Harsharan S. Bhatia

Regulation of prostaglandin E2 synthase expression in activated primary rat microglia: Evidence for uncoupled regulation of mPGES-1 and COX-2

Prostaglandin E2 (PGE2) is among the most important mediators involved in neuroinflammatory processes. The final step of its synthesis is regulated by enzymes termed prostaglandin E2 synthases (PGES). Three PGES are known, cytosolic (c)PGES, membrane‐associated (m)PGES‐1 and mPGES‐2. The expression of mPGES‐1 is induced by inflammatory stimuli such as lipopolysaccharide (LPS), interleukin (IL)‐1β, and tumor necrosis factor (TNF)‐α. Although some roles of mPGES‐1 have already been suggested, its function in the CNS and the signaling pathways involved in its upregulation are poorly understood. In this study, we examined the regulation of mPGES‐1 in primary rat microglia and the signaling pathways involved in its expression. Whereas the expression of cPGES and mPGES‐2 was not stimulated by LPS, low doses of LPS (0.1–1 ng/mL) sufficiently stimulated mPGES‐1 mRNA expression. A corresponding protein synthesis, however, was obtained only with higher doses (10–100 ng/mL). The LPS‐induced increase of mPGES‐1 was inhibited by different signaling pathway inhibitors, such as SP600125, LY294002, GF109203X, and SC‐514, suggesting the involvement of c‐Jun N‐terminal kinase (JNK), phosphatidylinositol 3‐kinase (PI‐3K)/Akt, protein kinase C (PKC) pathways, and the nuclear factor (NF)‐κB, respectively. In contrast to other reports, LPS‐induced mPGES‐1 synthesis was not invariably coupled to the synthesis of COX‐2, since inhibition of PI‐3K with LY294002 decreased mPGES‐1 but increased COX‐2 levels. This detailed view of the intracellular signaling pathways involved in mPGES‐1 expression in activated microglia opens a new avenue in the search for novel potential therapeutic targets to reduce neuroinflammation, and demonstrates that mPGES‐1 expression is not strictly coupled to the expression of COX‐2.

Mangiferin inhibits cyclooxygenase-2 expression and prostaglandin E2 production in activated rat microglial cells

Mangiferin, a naturally occurring glucosylxanthone, has potent antioxidant and anti-inflammatory properties, as demonstrated in several reports. However, very limited information is available on the effects of this natural polyphenol on microglial activation. Thus, the aim of this study was to examine whether mangiferin is able to reduce prostaglandin E(2) (PGE(2)) and 8-iso-prostaglandin F(2alpha) (8-iso-PGF(2alpha)) production by lipopolysaccharide (LPS)-activated primary rat microglia. Microglial cells were stimulated with 10ng/ml of LPS in the presence or absence of different concentrations of mangiferin (1-50 microM). After 24h incubation, culture media were collected to measure the production of PGE(2) and 8-iso-PGF(2alpha) using enzyme immunoassays. Protein levels of cyclooxygenase (COX)-1 and COX-2 were studied by immunoblotting after 24h of incubation with LPS. Mangiferin potently reduced LPS-induced PGE(2) synthesis and the formation of 8-iso-PGF(2alpha). Interestingly, mangiferin dose-dependently reduced LPS-induced COX-2 protein synthesis without modifying COX-2 transcription. This was due to a decrease in COX-2 transcript stability. However, mangiferin did not modify LPS-mediated phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK), a key factor involved in enhancing COX-2 mRNA stability and COX-2 translation in primary microglia. Mangiferin had no effects on LPS-induced expression of inducible nitric oxide synthase (iNOS) or TNF-alpha production. Taken together, results from the present study indicate that mangiferin is able to limit microglial activation, in terms of attenuation of PGE(2) production, free radical formation and reduction in COX-2 synthesis induced by LPS. These data suggest that modulation of microglial activation might contribute to the mechanism of cerebral protection by mangiferin.

Pharmacological inhibition of Akt and downstream pathways modulates the expression of COX-2 and mPGES-1 in activated microglia

BackgroundMicroglia are considered a major target for modulating neuroinflammatory and neurodegenerative disease processes. Upon activation, microglia secrete inflammatory mediators that contribute to the resolution or to further enhancement of damage in the central nervous system (CNS). Therefore, it is important to study the intracellular pathways that are involved in the expression of the inflammatory mediators. Particularly, the role of the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) and glycogen synthase kinase-3 (GSK-3) pathways in activated microglia is unclear. Thus, in the present study we investigated the role of Akt and its downstream pathways, GSK-3 and mTOR, in lipopolysaccharide (LPS)-activated primary rat microglia by pharmacological inhibition of these pathways in regard to the expression of cyclooxygenase (COX)-2 and microsomal prostaglandin E synthase-1 (mPGES-1) and to the production of prostaglandin (PG) E2 and PGD2.FindingsWe show that inhibition of Akt by the Akt inhibitor X enhanced the production of PGE2 and PGD2 without affecting the expression of COX-2, mPGES-1, mPGES-2 and cytosolic prostaglandin E synthase (cPGES). Moreover, inhibition of GSK-3 reduced the expression of both COX-2 and mPGES-1. In contrast, the mTOR inhibitor rapamycin enhanced both COX-2 and mPGES-1 immunoreactivity and the release of PGE2 and PGD2. Interestingly, NVP-BEZ235, a dual PI3K/mTOR inhibitor, enhanced COX-2 and reduced mPGES-1 immunoreactivity, albeit PGE2 and PGD2 levels were enhanced in LPS-stimulated microglia. However, this compound also increased PGE2 in non-stimulated microglia.ConclusionTaken together, we demonstrate that blockade of mTOR and/or PI3K/Akt enhances prostanoid production and that PI3K/Akt, GSK-3 and mTOR differently regulate the expression of mPGES-1 and COX-2 in activated primary microglia. Therefore, these pathways are potential targets for the development of novel strategies to modulate neuroinflammation.

Ascorbic acid enhances the inhibitory effect of aspirin on neuronal cyclooxygenase-2-mediated prostaglandin E2 production

Inhibition of neuronal cyclooxygenase-2 (COX-2) and hence prostaglandin E2 (PGE2) synthesis by non-steroidal anti-inflammatory drugs has been suggested to protect neuronal cells in a variety of pathophysiological situations including Alzheimers disease and ischemic stroke. Ascorbic acid (vitamin C) has also been shown to protect cerebral tissue in a variety of experimental conditions, which has been attributed to its antioxidant capacity. In the present study, we show that ascorbic acid dose-dependently inhibited interleukin-1beta (IL-1beta)-mediated PGE2 synthesis in the human neuronal cell line, SK-N-SH. Furthermore, in combination with aspirin, ascorbic acid augmented the inhibitory effect of aspirin on PGE2 synthesis. However, ascorbic acid had no synergistic effect along with other COX inhibitors (SC-58125 and indomethacin). The inhibition of IL-1beta-mediated PGE2 synthesis by ascorbic acid was not due to the inhibition of the expression of COX-2 or microsomal prostaglandin E synthase (mPGES-1). Rather, ascorbic acid dose-dependently (0.1-100 microM) produced a significant reduction in IL-1beta-mediated production of 8-iso-prostaglandin F2alpha (8-iso-PGF2alpha), a reliable indicator of free radical formation, suggesting that the effects of ascorbic acid on COX-2-mediated PGE2 biosynthesis may be the result of the maintenance of the neuronal redox status since COX activity is known to be enhanced by oxidative stress. Our results provide in vitro evidence that the neuroprotective effects of ascorbic acid may depend, at least in part, on its ability to reduce neuronal COX-2 activity and PGE2 synthesis, owing to its antioxidant properties. Further, these experiments suggest that a combination of aspirin with ascorbic acid constitutes a novel approach to render COX-2 more sensitive to inhibition by aspirin, allowing an anti-inflammatory therapy with lower doses of aspirin, thereby avoiding the side effects of the usually high dose aspirin treatment.

Early Life Stress Differentially Modulates Distinct Forms of Brain Plasticity in Young and Adult Mice

Background Early life trauma is an important risk factor for many psychiatric and somatic disorders in adulthood. As a growing body of evidence suggests that brain plasticity is disturbed in affective disorders, we examined the short-term and remote effects of early life stress on different forms of brain plasticity. Methodology/Principal Findings Mice were subjected to early deprivation by individually separating pups from their dam in the first two weeks after birth. Distinct forms of brain plasticity were assessed in the hippocampus by longitudinal MR volumetry, immunohistochemistry of neurogenesis, and whole-cell patch-clamp measurements of synaptic plasticity. Depression-related behavior was assessed by the forced swimming test in adult animals. Neuropeptides and their receptors were determined by real-time PCR and immunoassay. Early maternal deprivation caused a loss of hippocampal volume, which returned to normal in adulthood. Adult neurogenesis was unaffected by early life stress. Long-term synaptic potentiation, however, was normal immediately after the end of the stress protocol but was impaired in adult animals. In the forced swimming test, adult animals that had been subjected to early life stress showed increased immobility time. Levels of substance P were increased both in young and adult animals after early deprivation. Conclusion Hippocampal volume was affected by early life stress but recovered in adulthood which corresponded to normal adult neurogenesis. Synaptic plasticity, however, exhibited a delayed impairment. The modulation of synaptic plasticity by early life stress might contribute to affective dysfunction in adulthood.

Inhibition of neuroinflammation in LPS-activated microglia by cryptolepine

Cryptolepine, an indoloquinoline alkaloid in Cryptolepis sanguinolenta, has anti-inflammatory property. In this study, we aimed to evaluate the effects of cryptolepine on lipopolysaccharide (LPS)- induced neuroinflammation in rat microglia and its potential mechanisms. Microglial activation was induced by stimulation with LPS, and the effects of cryptolepine pretreatment on microglial activation and production of proinflammatory mediators, PGE2/COX-2, microsomal prostaglandin E2 synthase and nitric oxide/iNOS were investigated. We further elucidated the role of Nuclear Factor-kappa B (NF-κB) and the mitogen-activated protein kinases in the antiinflammatory actions of cryptolepine in LPS-stimulated microglia. Our results showed that cryptolepine significantly inhibited LPS-induced production of tumour necrosis factor-alpha (TNFα), interleukin-6 (IL-6), interleukin-1beta (IL-1β), nitric oxide, and PGE2. Protein and mRNA levels of COX-2 and iNOS were also attenuated by cryptolepine. Further experiments on intracellular signalling mechanisms show that IκB-independent inhibition of NF-κB nuclear translocation contributes to the anti-neuroinflammatory actions of cryptolepine. Results also show that cryptolepine inhibited LPS-induced p38 and MAPKAPK2 phosphorylation in the microglia. Cell viability experiments revealed that cryptolepine (2.5 and 5 μM) did not produce cytotoxicity in microglia. Taken together, our results suggest that cryptolepine inhibits LPS-induced microglial inflammation by partial targeting of NF-κB signalling and attenuation of p38/MAPKAPK2.

Histone deacetylase inhibitors valproic acid and sodium butyrate enhance prostaglandins release in lipopolysaccharide-activated primary microglia

Modifications of histone deacetylases (HDACs) may be involved in microglia-driven neuroinflammatory responses. Recent studies suggest that several inflammatory molecules can regulate the extent of neurodegeneration and regeneration in the central nervous system (CNS). In the present study, we investigated the effects of HDAC inhibitors (HDACi) valproic acid (VPA) and sodium butyrate (NaBut) on the release of prostaglandins (PGs) in lipopolysaccharide (LPS)-activated microglia. We found that VPA and NaBut significantly enhanced LPS-induced release of PGE2, PGD2 and 8-iso-PGF2α. In addition, both compounds increased cyclooxygenase-2 and microsomal prostaglandin E synthase immunoreactivity and gene expression in LPS-stimulated microglia. Interestingly, treatment of activated microglia with HDACi also enhanced the gene expression and the release of different pro-inflammatory cytokines. Microglia activation with LPS leads to IκB-α degradation, as well as p38, ERK1/2 and JNK MAPKs phosphorylation and thus activation, which is not affected by treatment with VPA and NaBut. Furthermore, VPA and NaBut treatment induced histone acetylation at H3-K18 in microglia. We suggest that VPA and NaBut-driven increase in PGs release in LPS-activated microglia might be regulated at the transcriptional level and involves histone hyperacetylation. Our data demonstrate that VPA and NaBut are able to modulate microglia responses to inflammatory insults and thus possibly can regulate the CNS degenerative and regenerative processes.

Resveratrol inhibits prostaglandin formation in IL-1β-stimulated SK-N-SH neuronal cells

Resveratrol, a polyphenol present in grapes and red wine, has been studied due to its vast pharmacological activity. It has been demonstrated that resveratrol inhibits production of inflammatory mediators in different in vitro and in vivo models. Our group recently demonstrated that resveratrol reduced the production of prostaglandin (PG) E2 and 8-isoprostane in rat activated microglia. In a microglial-neuronal coculture, resveratrol reduced neuronal death induced by activated microglia. However, less is known about its direct roles in neurons. In the present study, we investigated the effects of resveratrol on interleukin (IL)-1β stimulated SK-N-SH cells. Resveratrol (0.1-5 μM) did not reduce the expression of cyclooxygenase (COX)-2 and microsomal PGE2 synthase-1 (mPGES-1), although it drastically reduced PGE2 and PGD2 content in IL-1β-stimulated SK-N-SH cells. This effect was due, in part, to a reduction in COX enzymatic activity, mainly COX-2, at lower doses of resveratrol. The production of 8-iso-PGF2α, a marker of cellular free radical generation, was significantly reduced by resveratrol. The present work provides evidence that resveratrol reduces the formation of prostaglandins in neuroblastoma cells by reducing the enzymatic activity of inducible enzymes, such as COX-2, and not the transcription of the PG synthases, as demonstrated elsewhere.

Identification of 5-HT3 receptors on human platelets: increased surface immunoreactivity after activation with adenosine diphosphate (ADP) and thrombin receptor-activating peptide (TRAP).

Identification of 5-HT3 receptors on human platelets: Increased surface immunoreactivity after activation with adenosine diphosphate (ADP) and thrombin receptor-activating peptide (TRAP) -

Anti-inflammatory effects of 5-HT3 receptor antagonists in interleukin-1beta stimulated primary human chondrocytes

BACKGROUND Chondrocytes are one of the main cell types involved in rheumatoid inflammation, releasing mediators which add to cartilage destruction, bone damage and consequently disability. Current evidence suggests that serotonin 5-HT(3) receptor antagonists (5-HT(3)RA) show anti-inflammatory and antioxidant properties in vitro and in vivo. Yet, the mechanisms of the anti-inflammatory effects of 5-HT(3)RA have not been elucidated in detail. METHODS Therefore, we examined in detail the effects of 5-HT(3)RA on inflammatory parameters in human primary chondrocytes in vitro by studying prostaglandin E2 (PGE2) and 8-isoprostane (8-iso-PGF2α) levels by EIA and interleukin-6 (IL-6) synthesis by ELISA. Cyclooxygenase-2 (COX-2) and microsomal prostaglandin E synthase-1 (mPGES-1) protein levels were analyzed by Western blot. RESULTS We found a significant reduction of IL-1β induced PGE2, 8-iso-PGF2β and IL-6 chondrocytes by 5-HT(3)RA especially by dolasetron. CONCLUSIONS This study provides additional support to the potential use of 5-HT(3)RAs as therapeutic agents to reduce joint inflammation.