Investigation On The Neuroprotective Effects And Mechanisms Of Inhibition Of RIPK1/RIPK3/MLKL Signaling Pathway By Perampanel In Rats After Traumatic Brain Injury

Objective:Traumatic brain injury(TBI)has been a common neurosurgical emergency.Perampanel is a new non-competitive inhibitor of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor(AMPAR),which is now widely used as an anti-epileptic drug.Our team constructed a rat controlled cortical impact model(CCI model)in a preliminary study.Through a series of inflammatory factors,oxidative enzymes assay,we demonstrated that perampanel has antioxidant,anti-inflammatory,reduce brain edema,reduce brain contusion volume and improve motor function score in protecting brain injury in rats.We also found that perampanel protected neurovascular units from traumatic injury by modulating Sirt3 in an isolated neuronal model.These results demonstrate that perampanel can promote neurological recovery and reduce secondary brain injury in rats with brain injury.Previous studies have shown that multiple signaling pathways are activated after TBI and interact with each other to determine the survival or death of neurons,and confirmed that programmed necrosis is one of the important ways of neuronal death caused by TBI,which is a reliable target to intervene in secondary brain injury after TBI.In addition,the RIPK1-RIPK3-MLKL necrosis-associated complex is a key factor in the process of programmed necrosis.Therefore,the aim of this study was to establish a rat CCI model and to verify whether perampanel attenuates secondary brain injury in TBI rats by modulating the RIPK1-RIPK3-MLKL signaling pathway.Methods:a cranial impactor was applied to establish the CCI model.Ninety-six adult healthy male SD rats(250-350g)were randomly divided into Sham group(n=24),TBI group(n=24),TBI + Perampanel group(n=24)and TBI+Perampanel+NSA group(n=24).Perampanel(5mg/kg)and Perampanel(5mg/kg)+NSA(Necrosulfonamide)(1.65mg/kg)were injected intraperitoneally 5 minutes after TBI in the third group and the fourth group,respectively.After successful CCI modeling,behavioral functional changes were observed in SD rats by balance beam stay and walk experiments and the neurological severity score(NSS score)was used to observe the mortality rate between the groups.The brain tissues of SD rats were collected at different time intervals,and the water content of the brain tissues was determined by wet and dry weight method;the apoptosis of neurons in the percussion side of the cerebral cortex was detected by TUNEL staining;the expression of receptor-interacting protein kinase-1(RIPK1),receptor-interacting protein kinase-3(RIPK3),mixed-spectrum kinase domain-like protein(MLKL)and phosphorylated MLKL were detected by Western blot.Phosphorylated MLKL expression;immunofluorescence detection of MLKL expression;ELSA assay of inflammatory factors TNF-α,IL-1β;brain tissue malondialdehyde(MDA)and 4-hydroxynonenal(4-HNE)content measurement to clarify lipid peroxidation levels.Results:The neurological function of SD rats after TBI was severely impaired;the degree of brain edema was increased;the levels of MDA and 4-HNE in brain tissue were significantly increased(P < 0.05);the neuronal cell death was significantly increased as seen by TUNEL staining;the levels of inflammatory factors TNF-α,IL-1β were significantly increased by ELSA method(P < 0.05).After TBI,the neurological function after TBI was significantly improved by intraperitoneal injection of perampanel scores,reduce brain edema,lower brain tissue MDA and 4-HNE levels,reduce apoptosis of neuronal cells,and attenuate neuroinflammatory response.Further investigating the mechanism,the expression of RIPK1,RIPK3,MLKL and phosphorylated MLKL,proteins related to programmed necrosis pathway,was significantly increased by Western blot at 72 h after TBI(P<0.05),and the expression of MLKL was also increased by immunofluorescence.the use of perampanel after TBI could down-regulate RIPK1,RIPK3,MLKL and phosphorylated MLKL.Conclusion:Our study showed that programmed necrosis occurred in rat neuronal cells after TBI and that the expression of programmed necrosis pathway-related proteins RIPK1,RIPK3,MLKL,and phosphorylated MLKL was significantly increased.Inhibition of programmed necrosis by perampanel effectively reduced neuronal cell death and improved neurological function in TBI rats,and the underlying mechanism may be related to the inhibition of programmed necrosis pathway-related proteins RIPK1,RIPK3,MLKL and phosphorylated MLKL.In the current study,we established a controlled cortical impact model in rats to confirm the effectiveness of perampanel in rats after TBI,and unravels the molecular mechanism that the neuroprotective effect of perampanel by performing a series of experimental assays,including the examination of the expression levels of RIPK1 、 RIPK3 、 MLKL and phosphorylated MLKL in the necroptosis pathway.