| In recent years,the incidence of stroke in our country has been increasing year by year.It has a high disability and fatality rates,which brings a serious burden to patients and their families.And,ischemic stroke accounts for more than 70% of stroke incidence.Clinically,intravenous thrombolysis is mainly used to restore reperfusion for ischemic stroke,but this process will cause cerebral ischemia/reperfusion（I/R）injury.The therapeutic effect of cerebral I/R injury is not ideal.Therefore,it is important to further clarify mechanisms of the pathological injury of cerebral I/R,so as to explore potential targets for the treatment of cerebral I/R injury.Ferroptosis is an iron-dependent cell death resulted in excessive accumulation of lipid peroxides.In recent years,a large number of studies have shown that inhibition of ferroptosis has become an important way to reduce cerebral I/R injury.CISD2 is one of the markers of early neuronal differentiation.Recently,the research of CISD2 in neurological diseases has become more and more extensive.At the same time,some studies have shown that up-regulation of CISD2 can reduce the risk of cerebral damage by inhibiting intracellular iron overload and reduced lipid peroxidation levels.However,the role of CISD2 in cerebral I/R injury still unclear.Objective:1.To investigate the expression changes of CISD2 in mice with cerebral I/R injury.2.To investigate the effect of up-regulating CISD2 with cerebral I/R injury in mice.3.To explore the specific mechanism of up-regulating CISD2 against cerebral I/R injury in mice.Methods:1.In vivo: To explore the role of CISD2 in anti-cerebral I/R injury in mice,ICR mice were randomly divided into 6 groups（n=10）: sham group,I/R group,I/R + AAVNC group,I/R + AAV-CISD2 group,I/R + LV-NC group and I/R + LV-sh RNA group.In order to explore the mechanism of CISD2-overexpression on cerebral I/R injury,ICR mice were randomly divided into 5 groups（n=10）: Sham group,I/R,I/R + AAV-CISD2,I/R + ML385,I/R + AAV-CISD2 + ML385.The model of cerebral I/R middle was prepared by middle cerebral artery occlusion（MACO）.Longa’s score was used to evaluate neurological dysfunction in mice.Transmission Electron Microscopy（TEM）was used to observe the morphology of mitochondria in nerve cells of cerebral tissue.The morphology and number of nerve cells were observed by Nissl Staining Solution.2,3,5-triphenyltetrazolium chloride（2,3,5-triphenyltetrazolium chloride,TTC）staining was used to detect the volume of cerebral infarction.The levels of ferrous ion,malondialdehyde and glutathione in tissue were detected by microplate assay.The expressions of CISD2,Nrf2,and ferroptosis-related proteins were detected by western blot.2.In vitro: HT22 cells were used for in vitro experiments.To explore the role of CISD2 in anti-OGD/R injury,HT22 cells were divided into six groups: control group,OGD/R group,OGD/R + OE-NC group,OGD/R + OE group,OGD/R +sh RNA-NC group,and OGD/R + sh RNA group.In order to explore the mechanism of CISD2-overexpression against OGD/R injury in HT22 cells,the cells were divided into 5 groups: control group,OGD/R group,OGD/R + ML385 group,OGD/R + OE group,and OGD/R + OE + ML385 group.Cell survival rates were detected by Calcein-AM double staining.Ferro Orange staining was used to detect intracellular ferrous ion levels.Intracellular Lipid Ros level was detected by flow cytometry.The expressions of intracellular CISD2,nuclear factor E2-related factor2（Nrf2）,and ferroptosis-related proteins were detected by western blot.Results:1.The neurobehavioral scores showed that compared with the sham group,behavioral score was significantly increased in the I/R group（p<0.001）,compared with the I/R group,the behavioral score was significantly reduced in the CISD2-overexpression group（p < 0.01）;compared with the I/R + LV-NC group,the behavioral score was significantly increased in the I/R + LV-CISD2 group（p<0.01）;compared with the I/R + AAV-CISD2 group,the behavioral score was significantly increased in I/R + AAV-CISD2 + ML385 group（p<0.01）.2.The results of cerebral infarction volume showed that: the mice had no cerebral infarction volume in the sham group;compared with the sham group,the cerebral infarction volume of the mice was significantly increased in the I/R group（p<0.001）;compared with the I/R + AAV-NC group,the volume of cerebral infarction was significantly reduced in I/R + AAV-CISD2 group（p<0.01）;compared with I/R+ LV-NC group,the volume of cerebral infarction was significantly increased in I/R+ LV-CISD2 group（p<0.01）;compared with I/R + AAV-CISD2 group,the volume of cerebral infarction was significantly increased in the I/R + AAV-CISD2 + ML385group（p<0.01）.3.The results of Nissl staining showed that the neurons were normal shape（tabbylike）and the number of neurons was abundant in the sham group;compared with the sham group,the neurons were atrophied and the number was significantly reduced in the I/R group;The morphology of neurons tended to improve,and the number of neurons increased significantly in the I/R + AAV-CISD2 group.4.The results of transmission electron microscopy showed that the mitochondrial morphology in the nerve cells of the mice was long rod-shaped and had abundant cristae structure in the sham group;compared with the sham group,the mitochondrial morphology was spherical,and the cristae structure on the inner membrane disappeared or decreased in the I/R group;compared with the I/R +AAV-NC group,the mitochondrial morphology of nerve cells in the cerebral tissue tended to normal shape,and the cristae structure on the membrane increased significantly in the I/R + AAV-CISD2 group;compared with the I/R + AAV-CISD2 group,the mitochondrial morphological damage was aggravated,and the cristae structure was significantly reduced in the I/R + AAV-CISD2 + ML385 group.5.The results of ferrous ion,MDA,and GSH in tissue by microplate assay showed that: compared with the sham group,the levels of ferrous ion and MDA were significantly increased in the I/R group（all p<0.01）,and the level of GSH was significantly decreased（p<0.01）;Compared with the I/R + AAV-NC group,the levels of ferrous ions and MDA were significantly decreased in the I/R + AAVCISD2 group（all p<0.01）,and the level of GSH was significantly increased（p<0.01）;compared with the I/R + AAV-CISD2 group,the levels of ferrous ions and MDA were significantly increased in the I/R + AAV-CISD2 + ML385 group（all p<0.05）,and the level of GSH was significantly decreased（p<0.01）.6.The results of protein expression in cerebral showed that compared with the sham group,the expressions of TFR1 and N-Nrf2 were significantly increased in the I/R group（all p<0.05）,and the expressions of CISD2,GPX4,x CT,and HO-1 were significantly decreased（all p<0.05）;compared with the I/R + AAV-NC group,the expression of TFR1 was significantly decreased in the I/R + OE group（all p<0.05）,and the expressions of N-Nrf2,GPX4,x CT,and HO-1 were significantly increased（all p<0.05）;compared with the I/R + AAV-CISD2 group,TFR1 expression was significantly increased（all p<0.01）,N-Nrf2,GPX4,x CT,and HO-1 protein expressions were significantly decreased in the I/R + AAV-CISD2 + ML385 group（all p<0.01）.7.The results of cell survival rates showed that compared with the control group,the survival rates of cells were significantly decreased in the OGD/R group（p<0.001）;compared with the OGD/R group,the survival rates of cells were significantly increased in the OGD/R +OE group（p<0.01）;compared with the OGD/R group;the survival rates of cells were further decreased in the OGD/R +sh RNA group（p<0.01）;compared with OGD/R + OE group,he survival rates of cells were significantly decreased in the OGD/R + OE + ML385 group（p<0.01）.8.The results of intracellular ferrous ion and Lipid ROS showed that: compared with the control group,the levels of intracellular ferrous ion and Lipid ROS were significantly increased in the OGD/R group（p<0.01）;compared with the OGD/R group,the levels of intracellular ferrous ion and Lipid ROS were significantly reduced in the OGD/R + CISD2-OE group（p<0.01）;compared with the OGD/R +CISD2-OE group,the levels of intracellular ferrous ion and Lipid ROS were significantly increased in the OGD/R + CISD2-OE + ML385 group（p<0.05）.9.The results of protein expression on cells: Compared with the control group,the expressions of TFR1 and N-Nrf2 were significantly increased in the OGD/R group（all p<0.01）,and the expressions of CISD2,GPX4,x CT and HO-1 were significantly decreased（all p < 0.01）;compared with the OGD/R group,the expression of TFR1 was significantly decreased in the OGD/R + OE group（all p<0.01）,and the expressions of N-Nrf2,GPX4,x CT,and HO-1 were significantly increased（all p<0.01）;compared with the OGD/R + CISD2-OE group,TFR1 expression was significantly increased（all p<0.05）and N-Nrf2,GPX4,x CT and HO-1 expressions were significantly decreased in the OGD/R + CISD2-OE +ML385 group（all p<0.05）.Conclusions:1.Up-regulation of CISD2 can significantly alleviate cerebral ischemia reperfusion injury in mice.2.The protective effects of CISD2 up-regulation on cerebral ischemia reperfusion injury in mice are associated with the inhibition of ferroptosis.3.The cerebral-protective effect of up-regulated CISD2 may be related to the activation of the Nrf2/HO-1 pathway. |
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