Mechanisms Of Protection Against Cerebral Ischemia-reperfusion Injury By Remimazolam Through Activation Of The Keap1/NRF2 Signaling Pathway

Objective:Ischemia-reperfusion injury causes great harm for patients in the disease.Remazolam,a newly marketed benzodiazepine sedative drug,has shown promising clinical applications.The aim of this paper is to investigate whether remimazolam functions by activating the Keap1/NRF2 pathway to attenuate hippocampal neuronal oxyglucose deprivation-reoxyglucose injury and its possible mechanisms.Methods:Hippocampal neurons of SD rat mammary rats were cultured until day 8,and brain ischemia-reperfusion injury was simulated by making an oxygen-glucose deprivation-reoxygenation modelTo investigate the effect of rimazolam treatment on neuronal viability and apoptosis rate in cerebral ischemia-reperfusion injury and to clarify whether it has a cerebral protective effect.A random number table method was used to divide into 5 groups: control group(group C),model group(group M),oxyglucose deprivation-reoxygenation-reoxygenation group + rimazolam group(group R),oxyglucose deprivation-reoxygenationreoxygenation-reoxygenation group + rimazolam + dimethylsulfoxide group(group V),oxyglucose deprivation-reoxygenation-reoxygenation group + rimazolam + opium bitter alcohol group(group B),a hypoxic reoxygenation model was established by reoxygenation for 20 h after 6 h of hypoxia,rimazolam was added at the time of reoxygenation The final concentration of rimazolam was added at the time of reoxygenation,with a final concentration of 100 μM,and opium bitterol and dimethyl sulfoxide were added 4 h before hypoxia,with a final concentration of 500 n M of opium bitterol and <0.1% of dimethyl sulfoxide.After reoxygenation,cell viability was measured using CCK8 and apoptosis rate was measured using flow cytometry for each group.The expression levels of NRF2,Keap1,BCL2,BAX protein and m RNA levels of NRF2 were detected in each group using Weatern Blot,and NRF2 nuclear translocation was detected by immunofluorescence.The DCFH-DA method was used to detect ROS levels,immunofluorescence was used to label the outer mitochondrial membrane and observe the mitochondrial morphology,and transmission electron microscopy was used to observe the mitochondrial microstructure.The levels of OPA1 and OMA1 protein were also detected using Western Blot.Results: 1,compared with group C,cell viability decreased and apoptosis rate increased in group M(p<0.01);compared with group M,cell viability increased and apoptosis rate decreased in group R(p<0.01);no statistically significant change in group V relative to group R;compared with group V,cell viability decreased and apoptosis rate increased in group B(p<0.01).2.Compared with group C,the levels of NRF2,Keap1 and BCL2 protein decreased,the m RNA level of NRF2 decreased and the level of BAX protein increased in group M(p<0.01);compared with group M,the levels of NRF2,Keap1 and BCL2 protein increased,the m RNA level of NRF2 increased and the level of BAX protein decreased in group V(p<0.01);there was no statistical difference between group V and group R;compared with group V,the levels of NRF2,Keap1 and BCL2 protein increased,the m RNA level of NRF2 increased and the level of BAX protein decreased.There was no statistical difference between group V and group R;compared with group V,the levels of NRF2,Keap1 and BCL2 protein decreased,the m RNA level of NRF2 decreased and the level of BAX protein increased in group B(p<0.01).Using immunofluorescence to observe the NRF2 nuclear translocation in each group,it was seen that rimazolam treatment caused a substantial increase in NRF2 nuclear translocation.3.The DCFH-DA method for ROS levels showed that rimazolam treatment reduced ROS accumulation,whereas in group B,which was pretreated with opium bitter alcohol,fluorescence was again increased,indicating that NRF2 activation affected ROS metabolism.Immunofluorescence observation of mitochondrial morphology and transmission electron microscopy showed that rimazolam treatment reduced mitochondrial fragmentation,maintained mitochondrial number and protected mitochondrial microstructure.This protective effect was eliminated by treatment with rabbitol.It is concluded that rimazolam reduces cerebral ischaemia/reperfusion injury,possibly by activating the Keap1/NRF2 pathway,which protects mitochondrial morphology and its microstructure by reducing ROS accumulation,or at least in part through this pathway.m.