The Mechanism Of RIP3,MLKL And CaMKⅡ In Necroptosis Of Intracerebral Hemorrhage Induced Secondary Brain Injury

Background:There is secondary brain injury after intracerebral hemorrhage(ICH),and cell necrosis is a very important part of it.Although there are many types of cell necrosis,its subtype-necroptosis is a regulated form of cell necrosis.Previous studies have suggested that receptor-interacting protein 1(RIP1)is involved in necroptosis.Recent studies have also confirmed that RIP1 can mediate the occurrence of necroptosis after ICH.At the same time,receptor-interacting protein 3(RIP3)has been found to have interactions with RIP1,but its role in secondary brain injury is still unclear.Previous studies have suggested that RIP3 activates mixed-lineage kinase domain-like protein(MLKL)to induce necroptosis,but in hypoxic cardiomyopathy models,it has been found that RIP3 activated Ca2+-calmodulin-dependent protein kinase(CaMKⅡ)to induce necroptosis in cardiomyocytes,which was not via MLKL.Therefore,the role of RIP3,MLKL and CaMKⅡ in the process of neuronal necroptosis after ICH is still controversial.Part Ⅰ The relationship between necroptosis and RIP3\MLKL\CAMKⅡ after ICH in ratsPurpose:To explore the relationship between necroptosis,RIP3,MLKL,CaMKⅡ and their phosphorylation.Method:Establish an animal model of ICH by injecting autologous blood into the basal ganglia area of rats and randomly divide the rats into seven groups(six rats each):Sham group and 6,12,24,48,72h,7d after ICH;Propidium iodide in vivo staining to detect the level of cell necrosis after ICH,and evaluate the degree of partial necroptosis surrounding the hematoma.Westernblotting to detect the protein levels of RIP3,MLKL,CaMKⅡ and the changes of their phosphorylation levels after ICH.Immunofluorescence to detect the cellular localization of phosphorylated protein and evaluate the target cells of RIP3,MLKL,and CaMKⅡ modification changes to provide support for further study of their interactions.Results:1.Compared to sham group and the contralateral side of the ICH group,the number of PI-positive cells in the brain tissue surrounding the hematoma at 72h after ICH was significantly increased(p<0.01);2.Compared to sham group,the protein level of RIP3 in ICH group significantly increased at 48h and 72h(p<0.01)with almost 2 fold-change,the protein level of MLKLand CaMKⅡ also increased at 72h after ICH(p<0.01);3.Compared to sham group,the protein levels of phosphorylated RIP3,MLKL,CaMKⅡ significantly increased 72 hours after ICH(p<0.01),and the results of immunofluorescence showed that the phosphorylation of RIP3,MLKL,CaMKⅡ also increased(p<0.01)and mainly expressed in neuron surrounding the hematoma.Conclusions:1.RIP3,MLKL,and CaMKⅡ in the brain tissue around the hematoma increased significantly after ICH;2.The phosphorylation levels of RIP3,MLKL,and CaMKⅡ also increased significantly;3.RIP3,MLKL,and CaMKⅡ phosphorylation were mainly expressed in the neurons surrounding the hematoma;4.There is a correlation between the changes of RIP3,MLKL,and CaMKⅡ protein levels and the occurrence of necroptosis.Necrosis was involved in secondary brain injury after ICH in rats.RIP3,MLKL and CaMKⅡ may also be involved in the brain injury after ICH,which correlated to necrosis.Phosphorylation may be an important form contributing to neuronal injury after ICH.Part Ⅱ The effects of inhibiting RIP3\MLKL\CaMKⅡ on the level of necroptosis and neurological functions after ICH.Purposes:Interventions of inhibiting RIP3,MLKL and CaMKⅡ and changes of the necroptosis after ICH,as well as the improvement of short-term and long-term neurological functions.Method:After ICH model was established,the rats were randomly divided into six groups(sixteen rats each)and different inhibitors were used to intervene RIP3,MLKL and CaMKⅡ:westemblotting to detect the protein levels of RIP3,MLKL and CaMKⅡproteins and phosphorylation after ICH;Propidium iodide in vivo staining to detect the level of cell necrosis after different interventions,which also indicated the effects of different interventions on necroptosis;Short-term and long-term neurological function tests to observe the effects of different interventions on the functional rehabilitation of rats after ICH.Results:1.Compared to ICH+vehicle group,interventions inhibited RIP3,MLKL,CaMKⅡ respectively(p<0.01);2.Inhibition of RIP3 also reduced the protein levels of MLKL(p<0.01)and CaMKⅡ(p<0.01)and their phosphorylation in the brain tissues surrounding the hematoma;3.Inhibitions of MLKL and CaMKⅡ had no significant effect on RIP3 and its phosphorylation(p>0.05);4.Inhibition of MLKL had no significant effect on RIP3/CaMKⅡ and their phosphorylation(p>0.05);5.Inhibition of CaMKⅡ had no significant effect on RIP3/MLKL and their phosphorylation(p>0.05);6.The number of PI-positive cells surrounding the hematoma significantly decreased,after interventions of inhibiting RIP3,MLKL and CaMKⅡ(p<0.01);7.Compared to ICH+vehicle group,the interventions of RIP3,MLKL and CaMKⅡ significantly improve the short-term and long-term neurological functions of rats after ICH(p<0.01).Conclusions:Intervention with RIP3,MLKL and CaMKⅡ can reduce cell necrosis after ICH.The long-term and short-term neurological dysfunction also were improved.RIP3 participates in RIP1-mediated necroptosis after ICH.MLKL and CaMKⅡ also decreased significantly after intervention of RIP3,which may be related to RIP3-mediated necroptosis.RIP3,MLKL and CaMKⅡ were all involved in the process of necrosis after ICH.MLKL and CaMKⅡ may be the downstream activators of RIP3 mediating necroptosis pathway by phosphorylation.Inhibition of RIP3(GSK’872),MLKL(NSA)and CaMKⅡ(KN-93)could reduce necrosis surrounding the hematoma and improve short and long-term neurological dysfunction after hemorrhage.Part Ⅲ:The effects of inhibiting RIP3\MLKL\CaMKⅡ on their interactions and secondary brain injury after ICH in ratsPurpose:The effects of different interventions on the interaction levels of RIP3-MLKL/CaMKⅡ and the improvement on secondary brain injury after ICH.Method:After the ICH model was established,the rats were randomly divided into six groups(six rats each)and different inhibitors were used to intervene RIP3,MLKL and CaMKⅡ:immunoprecipitation to detect interactions of RIP3-MLKL and CaMKⅡ after ICH with different interventions;,Detect albumin to evaluate the blood brain barrier after ICH;Nissl staining to detect neuronal damage after ICH with different interventions;The levels of tumor necrosis factor α(TNFα),lactate dehydrogenase(LDH)and oxidative stress(ROS)were detected to measuring secondary brain injury after ICH with different interventions.Results:1.Compared with sham group,RIP3 and MLKL,RIP3 and CaMKⅡ have significant direct interactions after ICH(p<0.05);2.Inhibition of RIP3 can reduce the interaction level of RIP3 and MLKL(p<0.05),RIP3 and CaMKⅡ(p<0.01);3.Inhibition of MLKL reduced the interaction between RIP3 and MLKL,and had no significant effect on the interaction between RIP3 and CaMKⅡ(p>0.05);4.Inhibition of CaMKⅡ reduced the interaction between RIP3 and CaMKⅡ(p<0.01),and had no significant effect on the interaction between RIP3 and MLKL(p>0.05);5.Compared to sham group,the protein level of albumin in the brain tissue was significantly increased after ICH(p<0.01),and interventions of RIP3,MLKL and CaMKⅡreduced the protein level of albumin(p<0.01);6.Compared to ICH+vehicle group,inhibitions of RIP3,MLKL and CaMKⅡ significantly reduced the levels of LDH(p<0.01),ROS(p<0.01)and TNFa(p<0.01)surrounding the hematoma after ICH in rats;7.The number of surviving neurons in the cortex(p<0.05)and hippocampus(p<0.05)surrounding hematoma significantly increased in Nissl staining.Conclusions:RIP3 was activated by phosphorylation after ICH,and further promoted the phosphorylation of MLKL and CAMKII,which resulted in the necroptosis and secondary brain injury.The intervention of necroptosis could alleviate secondary brain injury,including blood-brain barrier damage,neuron damage,cell injury,oxidative stress and inflammation.