成束蛋白-1对腰椎间盘突出大鼠髓核细胞铁死亡的影响及相关机制

The effect of Fascin-1 protein on ferroptosis in nucleus pulposus cells of rats with lumbar disc herniation and related mechanisms

中文关键词:  腰椎间盘突出  Fascin-1  xCT  铁死亡  髓核组织

英文关键词:Lumbar disc herniation  Fascin-1  xCT  Ferroptosis  Nucleus pulposus tissue

中文摘要:

  【摘要】 目的：探讨成束蛋白-1（Fascin-1）对腰椎间盘突出（lumbar disc herniation，LDH）大鼠髓核（nucleus pulposus，NP）细胞铁死亡的影响及相关机制。方法：采用随机数字表法将36只SPF级雄性SD大鼠随机分为假手术组、模型组、sh-NC组（慢病毒空白载体）、sh-Fascin-1组（含有sh-Fascin-1的慢病毒载体）、sh-Fascin-1+Erastin（铁死亡诱导剂）组和sh-Fascin-1+Erastin-V（Erastin溶剂对照）组，每组6只，其中sh-NC组、sh-Fascin-1组、sh-Fascin-1+Erastin组、sh-Fascin-1+Erastin-V组在建模前48h注射相应慢病毒载体。除假手术组外，其余5组均采用自体尾椎L5/6椎间盘NP移植神经根法建立LDH模型。造模后7d，各组大鼠腹腔注射相应药物，每天1次，连续7d。造模前、造模后3d、7d、14d、21d对大鼠进行行为学检测，评估缩足阈值（pain withdrawal threshold，PWT）、热撤足潜伏期（pain withdrawal latency，PWL）。造模后22d，苏木精-伊红（hematoxylin-eosin，HE）染色观察L5/6椎间盘NP组织病理变化、免疫组化检测Fascin-1阳性细胞表达、免疫共沉淀检测Fascin-1与胱氨酸-谷氨酸逆转运蛋白轻链亚基（the light-chain subunit of system xc-，xCT）的相互作用及xCT泛素化水平；Western blot（WB）检测Fascin-1、长链脂酰辅酶A合成酶4（acyl-coa synthetase long chain family member 4，ACSL4）、谷胱甘肽过氧化物酶4（glutathione peroxidase 4，GPX4）和xCT蛋白的表达；生化检测活性氧自由基（reactive oxygen species，ROS）、超氧化物歧化酶（superoxide dismutase，SOD）活性、丙二醛（maleic dialdehyde，MDA）、亚铁离子（Fe2+）含量和乳酸脱氢酶释放量。结果：模型组造模后3d、7d、14d、21d的PWT、PWL均显著性低于假手术组。造模后22d，假手术组大鼠NP细胞排列规则，纤维环结构完整；模型组大鼠NP细胞减少，出现空洞样，排列不规则且皱缩，纤维环破裂；相较于sh-NC组，sh-Fascin-1组大鼠NP细胞有所增加，排列相对规则；对比sh-Fascin-1+Erastin-V组，sh-Fascin-1+Erastin组大鼠NP细胞排列不规则且皱缩，纤维环破裂。与假手术组相比，模型组ROS、MDA、Fe2+含量、乳酸脱氢酶释放量、ACSL4、Fascin-1表达、xCT泛素化水平升高，SOD活性及GPX4及xCT表达降低；相较于sh-NC组，sh-Fascin-1组指标变化趋势与模型组相反；相较于sh-Fascin-1+Erastin-V组，sh-Fascin-1+Erastin组指标变化趋势与模型组一致。模型组Fascin-1与xCT存在相互作用。结论：Fascin-1通过调控xCT促进LDH大鼠NP组织铁死亡进而影响LDH大鼠的临床症状，可作为LDH治疗的潜在靶点。

英文摘要:

  【Abstract】 Objectives: To explore the effect of Fascin-1 protein on ferroptosis in nucleus pulposus(NP) cells in rats with lumbar disc herniation(LDH) and the related mechanisms. Methods: Using the random number table method, 36 male SPF-grade SD rats were randomly divided into a sham operation group, a model group, a sh-NC group(empty lentivirus vector), a sh-Fascin-1 group(lentivirus vector containing sh-Fascin-1), a sh-Fascin-1+Erastin group(ferroptosis inducer), and a sh-Fascin-1+Erastin-V group(Erastin solvent control). Each group consisted of 6 rats. The sh-NC group, the sh-Fascin-1 group, the sh-Fascin-1+Erastin group, and the sh-Fascin-1+Erastin-V group were injected with the corresponding lentivirus vectors 48h before modeling. Except for the sham operation group, the other 5 groups underwent LDH modeling by autologous L5/6 NP transplantation to the nerve root. At 7d after modeling, rats in each group received intraperitoneal injections of the corresponding drug once daily for 7 consecutive days. Behavioral assessments were conducted on the rats before modeling and at 3, 7, 14, and 21d post-modeling to evaluate the pain withdrawal threshold(PWT) and pain withdrawal latency(PWL). At 22d after modeling, hematoxylin-eosin(HE) staining was used to observe pathological changes in the NP of the L5/6 intervertebral disc, immunohistochemistry was used to detect Fascin-1 positive expression in cells, and co-immunoprecipitation was used to detect the interaction between Fascin-1 and the light-chain subunit of system xc-(xCT), as well as the ubiquitination levels of xCT. Western blot(WB) was used to detect the expressions of Fascin-1, acyl-coa synthetase long chain family member 4(ACSL4), glutathione peroxidase 4(GPX4), and xCT. Biochemical detection was performed to detect the activities of reactive oxygen species(ROS) and superoxide dismutase(SOD), and maleic dialdehyde(MDA) and ferrous ion(Fe2+) contents, as well as lactate dehydrogenase(LDH) release amount. Results: In the model group, both PWT and PWL were significantly lower than those in the sham operation group at 3, 7, 14, and 21d post-modeling. At 22d post-modeling, the NP cells of rats in the sham operation group were arranged regularly and the annulus fibrosus structure was intact; In the model group, the number of NP cells decreased, presenting a cavity-like appearance, with irregular and shriveled arrangement and ruptured annulus fibrosus. Compared to the sh-NC group, the number of NP cells in the sh-Fascin-1 group of rats increased and the arrangement was relatively regular; Compared to the sh-Fascin-1+Erastin-V group, the NP cells of rats in the sh-Fascin-1+Erastin group were irregularly arranged and shrunken, and the annulus fibrosus was ruptured. Compared with the sham operation group, the model group exhibited increased levels of ROS, MDA, Fe2+, LDH release, ACSL4, Fascin-1 expression, and xCT ubiquitination, as well as decreased SOD activity and GPX4 and xCT expression; Compared with the sh-NC group, trends in the sh-Fascin-1 group were opposite to those in the model group; Compared with sh-Fascin-1+Erastin-V group, the trends in the sh-Fascin-1+Erastin group were consistent with those in the model group. An interaction between Fascin-1 and xCT was observed in the model group. Conclusions: Fascin-1 regulates xCT to promote ferroptosis in NP of LDH rats, thereby influencing the clinical symptoms of LDH rats. Moreover, Fascin-1 can serve as a potential target for LDH treatment.