铁死亡在压力诱导的椎间盘退变中的作用及机制研究

Role of Ferroptosis in Mechanical Stress⁃induced Intervertebral Disc Degeneration and Underlying Mechanism

目的探究铁死亡在压力诱导的椎间盘退变中的作用及其分子作用机制。方法将髓核细胞分为对照组、加压组和加压+铁死亡抑制剂(Fer⁃1)组,采用CCK⁃8法检测细胞增殖活力,透射电镜观察细胞内部超微结构,丙二醛(MDA)试剂盒检测髓核细胞脂质氧化水平。运用免疫荧光染色和Western Blot检测髓核细胞中谷胱甘肽过氧化物酶4(GPX4)蛋白表达水平;运用TargetScan 8.0预测与GPX4靶向结合的miRNA及其位点,并检测该miRNA在髓核细胞中的表达水平。结果压力作用导致髓核细胞增殖活力下降,脂质氧化水平升高,线粒体萎缩且膜密度增高,而Fer⁃1可有效逆转压力对髓核细胞的上述作用。压力下调髓核细胞中GPX4蛋白表达,生物信息学预测结果显示miR⁃1224靶向结合于GPX4且RT⁃PCR结果显示压力上调髓核细胞中miR⁃1224的表达水平(P<0.05)。抑制髓核细胞中的miR⁃1224可显著升高GPX4蛋白表达,并有效逆转压力诱导的髓核细胞表型退变及脂质氧化水平升高,过表达miR⁃1224则起到相反作用。结论压力导致髓核细胞中的miR⁃1224表达水平升高,通过靶向结合抑制了GPX4蛋白的表达,从而促进髓核细胞铁死亡的发生并导致椎间盘退变。

Objective To investigate the role of ferroptosis in compression⁃induced intervertebral disc degeneration(IDD)and the mechanism.Methods The nucleus pulposus(NP)cells were divided into control group,compression group and compression+Fer⁃1 group.The proliferation of NP cells was detected by CCK⁃8 assay,and the ultrastructure of NP cells was observed by transmission electron microscopy.The contents of malondialdehyde(MDA)were detected by detection kit.Immunofluorescence staining and Western blotting assays were performed to detect the expression of glutathione peroxidase 4(GPX4).TargetScan 8.0 software was used to predict the miRNA targeting GPX4 mRNA sequence.Then RT⁃PCR was performed to detect the expression of miR⁃1224 within NP cells.Results Compression led to the decreased cell proliferation and the increased lipid oxidation level,and decreased volume and increased membrane density of mitochondria within NP cells.However,Fer⁃1 effectively reversed the above effects of compression on NP cells.Compression decreased the expression of GPX4 protein and increased the expression of miR⁃1224 within NP cells(P<0.05).The results of Bioinformatics prediction showed that there was a potential binding site for miR⁃1224 in the mRNA sequence of GPX4.Inhibition of miR⁃1224 significantly increased the expression of GPX4 protein and effectively reduced the compression⁃induced degeneration phenotype.Conclusion Compression increases the expression of miR⁃1224,which targets and inhibits the expression of GPX4,thus promoting the occurrence of ferroptosis within NP cells and leading to intervertebral disc degeneration.