[Research on ferroptosis induced by RSL3, Erastin, and TBHP in rat intervertebral disc annulus fibrosus cells].

OBJECTIVE: To explore the effects of RAS-selective lethal small molecule 3 (RSL3), Erastin, and tert-butyl hydroperoxide (TBHP) on ferroptosis of rat annulus fibrosus cells (AFCs), and to establish an effective in vitro model of ferroptosis of rat AFCs, providing an experimental basis and theoretical support for the subsequent research on the mechanism of ferroptosis.

METHODS: AFCs were isolated and cultured from the caudal vertebrae of 6-week-old female Sprague-Dawley rats by enzymatic hydrolysis. The optimal treatment concentrations of RSL3, Erastin, and TBHP (used for establishing an in vitro model of ferroptosis in rat AFCs) were screened by the cell counting kit 8 (CCK-8) method. According to the optimal concentrations of each inducer obtained through screening, rat AFCs were divided into control group, RSL3 group, Erastin group, and TBHP group. The expression levels of ferroptosis-related genes [glutathione peroxidase 4 (GPX4), ferritin heavy chain 1 (FTH1), acyl coenzyme A synthase long chain member 4 (ACSL4), solute carrier family 7, member 11 (SLC7A11), and prostaglandin-endoperoxide synthase 2 (PTGS2)], and cell proliferation-related genes [proliferating cell nuclear antigen (PCNA)] were detected by real-time fluorescence quantitative PCR (RT-qPCR), Western blot, and cell immunofluorescence staining; iron (Fe 2+) content and malondialdehyde (MDA) content kits were used to evaluate iron accumulation and lipid peroxidation. The levels of intracellular lipid droplets (LDs) and reactive oxygen species (ROS) were determined by cell fluorescence staining to evaluate the level of cellular oxidative stress.

RESULTS: The optimal concentrations for treating rat AFCs were screened by CCK-8 method and rat AFCs were divided into control group, RSL3 (0.1 μmol/L) group, Erastin (0.5 μmol/L) group, and TBHP (50 μmol/L) group. Both RSL3 group and TBHP group downregulated the mRNA expression levels of GPX4, FTH1, SLC7A11, and PCNA, upregulated the mRNA expression levels of ACSL4 and PTGS2, decreased the protein expression levels of GPX4 and FTH1, and increased the protein expression level of PTGS2 ( P<0.05); simultaneously, increased the intracellular Fe 2+ and MDA contents, LDs and ROS levels ( P<0.05), significantly induced the occurrence of ferroptosis. While the Erastin group upregulated the mRNA expression levels of FTH1, SLC7A11, and PTGS2 ( P<0.05); however, it had no significant effect on the mRNA expression levels of GPX4, ACSL4, and PCNA, nor on the protein expression levels of GPX4, FTH1, and PTGS2 ( P>0.05), although it increased the intracellular MDA content ( P<0.05), but did not significantly change the intracellular Fe 2+ content, LDs and ROS levels ( P>0.05), failed to induce ferroptosis significantly.

CONCLUSION: RSL3 and TBHP are suitable for the establishment of ferroptosis models in rat AFCs, which is helpful for the subsequent study of ferroptosis in intervertebral disc degeneration.