Neuroprotective Effect Of ?-caryophyllene On Cerebral Ischemia-reperfusion Injury Via Regulation Of Necroptotic Neuronal Death And HMGB1/TLR4 Pathway

Background: Necrotic cell death is a hallmark feature of ischemic stroke and it may facilitate inflammation by releasing intracellular components after cell-membrane rupture.Previous studies reported that ?-caryophyllene(BCP)mitigates cerebral ischemia-reperfusion(I/R)injury,but the underlying mechanism remains unclear.Objective: To investigated the effect of ?-caryophyllene(BCP)on cerebral ischemia-reperfusion(I/R)injury in vivo and in vitro.Methods:1.Primary neurons with and without BCP(0.2,1,5,25 ?M)treatment were exposed to oxygen-glucose deprivation and re-oxygenation(OGD/R).Neuron damage,neuronal death type and mixed lineage kinase domain-like(MLKL)protein expression were assessed at 48 h after OGD/R.2.Mice were subjected to I/R with or without BCP(8,24,72 mg/kg).At 48 h of reperfusion,ischemic degrees were determined according to neurologic dysfunction score and cerebral infarct volume.The neuronal death type was determined by co-staining of TUNEL and cleaved caspase-3.Ultrastructure of ischemic brain tissue was analyzed using transmission electron microscopy.The protein expression of RIPK1 and phosphorylation of MLKL were measured by western blot.RIPK1,RIPK3,MLKL mRNA levels were measured by q RT-PCR.3.Mice were subjected to I/R with or without BCP(8,24,72 mg/kg).The protein expression of TLR4 and phosphorylation of NF-?B(p65)were measured by western blot.Translation of NF-?B(p65)was measured by immunohistochemistry.Interleukin-1?(IL-1?),tumor necrosis factor-?(TNF-?)and serum high-mobility group box 1(HMGB1)levels were measured by ELISA.Results:1.BCP(5 ?M)significantly reduced necroptotic neurons and MLKL protein expression following OGD/R.2.Compared to the I/R group,BCP(24,72 mg/kg)reduced the neurological score and cerebral infarct volume and necrosis in mice brain subjected to cerebral I/R.BCP(24,72 mg/kg)reduced increased receptor-interaction protein kinase-1(RIPK1),and MLKL phosphorylation expression after cerebral I/R injury.3.Compared to the I/R group,BCP also inhibited the activation of NF-?B pathway and decreased increases in TLR4,HMGB1,TNF-?,IL-1? levels induced by cerebral I/R.Conclusion: BCP alleviates ischemic brain damage potentially by inhibiting necroptotic neuronal death and HMGB1/TLR4/NF-?B pathway.