The Neuroprotective Effect Of EX-4 On BV2 Microglial Cell Induced By Oxygen-glucose Deprivation / Reoxygenation Injury

BackgroundIschemic stroke is one of the diseases with high morbidity,morbidity and fatality rate in the world.Thrombolysis and thrombectomy(ie,early reperfusion)is the most effective treatment for cerebral infarction.However,reperfusion injury occurs when the brain tissue resumes reperfusion after continuous ischemia and hypoxia for a certain period of time,resulting in reduced efficacy of patients.At present,there is no effective treatment.In recent years,clinical studies have found that activation of Glucagon-Like Peptide-1 Receptor(GLP-1R)can reduce cerebral ischemia-reperfusion injury.GLP-1 is an intestinal hormone expressed by the proglucagon gene and secreted by L cells in the small intestine,and exerts various pleiotropic effects in the body.Microglia are intrinsic immune cells of the central system,whose activation signals the occurrence of inflammation.Studies have confirmed that inflammatory reactions mediated by microglial pyrolysis can exacerbate cerebral ischemia-reperfusion injury,and the inhibition of pyroptosis of microglia is expected to provide a new idea for cerebral ischemia-reperfusion injury.ObjectiveThis study intends to use BV2 microglia cultured in vitro and establish an OGD / R cell model to simulate cerebral ischemia-reperfusion injury.On this basis,it is determine that whether activating GLP-1R has a protective effect on the damage model by inhibiting cell pyrolysis.Provide new ideas and new targets for the prevention and treatment of cerebral is chemia-reperfusion injury.At the same time,it provides a reliable experimental basis for the next step to study the neuroprotective effect of GLP-1R and related molecular mechanisms in vivo and in vitro.MethodsBy using normal microglia(BV2 cells),and the ODG/R conditions were simulated by using a three-gas incubator and sugar-free medium.According to the references,the oxygenand sugar deprivation time was 4h,6h,8h and 10 h,and the oxygen and sugar reoxygenation time was 0h,6h,12 h,18h and 24 h.After OGD / R treatment,the cell survival rate was detected by CCK8,and the combination time of about 50% survival rate was selected as the condition for establishing OGD / R model.Acco rding to the comprehensive reference,the exendin-4 of GLP-1 receptor agonists with dosages of 0n M,25 n M,50 n M,100 n M,200 n M,and 400 n M were used to pretreat the experimental cells for 24 hours,and were subje cted to ODG / R treatment.The optimal dose was selected by CCK8 to detect cell survival rate.After determining the time combination for establishing the OGD / R model and the optimal EX-4 concentration,BV2 was divided into experimental groups:(1)blank control group was normal BV2 cells;(2)OGD / R group: only OGD / R treatment BV2 cells;(3)EX-4 + OGD / R group: BV2 was pre-treated with GLP-1R agonist for OGD / R treatment;(4)BV2 undergoes OGD/R after pretreatment with G LP-1R antagonist.Total protein and cell culture medium supernatant were extracted from the cells,and the expression levels of NLRP3 and caspase-1 were detected by Western Blot.The secretion of IL-1β and IL-18 was detected by ELISA.The expression levels of NLRP3 were analyzed by immunofluorescence.Results1.The OGD / R time combination of 6h oxygen deprivation time and 12 h reoxygenati-on time showed that the cell survival rate was the closest to 50%,and this survival rate decreases significantly after the restoration of oxygen and sugar supply,which can effectively reflect cerebral ischemia reperfusion Damaged time combination model.2.The OGD / R group NLRP3,caspase-1 protein expression levels a nd IL-1β,IL-18 secretion levels were significantly higher than the blank control group(The NLRP3,caspase-1 protein expression levels and IL-1β secretion level compared with the blank control group P<0.001,IL-18 s-ecretion level is P<0.01),indicating that the inflammatory reaction mediat-ed by cell pyrolysis may aggravate cerebral ischemia Reperfusion injury.3.The expression levels of NLRP3 and caspase-1 protein and the secretion levels of IL-1β and IL-18 in EX-4 pretreatment + OGD / R group were lower than those in the OGD / R group(The NLRP3 protein expression level and the IL-1β secretion Compared with OGD/R group,P<0.001,the caspase-1 protein expression level and the IL-18 secretion level w-ere P<0.01).And the expression levels of NLRP3 and caspase-1,IL-1β and IL-18 in BV2 cells treated with GLP-1R antagonist EX-9-39 showed no significant difference compared with OGD/R(P>0.05),Indicating that activation of GLP-1R can inhibit the expression of NLRP3 inflammatory bodies in microglia.Conclusion1.BV2 cells establish an ischemia-reperfusion model,which is closest to the state of cells after cerebral ischemia-reperfusion in humans at 6h of oxygen deprivation and 12 h of reoxygenation.2.EX-4 can play a protective role by inhibiting the expression of NLRP3 inflammatory bodies in BV2 cells.