The Role Of MLKL On The Cerebral Ischemic Reperfusion Injury

Objective: 1. Observe the change of Mixed Lineage Kinase Domain-like(MLKL)protein after cerebral ischemic-reperfusion injury in mice.2. Observe the effect of Necrosulfonamide(NSA) on the cerebral ischemic-reperfusion injury in mice.3. To investigate the influence of NSA on the MLKL after cerebral ischemic-reperfusion injury in mice; research the mechanisms of MKL on the cerebral ischemic reperfusion injury.Methods: Male ICR mice were randomly divided into sham group and ischemic reperfusion(I/R)group, I/R group for setting up the middle cerebral artery occlusion(MCAO) model. Ischemic tissues were collected at different time points after I/R and the change of MLKL after I/R injury.Mice were randomly divided into sham group,sham+NSA group,I/R group and I/R+NSA group.The effects of the treatment of NSA on the mice cerebral infarction volume and the neurobehavioral function were detected.Infarct volume was determined by TTC staining;Neurological deficits were evaluated by a5-point score system;The expression levels of MLKL and PARP-1 after I/R injury were detected by Western blot analysis and immunofluorescence assay;The m RNA levels of MLKL detected by RT-PCR;Co-mmunoprecipitation was used to investigate the ubiquited MLKL in brain tissues.Results: Compared with Sham group,MLKL proteins of I/R group increase 6 h after I/R injury,increased significantly at 24 h after I/R injury and markedly increased thereafter,at48 h after I/R injury,MLKL level was reached the peak(P<0.05), and gradually decreased at 72 h and 7 d. Compared with I/R group, TTC staining showed that NSA markedly reduced infarct volume;NSA interventions can significantly decreased the neurological deficit score after I/R injury compared with I/R group;Compared with I/R group, NSA decreased MLKL expression after cerebral I/R injury, but did not affect the m RNA level of MLKL; NSA treatment significantly increased ubiquitination MLKL level and cleaved PARP-1 after I/R injury(P<0.05).Conclusions:Our data for the first time demonstrated that MLK protein plays an important role in the in cerebral I/R injury. MLKL expression was increased in ischemic brains after cerebral I/R injury,while MLKL inhibitor NSA had significantly neuroprotective effects;Our findings indicated that NSA decreased the level of MLKL after I/R injury by promoting degradation of MLKL mainly via the ubiquitination proteasome pathway.However, NSA also increased cleaved PARP-1 level. This study demonstrates that MLKL may represent a novel avenue for stroke treatment.