miR-24-3p靶向GSK-3β抑制铁死亡促进大鼠脊髓损伤修复

miR-24-3p promotes spinal cord injury repair in rats by inhibiting ferroptosis via targeting GSK-3β

摘要

目的 探讨miR-24-3p促进大鼠脊髓损伤（SCI）修复的分子机制。 方法 构建SD大鼠SCI模型，通过qRT-PCR检测SCI后miR-24-3p的表达变化。采用立体定位仪，通过尖端固定有毛细玻璃管的微量注射器于SCI头、尾各3 mm处注射miR-24-3p agomir或阴性对照agomir。使用BBB评分评估SCI大鼠运动功能恢复情况，HE染色观察脊髓组织形态学改变，试剂盒检测SCI区域Fe²⁺含量，普鲁士蓝+DAB染色检测SCI区域铁沉积变化。采用Western blotting和免疫荧光染色检测SCI区域组织GSK-3β及铁死亡相关蛋白xCT、GPX4的表达水平。双荧光素酶报告实验验证miR-24-3p与GSK-3β的靶向关系。体外构建Erastin诱导的PC12细胞铁死亡模型，检测MDA含量变化， Western blotting和免疫荧光染色检测细胞内GSK-3β、xCT和GPX4的蛋白表达水平变化。 结果 在SD大鼠SCI模型中，SCI区域miR-24-3p、xCT和GPX4蛋白表达在损伤后显著降低（P<0.05）。给予miR-24-3p agomir干预后，SCI大鼠后肢运动功能明显恢复，脊髓组织病理形态得到改善，Fe²⁺含量及铁沉积减少，GSK-3β蛋白表达降低，xCT和GPX4蛋白表达增加（P<0.05）。双荧光素酶报告实验证实miR-24-3p与GSK-3β之间存在靶向关系并抑制GSK-3β的表达。PC12细胞铁死亡模型中，转染miR-24-3p mimics可显著降低细胞内MDA含量和GSK-3β蛋白表达水平，并升高xCT和GPX4的蛋白表达水平；联合转染GSK-3β抑制剂后，上述效应进一步增强。 结论 miR-24-3p通过抑制GSK-3β抑制铁死亡，从而促进大鼠SCI的修复。

Abstract

Objective To investigate the molecular mechanism by which miR-24-3p promotes spinal cord injury (SCI) repair in rats. Methods The changes in spinal cord miR-24-3p expression was detected in a SD rat model of SCI with qRT-PCR. Using a stereotaxic apparatus, miR-24-3p agomir or a negative control reagent was microinjected at 3 mm rostral and caudal to the SCI epicenter of the rats. Motor function recovery of the SCI rats was evaluated with BBB scores, histopathological changes and Fe²⁺ content in the SCI area were examined with HE staining and a commercial assay kit, and the changes in iron deposition in the SCI area was observed using Prussian blue staining with DAB. Western blotting and immunofluorescence staining were used to detect expressions of glycogen synthase kinase-3β (GSK-3β) and ferroptosis-related proteins xCT and GPX4 in the injured tissue. The targeted regulatory relationship between miR-24-3p and GSK-3β was verified using dual-luciferase reporter assay. In PC12 cells with erastin-induced ferroptosis, malondialdehyde (MDA) content was detected and the expression levels of GSK-3β, xCT and GPX4 proteins were determined by Western blotting and immunofluorescence staining. Results The expression of miR-24-3p and protein levels of xCT and GPX4 were significantly decreased in the SCI area of the rat models. Treatment with miR-24-3p agomir significantly improved hindlimb motor function of the SCI rats, alleviated spinal cord pathologies, reduced Fe²⁺ content, iron deposition and GSK-3β protein expression, and upregulated xCT and GPX4 protein expressions in the SCI area. Dual-luciferase reporter assay confirmed targeted inhibition of GSK-3β by miR-24-3p. In erastin-induced PC12 cells, transfection with miR-24-3p mimics significantly decreased intracellular MDA content and GSK-3β protein expression and increased xCT and GPX4 protein levels, and these effects were enhanced by co-transfection with GSK-3β inhibitor. Conclusion miR-24-3p promotes repair of SCI in rats by inhibiting ferroptosis via targeted suppression of GSK-3β.

关键词

脊髓损伤 / miR-24-3p / GSK-3β / 铁死亡

Key words

spinal cord injury / miR-24-3p / glycogen synthase kinase-3β / ferroptosis

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