luteolin concentration-dependently abolished LPS/IFN-γ-induced NO, tumor necrosis factor α (TNF-α) and interleukin 1ß (IL-1ß) production as well as inducible nitric oxide synthase (iNOS) protein and mRNA expression. Luteolin exerted an inhibitory effect on transcription factor activity including nuclear factor κB (NF-κB), signal transducer and activator of transcription 1 (STAT1) and interferon regulatory factor 1 (IRF-1) in LPS/IFN-γ-activated BV-2 microglial cells. Biochemical and pharmacological studies revealed that the anti-inflammatory effect of luteolin was accompanied by down-regulation of extracellular signal-regulated kinase (ERK), p38, c-Jun N-terminal kinase (JNK), Akt and Src. Further studies have demonstrated that the inhibitory effect of luteolin on intracellular signaling execution and proinflammatory cytokine expression is associated with resolution of oxidative stress and promotion of protein phosphatase activity. Together, these results suggest that luteolin suppresses NF-κB, STAT1 and IRF-1 signaling, thus attenuating inflammatory response of brain microglial cells.

TNF-α and IL-1ß mediate Japanese encephalitis virus-induced RANTES gene expression in astrocytes.

Infection with Japanese encephalitis virus (JEV) causes neuroinfection and neuroinflammation characterized by profound neuronal destruction/dysfunction, concomitant microgliosis/astrogliosis, and production of various molecules that initiate the recruitment of immune cells to the sites of infection. Previously, we reported that glial cells expressed RANTES (regulated upon activation, normal T cell expressed and secreted) with chemotactic activity in response to JEV infection. In this study, we further demonstrated that JEV-infected microglia had an additional activity in regulating RANTES production. Both astrocytes and microglia responded to JEV infection by releasing RANTES through a process likely related to viral replication. Independent of infectious virus, supernatants of JEV-infected microglia, but not JEV-infected astrocytes, caused additional RANTES production from astrocytes. Antibody neutralization studies suggested the potential involvement of tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1ß) in mediating additional RANTES production. Treatment of astrocyte cultures with TNF-α and IL-1ß caused activation of several signaling molecules and transcription factors crucial to RANTES gene expression, including reactive oxygen species, extracellular signal-regulated kinase, NF-κB, and NF-IL6, increased RANTES gene promoter activity, and provoked RANTES production. As with RANTES, neutralization of bioactive TNF-α and IL-1ß caused an attenuation of chemotactic activity from supernatants of mixed glia containing astrocytes and microglia during the course of JEV infection. In conclusion, TNF-α and IL-1ß produced by JEV-infected microglia might trigger another mechanism which induces a secondary wave of RANTES gene expression by activating astrocytes. The released RANTES from glial cells might play a role in the recruitment of immune cells during JEV infection.

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.

Graptopetalum paraguayense E. Walther leaf extracts protect against brain injury in ischemic rats.

As practice in folk medicine, Graptopetalum paraguayense E. Walther possesses several biological/pharmacological activities including hepatoprotective, anti-oxidant, and anti-inflammatory. We investigated the neuroprotective potential of Graptopetalum paraguayense E. Walther leaf extracts on inflammation-mediated ischemic brain injury. Water (GWE), 50% alcohol (GE50) extracts of Graptopetalum paraguayense E. Walther, and extracts obtained from further extraction of GE50 with ethyl acetate (GEE) were used. Oral administration of GEE, but not GWE or GE50, for 2 weeks protected animals against cerebral ischemia/reperfusion brain injury. The neuroprotective effect of GEE was accompanied by reductions in brain infarction, neurological deficits, caspase-3 activity, malondialdehyde content, microglia activation, and inducible nitric oxide synthase (iNOS) expression. Since microglia-mediated inflammation plays critical roles in ischemic brain injury, anti-inflammatory potential of Graptopetalum paraguayense E. Walther leaf extracts was further investigated on lipopolysaccharide (LPS)/interferon-gamma (IFN-gamma-activated BV-2 microglial cells. GEE decreased H(2)O(2)- and LPS/IFN-gamma-induced free radical generation and LPS/IFN-gamma-induced iNOS expression. Mechanistic study revealed that the neuroactive effects of GEE were markedly associated with anti-oxidative potential, activation of serine/threonine and tyrosine phosphatases, and 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-kappaB and might be attributed to the presence of polyphenolic compounds such as gallic acid, genistin, daidzin, and quercetin. Together, our findings point out its potential therapeutic strategies that target microglia activation, oxidative stress, and iNOS expression to reduce ischemic brain injury and suggest that Graptopetalum paraguayense E. Walther leaf extracts represent a valuable source for the development of neuroprotective agents.

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-kappaB. 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.

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 3weeks. Daily administration of stearic acid was started 2weeks before injury and lasted for 5weeks. 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-kappaB 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.

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)-1beta, 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-1beta, 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-1beta 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-1beta, 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.

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 as 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, upregulation 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.

Protective effect of docosahexaenoic acid against brain injury in ischemic rats.

Evidence suggests that inactivation of cell-damaging mechanisms and/or activation of cell-survival mechanisms may provide effective preventive or therapeutic interventions to reduce cerebral ischemia/reperfusion (I/R) injuries. Docosahexaenoic acid (DHA) is an essential polyunsaturated fatty acid in the central nervous system that has been shown to possess neuroprotective effects. We examined whether different preadministrative protocols of DHA have effects on brain injury after focal cerebral I/R and investigated the potential neuroactive mechanisms involved. Sprague-Dawley rats were intraperitoneally pretreated with DHA once 1 h or 3 days being subjected to focal cerebral I/R or daily for 6 weeks before being subjected to focal cerebral I/R. Reduction of brain infarction was found in all three DHA-pretreated groups. The beneficial effect of DHA on the treatment groups was accompanied by decreases in blood-brain barrier disruption, brain edema, malondialdehyde (MDA) production, inflammatory cell infiltration, interleukin-6 (IL-6) expression and caspase-3 activity. Elevation of antioxidative capacity, as evidenced by decreased MDA level and increased superoxide dismutase activity and glutathione level, was detected only in the chronic daily-administration group. The two single-administration groups showed increased phosphorylation of extracellular-signal-regulated kinase (ERK). Elevation of Bcl-2 expression was detected in the chronic daily-administration and 3-day-administration groups. In vitro study demonstrated that DHA attenuated IL-6 production from stimulated glial cells involving nuclear factor kappaB inactivation. Therefore, the data suggest that the neuroprotective mechanisms of DHA pretreatment are, in part, mediated by attenuating damaging mechanisms through reduction of cytotoxic factor production and by strengthening survival mechanisms through ERK-mediated and/or Bcl-2-mediated prosurvival cascade.

Detrimental effects of post-treatment with fatty acids on brain injury in ischemic rats.

Studies have illustrated that fatty acids, especially polyunsaturated fatty acids (PUFA), have a role in regulating oxidative stress via the enhancement of antioxidative defense capacity or the augmentation of oxidative burden. Elevated oxidative stress has been implicated in the pathogenesis of brain injury associated with cerebral ischemia/reperfusion (I/R). The objective of this study was to assess whether treatment with fatty acids after focal cerebral I/R induced by occlusion of the common carotid arteries and the middle cerebral artery has effects on brain injury in a rat model. PUFA, including arachidonic acid (AA) and docosahexaenoic acid (DHA), and the saturated fatty acid, stearic acid (SA), were administrated 60 min after reperfusion via intraperitoneal injection. AA and DHA aggravated cerebral ischemic injury, which manifested as enlargement of areas of cerebral infarction and increased impairment of motor activity, in a concentration-dependent manner. However, there were no remarkable differences in post-ischemic alterations between the SA and saline groups. The post-ischemic augmentation of injury in AA and DHA treatment groups was accompanied by increases in the permeability of the blood-brain barrier (BBB), brain edema, metalloproteinase (MMP) activity, inflammatory cell infiltration, cyclooxygenase 2 (COX-2) expression, caspase 3 activity, and malondialdehyde (MDA) production, and by a decrease in the brain glutathione (GSH) content. Furthermore, we found that either AA or DHA alone had little effect on free radical generation in neuroglia, but they greatly increased the hydrogen peroxide-induced oxidative burden. Taken together, these findings demonstrate the detrimental effect of PUFA such as AA and DHA in post-ischemic progression and brain injury after cerebral I/R is associated with augmentation of cerebral I/R-induced alterations, including oxidative changes.