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

doi:10.13481/j.1671-587X.20260307

摘要/Abstract

摘要：

目的探讨沉默X盒结合蛋白1（XBP1）对脊髓损伤（SCI）大鼠神经功能恢复的影响，并阐明其可能的作用机制。方法将75只体质量为200~240 g的雄性SD大鼠随机分为假手术组、模型组（SCI组）、阴性对照慢病毒组（sh-NC组）、XBP1基因沉默组（sh-XBP1组）和sh-XBP1+核因子E2相关因子2（Nrf2）抑制剂ML385组（sh-XBP1+ML385组），每组15只。采用改良Allen’s法制备大鼠SCI模型，给予脊髓内注射相应慢病毒和腹腔注射ML385干预。采用BBB运动功能评分和旷场实验评估各组大鼠后肢运动功能，HE染色观察各组大鼠脊髓组织病理形态表现，免疫荧光法检测各组大鼠脊髓组织中神经元特异性标志物神经元核蛋白（NeuN）表达水平，试剂盒法检测各组大鼠脊髓组织中丙二醛（MDA）和谷胱甘肽（GSH）水平及超氧化物歧化酶（SOD）活性，试剂盒检测各组大鼠脊髓组织中亚铁离子（Fe²⁺）水平，Western blotting法检测各组大鼠脊髓组织中XBP1、Nrf2、谷胱甘肽过氧化物酶4（GPX4）和溶质载体超家族7成员11（SLC7A11）蛋白表达水平。结果 BBB运动功能评分和旷场实验，与假手术组比较，术后第7、14、21和28天SCI组大鼠BBB运动功能评分和旷场实验总移动距离明显降低（P<0.01）；与SCI组比较，术后第14、21和28天sh-XBP1组大鼠BBB运动功能评分和旷场实验总移动距离明显升高（P<0.05或P<0.01）；与sh-XBP1组比较，术后第21和28天sh-XBP1+ML385组大鼠BBB运动功能评分和旷场实验总移动距离明显降低（P<0.01）。HE染色，假手术组大鼠脊髓组织结构完整，染色均匀清晰；SCI组、sh-NC组和sh-XBP1+ML385组大鼠脊髓组织损伤严重，有水肿现象，神经细胞分布杂乱，形态、数量异常；sh-XBP1组大鼠脊髓组织结构较完整，神经细胞状态明显改善。免疫荧光法，与假手术组比较，SCI组大鼠脊髓组织中NeuN蛋白表达水平明显降低（P<0.01）；与SCI组比较，sh-XBP1组大鼠脊髓组织中NeuN蛋白表达水平明显升高（P<0.01）；与sh-XBP1组比较，sh-XBP1+ML385组大鼠脊髓组织中NeuN蛋白表达水平明显降低（P<0.05）。试剂盒检测法，与假手术组比较，SCI组大鼠脊髓组织中MDA和Fe²⁺水平明显升高（P<0.01），GSH水平和SOD活性明显降低（P<0.01）；与SCI组比较，sh-XBP1组大鼠脊髓组织中MDA和Fe²⁺水平明显降低（P<0.01），GSH水平和SOD活性明显升高（P<0.01）；与sh-XBP1组比较，sh-XBP1+ML385组大鼠脊髓组织中MDA和Fe²⁺水平明显升高（P<0.01），GSH水平和SOD活性明显降低（P<0.01）。Western blotting法，与假手术组比较，SCI组大鼠脊髓组织中XBP1蛋白表达水平明显升高（P<0.01），Nrf2、GPX4和SLC7A11蛋白表达水平明显降低（P<0.01）；与SCI组比较，sh-XBP1组大鼠脊髓组织中XBP1蛋白水平明显降低（P<0.01），Nrf2、GPX4和SLC7A11蛋白表达水平明显升高（P<0.01）；与sh-XBP1组比较，sh-XBP1+ML385组大鼠脊髓组织中Nrf2、GPX4和SLC7A11蛋白表达水平明显降低（P<0.01）。结论 XBP1基因沉默可以促进SCI大鼠神经功能恢复，其作用机制可能与上调Nrf2表达、抑制脊髓组织神经细胞铁死亡有关。

关键词: 脊髓损伤, X盒结合蛋白1, 铁死亡, 核因子E2相关因子2, 神经细胞

Abstract:

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.

Key words: Spinal cord injury, X-box binding protein 1, Ferroptosis, Nuclear factor E2-related factor 2, Nerve cells

中图分类号:

R744.9

谭俊杰,赵洁,刘申易,邓华阳,赵怡灯,朱拓,詹海兰,阮丽华. 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[J]. 吉林大学学报(医学版), 2026, 52(3): 641-650.

Junjie TAN,Jie ZHAO,Shenyi LIU,Huayang DENG,Yideng ZHAO,Tuo ZHU,Hailan ZHAN,Lihua RUAN. Effect of silencing XBP1 on recovery of neurological function in rats with spinal cord injury by regulating Nrf2-mediated ferroptosis[J]. Journal of Jilin University(Medicine Edition), 2026, 52(3): 641-650.

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Group	MDA[w_B/(μmol?g^－1)]	GSH[w_B/(μmol?g^－1)]	SOD[λ_B/(U?mg^-1)]
Sham operation	4.67±0.43	0.63±0.04	88.36±6.63
SCI	10.45±1.07^*	0.17±0.02^*	24.98±2.33^*
Sh-NC	10.93±1.42	0.18±0.02	25.21±2.16
Sh-XBP1	6.34±0.52^△	0.38±0.03^△	56.94±4.85^△
Sh-XBP1+ML385	9.05±0.71^#	0.26±0.02^#	40.62±3.72^#