HDAC4/5-HMGB1 signalling mediated by NADPH oxidase activity contributes to cerebral ischaemia/reperfusion injury.
Bottom Line: Our results showed that inhibition of NADPH oxidase activity ameliorated cerebral ischaemia/reperfusion (I/R) injury and among Zn(2+) -dependent HDACs, HDAC4 and HDAC5 were significantly decreased both in vivo and in vitro, which can be reversed by NADPH oxidase inhibitor apocynin.We further found that both HDAC4 and HDAC5 increased cell viability through inhibition of HMGB1, a central mediator of tissue damage following acute injury, expression and release in PC12 cells.Our results for the first time provide evidence that NADPH oxidase-mediated HDAC4 and HDAC5 expression contributes to cerebral ischaemia injury via HMGB1 signalling pathway, suggesting that it is important to elucidate the role of individual HDACs within the brain, and the development of HDAC inhibitors with improved specificity is required to develop effective therapeutic strategies to treat stroke.
Affiliation: Department of Pharmacology, Shandong University School of Medicine, Jinan, Shandong 250012, China.Show MeSH
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Mentions: As shown in Figure 1A, infarction volume at 24 hrs after reperfusion was 35.6 ± 4.1%. Neurological deficit score also confirmed the neurological dysfunction after MCAO (Fig. 1B). HE staining further showed morphological features of injured neurons after I/R in different specific brain regions including prefrontal cortex, striatum and hippocampus, all of which are predominantly injured in focal ischaemia. Arrowheads indicated the normal cells in sham-operated group and injured cells in I/R group. Significant cellular body shrinkage and condensation of dark nuclei were observed after I/R. To determine the expression patterns of HDAC family in ischaemic brain (ischaemic core and penumbra), real-time RT-PCR and Western blot analyses were performed. As shown in Figure 2A and B, among Zn2+-dependent HDACs, the expression of HDAC4 and HDAC5 was significantly decreased. The levels of HDAC1, 2, 3, 6, 7, 8, 10 and 11 had no significant difference at this experimental condition. Our immunohistochemical studies further confirmed the decreased levels of HDAC4 and HDAC5 in ischaemic core and penumbra of the ischaemic hemisphere (Fig. 2C). We then assessed the expression levels of HDAC4 and HDAC5 at different time-points after reperfusion, our results indicated that HDAC4 and HDAC5 expression still kept low expression at 2 weeks after reperfusion as shown in Figure 2D. To identify cell types in which HDAC4 and HDAC5 were expressed, dual immunofluorescent staining was performed. We identified neurons by staining for neuronal nuclear marker NeuN, astrocytes by GFAP and activated microglia/macrophages by CD11b. It was found that HDAC4 (Fig. 2E) and HDAC5 (data not shown) were predominantly expressed in neurons. No colocalization of HDAC4 or HDAC5 was observed in astrocytes or microglia. Collectively, these results suggest that neuron is a major resource of HDAC4 or HDAC5 in the brain. In addition, compared with sham group, we found that both HDAC4 and HDAC5 levels were significantly decreased in neurons of ischaemic brain.
Affiliation: Department of Pharmacology, Shandong University School of Medicine, Jinan, Shandong 250012, China.