In vitro and in vivo induction of heme oxygenase-1 in rat glial cells: Possible involvement of nitric oxide production from inducible nitric oxide synthase

To determine whether heme oxygenase-1 (HO-1) protein is induced by endogenous nitric oxide (NO) in rat glial cultures, we examined the effects of lipopolysaccharide (LPS), interferon-γ (IFN-γ), and NO donors such as S-nitroso-N-acetylpenicillamine (SNAP), in mixed glial cells and in vivo rat hippocampus. In cultured glial cells, treatment with LPS induced the expression of 130-kd inducible NO synthase (iNOS) after 6 h, and NO₂⁻accumulation and enhancement of the protein level of 33-kd HO-1 after 12 h. In addition, treatment with SNAP induced HO-1 expression after 6 h. Although NOS inhibitors such as N^G-nitro-L-arginine (NNA) and N^G-methyl-L-arginine did not change LPS-induced iNOS expression, these inhibitors suppressed both NO₂⁻ accumulation and the enhancement of HO-1. Immunocytochemistry showed that treatment with LPS for 24 h induced iNOS immunoreactivity predominantly in ameboid microglia, while this treatment induced HO-1-immunoreactivity in both microglia and astrocytes. In in vivo rat hippocampus, microinjection of LPS plus IFN-γ, or SNAP after 24 h also induced HO-1 immunoreactivity in reactive microglia and astrocytes. In addition, intraperitoneal administration of NNA inhibited HO-1 immunoreactivity induced by the microinjection of LPS plus IFN-γ. These results suggest that endogenous NO production by iNOS in microglia causes autocrine and paracrine induction of HO-1 protein in microglia and astrocytes in vitro and in rat brain. GLIA 22:138–148, 1998.© 1998 Wiley-Liss, Inc.     

Manganese potentiates lipopolysaccharide-induced expression of NOS2 in C6 glioma cells through mitochondrial-dependent activation of nuclear factor kappaB

Neuronal injury in manganese neurotoxicity (manganism) is thought to involve activation of astroglial cells and subsequent overproduction of nitric oxide (NO) by inducible nitric oxide synthase (NOS2). Manganese (Mn) enhances the effects of proinflammatory cytokines on expression of NOS2 but the molecular basis for this effect has not been established. It was postulated in the present studies that Mn enhances expression of NOS2 through the cis-acting factor, nuclear factor kappaB (NF-kappaB). Exposure of C6 glioma cells to lipopopolysaccharide (LPS) resulted in increased expression of NOS2 and production of NO that was dramatically potentiated by Mn and was blocked through overexpression of mutant IkappaBalpha (S32/36A). LPS-induced DNA binding of p65/p50 was similarly enhanced by Mn and was decreased by mutant IkappaBalpha. Phosphorylation of IkappaBalpha was potentiated by Mn and LPS and was not blocked by U0126, a selective inhibitor of ERK1/2. Mn decreased mitochondrial membrane potential and increased matrix calcium, associated with a rise in intracellular reactive oxygen species (ROS) that was attenuated by the mitochondrial-specific antioxidant, MitoQ. Blocking mitochondrial ROS also attenuated the enhancing effect of Mn on LPS-induced phosphorylation of IkappaBalpha and expression of NOS2, suggesting a link between Mn-induced mitochondrial dysfunction and activation of NF-kappaB. Overexpression of a dominant-negative mutant of the NF-kappaB-interacting kinase (Nik) prevented enhancement of LPS-induced phosphorylation of IkappaBalpha by Mn. These data indicate that Mn augments LPS-induced expression of NOS2 in C6 cells by increasing mitochondrial ROS and activation of NF-kappaB.     

Protective effect of the SOD/catalase mimetic MnTMPyP on inflammation-mediated dopaminergic neurodegeneration in mesencephalic neuronal-glial cultures

Reactive oxygen species (ROS) produced by activated microglia are deleterious to neurons. In this study, we studied the effect of Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP), a superoxide dismutase/catalase mimetic, on the lipopolysaccharide (LPS)-induced degeneration of dopaminergic neurons in rat mesencephalic neuroglia cultures. MnTMPyP exhibited a significantly protective effect against LPS (5 ng/ml)-induced neurotoxicity as determined by [3H]dopamine uptake and immunocytochemical analysis. MnTMPyP significantly attenuated LPS-induced production of superoxide free radical and prostaglandin E(2) (PGE(2)) in microglia. These results indicate that MnTMPyP may potentially be used for the treatment of inflammation-related degenerative neurological disorders.     

Luteolin inhibits microglial inflammation and improves neuron survival against inflammation

Microglia activation is one of the causative factors for neuroinflammation, which results in brain damage during neurodegenerative disease. Accumulating evidence has shown that the flavonoid luteolin (Lut) possesses potent anti-inflammatory properties; however, its effect on microglia inhibition is currently unknown. Moreover, it is not clear whether Lut also has indirect neuroprotective effects by reducing inflammatory mediators and suppressing microglia activation. In this study, we examined the effects of Lut on lipopolysaccharide (LPS)-induced proinflammatory mediator production and signaling pathways in murine BV2 microglia. In addition, we cocultured microglia and neurons to observe the indirect neuroprotective effects of Lut. Lut inhibited the LPS-stimulated expression of inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor alpha (TNF-α), and interleukin-1β (IL-1β) as well as the production of nitric oxide (NO) and prostaglandin E(2) (PGE(2)). Moreover, Lut blocked LPS-induced nuclear factor kappa B (NF-κB) activation. Preincubation of microglia with Lut diminished the neurotoxic effects, owing to the direct anti-inflammatory effects of the compound. Taken together, our findings suggest that Lut may have a potential therapeutic application in the treatment of neuroinflammatory disorders.     

Silymarin protects dopaminergic neurons against lipopolysaccharide-induced neurotoxicity by inhibiting microglia activation

An inflammatory response in the central nervous system mediated by activation of microglia is a key event in the early stages of the development of neurodegenerative diseases. Silymarin is a polyphenolic flavanoid derived from milk thistle that has anti-inflammatory, cytoprotective and anticarcinogenic effects. In this study, we first investigated the neuroprotective effect of silymarin against lipopolysaccharide (LPS)-induced neurotoxicity in mesencephalic mixed neuron-glia cultures. The results showed that silymarin significantly inhibited the LPS-induced activation of microglia and the production of inflammatory mediators, such as tumour necrosis factor-alpha and nitric oxide (NO), and reduced the damage to dopaminergic neurons. Therefore, the inhibitory mechanisms of silymarin on microglia activation were studied further. The production of inducible nitric oxide synthase (iNOS) was studied in LPS-stimulated BV-2 cells as a model of microglia activation. Silymarin significantly reduced the LPS-induced nitrite, iNOS mRNA and protein levels in a dose-dependent manner. Moreover, LPS could induce the activation of p38 mitogen-activated protein kinase (MAPK) and c-jun N-terminal kinase but not extracellular signal-regulated kinase. The LPS-induced production of NO was inhibited by the selective p38 MAPK inhibitor SB203580. These results indicated that the p38 MAPK signalling pathway was involved in the LPS-induced NO production. However, the activation of p38 MAPK was not inhibited by silymarin. Nevertheless, silymarin could effectively reduce LPS-induced superoxide generation and nuclear factor kappaB (NF-kappaB) activation. It suggests that the inhibitory effect of silymarin on microglia activation is mediated through the inhibition of NF-kappaB activation.     

Triptolide, a Chinese herbal extract, protects dopaminergic neurons from inflammation-mediated damage through inhibition of microglial activation

Mounting lines of evidence have suggested that brain inflammation participates in the pathogenesis of Parkinson's disease. Triptolide is one of the major active components of Chinese herb Tripterygium wilfordii Hook F, which possesses potent anti-inflammatory and immunosuppressive properties. We found that triptolide concentration-dependently attenuated the lipopolysaccharide (LPS)-induced decrease in [3H]dopamine uptake and loss of tyrosine hydroxylase-immunoreactive neurons in primary mesencephalic neuron/glia mixed culture. Triptolide also blocked LPS-induced activation of microglia and excessive production of TNFalpha and NO. Our data suggests that triptolide may protect dopaminergic neurons from LPS-induced injury and its efficiency in inhibiting microglia activation may underlie the mechanism.     

Agmatine suppresses nitric oxide production in microglia

We investigated the effect of agmatine, an arginine metabolite synthesized in the brain, in cultured microglia obtained from neonatal rat cerebral cortex. Agmatine (1–300 μM) did not affect viability of cultured microglia. Activation of microglia by lipopolysaccharide (LPS, 1 μg/ml) caused the expression of inducible nitric oxide synthase (iNOS) and the production of nitric oxide (NO) assessed as the accumulation of nitrite in the culture supernatants. Agmatine had no effect on the expression of iNOS, but significantly suppressed the LPS-induced NO production in a concentration-dependent manner. Agmatine was also effective in suppressing the production of NO induced by a combination of interferon-γ (500 U/ml) and amyloid β protein (10 μM). In co-cultures of rat cortical neurons and microglia, LPS caused significant loss of neuron viability. The LPS neurotoxicity was not observed in the absence of microglia, and was completely blocked by the NOS inhibitor diphenyleneiodoium chloride. The neuronal death induced by microglia-derived NO was significantly attenuated by the presence of agmatine. These results suggest that agmatine works to protect neurons by inhibiting the production of NO in microglia.     

<Emphasis Type="Italic">Withania somnifera</Emphasis> and Its Withanolides Attenuate Oxidative and Inflammatory Responses and Up-Regulate Antioxidant Responses in BV-2 Microglial Cells

Withania somnifera (L.) Dunal, commonly known as Ashwagandha, has been used in Ayurvedic medicine for promoting health and quality of life. Recent clinical trials together with experimental studies indicated significant neuroprotective effects of Ashwagandha and its constituents. This study is aimed to investigate anti-inflammatory and anti-oxidative properties of this botanical and its two withanolide constituents, namely, Withaferin A and Withanolide A, using the murine immortalized BV-2 microglial cells. Ashwagandha extracts not only effectively inhibited lipopolysaccharide (LPS)-induced nitric oxide (NO) and reactive oxygen species (ROS) production in BV-2 cells, but also stimulates the Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathway, leading to induction of heme oxygenase-1 (HO-1), both in the presence and absence of LPS. Although the withanolides were also capable of inhibiting LPS-induced NO production and stimulating Nrf2/HO-1 pathway, Withaferin A was tenfold more effective than Withanolide A. In serum-free culture, LPS can also induce production of long thin processes (filopodia) between 4 and 8 h in BV-2 cells. This morphological change was significantly suppressed by Ashwagandha and both withanolides at concentrations for suppressing LPS-induced NO production. Taken together, these results suggest an immunomodulatory role for Ashwagandha and its withanolides, and their ability to suppress oxidative and inflammatory responses in microglial cells by simultaneously down-regulating the NF-kB and upregulating the Nrf2 pathways.     

Protein tyrosine kinase inhibitors suppress the production of nitric oxide in mixed glia, microglia-enriched or astrocyte-enriched cultures

Nitric oxide (NO) produced by glial cells has been implicated in the neuropathogenesis of various diseases. However, the signaling transduction pathway(s) for the production of NO in these cells is not well understood. To test whether protein tyrosine kinases (PTKs) are required for signaling events of NO production in glial cells, this study examined the effects of genistein and tyrphostin A25, two potent inhibitors of PTKs, on the production of NO in mouse primary mixed glia, microglia-enriched or astrocyte-enriched cultures exposed to lipopolysaccharide (LPS) or a combination of LPS and interferon-γ (IFNγ). LPS induced a dose-dependent increase in NO production from the mixed glia cultures. The LPS-induced NO production was significantly enhanced by stimulating the cells with IFNγ. Genistein or tyrphostin A25 inhibited the production of NO in both LPS- and IFNγ/LPS-stimulated mixed glia cultures. The production of NO in the stimulated microglia-enriched or astrocyte-enriched cultures was also inhibited by tyrphostin A25. To verify the cellular sources of NO, immunocytochemical staining of inducible NO synthase (iNOS) was followed by staining with the microglia marker Mac-1 or the astrocyte marker glial fibrillary acid protein (GFAP) in microglia-enriched or astrocyte-enriched cultures. The expression of iNOS and the production of NO in microglia-enriched cultures were significantly higher than those in the identically stimulated astrocyte-enriched cultures. These results demonstrate that PTKs are involved in the signaling events of LPS-induced NO production in microglia and astrocytes, and that microglia are more responsive than astrocytes to stimuli which induce NO. These results may provide insights into therapeutic interventions in the pathway for NO production in the brain.     

Alismol,a Sesquiterpenoid Isolated from <Emphasis Type="Italic">Vladimiria souliei</Emphasis>, Suppresses Proinflammatory Mediators in Lipopolysaccharide-Stimulated Microglia

Microglia activation plays an important role in the pathogenesis of various neurodegenerative diseases by producing neurotoxic factors, such as proinflammatory cytokines and nitric oxide (NO); therefore, suppression of microglia activation is a potential therapeutic approach against these diseases. Previous study showed that alismol, a sesquiterpenoid isolated from the roots of Vladimiria souliei inhibits interferon-γ-induced NO production in murine macrophage RAW264.7 cells. In the present study, we found that alismol reduced NO and prostaglandin E₂ (PGE₂) levels and suppressed the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in lipopolysaccharide (LPS)-stimulated primary and cultured microglia. Alismol also inhibited the mRNA and protein expression of proinflammatory cytokines including interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α. Further mechanistic studies revealed that alismol inhibited LPS-induced nuclear factor-κB (NF-κB) activation but not mitogen-activated protein kinase (MAPK) pathway. Finally, we demonstrated the neuroprotective effects of alismol in microglia-neuron coculture systems. Collectively, these results suggest that the inhibition of microglia activation by alismol may provide potential therapeutic strategy for various neuroinflammatory diseases.     

Protective role of heme oxygenase-1 in oxidative stress-induced neuronal injury.

Heme oxygenase-1 (HO-1) is a stress protein induced in response to a variety of oxidative challenges. After treatment of the hybrid septal cells SN 56 with beta-amyloid peptide (beta-AP1-40) or hydrogen peroxide (H2O2), we detected high levels of reactive oxygen species, accompanied by a significant elevation in HO-1 expression. Levels of HO-1 increased and then decreased following cell loss. Pretreatment of SN 56 cells with HO-1 antisense oligonucleotides dramatically decreased the immunoreactivity of HO-1 and significantly enhanced the cytotoxicity of beta-AP1-40 and H2O2. In contrast, pretreatment with hemin, an HO-1 inducer, increased the expression of HO-1 and decreased the beta-AP1-40- and H2O2-induced cytotoxicity. These findings support the importance of HO-1 in protecting neurons against oxidative stress-induced injury.     

Carbon monoxide (CO)-releasing molecule-derived CO regulates tissue factor and plasminogen activator inhibitor type 1 in human endothelial cells

Introduction

Heme oxygenase-1 (HO-1) is the rate limiting enzyme that catalyzes the conversion of heme into biliverdin, free iron, and carbon monoxide (CO). The first human case of HO-1 deficiency showed abnormalities in blood coagulation and the fibrinolytic system. Thus, HO-1 or HO-1 products, such as CO, might regulate coagulation and the fibrinolytic system. This study examined whether tricarbonyldichlororuthenium (II) dimer (CORM-2), which liberates CO, modulates the expression of tissue factor (TF) and plasminogen activator inhibitor type 1 (PAI-1) in human umbilical vein endothelial cells (HUVECs), and TF expression in peripheral blood mononuclear cells (PBMCs). Additionally, we examined the mechanism by which CO exerts its effects.

Materials and Methods

HUVECs were pretreated with 50 μM CORM-2 for 3 hours, and stimulated with tumor necrosis factor-α (TNF-α, 10 ng/ml) for an additional 0-5 hours. PBMCs were pretreated with 50-100 μM CORM-2 for 1hour followed by stimulating with lipopolysaccharid (LPS, 10 ng/ml) for additional 0-9 hours. The mRNA and protein levels were determined by RT-PCR and western blotting, respectively.

Results

Pretreatment with CORM-2 significantly inhibited TNF-α-induced TF and PAI-1 up-regulation in HUVECs, and LPS-induced TF expression in PBMCs. CORM-2 inhibited TNF-α-induced activation of p38 MAPK, ERK1/2, JNK, and NF-κB signaling pathways in HUVECs.

Conclusions

CORM-2 suppresses TNF-α-induced TF and PAI-1 up-regulation, and MAPKs and NF-κB signaling pathways activation by TNF-α in HUVECs. CORM-2 suppresses LPS-induced TF up-regulation in PBMCs. Therefore, we envision that the antithrombotic activity of CORM-2 might be used as a pharmaceutical agent for the treatment of various inflammatory conditions.     

IL-1beta, an immediate early protein secreted by activated microglia, induces iNOS/NO in C6 astrocytoma cells through p38 MAPK and NF-kappaB pathways

In the present study we sought to examine cell-cell interactions by investigating the effects of factors released by stimulated microglia on inducible nitric oxide (NO) synthase (iNOS) induction in astrocytoma cells. After examining the temporal profiles of proinflammatory molecules induced by lipopolysaccharide (LPS) stimulation in BV2 microglial cells, iNOS and IL-1beta were observed to be the first immediate-response molecules. Removal of LPS after 3 hr stimulation abrogated NO release, whereas a full induction of IL-1beta was retained in BV2 cells. We observed consistently that conditioned medium (CM) from activated microglia resulted in the induction of iNOS in C6 cells, and IL-1beta was shown to be a key regulator of iNOS induction. An IL-1beta-neutralizing antibody diminished NO induction. Incubation with recombinant IL-1beta stimulated NO release to a lesser extent compared to microglial CM; co-treatment of LPS and IL-1beta had a potent, synergistic effect on NO release from C6 cells. Transient transfection with MEK kinase 1 (MEKK1) or nuclear factor-kappa B (NF-kappaB) expression plasmids induced iNOS, and IL-1beta further enhanced the MEKK1 response. Furthermore, IL-1beta-mediated NO release from C6 cells was significantly suppressed by inhibition of p38 mitogen activated protein kinase (MAPK) or NF-kappaB by specific chemical inhibitors. Both IL-1beta and MEKK1 stimulated p38 and JNK MAPKs, as well as the NF-kappaB pathway, to induce iNOS in C6 cells. Microglia may represent an anti-tumor response in the central nervous system, which is potentiated by the local secretion of immunomodulatory factors that in turn affects astrocytoma (glioma) cells. A better understanding of microglia-glioma or microglia-astrocyte interactions will help in the design of novel immune-based therapies for brain tumors or neuronal diseases.     

Cyclopentenone prostaglandin 15-deoxy-Delta(12,14)-prostaglandin J(2) acts as a general inhibitor of inflammatory responses in activated BV-2 microglial cells

15-deoxy-Delta(12,14)-PGJ(2), a cyclopentenone derivative of PGD(2), was recently reported [Petrova et al., Proc. Natl. Acad. Sci. USA 96 (1999) 4668-4673] to suppress inducible nitric oxide synthase (iNOS) production in microglia and mixed glial cultures stimulated with lipopolysaccharide (LPS). We report here that in addition to suppressing iNOS production, 15d-PGJ(2) also decreases the production of tumor necrosis factor alpha (TNFalpha), interleukin-1 beta (IL-1beta) and cyclooxygenase-2 (COX-2) in LPS-stimulated BV-2 microglial cells, thereby acting as a general inhibitor of microglial activation. Concomitantly, 15d-PGJ(2) itself up-regulates the production of the antioxidant enzyme heme oxygenase-1 (HO-1) and increases intracellular total glutathione levels. To test if increased HO-1 levels were involved in the ability of 15d-PGJ(2) to block microglial activation, we used a HO-1 inhibitor that could block the activity of HO-1. The presence of the HO-1 inhibitor did not alter the 15d-PGJ(2)-induced inhibition of LPS-stimulated iNOS and TNFalpha protein levels, and led to only a partial reduction in the protection offered by 15d-PGJ(2) against LPS-induced nitrite production. These results suggest that HO-1 upregulation by 15d-PGJ(2) is not the primary pathway responsible for the anti-inflammatory action of 15d-PGJ(2) in microglial cells.