| Objective:Paraplegia was the most serious complication of spinal cord ischemia-reperfusion injury(SCIRI),which often occured after spinal surgery and cardiovascular surgery.It had brought great pain to patients.The mechanism of SCIRI was complex,and a variety of signal pathways were involved in it,leading to death of motor neurons and paraplegia.There were many types of neuronal death.Howerver,it could not completely be inhibited by blocking one of the specific death mechanisms.Therefore,it was urgent to study the mechanism of neuronal death and use all means to save cell,improve neural function.Froptosis was a new type of cell death discovered recently.It was characterized by iron accumulation and lipid peroxidation.Some studies have found that ferroptosis was involved in ischemia-reperfusion injury.However,it has not been reported in SCIRI.The purpose of this study was to explore the role of ferroptosis in rat SCIRI.miR-21-5p is an anti-inflammatory and anti-apoptotic miRNA,which could reduce the degree of lipid peroxidation in oxygen-glucose deprivation model.Lipid peroxidation was an important feature of ferroptosis.As a predicted target gene,EphA4 was abundantly expressed in the nervous system.EphA4-mediated signaling pathway was the main mechanism of excitotoxic neuronal death induced by glutamate.Glutamate excitotoxicity could induce ferroptosis of neurons by inhibiting the function of system XC~-.Accordingly,another aspect of the present study was to explore the role of miR-21-5p/EphA4 in SCIRI and to determine whether it was related to ferroptosis.Sevoflurane,as the most commonly used inhalation anesthetic,had a protective effect on ischemia-reperfusion injury of important organs.But the role of miR-21-5p in sevoflurane prediction has not been reported.Therefore,the third aspect of this study was to clarify the protective effect of sevoflurane preconditioning on SCIRI through miR-21-5p/EphA4 and its downstream pathway involved in it.Methods:1.The rat SCIRI model was established by clamping the aortic arch for 14minutes.The related indexes of ferroptosis including iron,MDA,GSH,GPX activity were detected.It was evaluated that the effects of Fer-1 on the related indexes of ferroptosis,the number of electron microscope mitochondria with ferroptosis charactors,neurological function of lower extremities,histological evaluation,the permeability of BSCB,the inflammatory level and the key proteins of ferroptosis.2.The expression of miR-21-5p and EphA4 were detected after SCIRI.The effect of mimic or inhibitor or NC on the expression of EphA4 was assessed.The targeting relationship between miR-21-5p and EphA4 was determined by double luciferase report assays.The efficiency of inhibiting EphA4 expression was detect.It was evalutaded that the effects of mimic,inhibitor,NC,AV-sh-EphA4,AV-NC,mimic+AV-EphA4-over and AV-sh-EphA4+Erastin on the related indexes of ferroptosis,the number of electron microscope mitochondria with ferroptosis charactors,neurological function of lower extremities,histological evaluation,the permeability of BSCB and the inflammatory level.Performing IP with EphA4 as bait protein.The related protein Erk1/2 and Bcelin1were screened out by mass spectrometry.Bcelin1,p-Erk1/2 and Erk1/2 were detected in IP complex with EphA4 as bait protein.After performing IP using Beclin1 as bait protein,XCT was assessed.The effects of U0126 on p-Erk1/2,c-Myc,TFR1and Erk1/2 were detected.The effects of mimic,inhibitor,NC,AV-sh-EphA4,AV-NC and mimic+AV-EphA4-over on p-Bcelin1,p-Erk1/2,c-Myc,TFR1 and Erk1/2 were detected.Immunofluorescence double labeling of EphA4 and p-Bcelin1,p-Erk1/2 was performed.3.The expression of miR-21-5p was detected after sevoflurane preconditioning.After sevoflurane preconditioning and combined with inhibitor,the expression of EphA4 was detected.After performing sevoflurane preconditioning,combined with inhibitor,combined with AV-EphA4-over,the effects on the related indexes of ferroptosis,the number of electron microscope mitochondria with ferroptosis charactors,neurological function of lower extremities,histological evaluation,the permeability of BSCB and the inflammatory level were assessed.p-Bcelin1,p-Erk1/2,c-Myc,TFR1 and Erk1/2 were detected.Results:1.The related indexed of ferroptosis in rat SCIRI model changed significantly.Intrathecal administration of Fer-1 could significantly improve the related indexes of ferroptosis,reduce the number of mitochondria with the characteristics of ferroptosis,improve the neurological score of lower limbs,increase the number of intact motor neurons in the anterior horn of the spinal cord,reduce the destruction of BSCB,diminish the overall inflammatory level,increase the expression of GPX4 and decreased the expression of XCT in the spinal cord.2.In SCIRI,miR-21-5p decreased remarkably and EphA4 expression increased significantly.miR-21-5p mimic could significantly reduce the expression of EphA4.Double luciferase report assays showed that there was a targeting relationship between miR-21-5p and EphA4.Intrathecal administration of miR-21-5p mimic,AV-sh-EphA4significantly improved the changes of indexes related to ferroptosis caused by SCIRI,reduced the number of mitochondria with ferroptosis character,improved the neurological score of lower limbs,increased the number of intact motor neurons in the anterior horn of the spinal cord,reduced the destruction of BSCB and diminished the overall inflammatory level.Overexpression of EphA4 could counteracted the effects of miR-21-5p mimic.Erastin could counteract the effects of AV-sh-EphA4.SCIRI induced an increase in the binding of EphA4 to Beclin1.Knocking down EphA4 decreased the increase in p-Beclin1 induced by SCIRI.Using Beclin1 as a bait protein,knocking down EphA4 could reduce the formation of Beclin1 and XCT complex caused by SCIRI.SCIRI caused a significant increase in the level of p-Erk1/2 bound to EphA4.Intrathecal injection of U0126 inhibited the activation of Erk1/2 and decreased the expression of c-Myc and TFR1.miR-21-5p mimic and AV-sh-EphA4 significantly reduced the increase of p-Erk1/2,p-Beclin1,c-Myc and TFR1 expression induced by SCIRI.AV-EphA4-over could counteract the inhibition to the expression of downstream related proteins induced by miR-21-5p mimic.In the fluorescence double labeling experiment,we also found that EphA4 was co-labeled with p-Beclin1 and p-Erk1/2.3.Sevoflurane pretreatment increased the expression of miR-21-5p and decreased the expression of EphA4 after SCIRI.miR-21-5p inhibitor counteracted the effect of pretreatment on EphA4.Sevoflurane pretreatment significantly improved the related indexes of ferroptosis,reduced the number of mitochondria with the characteristics of ferroptosis,improved the neurological score of lower limbs,increased the number of intact motor neurons in the anterior horn of the spinal cord,reduced the destruction of BSCB,diminished the overall inflammatory level and reduced the expression of p-Erk1/2,p-Beclin1,c-Myc,TFR1.Intrathecal administration of miR-21-5p inhibitor or AV-EphA4-over can counteract its effects.Conclusions:SCIRI caused ferroptosis of neurons in anterior horn of spinal cord.Sevoflurane preconditioning might inhibitor the formation of Beclin1-XCT complex and the activation of Erk/c-Myc/TFR1 pathway through miR-21-5p/EphA4,thereby reducing ferroptosis of motor neurons and having neuroprotective effect on nerve injury. |
