Role Of RIP3 In Early Brain Injury After Subarachnoid Hemorrhage In Rats: Inducing Necroptosis And Promoting Inflammation

Part I:Changes of protein level and distribution of RIP3 after subarachnoid hemorrhageObjective:To study the changes of RIP3 protein level and distribution in brain tissue after subarachnoid hemorrhage(SAH)and the level and mechanism of necroptosis in rats with subarachnoid hemorrhage(SAH).Methods:48rats were randomly divided into 8 groups:Sham group and 3h,6h,12h,24h,48h,72h,7d after SAH,with 6 rats in each group.All rats were killed at the corresponding time point after SAH.The protein level and distribution of RIP3 were detected by immunofluorescence and Western blot technique,and the level of necroptosis was analyzed by PI staining technique.Immunoprecipitation analysis was used to analyze the mechanism of necroptosis.Results:1.The level of RIP3 in brain tissue of rats after SAH increased first and then decreased,and the level of RIP3 protein increased most significantly in 24 hours after SAH(P<0.05).2.Immunofluorescence in vivo showed that the localization of RIP3 in the cells of 24h after SAH was significantly higher than that in Sham group(P<0.05).3.After SAH,the number of PI positive cells in brain tissue increased.Conclusion:The expression of RIP3 was significantly increased in 24 h after SAH,and the necroptosis in brain tissue was increased after SAH.Part II:Effect of regulating RIP3 on brain injury after Subarachnoid HemorrhageObjective:To explore the expression and phosphorylation of RIP3 and MLKL after subarachnoid hemorrhage(SAH)by intervention of RIP3 inhibitor(GSK872),TNF-αinhibitor(TNF-a inhibitor),RIP3 plasmid and small interfering RNA,And the role of its changes in the injury after SAH in rats.Methods:180 SD rats were randomly divided into 10 groups:sham operation group,SAH group,SAH+Vehicle group,SAH+GSK872 group,SAH+TNF-α inhibitor group,SAH+GSK872+TNF-a inhibitor group,SAH+Si-NC group,SAH+Si-RIP3 group,SAH+Vector group and SAH+Over-RIP3 group,with 18 rats in each group.The model was established 48 hours after intervention in GSK872,TNF-α inhibitor,transfection of RIP3 plasmid and small interfering RNA.The neurological function score was performed 24 hours after SAH,Western blot analysis,immunoprecipitation and PI staining were performed in bilateral temporal base brain tissue,and ELISA(TNF-α)test was performed in cerebrospinal fluid(CSF).Part III:The role of RIP3 in neurons after subarachnoid hemorrhage in vitro and its role in necroptosisObjective:The phosphorylation of RIP3 and MLKL was detected by GSK872 intervention in primary neurons,and the mechanism of necroptosis was verified in vitro.Methods:The primary neurons were divided into the following 8 groups:normal control group,Condition Medium,Condition Medium+Vehicle,Condition Medium+GSK872,by Western blot,immunoprecipitation to detect the mechanism of necroptosis and phosphorylation of RIP 1 and MLKL.Results:1.After Condition Medium stimulation,interactions of RIP3 and RIP 1,RIP3 and MLKL were significantly increased in neurons compared with Control group.2.The phosphorylation of RIP3 in Condition Medium group was significantly increased when compared with Control group.The phosphorylation of RIP3 in Condition Medium+GSK872 group was decreased compared with the Condition Medium+Vehicle group.3.The phosphorylation of MLKL in Condition Medium group was significantly increased compared with the Control group.The phosphorylation of MLKL in Condition Medium+GSK872 group was decreased compared with the Condition Medium+Vehicle groupConclusion:GSK872 inhibits phosphorylation of RIP3 and MLKL in vitro.