macelignan and Inflammation

Ischemia-reperfusion injury (IRI) refers to tissue damage that occurs when blood supply returns to tissue after a period of ischemia, anoxia or hypoxia. It occurs frequently during shock, organ transplantation and heart failure. It can cause impairment or even renal failure. Macelignan is a lignin isolated from the seeds of Myristica fragrans. It has been reported to inhibit neuroinflammation and oxidative toxicity. The preventive or therapeutic effects of macelignan on renal IRI has not been reported. The present study investigated the effects of macelignan on renal IRI in rats, and the underlying mechanism(s). Healthy adult male Sprague Dawley rats (n = 50) aged 7 - 9 weeks (mean weight = 220 ± 20 g) were used in this study. The rats were randomly assigned to five groups of 10 rats each: sham   treated group, IRI group and 40 mg macelignan/kg body weight (bwt) group, 80 mg macelignan/kg bwt group, and 160 mg macelignan/kg bwt group. Ischemia-reperfusion injury was induced in the rats using standard procedure. The results showed that serum levels of creatinine, blood urea nitrogen (BUN), interleukin-6 (IL-6), tumor necrosis factor α (TNF-α) and gamma interferon (IFN-γ) were significantly higher in IRI group than in sham treated group, but were significantly and dose-dependently reduced after treatment with macelignan (p < 0.05). The activities of catalase and superoxide dismutase (SOD), and reduced glutathione (GSH) level were significantly reduced in IRI group, when compared with sham treated group, but were significantly and dose-dependently increased after treatment with macelignan (p < 0.05). However, the level of malondialdehyde (MDA) was significantly higher in IRI group than in sham treated group, but treatment with macelignan reduced it significantly and dose-dependently (p < 0.05). Macelignan also significantly and dose-dependently inhibited IRI-induced apoptosis in epithelial cells of renal tubules (p < 0.05). The results of Western blotting showed that IRI significantly upregulated the expressions of bax and caspase-3, and down-regulated the expression of bcl-2 in epithelial cells of renal tubules (p < 0.05). However, treatment with macelignan significantly and dose-dependently down-regulated the expressions of bax and caspase-3 in these cells, but significantly and dose-dependently upregulated the expression of bcl-2. These results show that macelignan confers protection on renal IRI via mechanisms involving inhibition of infla

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Biomarkers; Blood Urea Nitrogen; Caspase 3; Catalase; Creatinine; Epithelial Cells; Glutathione; Inflammation; Interferon-gamma; Interleukin-6; Kidney; Lignans; Male; Malondialdehyde; Rats, Sprague-Dawley; Reperfusion Injury; Superoxide Dismutase; Tumor Necrosis Factor-alpha

Inflammatory events involving activated microglia have been recognized to play an important role in pathogenesis of various neurodegenerative disorders including Parkinson disease. Compounds regulating activation profiles of microglia may provide therapeutic benefits for Parkinson disease characterized by degeneration of midbrain dopaminergic neurons. Here we examined the effect of macelignan, a compound derived from nutmeg, on inflammatory degeneration of midbrain dopaminergic neurons. Treatment of midbrain slice cultures with interferon (IFN)-γ and lipopolysaccharide (LPS) caused a substantial decrease in viable dopaminergic neurons and an increase in nitric oxide (NO) production indicated by extracellular nitrite accumulation. Application of macelignan (10 μM) concomitantly with LPS prevented the loss of dopaminergic neurons. Besides nitrite accumulation, up-regulation of inducible NO synthase protein expression in response to IFN-γ/LPS was confirmed by Western blotting, and immunohistochemical examination revealed expression of inducible NO synthase in a subpopulation of Iba-1-poitive microglia. However, macelignan did not affect any of these NO-related parameters. On the other hand, macelignan promoted expression of arginase-1 in midbrain slice cultures irrespective of the presence or the absence of IFN-γ/LPS treatment. Arginase-1 expression was mainly localized in a subpopulation of Iba-1-positive cells. Importantly, the neuroprotective effect of macelignan was antagonized by N(ω)-hydroxy-nor-L-arginine, a specific arginase inhibitor. The neuroprotective effect of macelignan was also prevented by GW9662, a peroxisome proliferator-activated receptor γ (PPARγ) antagonist. Overall, these results indicate that macelignan, a compound with PPARγ agonist activity, can provide neuroprotective effect on dopaminergic neurons in an arginase-dependent but NO-independent manner.

Topics: Animals; Animals, Newborn; Arginase; Dopaminergic Neurons; Dose-Response Relationship, Drug; Gene Expression Regulation, Enzymologic; Inflammation; Lignans; Mesencephalon; Microglia; Nerve Degeneration; Neuroprotective Agents; Organ Culture Techniques; Rats; Rats, Wistar

This protocol describes microsphere-based protease assays for use in flow cytometry and high-throughput screening. This platform measures a loss of fluorescence from the surface of a microsphere due to the cleavage of an attached fluorescent protease substrate by a suitable protease enzyme. The assay format can be adapted to any site or protein-specific protease of interest and results can be measured in both real time and as endpoint fluorescence assays on a flow cytometer. Endpoint assays are easily adapted to microplate format for flow cytometry high-throughput analysis and inhibitor screening.

Topics: Animals; Biotinylation; Flow Cytometry; Fluorescence Resonance Energy Transfer; Green Fluorescent Proteins; High-Throughput Screening Assays; Humans; Inflammation; Kinetics; Microspheres; Peptide Hydrolases; Peptides; Reproducibility of Results; Temperature

UVB irradiation (290-320 nm) is the most damaging component of the UV spectrum and causes both direct and indirect damage to the basal cell layer of the epidermis; this results in the activation of a number of signaling pathways involved in pathophysiological processes in the skin, such as photoaging and inflammation. In photoaging UVB irradiation promotes degradation of the extracellular matrix (ECM) by matrix metalloproteinases (MMPs) and, in inflammation, UVB irradiation promotes the expression of inducible cyclooxygenase (COX-2), leading to overproduction of inflammatory mediators.. We first investigated the protective effects of macelignan from Myristica fragrans Houtt. on immortalized human keratinocytes (HaCaT) against UVB damage. We then explored the inhibitory effects of macelignan on UVB-induced MMP-9 and COX-2 and investigated the molecular mechanism underlying those effects.. HaCaT cells were treated with macelignan for the indicated times followed by irradiation with UVB. Secretion of MMP-9 was measured by gelatin zymography. Expression of COX-2, mitogen-activated protein kinases (MAPKs), phosphatidylinositol 3-kinase/Akt (PI3K/Akt), c-Fos, c-Jun, and CREB were assayed by western analysis.. Macelignan at a concentration of 0.1-1 microM increased the viability of HaCaT cells following UVB irradiation and inhibited MMP-9 secretion and COX-2 expression in a concentration-dependent manner. An inhibitory effect was also seen in the signal transduction network, where macelignan treatment reduced the activation of UVB-induced MAPKs, PI3K/Akt, and their downstream transcription factors.. These results suggest that macelignan protects skin keratinocytes from UVB-induced damage and inhibits MMP-9 and COX-2 expression by attenuating the activation of MAPKs and PI3K/Akt.

Topics: Cell Line, Transformed; Cell Survival; Cyclic AMP Response Element-Binding Protein; Cyclooxygenase 2; Enzyme Activation; Humans; Inflammation; Keratinocytes; Lignans; Matrix Metalloproteinase 9; Mitogen-Activated Protein Kinases; Myristica; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; Signal Transduction; Ultraviolet Rays

Previous studies have shown that macelignan has anti-inflammatory and neuroprotective effects. Subsequently, in the current study, we demonstrate that oral administrations of macelignan reduce the hippocampal microglial activation induced by chronic infusions of lipopolysaccharide (LPS) into the fourth ventricle of Fisher-344 rat brains. A Morris water maze was used to evaluate the status of the hippocampal-dependent spatial learning in control rats with an artificial cerebrospinal fluid infusion, rats with chronic LPS infusions, and rats with chronic LPS infusions and oral administrations of macelignan. The rats with chronic LPS infusions showed spatial memory impairments relative to the control rats in the performance of the memory task. Daily administration of macelignan reduced the spatial memory impairments induced by the chronic LPS infusions. The results indicate that macelignan may possess therapeutic potential for the prevention of Alzheimer's disease.

Topics: Analysis of Variance; Animals; Avoidance Learning; Behavior, Animal; Histocompatibility Antigens Class II; Inflammation; Learning Disabilities; Lignans; Lipopolysaccharides; Male; Maze Learning; Microglia; Rats; Rats, Inbred F344; Reaction Time; Space Perception