| Objective:Remimazolam is a novel benzodiazepine agonist targeting the gammaaminobutyric acid a(GABAa)receptor.Recent studies have shown that remimazolam is protective against cerebral ischemia/reperfusion injury.However,the protective mechanism of remimazolam against cerebral ischemia/reperfusion injury needs to be further investigated.The aim of this study was to investigate the neuroprotective effects of remimazolam against oxygen glucose deprivation/reoxygenation(OGD/R)injury in vitro and brain I/R injury in vivo,and to investigate whether this mechanism involves enhancement of nuclear factor erythropoietin 2-related factor 2(Nrf-2)expression via the protein kinase B/glycogen synthase kinase-3β(AKT/GSK-3β)pathway.Methods:Both in vivo and in vitro experiments were performed in two parts.The first part of the in vivo experiment was grouped into a sham-operated group(Sham group),a remimazolam group(RE group),an I/R model group(I/R group)and an I/R +remimazolam group(I/R + RE group).The rats in the sham-operated group received only isolation of the common carotid artery,internal carotid artery,and external carotid artery without occlusion of the middle cerebral artery;the rats in the remimazolam group received the same surgery as the sham-operated group and were injected intraperitoneally with 16 mg/kg of remimazolam 2 h after surgery;the rats in the I/R group underwent middle cerebral artery occlusion and reperfusion surgery(MCAO/R),i.e.,the middle cerebral artery was occluded for 2 h and then reperfused for 24 h In the I/R+RE group,16 mg/kg of remimazolam was injected intraperitoneally into the rats immediately after reperfusion.The first part of the in vitro experiment was grouped into control group(Control group),remimazolam group(RE group),cellular oxygen glucose deprivation reperfusion group(OGD/R group)and cellular oxygen glucose deprivation reperfusion plus rimazolam group(OGD/R+RE group).The control group cultured the cells routinely;the remimazolam group cultured the cells routinely and added 100ug/ml of remimazolam to the medium;the OGD/R group placed the cells under hypoxic glucose-free conditions for 6h and then reoxygenated and reperfused for24h;the OGD/R+RE group added 100ug/ml of remimazolam to the medium immediately after the cells were reoxygenated and reperfused.For the first part of the experiments,in vivo experiments used TTC staining,neurological deficit scoring and HE staining to explore the neuroprotective effects of remimazolam,while in vitro experiments used CCK-8 assay to detect the effects of remimazolam on the viability of OGD/R-treated SH-SY5 Y cells.Then,in vivo experiments were performed to detect the expression of apoptotic proteins Bax,Bcl2 and Cleaved-Caspase3 by Western blot method,and in vitro experiments were performed to detect the apoptotic rate of remimazolam on OGD/R-treated SH-SY5 Y cells by flow cytometry to explore the effect of remimazolam apoptosis of brain ischemia-reperfusion injury cells.After that,in vitro experiments were performed using DCFH-DA staining and flow cytometry to detect intracellular reactive oxygen species(ROS)content,and both in vivo and in vitro experiments were performed using kits to detect the content of malondialdehyde(MDA),a marker of membrane lipid peroxidation,the antioxidant enzyme superoxide dismutase(SOD)and glutathione peroxidase(GSH-PX),to explore the effects of remimazolam in cerebral ischemia-reperfusion injury on antioxidant effects.Finally,both in vivo and in vitro experiments were performed to detect the protein expression of AKT,phosphorylated AKT(p-AKT),glycogen synthase kinase-3β(GSK-3β),phosphorylated GSK-3β(p-GSK-3β),nuclear factor red lineage-derived factor 2-related factor(Nrf2),and heme oxygenase 1(HO-1)by Western blot method.In the second part of the experiment,in vivo experiments were grouped into sham-operated group(Sham group),I/R model group(I/R group),I/R+ remimazolam group(I/R+RE group),I/R+ remimazolam +LY294002 group(I/R+RE+LY group)and I/R+LY294002group(I/R+LY group).Rats in the sham-operated group did not undergo middle cerebral artery occlusion,and other surgical operations were the same as those in the I/R group;rats in the I/R group underwent MCAO/R;rats in the I/R+RE group were injected with 16 mg/kg of remimazolam via their intraperitoneal cavity immediately after reperfusion;rats in the I/R+RE+LY group were injected with PI3K/AKT inhibitor through the lateral ventricle 30 min before MCAO/R LY294002(50 mmol/L,10 ul/per rat),and 16 mg/kg of remimazolam was injected via their intraperitoneal cavity immediately after reperfusion;rats in the I/R+RE+LY group were injected with LY294002(50 mmol/L,10 ul/per rat)via the lateral ventricle 30 min before ischemia.The second part of the in vitro experiments were grouped into control group(Control group),cytosolic oxygen glucose deprivation reperfusion group(OGD/R group),cytosolic oxygen glucose deprivation reperfusion + remimazolam group(OGD/R + RE group),cytosolic oxygen glucose deprivation reperfusion + rimazolam + LY294002group(OGD/R + RE + LY group)and cytosolic oxygen glucose deprivation reperfusion+ LY294002 group(OGD/R + LY group).Cells in the control group were cultured routinely;in the OGD/R group,cells were placed under anaerobic and sugar-free conditions for 6 h and then reoxygenated and re-glycosylated for 24 h;in the OGD/R +RE group,100ug/ml of remimazolam was added to the culture medium immediately after reperfusion;in the OGD/R + RE + LY group,10 μmol/L of LY294002 was added to the culture medium 24 h before oxygen and sugar deprivation and immediately after reperfusion For the second part of the in vivo and in vitro experiments,AKT,p-AKT,GSK-3β,p-GSK-3β,NRF and NRF were first measured using Western blot method in each group of rat brain tissue and each group of cells.GSK-3β,NRF2,HO-1 protein expression and Nrf2 nuclear translocation by immunofluorescence to investigate whether remimazolam activates the AKT/GSK-3β/NRF2 signaling pathway.Then the levels of MDA,SOD and GSH-Px in brain tissue and cells of each group were measured using the kit,and the reactive oxygen species content in each group was detected by DCFH-DA probe and flow cytometry to investigate whether the antioxidant effect induced by remimazolam was associated with the AKT/GSK-3β/NRF2 signaling pathway.Finally,the expression of apoptotic proteins Bax,Bcl2 and Cleaved-Caspase3 in the brain tissues of each group of rats was detected using Western blot method and the apoptosis rate of each group of cells was detected by flow cytometry to investigate whether the anti-apoptotic effect induced by remimazolam was associated with AKT/GSK-3β/NRF2 signaling pathway.Results:1.In the first part of the in vivo experiment,the cerebral infarct area and neurological deficit score were reduced in the I/R+RE group compared with the I/R group(P<0.05),and the difference was statistically significant.HE staining showed that the brain cell damage was reduced in the I/R+RE group compared with the I/R group.The results of CCK8 experiments in the first part of the in vitro experiment showed that remimazolam attenuated the damage of SH-SY5 Y cells by OGD/R and increased cell viability.2.In the first part of in vivo experiment,Bcl-2 protein expression was upregulated and Bax and Cleaved-Caspase3 expression was decreased in the I/R+RE group compared with the I/R group(P<0.05),and the difference was statistically significant.The flow cytometry results of the first part of the in vitro experiment showed that remimazolam reduced the apoptosis induced by OGD/R.3.In the first part of the in vitro experiment,ROS was reduced in the OGD/R+RE group compared with the OGD/R group(P<0.05),and the difference was statistically significant.In the first part of the in vitro and in vivo experiment,remimazolam decreased the expression of MDA and increased the expression of SOD and GSH-Px.4.In the first part of the in vitro and in vivo experiment,remimazolam increased the expression of Nrf2,HO-1,p-Akt and p-GSK-3β protein.5.In the second part of the in vitro and in vivo experiment,LY294002,an inhibitor of PI3K/AKT,decreased the expression of Nrf2,HO-1,p-Akt and p-GSK-3β proteins,reversing the effect of remimazolam on the expression of these proteins.Moreover,remimazolam caused a shift of Nrf2 expression from the cytoplasm to the nucleus in brain I/R injury,which was reversed by LY294002.6.In the second part of the in vitro experiment,ROS was reduced in the OGD/R+RE+LY group compared with the OGD/R+RE group(P<0.05),and the difference was statistically significant.In the second part of the in vitro and in vivo experiment,LY294002,an inhibitor of PI3K/AKT,increased MDA,decreased SOD,CAT and GSH-Px,and reversed the antioxidant effect of remimazolam.7.In the second part of the in vivo experiment,the I/R+RE/+LY group decreased the expression of Bcl2 and increased the expression of Bax and Cleaved-Caspase3 compared with the I/R+RE group(P<0.05),and the difference was statistically significant.In the second part of the in vitro experiment,LY294002,an inhibitor of PI3K/AKT,increased the number of apoptotic cells and reversed the anti-apoptotic effect of remimazolam.Conclusion:This study suggests that remimazolam has a neuroprotective effect,possibly through activation of the AKT/GSK-3β/NRF2 pathway,attenuating oxidative stress and apoptosis,thereby reducing cerebral ischemia-reperfusion injury. |
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