RIP1/RIP3/MLKL-Mediated Necroptotic Cell Death In Mice Intracerebral Hemorrhage

Objectives Intracerebral hemorrhage(ICH)is the second most common cerebrovascular emergency.After onset of ICH,the brain undergoes a series of pathological processes,including brain edema,blood-brain barrier(BBB)permeability disruption,inflammatory reaction,and lipid peroxidation.These pathologic changes act together on brain cells and cause cellular death,eventually leading to neurological deficits.At present,literature concerning the mechanisms of cell death predominantly concentrated on apoptosis,little study has been conducted to explore the contribution of necrosis.Necroptosis is a recently proposed programmed cell death that has been reported to implicate in several animal models of brain injury in vivo and in vitro.However,the contribution of necroptosis to brain injury after ICH has not been fully illustrated.The aim of this study was to investigate whether the RIP1/RIP3/MLKL-mediated necroptotic activation accounts for ICH-induced brain injury,and to evaluate the potential effect of necrostatin-1 using a mouse model of collagenase-induced ICH.Methods 1.Detection the levels of necroptosis-associated proteins(RIP3 and MLKL)in mice after ICH by western blot and immunofluorescence:Forty-two healthy mice(C57BL/6)were randomly divided into sham group(n=9)and ICH group(n=33).ICH group was further divided into five subgroups according to the different time points(1,3,5,7,10),of which,9 mice in the ICH-3d subgroup,and 6 mice in other sungroups.ICH model was established by injection of 0.5 ul collagenase IV(0.05 U)into the basal ganglia.The sham group was injected with an equal amount of normal saline instead of collagenase.Six mice in each subgroup were used for western blot.The relative densities of RIP3 and MLKL were applied for statistic analyses by using GAPDH as standardized internal protein.Brains from 3 days post-ICH were processed for frozen sections,which were observed under a microscope for determination of the expressions and distributions of RIP3 and MLKL.2.Seventy-two male mice were randomly divided into 4 groups,18 mice per group.A group of mice were used for the assessments of neurological deficit scores and brain water content;A group of mice were used for measuring the Evans blue extravasation to explore the permeability of blood brain barrier;A group of mice were used for western blot assay to determine the expression of RIP1,RIP3 and MLKL and to explore the inhibition role of necrostatin-1.Another group was used to measure the content of malondialdehyde and the concentrations of inflammation factors.Eighteen mice were randomly divided into sham group,ICH + DMSO group,and ICH + Nec-1 group(n=6 per group).Establishment of ICH model is identical with the first part of experiment.A total of 80 nmol Nec-1 dissolved in 1 ul DMSO was infused into the right ventricle 15 minutes before the surgery.The ICH + DMSO group received an equal amount of DMSO.3.Statistical analysis:Data were presented as mean ± SEM.One-way ANOVA followed by the Tukey test was used for statistical analyses except behavioral data which was analyzed with Kruskal-Wallis test.Statistical analyses were performed with SPSS 17.0 and Graph Pad Prism 5.P < 0.05 was considered statistically significant.Results 1.Western blot: MLKL level was significantly increased at 1 day after mice subjected to ICH as compared to the sham group,and peaked at 3 days,then decreased successionally to the end point of observation.In addition,the expression of RIP3 was also elevated notably at 1 day post-ICH,and maintained a high level for at least 7 days when compared with the sham group.Consistent with the western blot analysis,immunofluorescence further confirmed the ICH-induced enhance in the protein level of RIP3 and MLKL in mice following ICH.Moreover,both proteins primarily distributed in cytoplasm.2.Nec-1 inhibited the activation of necroptosis in mice after ICH 2.1 The level of RIP1 was markedly elevated in the ICH + DMSO group as compared to the sham group while it was greatly reduced by inhibitor Nec-1.Additionally,similar tendencies were also observed in the expression of downstream RIP3 and MLKL.Both proteins increased significantly in the peri-hematoma brain tissues and were decreased clearly after Nec-1 pretreatment.2.2 Obvious neurological dysfunction was found in mice subjected to ICH following treatment with vehicle as compared to the sham group.Of interest,the neurological deficits were significantly improved after Nec-1 treatment.Our results verified that BBB disruption appeared in the ipsilateral and contralateral hemispheres after ICH induction,which were dramatically ameliorated by Nec-1 administration.Brain water content also increased clearly in ipsilateral hemisphere in the absence of significant alternation in other brain regions.Likewise,pretreatment with Nec-1 also decreased brain edema obviously in the ICH + Nec-1 group when compared with the ICH + DMSO group.2.3 Inflammatory mediators(TNF-α and IL-1β)were detected in the peri-hematoma regions.Our results indicated an increasing of inflammatory cytokines after ICH.Similarly,both inflammatory factors were also downregulated after Nec-1 treatment.2.4 we found that MDA content was slightly expressed in the sham group while obviously enhanced in the ICH+DMSO group.However,MDA level was downregulated after treating with Nec-1.Conclusion 1.Necroptosis is activated in brain tissue around hematoma in mice after ICH.The expression of necroptosis-associated proteins were elevated notably at 1 day post-ICH,and maintained at least 7 days when compared with the sham group.RIP3 and MLKL mainly distributed in cytoplasm.2.Nec-1 pretreatment inhibits the RIP1/RIP3/MLKL-mediated necroptosis,and decreases the expression of its signaling proteins.3.Nec-1 inhibits the release of inflammatory factors(IL-1β and TNF-α),and reduces the oxidative damage,brain edema,disruption of blood brain barrier,then improving the functional recovery.