The Protection Of MLKL Inhibitor NSA On Focal Ischemia/Reperfusion Injury In Rats And Its Mechanism

Objective:Necroptosis is a type of cell death described as a regulated necrosis characterized by cell membrane disruption mediated by phosphorylated mixed lineage kinase like protein(MLKL),and significantly contributes to negative events occurring with the ischemic stroke.We previously demonstrated that necroptosis related protein RIP1K(receptor-interacting protein kinase 1)knockdown or Nec-1(necrostatin-1,a specific inhibitor of RIP1K)treatment reduced infarct volume,improved neurological deficits,and attenuated neuronal or astrocytic necrotic cell death in the ischemic cortex.Whether the MLKL inhibitor NSA(necrosulfonamide)is effective for astrocytes has not been studied.This study is aim to explore the protection of NS A in ischemic stroke and its mechanisms.Methods:In vivo,rats were subjected to the transient middle cerebral artery occlusion(tMCAO).After cerebral ischemia for 90 min followed by reperfusion 24 h,TTC staining was used to detect the cerebral infarction volume;Neurologic deficit scores and right forelimb use were detected to evaluate the neurologic behavior.The distribution of necroptosis related proteins after tMCAO were observed in cytoplasmic,nuclear and nuclear envelope fraction with western blotting.In vitro,the primary astrocytes and Human Astrocytes were subjected to oxygen glucose deprivation(OGD)followed by 24 h reoxygenation.The lactate dehydrogenase(LDH)leakage detection kit was used to detect the protection of NSA after OGD/Re injury in astrocytes and Human Astrocytes.The levels of necroptosis related proteins were detected with Western Blotting analysis.Results:In vivo,NSA decreased the cerebral infarction volume and improved neurological deficits in tMCAO rats.In vitro,NSA increased the number of astrocytes and Human Astrocytes,decreased the LDH leakage induced by OGD/Re.Western Blotting showed that NSA reduced the levels of necroptosis related protein MLKL,p-MLKL,RIP3K,p-RIP3K,RIP1K,p-RIP1K in astrocytes after OGD/Re injury.The nuclear protein level and the nuclear envelope level of MLKL,p-MLKL,RIP3K,p-RIP3K were indeed increased greatly in the peri-infarct region after I/R injury,but RIP1K weren't.NSA administrated treatment significantly decreased the nuclear protein level and the nuclear envelope level of MLKL,p-MLKL,RIP3K,p-RIP3K.Conclusion:(1)NSA has a protective effect on rat's tMCAO-induced injury.(2)NSA protects astrocytes against OGD/Re-induced injury and reduces the levels of necroptosis related proteins in astrocytes after OGD/Re injury.(3)MLKL and RIP3K were translocated to nuclears in the peri-infarct region after I/R injury.NSA blocks the combination and the nuclear envelope localization of MLKL and RIP3K.