Abstract
Microglia-mediated inflammation is implicated in pathogenesis of neurodegenerative diseases. Oroxylin A, a flavonoid isolated from Scutellariae baicalensis, has been shown to ameliorate microglia activation-mediated neurodegeneration in vivo. The molecular mechanism underlying the inhibitory effects of oroxylin A on microglia activation, however, remains unknown. In the present study, effects of oroxylin A co-treated with lipopolysaccharide (LPS, 100 ng/ml) on LPS-induced activation of cultured microglial BV-2 cells were examined. Nitric oxide (NO) production was determined by Greiss method. Expression of inducible nitric oxide synthase (iNOS), interleukin (IL)-1β and IL-6 was assessed using real-time RT-PCR or Western blot analysis. Furthermore, activation of the nuclear factor κB (NFκB) and the signal transducer and activator of transcription 1 (STAT1) was examined by Western blot analysis and transcription factor DNA-binding activity assay. Our results indicated that oroxylin A (10–100 µM) in a concentration-dependent manner inhibited LPS-induced NO production via blocking iNOS expression at both mRNA and protein levels without affecting the degradation rate of iNOS mRNA. Moreover, oroxylin A significantly attenuated LPS-induced late expression (20 hours after LPS challenge) of IL-1β and IL-6. Furthermore, oroxylin A significantly suppressed LPS-induced JAK2-mediated STAT1 phosphorylation without affecting LPS-induced NFκB-p65 nuclear translocation or NFκB-p65 DNA-binding activity. This is consistent with the finding that AG490, a specific JAK2 inhibitor, significantly inhibited LPS-induced STAT1 phosphorylation with almost completely diminished iNOS expression. These results suggest that oroxylin A, via suppressing STAT1 phosphorylation, inhibits LPS-induced expression of pro-inflammatory genes in BV-2 microglial cells.
Highlights
Accumulating evidence suggests that inflammation play a critical role in neurodegenerative diseases, including Parkinson’s disease [1], Alzheimer’s disease [2], Huntington’s disease [3] and multiple sclerosis [4]
Our results indicated that oroxylin A, via inhibiting signal transducer and activator of transcription 1 (STAT1) phosphorylation, blocked LPS-induced expression of pro-inflammatory genes, including inducible nitric oxide synthase (iNOS), IL-1b and IL-6
LPS-induced upregulation of iNOS mRNA was suppressed by oroxylin A in a concentration dependent manner with maximum suppression at 50 mM (Fig. 1C)
Summary
Accumulating evidence suggests that inflammation play a critical role in neurodegenerative diseases, including Parkinson’s disease [1], Alzheimer’s disease [2], Huntington’s disease [3] and multiple sclerosis [4]. Activation of microglia is indispensible for clearance of cell debris or invading pathogens [5,7]. The prolonged and massive activation of microglia with excessive production of pro-inflammatory factors is thought, in part, responsible for inflammation-induced neurodegeneration [6,8]. Nitric oxide (NO), produced by inducible nitric oxide synthase (iNOS) in microglia, is one of the best characterized proinflammatory factors that induce neuronal death. Inhibition of iNOS prevented microglia-mediated neuronal death, indicating that NO plays a pivotal role in microglia-mediated neurotoxicity [10]. In addition to NO, interleukins (ILs) such as IL-1b and IL-6 play critical roles in microglia-mediated neurodegeneration [8,11,12]
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