The Study On Repairing Spinal Cord Injury By Deferoxamine Through Inhibiting Ferroptosis

【Objective】 Spinal cord injury(SCI)is a devastating neurological disorder with high morbidity,mortality and disability rate.Numerous studies have investigated the mechanisms of cell death including apoptosis,necrosis and autophagy and have developed novel therapies in SCI.Ferroptosis is an iron-dependent novel cell death pathway characterized by lipid peroxidation.Deferoxamine(DFO)is mainly used as a treatment for diseases with iron overload,as well as a ferroptosis inhibitor,has been reported to promote SCI repair.However,the underlying molecular mechanisms of DFO repairing SCI is limited.In this study,we aim to explore whether ferroptosis occurs after SCI and clarify whether DFO could perform protective effects on SCI through inhibiting ferroptosis.【Methods】 In vivo study: Female Wistar rats weighing 240 ± 10 g were randomly assigned to the following groups: the Sham group,the SCI group and the DFO group.Spinal cord tissues of rats were collected and processed for further analysis: TEM was performed to observe the morphology of mitochondria after SCI;Western blot was conducted to detect the expression of ferroptosis markers GPX4 and xCT;Total GSH Assay Kit was used to detect the GSH level;RT-PCR was implemented to detect the mRNA expression level of ferroptosis-related mitochondria genes cs,ttc35,rpl8,atp5g3,acsf2 and ireb2;Immunofluorescence(IF)staining of NeuN was used to observe the protective effects on neural survival of DFO;BBB locomotor scores was applied to perform locomotor function recovery.In vitro study: In the first part,the neurons isolated from cerebral cortices of 16 th gestational day mice fetuses were randomly divided into 6 groups: PBS group,DMSO group and different concentrations of erastin group.The optimum concentration of erastin was obtained through the detection of cell viability,neural survival,neurite length,ROS levels and the expression of ferroptosis markers.After optimizing the potency of erastin,the second part was to explore the specificity of erastin-induced ferroptosis in neurons and whether DFO could protect the erastin-induced cellular damage by dividing the neurons into 6 groups: PBS group,DMSO group,erastin group,erastin + Z-DEVD-FMK group,erastin + Necrostatin-1 group and erastin + DFO group.The second part shared the same methods with the previous part.【Results】 In vivo study: TEM results showed hemolysis and shrunken mitochondria in injured spinal cord,instead of the Sham tissues;Iron level showed an obvious increase after injury(P < 0.001),DFO treatment significantly decreased the iron concentration(P < 0.001);The expression of negative regulatory factors xCT and GPX4,was decreased post SCI(P < 0.05),whereas DFO effectively upregulated the expression of GSH,xCT and GXP4(P < 0.001);The mRNA levels of ferroptosis specific genes cs,ttc35,rpl8,and atp5g3 were not increased after SCI,only ireb2 and acsf2 were upregulated(P<0.05),DFO treatment significantly decreased the mRNA expression of ireb2 and acsf2(P<0.05);The number of NeuN+ cells was decreased post SCI,DFO increased the number of NeuN+ cells;BBB scores showed better hindlimb locomotive function in DFO-treated SCI rats compared with the SCI group.In vitro study: Based on the results of cell viability,neural survival,neurite length,ROS levels and the expression of ferroptosis markers,ferroptosis was induced when primary cortical neurons exposed to erastin for 48 hours at the concentration of 50 uM.In the second part,compared with the erastin group,erastin + Z-DEVD-FMK group and erastin + Necrostatin-1 group,more survival neurons,higher cell viability,less neurite collapse,lower ROS level were observed,and DFO treatment also upregulated the expression of xCT and GPX4.【Conclusions】 1.Ferroptosis plays a critical role in SCI due to our detection outcomes of increased iron levels,shrunken mitochondria and activation of ferroptosis signaling pathway;2.DFO performs protective effects on repairing SCI through reducing the iron level and inhibiting ferroptosis pathway;3.Neuronal ferroptosis was induced when primary cortical neurons exposed to erastin for 48 hours at the concentration of 50 uM.Ferroptosis inhibitor DFO attenuates erastin-induced neuronal damage and promotes the neuronal survival through inhibiting ferroptosis in neurons.