Abstract
BackgroundHeme oxygenase (HO)-1 has been shown to attenuate oxidative injury and reduce apoptosis. HO-1 can be induced by various stimuli released during cellular injury, such as heme. Deleterious free heme is degraded by HO-1 to carbon monoxide, iron and biliverdin, which have potent anti-oxidant and anti-inflammatory properties. In this study, we tested the hypothesis that upregulation of HO-1 would inhibit production of the free radical (NO) by interlukin (IL)-1β-activated human astrocytes.MethodsTo measure NO production, inducible NO synthase (iNOS), HO-1 expression and mitogen-activated protein (MAP) kinase activation we used hemin as an HO-1 inducer and tin protoporphyrin (SnPP) IX as an inhibitor of HO-1 activity in human astrocyte cultures prior to IL-1β exposure. Transfection of astrocyte cultures was performed using a pLEX expression vector carrying the human HO-1 sequence prior to IL-1β treatment. Supernatants of astrocyte cultures pretreated with inhibitors of p38 MAPK or MEK1/2 prior to IL-1β exposure were collected for NO assay.ResultsIL-1β treatment of astrocytes alone induced undetectable amounts of HO-1 protein by western blot. However, HO-1 mRNA expression was modestly up-regulated in response to IL-1β stimulation. Pretreatment with hemin alone substantially induced both HO-1 mRNA and protein expression, and HO-1 mRNA expression was further enhanced when hemin was combined with IL-1β treatment. In contrast, IL-1β-induced iNOS mRNA expression and NO production were markedly inhibited by hemin treatment. When pretreated with SnPP, the inhibitory effect of hemin on IL-1β-induced NO production and iNOS expression was reversed, suggesting the involvement of HO-1. IL-1β-induced p38 MAPK activation, which is known to be required for NO production, was also down-regulated by hemin.ConclusionThese findings support the hypothesis that up-regulation of HO-1 in astrocytes is associated with down-regulation of iNOS expression and thereby NO production, an effect that involves the p38 MAPK signaling pathway, which suggests that this glial cell response could play an important protective role against oxidative stress in the brain.
Highlights
Heme oxygenase (HO)-1 has been shown to attenuate oxidative injury and reduce apoptosis
Several signaling pathways are activated by IL-1b in astrocytes, we focused on mitogen activated protein kinases (MAPKs) to determine if the effect of hemin on IL-1b-stimulated astrocytes is mediated through a MAPK signaling pathway
Inhibition of inducible nitric oxide (NO) synthase (iNOS) mRNA expression and NO production To test the hypothesis that hemin would inhibit iNOS expression, human astrocyte cultures were pretreated with hemin for 24 h followed by IL-1b treatment for 4 h or 24 h for total RNA isolation
Summary
Heme oxygenase (HO)-1 has been shown to attenuate oxidative injury and reduce apoptosis. Deleterious free heme is degraded by HO-1 to carbon monoxide, iron and biliverdin, which have potent anti-oxidant and anti-inflammatory properties. Hemin (ferriprotoporphyrin IX chloride), the oxidized form of the heme moiety of hemoglobin and a constituent of many enzymes, is degraded by heme oxygenase (HO)-1, which in turn generates carbon monoxide (CO), iron and biliverdin. While CO and biliverdin each have cytoprotective and anti-inflammatory properties, iron is sequestered by ferritin to reduce free radical formation and later utilized to maintain iron homeostasis for gene regulation. Hemin has been reported to suppress human immunodeficiency virus (HIV)-1 infection of human monocytes through HO-1 induction [1], but has been reported to induce necroptosis of murine cortical astrocytes [2] and oxidative injury to human neurons [3]. HO-1 deficiency in humans results in severe abnormal growth and development [16]
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