Humulene derivatives from Zingiber zerumbet with the inhibitory effects on lipopolysaccharide-induced nitric oxide production.

A new humulene sesquiterpene, 5-hydroxyzerumbone (5-hydroxy-2E,6E,9E-humulatrien-8-one) (1) and a known compound, zerumboneoxide (2) were isolated from the rhizomes of Zingiber zerumbet (Zingiberaceae), and found to inhibit lipopolysaccharide-induced nitric oxide production in murine macrophage RAW 264.7 cells with IC50 values of 14.1 and 23.5 microM, respectively, by bioassay-guided fractionation (positive control: N(omega)-monomethyl-L-arginine, IC50=21.3 microM). The structure of 1 was determined by spectroscopic methods including 1D and 2D-NMR.

A new cytotoxic phenylbutenoid dimer from the rhizomes of Zingiber cassumunar.

A new phenylbutenoid dimer, (+/-)- trans-3-(4-hydroxy-3-methoxyphenyl)-4-[(E)-3,4-dimethoxystyryl]cyclohex-1-ene, was isolated from the rhizomes of Zingiber cassumunar along with the three known compounds, (+/-)- trans-3-(3,4-dimethoxyphenyl)-4-[(E)-3,4-dimethoxystyryl]cyclohex-1-ene, 4-(3,4-dimethoxyphenyl)but-1,3-diene, and 4-(2,4,5-trimethoxyphenyl)but-1,3-diene by bioassay-directed fractionation using the A549 human cancer cell line cytotoxicity assay. Structure of the new compound was elucidated by spectral analysis, including 1D and 2D NMR experiments.

Serum-dependence of LPS-induced neurotoxicity in rat cortical neurons.

Previous studies have shown that the bacterial endotoxin, lipopolysaccharide (LPS), is neurotoxic both in vitro and in vivo. The rate of binding of LPS to a target cell is greatly enhanced by serum in general and by LPS binding protein (LBP) in particular. The purpose of the study described in this paper was to determine if microglia activation and LPS-induced neurotoxicity is serum or LBP dependent. A murine microglial cell line, BV2, was used to assess the serum dependence of nitric oxide production and tumor necrosis factor a release in microglia. Embryonic rat cortical neuron/glia mixed cultures were used to determine the serum dependence of LPS-induced neurotoxicity. Our results from both cell culture systems show that LPS-induced inflammatory responses are serum dependent at lower doses of LPS and progressively become serum independent above 10 ng/ml. Purified human recombinant LBP reconstitutes the lost LPS-induced inflammatory responses in primary and immortalized cell cultures treated with heat-denatured serum and appears to account for the serum dependence. These data suggest that the cell surface signaling receptor for LPS at the low and high concentrations are likely to differ, consistent with the existence of a variety of LPS receptors.

Role of nitric oxide in inflammation-mediated neurodegeneration.

Increasing evidence has suggested that inflammation in the brain is closely associated with the pathogenesis of several degenerative neurologic disorders, including Parkinson's disease, Alzheimer's diseases, multiple sclerosis, amyotrophic lateral sclerosis, and AIDS dementia. The hallmark of brain inflammation is the activation of glial cells, especially that of microglia that produce a variety of proinflammatory and neurotoxic factors, including cytokines, fatty acid metabolites, free radicals--such as nitric oxide (NO) and superoxide. Excessive production of NO, as a consequence of nitric oxide synthase induction in activated glia, has been attributed to participate in neurodegeneration. Using primary mixed neuron-glia cultures and glia-enriched cultures prepared from embryonic rodent brain tissues, we have systemically studied the relationship between the production of NO and neurodegeneration in response to stimulation by the inflammagen lipopolysaccharide. This review summarizes our recent findings on the kinetics of NO generation, the relative contribution of microglia and astrocytes to NO accumulation, the relationship between NO production and neurodegeneration, and points of intervention along the pathways associated with NO generation to achieve neuroprotection. We also describe our results relating to the effect of several opioid-related agents on microglial activation and neuroprotection. Among these agents, the opioid receptor antagonist naloxone, especially its non-opioid enantiomer (+)-naloxone, promises to be of potential therapeutic value for the treatment of inflammation-related diseases.

Gö6976 protects mesencephalic neurons from lipopolysaccharide-elicited death by inhibiting p38 MAP kinase phosphorylation.

Glial activation is associated with inflammation-related neuron degeneration in the brain. A variety of protein kinases are assumed to contribute to the expression of inflammation-related products, such as nitric oxide (NO) and proinflammatory cytokines, however, the mechanisms of glial activation and glia-mediated neurotoxicity remain unclear. We found that the indolocarbazole, Gö6976, originally known as a selective protein kinase C (PKC) inhibitor, protects neurons from glia-mediated damage and suppresses lipopolysaccharide (LPS)-induced microglial production of inflammatory factors. The purpose of the study we report here was to determine the mechanism underlying the neuroprotective effect of Gö6976 in mesencephalic neuron/glia cultures. Gö6976 suppressed LPS-induced neurotoxicity in mesencephalic neuron/glia cultures and the protective effect of Gö6976 paralleled the suppression of p38 mitogen activated protein kinase (MAPK) activation and inhibition of NO production. Gö6976 did not directly inhibit the activity of p38 MAPK; rather, the inhibitor suppressed the phosphorylation of p38 MAPK, suggesting that the target of Gö6976 is a signaling event upstream of p38 MAPK. Although Gö6976 was originally known to be a selective PKC inhibitor, the neuroprotection was not mediated through its reputed effects on PKC activity. This paper demonstrates that the neuroprotective effect of Gö6976 against LPS-induced damage is mediated through the inhibition of proinflammatory factors, such as NO from microglia, by inhibiting the phosphorylation of p38 MAPK.

p38 MAP kinase is involved in lipopolysaccharide-induced dopaminergic neuronal cell death in rat mesencephalic neuron-glia cultures.

Immune stimulants, such as the bacterial endotoxin, lipopolysaccharide (LPS), the human immunodeficiency virus-1 coat protein gp120, or beta-amyloid peptides, lead to glial activation and production of various immune mediators, such as nitric oxide (NO) and proinflammatory cytokines in the brain. These mediators appear to contribute to neuronal cell death in neurodegenerative diseases. However, the signaling pathways, which mediate the neurotoxic effect by the endotoxin, are not understood. The purpose of this study was to determine the role of mitogen-activated protein kinase (MAPK) in LPS-induced neurodegeneration using mesencephalic dopaminergic neuron/glia cultures. We have found that the p38 MAPK is important in LPS-induced death of mesencephalic neurons in rat neuron-glia mixed cultures. Upon treatment with 10 ng/ml LPS, the number of dopaminergic neurons decreased by 80% within 48 h, preceded by a significant production of NO by glia. Neuroprotection by selective inhibition of p38 MAPK activity paralleled a decrease in LPS-induced inducible nitric oxide synthase (iNOS) expression. These events were significantly reduced by the selective p38 MAPK inhibitor, SB202190, but not by the inactive analogue SB202474. Inhibition of iNOS activity and NO production by treatment with GW274150 was also neuroprotective. Although the p38 MAPK inhibitor afforded significant neuroprotection from LPS toxicity in the neuron-glia mixed culture, it failed to protect dopaminergic neurons from 6-hydroxy-dopamine-induced toxicity, which acts directly on dopaminergic neurons by inducing hydroxyl radical formation from the mitochondria. The results suggest that p38 MAPK in glia plays a significant role in the LPS-induced death of mesencephalic neurons through induction of nitric oxide synthase and resulting NO production.