Liproxstatin-1 Protects Vascular Endothelial Cells Via Ferroptosis Inhibition And Maintain S Blood-spinal Cord Barrier After Spinal Cord Injury

Background and purposeSpinal cord injury(SCI),an extremely serious traumatic disease of the central nervous system,leads to permanent neurological dysfunction and movement impairment in patients,and there is currently no effective treatment.The blood-spinal cord barrier(BSCB)is a barrier between the blood and the spinal cord parenchyma that prevents harmful substances from entering the spinal cord.It is important to protect the BSCB at the early stage to limit the secondary destruction and promote the repair of SCI.Ferroptosis mainly occurs in the early stage of SCI and plays a key role in the imbalance of the spinal microenvironment.Our previous studies have shown that systemic inhibition of ferroptosis can effectively improve the longterm recovery effect of SCI.However,the relationship between ferroptosis and secondary injury of the BSCB remains unclear.Therefore,this study further explored the role of ferroptosis in the BSCB secondary injury,revealed the key pathway of ferroptosis in vascular endothelial cells(ECs),clarified the key mechanism of early delivery of ferroptosis inhibitor to protect the BSCB from secondary damage,and provided new targets and new ideas for SCI repair.Methods1.The model of moderate spinal cord contusion in rats was established by using Impactor Model Ⅲ with the impact height of 25 mm and weight of 10 g.8-week-old female Wistar rats with 220-240 g were randomly divided into 3 groups:Sham group,SCI group,and liproxstatin1(Lip-1)group.1 mL of Lip-1 was administered by intraperitoneal injection 30 min after SCI,once daily for 3 days.2.The BSCB permeability was determined by Evans blue(EB)leakage test.Immunofluorescence staining of Zonula occludens-1(ZO-1)was used to detect the function of BSCB.3.Ferroptosis model of brain microvascular endothelial cells(bEnd.3)was established to detect the content of ferroptosis related proteins and the expression changes of tight junction protein in bEnd.3 cells.Based on the rat model of SCI,immunofluorescence staining was performed on ECs(marker:RECA1)and ferroptosis key enzymes ACSL4 and 15-LOX in the spinal cord of rats,respectively.Through co-localization analysis of ECs and ferroptosis key enzymes,the inhibitory effect of Lip-1 on ferroptosis of ECs was revealed.4.Macrophage(marker:CD68)and neutrophil(marker:MPO)in the spinal cord of rats were stained with immunofluorescence to detect the infiltration of immune cells at the injured site.5.The neuroprotective effect of Lip-1 was detected by immunofluorescence staining of GFAP and NeuN in rat spinal cord.The repair effect of Lip-1 was observed by behavioral evaluation(BBB score and CatWalk gait analysis),electrophysiological detection(sensory evoked potential and motor evoked potential),and histological experiment(H&E staining).Results1.Ferroptosis inhibitor Lip-1 alleviates secondary disruption of the blood-spinal cord barrier after spinal cord injury in rats.EB staining results showed obvious blue dye leakage in the spinal cord tissue 3 days after injury,and the BSCB leakage was significantly reduced after Lip-1 treatment.Immunofluorescence staining of ECs(RECA1)and tight junction protein(ZO-1)showed decreased expression of ZO-1 in ECs at 3 days after SCI,and Lip-1 increased the expression of ZO-1 in ECs.2.Lip-1 inhibits ferroptosis and increases the expression of tight junction protein in bEnd.3 cells.7.66 μM of RSL3(CC50)was used to induce ferroptosis in bEnd.3 cells.The results showed that GSH content and GPX4 expression decreased.ACSL4 and 15-LOX expression increased.The expression of BODIPY 581/591 C11 increased in oxidation state.Moreover,the expression of ZO-1 in ferroptotic bEnd.3 cells was also significantly reduced.bEnd.3 cells were treated with RSL3+Lip-1,and then GSH content and GPX4 content were increased.ACSL4 and 15-LOX expression was decreased.Reduced-state BODIPY 581/591 C11 was increased.Most importantly,ZO-1 expression was increased in bEnd.3 cells.3.Lip-1 inhibits ferroptosis of vascular endothelial cells after spinal cord injury in rats.At 3 days after injury,the results of immunofluorescence staining of spinal cord tissue showed that the co-localization of RECA1 with ACSL4 and 15-LOX in the SCI group was higher than that in the Sham group,that is,the expression of ACSL4 and 15-LOX in ECs increased at 3 days after injury.After Lip-1 treatment,the co-localization of RECA1 with ACSL4 and 15-LOX decreased,that is,Lip-1 reduced the levels of ACSL4 and 15-LOX in ECs.4.Lip-1 alleviates infiltration of macrophages and neutrophils after spinal cord injury.Tissue immunofluorescence showed an increase in CD68 fluorescence intensity at 7 days after SCI and MPO fluorescence intensity at 3 days after injury.In contrast,the fluorescence intensity of CD68 and MPO in Lip-1 group was significantly weaker than that of SCI group,indicating that Lip-1 could inhibit the infiltration of macrophages and neutrophils in spinal cord tissues after injury.5.Lip-1 reduces the production of glial scar and increases the number of neurons after spinal cord injury.The immunofluorescence results showed that the fluorescence intensity of GFAP in the SCI group was significantly higher than that in the Sham group,indicating a large number of glial scar at 8 weeks after injury.After Lip-1 treatment,the intensity of GFAP-positive fluorescence was reduced,indicating that Lip-1 reduced glial scar at 8 weeks after injury.In addition,the number of NeuN-positive cells in the Lip-1 group was significantly more than that in the SCI group,suggesting that Lip-1 could increase the number of neurons in the spinal cord.6.Lip-1 promotes functional recovery after spinal cord injury in rats.H&E staining showed that rats in the Lip-1 group spared more spinal cord tissue than the SCI group at 8 weeks after injury.BBB scores of rats at 8 weeks after SCI showed that the scores in the Lip-1 group were higher than those in the SCI group,and the difference was statistically significant.The footprints of CatWalk gait analysis showed that the double hind limbs in the Lip-1 group were significantly clearer and more complete than those in the SCI group,that is,the hind limbs had better recovery.Electrophysiological test results showed that the latency of motor evoked potential and sensory evoked potential in the Lip-1 group was shorter and the amplitude was higher than SCI group.These results indicate that Lip-1 can effectively improve the functional recovery of rats after SCI.Conclusions1.Ferroptosis inhibitor Lip-1 alleviated secondary damage of the BSCB,reduced infiltration of peripheral inflammatory cells,and reduced microenvironmental imbalance mediated by secondary injury of SCI.2.Lip-1 inhibited ferroptosis of vascular ECs through lipid peroxidation pathway and stabilizes the BSCB structure.3.Lip-1 effectively inhibited the formation of glial scar,protected the remaining neurons,and repaired the motor function and nerve conduction function of rats after SCI.