The Effect And Mechanism Of Dexmedetomidine Against Cerebral Ischemia Reperfusion Injury Based On Ferroptosis

With the acceleration of the aging process of the population,stroke has became the main cause of death.Among them,stroke caused by ischemia accounts for about85%.At present,the main method for the treatment of ischemic stroke is to restore blood reperfusion in the ischemic area.However,in the process of blood reperfusion,it will further lead to brain tissue damage in the ischemic area,which called ischemia /reperfusion injury(I/R).In recent years,it has become a research hotspot to explore the mechanism of cerebral ischemia reperfusion and to develop new effective drugs for the treatment of cerebral ischemia reperfusion.Previous studies have implied dexmedetomidine(DEX)has the effects of attenuating cerebral ischemia reperfusion,but its mechanism remains to be further studied.Iron exists widely in brain tissue.Under stress conditions,iron accumulates excessively in cells.Excess iron produces highly oxidizing hydroxyl radicals through Fenton reaction,which can induce cell membrane lipid peroxidation and eventually lead to ferroptosis.Studies have shown that increased expression of Nrf2 in tumor cells can inhibit the occurrence of ferroptosis.In liver injury,the use of dexmedetomidine can increase the expression of Nrf2 protein.However,it is not clear whether dexmedetomidine attenuate cerebral ischemia reperfusion through Nrf2 regulate ferroptosis in mice.Therefore,in this study,the role and mechanism of ferroptosis and related pathways in the protective effect of dexmedetomidine on cerebral ischemia reperfusion were investigated in vivo and in vitro experiments.Objective: 1.To explore the role and mechanism of ferroptosis in dexmedetomidine against cerebral ischemia reperfusion injury in mice.Methods:1.In vivo: The model of middle cerebral artery occlusion(MCAO)was established by thread embolism method;In order to explore the optimal concentration of the protective effect of dexmedetomidine on the brain,low(25 μg/kg),middle(50 μg/kg)and high(100 μg/kg)doses of dexmedetomidine were given,and the final dose of dexmedetomidine was selected by observing changes in the neurobehavioral scores and volume of cerebral infarction in mice.For the purpose of further exploring the mechanism of dexmedetomidine neuroprotection,eighty male ICR mice were divided into four groups: sham group,I/R group,I/R+DEX50 group,middle-dose dexmedetomidine + Nrf2 inhibitor group(I/R+DEX50+ML385).The neurological impairment of mice in each experimental group was evaluated by Longa scores;the brain slices of the mice in each group were performed by the TTC staining,meanwhile,calculating the percentage of cerebral infarction volume;observing the mitochondrial morphology of mice in each group by transmission electron microscope;detecting the content of MDA,Fe2+,and GSH in mouse brain tissue by thiobarbituric acid,colorimetric and microplate method;the expression of Nrf2,TFR1,SLC7A11,GPX4 were detected by western blot.2.In vitro: HT22 cells was used to prepare hypoxia reoxygenation injury model.In order to explore the optimal concentration of dexmedetomidine for the protection of cell hypoxia reoxygenation injury,the HT22 cells was divided into five groups,which included control group(control),hypoxia reoxygenation group(H/R),low-concentration(H/R+DEX2.5,2.5μM),middle-concentration(H/R+DEX5,5μM)and high-concentration(H/R+DEX10,10μM)dexmedetomidine intervention hypoxia reoxygenation group.To further explore the mechanism of the protective effect of dexmedetomidine on hypoxia reoxygenation of HT22 cells,the cells was randomly divided into four groups: control group,H/R group,H/R+DEX5 group,and dexmedetomidine middle dose +Nrf2 inhibitor group(H/R+DEX5+ML385).The MTT was used to detect the cell surviving fraction in each group;the Ferro Orange fluorescent probe was used to detect the change of Fe2+ in the cell;the C11 BODIPY581/591 was used to detect the change of Lipid peroxidation(Lip ROS)in the cell;thiobarbituric acid and microplate method were employed to detect the content of MDA and GSH in cells;the proteins expression of Nrf2,TFR1,SLC7A11,GPX4 were detected by western blot.Results: 1.The results of the neurobehavioral score and the percentage of cerebral infarction volume showed that,compared with the mice in the I/R group,the neurobehavioral score and the percentage of cerebral infarction volume of the mice in the low,medium and high dose treatment groups were reduced.Besides,the medium-dose and high-dose treatment groups had better effects;compared with the I/R+DEX50 group,the neurobehavioral scores and the percentage of cerebral infarction volume of the mice in the I/R+DEX50+ML385 group increased significantly.2.Results obtained by transmission electron microscope showed that the mitochondria were regular shape,complete structure in the sham group.Compared with the sham group,the mitochondria of the I/R group showed characteristic changes of ferroptosis,including mitochondrial shrinkage,reduction or disappearance of mitochondrial cristae,and increased mitochondrial membrane density.After dexmedetomidine treatment,the mitochondrial damage were reduced,compared with the I/R group.After using the Nrf2 inhibitor ML385 on the basis of dexmedetomidine,the protective effect of dexmedetomidine on mitochondria was decreased.3.The results of the content of MDA,GSH and Fe2+ in the brain tissue of mice showed that,after cerebral I/R injury,the content of MDA,Fe2+ in the brain tissue of mice in I/R group increased while the content of GSH decreased.;after dexmedetomidine treatment,the content of MDA and Fe2+ decreased,and the content of GSH increased compared with the I/R group;after using the Nrf2 inhibitor ML385 on the basis of dexmedetomidine,compared with the I/R+DEX50 group,the content of MDA and Fe2+ increased,while the content of GSH decreased.4.The results of Nrf2,TFR1,SLC7A11 and GPX4 protein expression in mouse brain tissue showed that after cerebral I/R injury,the Nrf2,GPX4,SLC7A11 protein expression in I/R group was significantly reduced,and the TFR1 protein expression was significantly increased;after dexmedetomidine treatment,compared with the I/R group,the expression of Nrf2,GPX4,and SLC7A11 protein were significantly increased,while the expression of TFR1 protein was significantly reduced;after using the Nrf2 inhibitor ML385 on the basis of dexmedetomidine,compared with the I/R+DEX50 group,the expression of Nrf2,GPX4,SLC7A11 protein in the brain tissue of mice was significantly reduced,and the expression of TFR1 protein was significantly increased.5.The cell surviving fraction results showed that,compared with the H/R group,the cell surviving fraction of the H/R+DEX2.5,H/R+DEX5,H/R+DEX10 groups increased;but the Nrf2 inhibitor ML385 weakened the protective effect of dexmedetomidine and the cell surviving fraction decreased.6.The Fe2+,Lip ROS,MDA,GSH detection results showed that compared with the control group,the Lip ROS,MDA,Fe2+ content in the H/R group increased,and the GSH content decreased;after dexmedetomidine treatment,the Lip ROS,MDA,Fe2+ content decreased,and GSH content increased compared with H/R group;after using Nrf2 inhibitor ML385 on the basis of dexmedetomidine,compared with H/R+DEX5 group,the content of Lip ROS,MDA,Fe2+ increased,and the content of GSH decreased.7.The results of Nrf2,TFR1,SLC7A11 and GPX4 protein expression in cells showed that,compared with the control group,the Nrf2,GPX4,SLC7A11 protein expression in H/R group was significantly reduced,and the TFR1 protein expression was significantly increased;after dexmedetomidine treatment,compared with the H/R group,the expression of Nrf2,GPX4,SLC7A11 protein was significantly increased,and the expression of TFR1 protein was significantly reduced;after using the Nrf2 inhibitor ML385 on the basis of dexmedetomidine,the expression of Nrf2,GPX4,SLC7A11 protein was significantly reduced,and the expression of TFR1 protein was significantly increased in HT22 cells.Conclusions: Dexmedetomidine has an inhibitory effect on ferroptosis in the brain of mice with cerebral ischemia reperfusion injury.The mechanism may be related to the regulation of iron metabolism and lipid peroxidation regulated by Nrf2.