沉默XBP1通过调控Nrf2介导的铁死亡对脊髓损伤大鼠神经功能恢复的影响OA

Objectives:To discuss the effect of silencing X-box binding protein 1(XBP1)on neurological functional recovery in the rats with spinal cord injury(SCI),and to clarify its possible mechanism.Methods:Seventy-five male SD rats with body mass of 200-240 g were randomly divided into sham operation group,model group(SCI group),negative control lentivirus group(sh-NC group),XBP1 gene silencing group(sh-XBP1 group),and sh-XBP1+nuclear factor E2-related factor 2(Nrf2)inhibitor ML385 group(sh-XBP1+ML385 group),with 15 rats in each group.The modified Allen's method was used to establish the SCI models of rats;the corresponding lentivirus were injected into the spinal cord and ML385 was intraperitoneally injected for intervention.BBB motor function score and open field test were used to evaluate the hindlimb motor function of the rats in various groups;HE staining was used to observe the histopathological morphology of spinal cord tissue of the rats in various groups;immunofluorescence method was used to observe the expression levels of neuronal specific marker neuronal nuclear protein(NeuN)in spinal cord tissue of the rats in various groups;kits was used to detect the levels of malondialdehyde(MDA)and glutathione(GSH)and the activities of superoxide dismutase(SOD)in spinal cord tissue of the rats in various groups;a kit was used to detect the levels of ferrous ion(Fe2+)in spinal cord tissue of the rats in various groups;Western blotting method was used to detect the expression levels of XBP1,Nrf2,glutathione peroxidase 4(GPX4),and solute carrier super family 7 member 11(SLC7A11)proteins in spinal cord tissue of the rats in various groups.Results:The BBB motor function score and open field test results showed that compared with sham operation group,the BBB motor function scores and total moving distances in open field test of the rats in SCI group were significantly decreased at 7,14,21,and 28 d after surgery(P<0.01);compared with SCI group,the BBB motor function scores and total moving distances in open field test of the rats in sh-XBP1 group were significantly increased at 14,21,and 28 d after surgery(P<0.05 or P<0.01);compared with sh-XBP1 group,the BBB motor function scores and total moving distances in open field test of the rats in sh-XBP1+ML385 group were significantly decreased at 21 and 28 d after surgery(P<0.01).The HE staining results showed that in sham operation group,the spinal cord tissue structure of the rats was intact,with uniform and clear staining;in SCI group,sh-NC group,and sh-XBP1+ML385 group,the spinal cord tissue of the rats was severely injured,with edema,disordered distribution of nerve cells,and abnormal morphology and number;in sh-XBP1 group,the spinal cord tissue structure of the rats was relatively intact,and the state of nerve cells was significantly improved.The immunofluorescence method results showed that compared with sham operation group,the expression level of NeuN protein in spinal cord tissue of the rats in SCI group was significantly decreased(P<0.01);compared with SCI group,the expression level of NeuN protein in spinal cord tissue of the rats in sh-XBP1 group was significantly increased(P<0.01);compared with sh-XBP1 group,the expression level of NeuN protein in spinal cord tissue of the rats in sh-XBP1+ML385 group was significantly decreased(P<0.05).The ELISA and kit results showed that compared with sham operation group,the levels of MDA and Fe2+in spinal cord tissue of the rats in SCI group were significantly increased(P<0.01),and the level of GSH and the activity of SOD were significantly decreased(P<0.01);compared with SCI group,the levels of MDA and Fe2+in spinal cord tissue of the rats in sh-XBP1 group were significantly decreased(P<0.01),and the level of GSH and the activity of SOD were significantly increased(P<0.01);compared with sh-XBP1 group,the levels of MDA and Fe2+in spinal cord tissue of the rats in sh-XBP1+ML385 group were significantly increased(P<0.01),and the level of GSH and the activity of SOD were significantly decreased(P<0.01).The Western blotting method results showed that compared with sham operation group,the expression level of XBP1 protein in spinal cord tissue of the rats in SCI group was significantly increased(P<0.01),and the expression levels of Nrf2,GPX4,and SLC7A11 proteins were significantly decreased(P<0.01);compared with SCI group,the expression level of XBP1 protein in spinal cord tissue of the rats in sh-XBP1 group was significantly decreased(P<0.01),and the expression levels of Nrf2,GPX4,and SLC7A11 proteins were significantly increased(P<0.01);compared with sh-XBP1 group,the expression levels of Nrf2,GPX4,and SLC7A11 proteins in spinal cord tissue of the rats in sh-XBP1+ML385 group were significantly decreased(P<0.01).Conclusion:Silencing of XBP1 gene can promote neurological functional recovery in the rats with SCI,and its mechanism may be related to up-regulation of Nrf2 expression and inhibition of ferroptosis in nerve cells of spinal cord tissue.