Anti-Necroptosis Chemical Necrostatin-1 Can Also Suppress Apoptotic and Autophagic Pathway to Exert Neuroprotective Effect in Mice Intracerebral Hemorrhage Model

Abstract

Necroptosis was recently discovered as one form of programmed cell death (PCD) and could be specifically inhibited by necrostatin-1. The aim of this study was to examine the effect of necrostatin-1 on brain injury and investigate the role of necrostatin-1 on the other two types PCD (apoptosis and autophagic cell death) in a mouse intracerebral hemorrhage (ICH) model. Male ICR mice received an infusion of type IV collagenase to induce ICH or saline as control into the left striatum. In the presence of vehicle, 3-MA, zVAD, and necrostatin-1 were pretreated with a single intracerebroventricular (i.c.v.) injection in the ipsilateral ventricle 15 min before ICH, respectively. Compared with vehicle groups, necrostatin-1 treatment significantly reduced injury volume and propidium iodide-positive cells at 24 and 72 h after ICH. Immunoblotting analysis showed that necrostatin-1 treatment suppressed autophagic-associated proteins (LC3-II, Beclin-1) and maintained p62 at normal level at 24 and 72 h after ICH. In addition, necrostatin-1 treatment enhanced the protein level of Bcl-2 and decreased the protein level of cleaved caspase-3 and the Beclin-1/Bcl-2 ratio at 24 and 72 h after ICH. Moreover, both 3-MA and necrostatin-1 treatment could suppress cleaved caspase-3 and LC3-II production, whereas zVAD treatment could inhibit caspase-3 cleavage but increased LC3-II protein levels at 72 h after ICH. Taken together, the data demonstrated for the first time that the specific inhibitor necrostatin-1 suppressed apoptosis and autophagy to exert these neuroprotective effects after ICH and that there existed a cross-talk among necroptosis, apoptosis, and autophagy after ICH.

Necrostatin-1 Reduces Neurovascular Injury after Intracerebral Hemorrhage

“…Our findings, which are in agreement with a recent report showing Nec-1 on apoptotic and autophagic cell death after collagenase-induced ICH [65], extend these findings using clinically relevant endpoints and suggest a potential role for targeting cell death pathways after ICH. Although the mechanism(s) whereby Nec-1 limited neurological injury were not explored in this study, it is notable that the biologically inactive, structural analog of Nec-1 did not exert the same protective effects of Nec-1 with respect to BBB opening, edema development, and behavioral outcomes; however, it is noteworthy that Nec-1 inactive attenuated hematoma volume to a similar magnitude as compared to Nec-1.…”

Neuronal Death After Hemorrhagic Stroke In Vitro and In Vivo Shares Features of Ferroptosis and Necroptosis

“…46 Furthermore, ICH induced immunohistochemical expression of the autophagosome marker LC3-II in mice. 38 Similarly, He and colleagues reported an increase in LC3-II, the lysosomal marker cathepsin D, and vacuole formation after ICH in rats. 47 …”

“…37 In a mouse model of ICH, intracerebroventricular administration of necrostatin-1 reduces cell death, brain edema, hematoma volumes, and neurologic score deficits. 38 Moreover, expression of RIP3 increases following ICH, while pretreatment with Necrostatin-1 diminishes the interaction of RIP3 with RIP1 and ameliorates cell death following ICH. 39 Mice deficient in RIP3 show decreased cell death following ICH.…”

“…42 While, hemoglobin treatment of rat cortical neurons induced upregulation of cleaved caspase-3 and increased caspase-3-like enzyme activity, pretreatment with z-VAD-fmk and other caspase inhibitors did not prevent cell death. 43 After ICH in vivo, immunohistochemical expression of cleaved caspase-3 increased in rats 44 and mice 38 as did expression of apoptosis-related genes in rats. 45 …”

RIP 1 negatively regulates basal autophagic flux through TFEB to control sensitivity to apoptosis

“…RIP1 is essential for necrotic cell death induced by death receptor agonists such as TNF or FasL, where its kinase activity is important for the formation of a downstream complex with RIP3 [7]. Necrostatin-1 inhibits RIP1 kinase activity and necrotic cell death [14] and was previously reported to repress autophagy in certain contexts [15,16]. However, it had no effect on basal autophagy ( Supplementary Fig S8A and B), suggesting that the kinase activity of RIP1 is not required for suppression of autophagy.…”