Abstract
Cell death is a hallmark of second brain injury after intracerebral hemorrhage (ICH); however, the mechanism still has not been fully illustrated. In this study, we explored whether necroptosis, a type of regulated necrosis, has an essential role in brain injury after ICH. We found that inhibiting receptor-interacting protein 1 (RIP1) – a core element of the necroptotic pathway – by a specific chemical inhibitor or genetic knockdown attenuated brain injury in a rat model of ICH. Furthermore, necroptosis of cultured neurons could be induced by conditioned medium from microglia stimulated with oxygen hemoglobin, and this effect could be inhibited by TNF-α inhibitor, indicating that TNF-α secreted from activated microglia is an important factor in inducing necroptosis of neurons. Undoubtedly, overexpression of RIP1 increased conditioned medium-induced necroptosis in vitro, but this effect was partially diminished in mutation of serine kinase phosphorylation site of RIP1, showing that phosphorylation of RIP1 is the essential molecular mechanism of necroptosis, which was activated in the in vitro model of ICH. Collectively, our investigation identified that necroptosis is an important mechanism of cell death in brain injury after ICH, and inhibition of necroptosis may be a potential therapeutic intervention of ICH.
Highlights
Intracerebral hemorrhage (ICH) is the second largest type of stroke, accounting for ~ 15% of all patients with stroke.[1,2] ICH is associated with fast progression, high mortality and high morbidity
We determined the expression of RIP1, which is a major regulator for necroptosis in brain tissues; the results of western blot suggested that the expression levels of RIP1 were progressively upregulated and peaked at 24 h after ICH (P = 0.0003 versus Sham group, n = 3; Figures 1c and d)
We further evaluated the formation of necrosome, which is an important hallmark of activation of necroptosis in brain tissues after ICH; anti-RIP1 antibody was used by immunoprecipitation (IP), and RIP3, MLKL and caspase-8 were detected by immunoblotting
Summary
Intracerebral hemorrhage (ICH) is the second largest type of stroke, accounting for ~ 15% of all patients with stroke.[1,2] ICH is associated with fast progression, high mortality and high morbidity. The membrane rupture caused a large number of cytoplasmic components to leak from the cell and induced inflammation in the surrounding tissues.[8] Previous studies suggested that necrosis is an occasional and irregular event and is difficult to study.[9] Yuan and co-workers[10] reported, for the first time, necroptosis as a form of necrosis that can be regulated.[10] Necroptosis has similar morphological characteristics (including early membrane integrity, cell and intracellular organelle swelling) with necrosis, but it is caspase-independent programmed cell death.[11]. Cell Death and Disease we studied the role of necroptosis, a type of regulated necrosis, in ICH
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