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

English Keywords:Lumbar disc herniation  Fascin-1  xCT  Ferroptosis  Nucleus pulposus tissue

English Abstract:

  【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.