Role For RIP1 In Mediating Necroptosis In Experimental Intracerebral Hemorrhage Model

Part Ⅰ The Study on necroptosis in rats’ ICH model.Objective: To study different time points of necroptosis after ICH on rats.Methods: 1.12 healthy male SD rats were randomly divided into two groups with six rats per group.24 hours after ICH establishment,the rats were sacrificed through circulation perfusion with PBS.The brain cortex of 6 rats in each group was extracted and used for(propidium iodide)PI staining analysis.2.54 healthy male SD rats were randomly divided into night groups with six rats per group.Rats were killed at the indicated time pointand and brain cortex was extracted and used for western blot analysis.3.24 healthy male SD rats were randomly divided into two groups with twelve rats per group.Rats were killed 24 hours after ICH and brain cortex was extracted and used for PI fluorescence,immunoprecipitation(IP)and double immunofluorescence analysisResults: 1.24 hours after ICH,PI staining positive rate increased significantly.2.Expression of RIP1 increased after ICH and reached peak at 24 h.3.24 hours after ICH,expression of necroptosis related protein invreased significantly.Conclusions: The data showed that necroptosis activated after ICH and expression of its related protein reached peak at 24 h.Part ⅡEffect of drug inhibition and gene intervention on necroptosis after ICH.Objective: To study the effects of necrostatin-1(Nec-1,necroptosis inhibitor)and gene intervention on necroptosis and outcome in rats ICH model.Methods: 1.108 healthy male SD rats were randomly divided into six groups with 18 rats in each group: sham,ICH,ICH+Vehicle,ICH+Nec-1,ICH+z-VAD and ICH+ Nec-1+z-VAD groups.Firstly evaluated neurological scores,then brains were extracted for western blot,IP,PI staining,terminal deoxynucleotidyl transferase-mediated d UTP nick end labeling(TUNEL)staining,brain edema detection and ELISA.2.108 healthy male SD rats were randomly divided into six groups with 18 rats in each group: sham,ICH,ICH+Si-NC,ICH+Si-RIP1,ICH+Ad-GFP and ICH+Ad-RIP1 groups.Firstly evaluated neurological scores,then brains were extracted for western blot,IP,PI staining,brain edema detection and ELISA.Results: 1.Nec-1 could effectively reduce levels of necroptosis related protein,albumin,brain edema and TNF-α while improve neurological scores 24 h after ICH.2.Levels of necroptosis related protein,albumin and TNF-α were decreased while level of neurological scores was increased in ICH+Si-RIP1 group,oppositing to ICH+Ad-RIP1 group.Conclusions: 1.24 hours after ICH,Nec-1 could effectively inhibit necroptosis,inflammation,brain edema and improve blood-brain barrier(BBB),neurological outcome.2.24 hours after ICH,gene disturbance could effectively inhibit necroptosis,inflammation,brain edema and improve blood-brain barrier(BBB),neurological outcome,oppositing to gene overexpression.Part Ⅲ Mechanisms of necroptosis after ICH model in vitro.Objective: Mimic ICH model in vitro and to study mechanisms of necroptosis after ICH.Methods: 1.Used two in vitro models of ICH: one is Oxy Hb to deal directly with the neurons,the other is pre-stimulate microglia with Oxy Hb,and collect the supernatant as condition medium,then treat neurons with the condition medium.After two processing,neurons were digested by trypsin into cell suspension,extracted for western blot,IP,double immunofluorescent staining or stained with Annexin V and PI and then detected by flow cytometry.2.Detected the phosphorylation of RIP1 by IP using anti-RIP1 antibody followed by immunoblotting for anti-phosphorylation serine(p-Ser)antibody.We used over-expression with a mutation of phosphorylation site(S166A)of RIP1 and digested by trypsin into cell suspension,extracted for western blot,IP,double immunofluorescent staining or stained with Annexin V and PI and then detected by flow cytometry.Results: 1.There was a high apoptotic ratio in Oxy Hb directly stimulated neurons group,while there was a higher necroptotic ratio in condition medium treatment group.And TNF-α inhibitor pretreatment significantly reduced the percentage of necroptotic neurons.Interactions of RIP1 and RIP3,RIP1 and MLKL,and RIP1 and caspase-8 were significantly increased by treatment with condition medium and could be remarkably inhibited by TNF-α inhibitor pretreatment.2.Phosphorylation(serine site)of RIP1 was obviously increased in condition medium treatment group and could be inhibited by TNF-α inhibitor pretreatment.And flow cytometry revealed that upregulated the expression of RIP1 could increase the percentage of necroptosis,but over-expression of mutation of phosphorylation site(S166A)of RIP1 has no obvious effect in inducing necroptosis of neurons.Interactions of RIP1 and RIP3,RIP1 and MLKL,and RIP1 and caspase-8 were significantly increased by over-expression of RIP1 and could be remarkably inhibited by mutation of phosphorylation site(S166A)of RIP1.Conclusions: 1.TNF-α in condition medium may be an important factor of inducing necroptosis in neurons.2.phosphorylation in 166 th site of RIP1 may be an important element of inducing necroptosis in neurons.