A Study Of Spinal Cord Injury Repair By Inhibiting Ferroptosis With U0126

【Objective】Spinal cord injury is one serious complication of spinal injury with the characteristics of high morbidity,high disability,high mortality and many complications.The recovery of motor and sensory function after spinal cord injury is a worldwide difficult problem.The formation of local inhibitory microenvironment which aggravated secondary injury leads to the death of neurons and various support cells after spinal cord injury.Our group found that there is a completely new programmed cell death pathway–ferroptosis,which is different from the traditional sense of necrosis,apoptosis,necroptosis and pyroptosis and could promote neuronal death and participate in secondary injury.This study aimed to explore the existence of ferroptosis in the process of spinal cord injury,and reveal the internal mechanism of its specific role in the secondary injury.In addition we tried to reverse the ferroptosis by the treatment of U0126 and to clarify the specific mechanism of the repair of spinal cord injury.【Methods】1.Explore whether ferroptosis is included in the process of secondary injury.Female Wistar rats(240g+10g,10 weeks old)were randomly divided into sham group and Injury group.Sham group take the T10 segment lamina enucleation while Injury group take the spinal cord injury with NYU IMPACTOR MODEL-II.Detect the expression of ferroptosis marker xCT,GSH,GPX4 and 4HNE using Western Blotting and GSH detection kit 6 hours after surgery.Detect the change of mitochondria by electron microscopy and figure out that whether there is the ferroptosis characteristic changes-mitochondrial shrinkage,membrane thickening and mitochondrial crista disappear 15 minutes,1 hour,4 hours and 24 hours after the operation.These two parts aim to explore whether there is ferroptosis in the process of spinal secondary injury.2.Detecting the effects of U0126 on ferroptosis and figure out the mechanism of U0126 on repairing spinal cord injury.Female Wistar rats(240g+10g,10 weeks old)were randomly divided into Sham group,Injury group and U0126 group.Rats in the Sham group take the T10 segment lamina enucleation while Injury group and U0126 group take the spinal cord injury with NYU IMPACTOR MODEL-II.Detecting the ferroptosis markers(xCT,GSH,GPX4,4HNE),and inflammation factors,activated astrocytes,survived neurons and hindlimb movement changes by Western Blotting,HE staining,immunofluorescence,BBB score test to demonstrate the effect of U0126 on ferroptosis through molecular,organizational and behavioral levels,so as to clarify the effectiveness and specific mechanism of spinal cord injury repairing.【Results】1.Ferroptosis is included in the process of secondary injury.We found that the expression of xCT,GSH and GPX4 in Injury group was lower than Sham group while the expression of 4HNE was opposite.And the characteristic changes of mitochondrial included mitochondrial shrinkage,mitochondrial membrane thickening and mitochondrial cristae disappeared was happened at the time of 15 minutes,1 hour,4 hours,24 hours after spinal cord injury which proves that the ferroptosis exists in the process of the spinal cord injury.2.U0126 impair the spinal cord injury through inhibiting ferroptosis.We found that after spinal cord injury,treatment with U0126 could increase xCT,GSH and GPX4 expression and reduced the expression of 4HNE,and significantly improved organization structure,inhibited glial scar formation,protected neuron survival,inhibited inflammation and chemokine expression and improve the function of rat hind leg movement using Western Blotting,electron microscope,immunofluorescence and BBB score methods,which proving that U0126 could inhibit ferroptosis to repair spinal cord injury.【Conclusions】1.Ferroptosis is included in the pathological mechanism of spinal cord injury.This experiment research detecting ferroptosis markers xCT,GSH,GPX4 and 4HNE changes and observation mitochondria microstructure characteristic by Western Blotting,electron microscopy and other means preliminary evident that ferroptosis is included in the process of spinal cord injury.2.Ferroptosis inhibitor could impair the spinal cord injury through inhibiting ferroptosis.This study found that U0126 could reverse the expression of ferroptosis markers after spinal cord injury and can significantly improve the organizational structure,inhibit glial scar formation after spinal cord injury,protect neuron survival and inhibit inflammation and chemokine expression,thus improve the rat hind limbs motor function,achieve the purpose of repair of spinal cord injury.This finding provides one new target for the treatment of spinal cord injury.