The Mechanism Of RIPC Attenuating Cerebral Ischemia-Reperfusion Injury By Pre-activating Notch1 Signaling Pathway

Background:Remote ischemic preconditioning(RIPC)initiates endogenous protective pathways in the brain through transient,sublethal distal limb ischemia-reperfusion,which effectively induces the formation of ischemic tolerance and reduces subsequent cerebral Ischemia-reperfusion injury,thereby improving the prognosis of patients with ischemic stroke.Because of its safety,noninvasion,effectiveness,and simplicity,RIPC is considered to be a promising pretreatment paradigm with good clinical transformation prospects.It has a good application prospect for acute,subacute and chronic neurological diseases related to ischemia or inflammation.Evidence suggests that the protective effects of RIPC against ischemia are mainly related to neuronal,humoral and immunity-related pathways,as well as a variety of biomolecules and signaling pathways.However,the underlying mechanism of RIPC-mediated cerebral ischemia tolerance is complicated and not well understood.It's important to elucidate the mechanism of RIPC-induced cerebral ischemic tolerance for the treatment of stroke and other cerebral ischemic diseases.Our previous study found that CIP(cerebral ischemic preconditioning,CIP)could induce neuroprotective effects against cerebral ischemia-reperfiision injury by pre-activate the Notch1 signaling in the cerebral ischemic penumbra.This study was to investigate the neuroprotective effects of RIPC on cerebral ischemia-reperfusion injury by pre-activate the Notch1 signaling and to elucidate the role of Notch1 and NF-?B signaling pathways in RIPC-induced ischemic tolerance.Methods:Middle cerebral artery occlusion and reperfusion(MCAO/R)in SD rats and oxygen-glucose deprivation and reoxygenation(OGD/R)in primary hippocampal neurons were used as models of I/R injury in vivo and in vitro,respectively.The RIPC protocol consisted of 4 cycles per day of 5 min of ischemia followed by 5 min of reperfusion for 3 days before middle cerebral artery occlusion and reperfusion.Intracerebroventricular DAPT injection and sh-Notch lentivirus interference were used to inhibit the Notch signaling pathway.After 24 h of reperfusion,neurological deficit scores,cerebral infarct volume,neuronal apoptosis,and cell viability were assessed respectively.The protein expression levels of NICD,Hesl,p-Ikk?/?,p-NF-?B p65,Bcl-2 and Bax were assessed by Western Blotting.Results:Part 1 Animal experiment 1:RIPC significantly attenuated the MCAO/R injury,improve the neurological function score and reduced the volume of cerebral infarction(P<0.01).RIPC significantly upregulated the expression of NICD,Hesl,IKKp,and NF-?B p65 in the RIPC+MCAO/R group.Part 2 Cell experiment:OGD preconditioning(OGD-Pre)significantly increased cell viability after OGD/R injury on day3 and day7 and reduced neuronal apoptosis on day3 and day7 after OGD/R(P<0.05).However,the neuroprotective effect was attenuated by Notch1-RNAi,but the neuroprotective effect was attenuated by Notch1-RNAi(P<0.05).Notch1-RNAi significantly increased hippocampal neuronal cell viability and reduced neuronal apoptosis on day 3 and day 7 following direct lethal OGD/R injury in vitro.OGD-Pre significantly upregulated the expression of NICD,Hesl,p-IKK ?/?,p-NF-?B p65,and Bcl-2 in the OGD+OGD/R group.The expression of p-IKK ?/?,p-NF-?B p65 was suppressed by Notch1-RNAi,suggesting that the NF-?B signaling pathway was regulated by the Notch1 signaling pathway.Part 3 Animal experiment 2:RIPC significantly reduced hippocampal neuronal apoptosis in rats after MCAO/R.The neuroprotective effects of RIPC was reversed by DAPT,inhibitor of the Notch signaling pathway.DAPT significantly increased neurological scores and reduced infarct volume and neuronal apoptosis after direct lethal MCAO/R injury in vivo.RIPC significantly upregulated the expression of NICD,Hesl,p-IKK ?/?,p-NF-?B p65,and Bcl-2.The expression of p-DCK ?/?,p-NF-?B p65 was suppressed by DAPT,suggesting that the NF-?B signaling pathway was regulated by the Notch1 signaling pathway.Conclusions:The neuroprotective effect of RIPC against cerebral I/R injury was associated with preactivation of the Notch1 and NF-?B pathways in neurons.The NF-?B pathway is a downstream target of the Notch1 pathway in RIPC and helps protect against focal cerebral I/R injury.