The Study On Repairing Spinal Cord Injury By Inhibiting Ferroptosis With Liproxstatin-1

【Objective】 Spinal cord injury(SCI)is a serious traumatic disease of the central nervous system.It has the characteristics of high morbidity,high disability,high mortality and many complications.It has imposed a heavy financial burden on patients,families and society.Our prophase research found that ferroptosis was involved in the secondary injury stage following SCI.Ferroptosis is a newly discovered non-apoptotic form of cell death characterized by the accumulation of iron-dependent lipid peroxides and lipid reactive oxygen species(ROS),which is morphologically,biochemical,and genetically distinct from necrosis,apoptosis,and autophagy.Liproxstatin-1(Lipro-1)inhibits ferroptosis more effectively than other inhibitors,such as deferoxamine,vitamin E and ferrostatin-1.However,studies on the effectiveness and mechanism of Lipro-1 in rats contusion SCI have not been carried out.The purpose of this study was to investigate the effectiveness of ferroptosis inhibitor Lipro-1 in repairing spinal cord injury,and to explore the underlying mechanism of Lipro-1,so as to provide new therapeutic strategies for the treatment of SCI.【Methods】 1.In vitro experiment: to explore whether lipro-1 can inhibit RSL-3-induced ferroptosis in SH-SY5 Y cells.(1)Experimental grouping: PBS group,DMSO group,RSL-3 group,RSL-3 + Lipro-1 group,Lipro-1 group;(2)Detection of cell activity: SH-SY5 Y cells were treated with RSL-3 at a concentration gradient for 18 h,and cell activity was measured by CCK8,and the IC50 of RSL-3 was calculated;SH-SY5 Y cells were treated with RSL-3 and concentration gradient Lipro-1 for 18 hours.Cell activity was measured by CCK8 to select the optimal intervention concentration of Lipro-1.(3)PI/Hoechst staining: the number of PI-positive cells in each group was observed by PI/Hoechst staining to verify the ability of Lipro-1 to protect cells;(4)Western blot: The expression of ferroptosis key proteins GPX4,5-LOX and ACSL4 was detected by Western blot;(5)Detection of mitochondrial lipid peroxidation and cell MDA content: mitochondrial lipid peroxidation and cell MDA content were detected by mitochondrial specific fluorescence dye Mito Pe DPP and MDA content detection kits to reflect the ability of Lipro-1 to remove lipid peroxidation.2.In vivo experiments: to investigate the effectiveness and mechanism of Lipro-1 in repairing spinal cord injury.(1)Experimental group: female Wistar rats(8 weeks,150 g-180 g)were randomly divided into 3 groups: Sham group,SCI group and Lipro-1 group(30 per group).In the Sham group,only T10 laminectomy was performed without hitting the spinal cord.After removing the T10 lamina in the SCI group and the Lipro-1 group,the Impactor Model-III was used to prepare the moderate contusion SCI model(25mm);(2)Dosage regimen: after the contusion SCI model was successfully prepared,rats in the Lipro-1 group were intraperitoneally injected with a 10 mg/kg Lipro-1,and then injected once a day until 3 days after the injury.Rats in the SCI group were intraperitoneally injected with the same dose of normal saline;(3)Behavioral evaluation: before SCI and 1d,1w,2w,3w and 4w after SCI,the BBB scoring scale and inclined plate test were used to evaluate the recovery of the motor function of the hind limb.(4)Neural electrophysiological detection: 4 weeks after SCI,the sensory evoked potential(SEP)and motor evoked potential(MEP)were used to evaluate the recovery of nerve conduction function;(5)Western blot: the expression of ferroptosis key proteins GPX4,Tf,Tf R,FTL,5-LOX and ACSL4 in spinal cord tissues were detected by Western blot 1 and 3 days after SCI.(6)ELISA test: the contents of MDA and 4-HNE in spinal cord tissue were measured by ELISA 2 days after SCI;(7)Immunofluorescence staining: the survival of Neu N-positive neurons and Olig2-positive oligodendrocytes in spinal cord tissues was detected by immunofluorescence staining 3 and 7 days after SCI.At 4w after SCI,GFAP immunofluorescence intensity was detected to reflect the activation degree of astrocytes.(8)HE staining and LFB staining: 4 weeks after SCI,the integrity of the spinal cord tissue structure was detected by HE staining and LFB staining.【Results】 1.In vitro experiments: Lipro-1 inhibits RSL-3-induced ferroptosis in SH-SY5 Y cells.SH-SY5 Y cells were treated with RSL-3 at a concentration gradient for 18 h.The cell activity was detected by CCK8,and the IC50 of RSL-3 was calculated to be 4.083μM;SH-SY5 Y cells were co-treated with 4μM RSL-3 and concentration gradient Lipro-1 for 18 h,and the cell activity was detected by CCK8.The results showed that the EC50 of Lipro-1 was 2.295 n M,and when the concentration was 100 n M,the cell activity was nearly 100%.PI/Hoechst staining results showed that the number of PIpositive cells in the RSL-3 group increased significantly compared with the DMSO group(P<0.05);Compared with RSL-3 group,the number of PI-positive cells in RSL-3+Lipro-1 group was significantly reduced(P<0.05);Western blot results showed that the expression of GPX4 in RSL-3 group was significantly reduced,and the expression of 5-LOX and ACSL4 increased significantly(P<0.05).The expression of GPX4 significantly increased after Lipro-1 treatment,and the expression of 5-LOX and ACSL4 decreased significantly(P<0.05).The MDA content and mitochondrial lipid peroxidation increased significantly in the RSL-3 group(P < 0.05),while MDA content and mitochondrial lipid peroxidation decreased significantly after Lipro-1 treatment(P<0.05).2.In vivo experiments: Liproxstatin-1repairs SCI by inhibiting ferroptosis.After SCI,the BBB scoring scale and inclined plate test were used to evaluate the recovery of the motor function of the hind limb.The results showed that compared with the SCI group,the hind limb motor function of the rats of Lipro-1 group was better,and the difference was statistically significant(P<0.05);Neurophysiological tests showed that the latency of SEP in the Lipro-1 group was shorter than that in the SCI group,and the difference was statistically significant(P<0.05).The amplitude of MEP in Lipro-1 group was higher than that in SCI group,and the difference was statistically significant(P<0.05).After SCI,the expression of ferroptosis negative regulator GPX4 decreased significantly(P<0.05),and the expression of positive regulators Tf,Tf R,FTL,5-LOX and ACSL4 increased significantly(P<0.05).However,after treatment with Lipro-1,the expression of GPX4 increased significantly(P<0.05),and the expression of Tf,Tf R,FTL,5-LOX and ACSL4 decreased significantly(P<0.05).Compared with Sham group,the contents of MDA and 4-HNE in SCI group increased significantly(P<0.05),and decreased significantly after treatment with Lipro-1(P<0.05);Compared with Sham group,the number of neurons and oligodendrocytes in SCI group decreased significantly(P<0.05),and the number of neurons and oligodendrocytes increased significantly after Lipro-1 treatment(P<0.05).At 4w after SCI,the number of GFAP+ astrocytes increased significantly(P<0.05),and decreased significantly after Lipro-1 treatment(P<0.05).The results of HE staining and LFB staining showed that Lipro-1 can reduce the cavity area of spinal cord tissue and reduce the degree of demyelination(P<0.05).【Conclusions】 1.Liproxstatin-1 up-regulated the expression of GPX4,down-regulated the expression of ACSL4 and 5-LOX,alleviated mitochondrial lipid peroxidation,and further inhibited ferroptosis induced by RSL-3 in SH-SY5 Y cells.2.Liproxstatin-1 inhibited ferroptosis in the acute stage of SCI,reduced lipid peroxidation,protected neurons and oligodendrocytes,reduced glial scar hyperplasia,improved the sensory and motor functions of rats,and thus promoted the repair of spinal cord injury.