ABSTRACT
Cerebral ischemia/reperfusion involves inflammatory process and naloxone is able to reduce infarct volume and has been used as a therapeutic agent for brain injury. Hypoxia induces the immediate early genes (IEGs) rapidly and transiently that may initiate a cascade of cellular responses that are necessary for survival and normal function. However, the protective effect of naloxone on ischemic/hypoxic neuronal cells was only partly studied. Thus, the effects of naloxone on oxygen- and glucose-deprivation (OGD) and OGD followed by reoxygenation (OGD/R) on the expression of IEGs were examined in PC12 cells. The result showed that lactate dehydrogenase (LDH) released in the media was reduced by naloxone. The temporal response of IEG mRNA encoding c-fos, c-jun, nur77, and zif268 was induced with different degree of intensity following hypoxia, whereas the level of GAPDH mRNA was relatively constant. However, these signals of c-fos, c-jun, and nur77 by hypoxia were reduced significantly by naloxone. Treatment with OGD also activated mitogen-activated protein kinase (MAPK) pathway. The induction of c-fos, c-jun, nur77, and zif268 by hypoxia was inhibited by naloxone (0.1 microM) and MAPK inhibitors (10 microM of U0126, D98059, SB203580). However, naloxone increased the expression of ERK1/2 by OGD concomitantly diminished the LDH release. Thus, the present studies demonstrated that OGD induced IEGs including c-fos, c-jun, nur77, and zif268 and MAPK signaling pathways were regulated differently by naloxone.
Subject(s)
Gene Expression Regulation/drug effects , Genes, Immediate-Early/drug effects , Hypoxia-Ischemia, Brain/drug therapy , Hypoxia-Ischemia, Brain/metabolism , Naloxone/pharmacology , Neurons/drug effects , Animals , Brain Infarction/drug therapy , Brain Infarction/metabolism , Brain Infarction/physiopathology , Cytoprotection/drug effects , Cytoprotection/physiology , DNA-Binding Proteins/genetics , Encephalitis/drug therapy , Encephalitis/metabolism , Encephalitis/physiopathology , Enzyme Inhibitors/pharmacology , Genes, Immediate-Early/genetics , Hypoxia-Ischemia, Brain/physiopathology , L-Lactate Dehydrogenase/drug effects , L-Lactate Dehydrogenase/metabolism , MAP Kinase Signaling System/drug effects , MAP Kinase Signaling System/genetics , Mitogen-Activated Protein Kinase 3/drug effects , Mitogen-Activated Protein Kinase 3/metabolism , Naloxone/therapeutic use , Narcotic Antagonists/pharmacology , Narcotic Antagonists/therapeutic use , Neurons/metabolism , Neuroprotective Agents/pharmacology , Neuroprotective Agents/therapeutic use , Nuclear Receptor Subfamily 4, Group A, Member 1 , Oxidative Stress/drug effects , Oxidative Stress/physiology , PC12 Cells , Proto-Oncogene Proteins c-fos/genetics , Proto-Oncogene Proteins c-jun/genetics , Rats , Receptors, Steroid/genetics , Reperfusion Injury/drug therapy , Reperfusion Injury/metabolism , Reperfusion Injury/physiopathologyABSTRACT
The alpha-ketoglutarate dehydrogenase complex (KGDHC) is a mitochondrial enzyme in the TCA cycle. Inhibition of KGDHC activity by alpha-keto-beta-methyl-n-valeric acid (KMV) is associated with neuron death. However, the effect of KMV in microglia is unclear. Therefore, we investigated the effect of KMV on BV-2 microglial cells exposed to hypoxia or oxidative stress. The results showed that KMV (1-20 mM) enhanced the cell viability under hypoxia. KMV dose-dependently reduced ROS and LDH releases from hypoxic BV-2 cells. KMV also reduced ROS production and enhanced the cell viability under H2O2 but failed to reduce the SIN-1 and sodium nitroprusside (SNP) toxicity. KMV also reduced caspase-3 and -9 activation under stress. These results suggest that KMV protects BV-2 cells from stress and acts by reducing ROS production through inhibition of KDGHC.
Subject(s)
Cell Hypoxia/physiology , Cytoprotection/drug effects , Keto Acids/pharmacology , Microglia/drug effects , Oxidative Stress , Apoptosis/drug effects , Cell Hypoxia/drug effects , Cell Line , Humans , Hydrogen Peroxide/pharmacology , Microglia/cytology , Microglia/metabolism , Reactive Oxygen Species/metabolismABSTRACT
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.
