Research on ferroptosis induced by RSL3, Erastin, and TBHP in rat intervertebral disc annulus fibrosus cells_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

PAN Xiaolin , TAN Jing , LIU Yuying , LEI Guihua ,  Lü Fengjuan

SCUT-UWA Joint Research Centre for Regenerative Medicine, School of Medicine, South China University of Technology, Guangzhou Guangdong, 510006, P. R. China;

Corresponding?author：

Lü Fengjuan, Email: Lufj0@scut.edu.cn

Keywords：

Intervertebral disc degeneration; annulus fibrosus; ferroptosis; RAS-selective lethal small molecule 3; tert-butyl hydroperoxide; Erastin; modeling

DOI：

10.7507/1002-1892.202601097

Video：

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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 (Fe²⁺) 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 Fe²⁺ 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 Fe²⁺ 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.

Citation： PAN Xiaolin, TAN Jing, LIU Yuying, LEI Guihua, Lü Fengjuan. Research on ferroptosis induced by RSL3, Erastin, and TBHP in rat intervertebral disc annulus fibrosus cells. Chinese Journal of Reparative and Reconstructive Surgery, 2026, 40(7): 1110-1120. doi: 10.7507/1002-1892.202601097 Copy

1.	Hadjipavlou AG, Tzermiadianos MN, Bogduk N, et al. The pathophysiology of disc degeneration: a critical review. J Bone Joint Surg (Br), 2008, 90(10): 1261-1270.
2.	Peng Y, Lv FJ. Symptomatic versus asymptomatic intervertebral disc degeneration: Is inflammation the key? Crit Rev Eukaryot Gene Expr, 2015, 25(1): 13-21.
3.	Lv FJ, Peng Y, Lim FL, et al. Matrix metalloproteinase 12 is an indicator of intervertebral disc degeneration co-expressed with fibrotic markers. Osteoarthritis Cartilage, 2016, 24(10): 1826-1836.
4.	Liu C, Liang G, Deng Z, et al. The upregulation of COX2 in human degenerated nucleus pulposus: The association of inflammation with intervertebral disc degeneration. Mediators Inflamm, 2021, 2021: 2933199. doi: 10.1155/2021/2933199.
5.	Lyu FJ, Cui H, Pan H, et al. Painful intervertebral disc degeneration and inflammation: from laboratory evidence to clinical interventions. Bone Res, 2021, 9(1): 7. doi: 10.1038/s41413-020-00125-x.
6.	Ye F, Lyu FJ, Wang H, et al. The involvement of immune system in intervertebral disc herniation and degeneration. JOR Spine, 2022, 5(1): e1196. doi: 10.1002/jsp2.1196.
7.	Cachot U, Kandil K, Za?ri F, et al. Role of mechanical representativity in multiaxial and transverse mechanics of human annulus fibrosus: A microstructure-based biphasic finite element study. Acta Biomater, 2025, 197: 266-282.
8.	Fan C, Chu G, Yu Z, et al. The role of ferroptosis in intervertebral disc degeneration. Front Cell Dev Biol, 2023, 11: 1219840. doi: 10.3389/fcell.2023.1219840.
9.	Dixon SJ, Lemberg KM, Lamprecht MR, et al. Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell, 2012, 149(5): 1060-1072.
10.	Sun K, Shi Y, Yan C, et al. Glycolysis-derived lactate induces ACSL4 expression and lactylation to activate ferroptosis during intervertebral disc degeneration. Adv Sci (Weinh), 2025, 12(21): e2416149. doi: 10.1002/advs.202416149.
11.	Chen J, Yang X, Feng Y, et al. Targeting ferroptosis holds potential for intervertebral disc degeneration therapy. Cells, 2022, 11(21): 3508. doi: 10.3390/cells11213508.
12.	Yang S, Zhu Y, Shi Y, et al. Screening of NSAIDs library identifies Tinoridine as a novel ferroptosis inhibitor for potential intervertebral disc degeneration therapy. Free Radic Biol Med, 2024, 221: 245-256.
13.	Chen C, Wang X, Zhao Y, et al. Exosomes inhibit ferroptosis to alleviate intervertebral disc degeneration via the p62-KEAP1-NRF2 pathway. Free Radic Biol Med, 2025, 232: 171-184.
14.	王澤, 于涵, 焦磊, 等. 蘿卜硫素通過增強抗氧化能力抑制髓核細胞鐵死亡. 解放軍醫學院學報, 2025, 46(4): 354-361.
15.	諶超, 胡耀全, 呂正品, 等. 叔丁基氫過氧化物可誘導髓核細胞發生鐵死亡. 中國組織工程研究, 2025, 29(32): 6858-6865.
16.	Zhang J, He L, Li Q, et al. EGR1 knockdown confers protection against ferroptosis and ameliorates intervertebral disc cartilage degeneration by inactivating the MAP3K14/NF-κB axis. Genomics, 2023, 115(5): 110683. doi: 10.1016/j.ygeno.2023.110683.
17.	Wang H, Zhou Z, Wu T, et al. Deferoxamine improves intervertebral disc degeneration by activating HIF-1α/BNIP3-mediated mitophagy and inhibiting ferroptosis. Int Immunopharmacol, 2025, 166: 115583. doi: 10.1016/j.intimp.2025.115583.
18.	Liang G, Tan J, Chen C, et al. Single nuclear-spatial transcriptomic sequencing reveals distinct puncture-induced cell subpopulations in the intervertebral disc of a rat model. Clin Transl Med, 2025, 15(6): e70370. doi: 10.1002/ctm2.70370.
19.	Wang X, Wang H, Yang H, et al. Tumor necrosis factor-α- and interleukin-1β-dependent matrix metalloproteinase-3 expression in nucleus pulposus cells requires cooperative signaling via syndecan 4 and mitogen-activated protein kinase-NF-κB axis: implications in inflammatory disc disease. Am J Pathol, 2014, 184(9): 2560-2572.
20.	Yang WS, Kim KJ, Gaschler MM, et al. Peroxidation of polyunsaturated fatty acids by lipoxygenases drives ferroptosis. Proc Natl Acad Sci U S A, 2016, 113(34): E4966-75.
21.	Xu Y, Zhang L, Ji M, Lu J. Prominin-2/FBXO22/BACH1 axis protects bone marrow mesenchymal stem cells against TBHP-induced ferroptosis and ameliorates intervertebral disc degeneration. Stem Cell Res Ther, 2025, 16(1): 340. doi: 10.1186/s13287-025-04453-9.
22.	Koppula P, Zhang Y, Zhuang L, et al. Amino acid transporter SLC7A11/xCT at the crossroads of regulating redox homeostasis and nutrient dependency of cancer. Cancer Commun (Lond), 2018, 38(1): 12. doi: 10.1186/s40880-018-0288-x.
23.	Bersuker K, Hendricks JM, Li Z, et al. The CoQ oxidoreductase FSP1 acts parallel to GPX4 to inhibit ferroptosis. Nature, 2019, 575(7784): 688-692.
24.	Dodson M, Castro-Portuguez R, Zhang DD. NRF2 plays a critical role in mitigating lipid peroxidation and ferroptosis. Redox Biol, 2019, 23: 101107. doi: 10.1016/j.redox.2019.101107.
25.	Gai C, Liu C, Wu X, et al. MT1DP loaded by folate-modified liposomes sensitizes erastin-induced ferroptosis via regulating miR-365a-3p/NRF2 axis in non-small cell lung cancer cells. Cell Death Dis, 2020, 11(9): 751. doi: 10.1038/s41419-020-02939-3.
26.	Wang C, Wang Z, Li B, et al. Lipid droplet-targeting fluorescent probes for monitoring hydrogen sulfide in ferroptosis. Anal Chim Acta, 2025, 1362: 344156. doi: 10.1016/j.aca.2025.344156.
27.	Dong S, Li X, Xu G, et al. Quercetin attenuates the symptoms of osteoarthritis in vitro and in vivo by suppressing ferroptosis via activation of AMPK/Nrf2/Gpx4 signaling. Mol Med Rep, 2025, 31(3): 60. doi: 10.3892/mmr.2024.13425.
28.	Zhang T, Zhang Y, Xie J, et al. Ferroptosis in neurodegenerative diseases: mechanisms and therapeutic potential of stem cell derivatives. Front Cell Dev Biol, 2025, 13: 1577382. doi: 10.3389/fcell.2025.1577382.

1. Hadjipavlou AG, Tzermiadianos MN, Bogduk N, et al. The pathophysiology of disc degeneration: a critical review. J Bone Joint Surg (Br), 2008, 90(10): 1261-1270.
2. Peng Y, Lv FJ. Symptomatic versus asymptomatic intervertebral disc degeneration: Is inflammation the key? Crit Rev Eukaryot Gene Expr, 2015, 25(1): 13-21.
3. Lv FJ, Peng Y, Lim FL, et al. Matrix metalloproteinase 12 is an indicator of intervertebral disc degeneration co-expressed with fibrotic markers. Osteoarthritis Cartilage, 2016, 24(10): 1826-1836.
4. Liu C, Liang G, Deng Z, et al. The upregulation of COX2 in human degenerated nucleus pulposus: The association of inflammation with intervertebral disc degeneration. Mediators Inflamm, 2021, 2021: 2933199. doi: 10.1155/2021/2933199.
5. Lyu FJ, Cui H, Pan H, et al. Painful intervertebral disc degeneration and inflammation: from laboratory evidence to clinical interventions. Bone Res, 2021, 9(1): 7. doi: 10.1038/s41413-020-00125-x.
6. Ye F, Lyu FJ, Wang H, et al. The involvement of immune system in intervertebral disc herniation and degeneration. JOR Spine, 2022, 5(1): e1196. doi: 10.1002/jsp2.1196.
7. Cachot U, Kandil K, Za?ri F, et al. Role of mechanical representativity in multiaxial and transverse mechanics of human annulus fibrosus: A microstructure-based biphasic finite element study. Acta Biomater, 2025, 197: 266-282.
8. Fan C, Chu G, Yu Z, et al. The role of ferroptosis in intervertebral disc degeneration. Front Cell Dev Biol, 2023, 11: 1219840. doi: 10.3389/fcell.2023.1219840.
9. Dixon SJ, Lemberg KM, Lamprecht MR, et al. Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell, 2012, 149(5): 1060-1072.
10. Sun K, Shi Y, Yan C, et al. Glycolysis-derived lactate induces ACSL4 expression and lactylation to activate ferroptosis during intervertebral disc degeneration. Adv Sci (Weinh), 2025, 12(21): e2416149. doi: 10.1002/advs.202416149.
11. Chen J, Yang X, Feng Y, et al. Targeting ferroptosis holds potential for intervertebral disc degeneration therapy. Cells, 2022, 11(21): 3508. doi: 10.3390/cells11213508.
12. Yang S, Zhu Y, Shi Y, et al. Screening of NSAIDs library identifies Tinoridine as a novel ferroptosis inhibitor for potential intervertebral disc degeneration therapy. Free Radic Biol Med, 2024, 221: 245-256.
13. Chen C, Wang X, Zhao Y, et al. Exosomes inhibit ferroptosis to alleviate intervertebral disc degeneration via the p62-KEAP1-NRF2 pathway. Free Radic Biol Med, 2025, 232: 171-184.
14. 王澤, 于涵, 焦磊, 等. 蘿卜硫素通過增強抗氧化能力抑制髓核細胞鐵死亡. 解放軍醫學院學報, 2025, 46(4): 354-361.
15. 諶超, 胡耀全, 呂正品, 等. 叔丁基氫過氧化物可誘導髓核細胞發生鐵死亡. 中國組織工程研究, 2025, 29(32): 6858-6865.
16. Zhang J, He L, Li Q, et al. EGR1 knockdown confers protection against ferroptosis and ameliorates intervertebral disc cartilage degeneration by inactivating the MAP3K14/NF-κB axis. Genomics, 2023, 115(5): 110683. doi: 10.1016/j.ygeno.2023.110683.
17. Wang H, Zhou Z, Wu T, et al. Deferoxamine improves intervertebral disc degeneration by activating HIF-1α/BNIP3-mediated mitophagy and inhibiting ferroptosis. Int Immunopharmacol, 2025, 166: 115583. doi: 10.1016/j.intimp.2025.115583.
18. Liang G, Tan J, Chen C, et al. Single nuclear-spatial transcriptomic sequencing reveals distinct puncture-induced cell subpopulations in the intervertebral disc of a rat model. Clin Transl Med, 2025, 15(6): e70370. doi: 10.1002/ctm2.70370.
19. Wang X, Wang H, Yang H, et al. Tumor necrosis factor-α- and interleukin-1β-dependent matrix metalloproteinase-3 expression in nucleus pulposus cells requires cooperative signaling via syndecan 4 and mitogen-activated protein kinase-NF-κB axis: implications in inflammatory disc disease. Am J Pathol, 2014, 184(9): 2560-2572.
20. Yang WS, Kim KJ, Gaschler MM, et al. Peroxidation of polyunsaturated fatty acids by lipoxygenases drives ferroptosis. Proc Natl Acad Sci U S A, 2016, 113(34): E4966-75.
21. Xu Y, Zhang L, Ji M, Lu J. Prominin-2/FBXO22/BACH1 axis protects bone marrow mesenchymal stem cells against TBHP-induced ferroptosis and ameliorates intervertebral disc degeneration. Stem Cell Res Ther, 2025, 16(1): 340. doi: 10.1186/s13287-025-04453-9.
22. Koppula P, Zhang Y, Zhuang L, et al. Amino acid transporter SLC7A11/xCT at the crossroads of regulating redox homeostasis and nutrient dependency of cancer. Cancer Commun (Lond), 2018, 38(1): 12. doi: 10.1186/s40880-018-0288-x.
23. Bersuker K, Hendricks JM, Li Z, et al. The CoQ oxidoreductase FSP1 acts parallel to GPX4 to inhibit ferroptosis. Nature, 2019, 575(7784): 688-692.
24. Dodson M, Castro-Portuguez R, Zhang DD. NRF2 plays a critical role in mitigating lipid peroxidation and ferroptosis. Redox Biol, 2019, 23: 101107. doi: 10.1016/j.redox.2019.101107.
25. Gai C, Liu C, Wu X, et al. MT1DP loaded by folate-modified liposomes sensitizes erastin-induced ferroptosis via regulating miR-365a-3p/NRF2 axis in non-small cell lung cancer cells. Cell Death Dis, 2020, 11(9): 751. doi: 10.1038/s41419-020-02939-3.
26. Wang C, Wang Z, Li B, et al. Lipid droplet-targeting fluorescent probes for monitoring hydrogen sulfide in ferroptosis. Anal Chim Acta, 2025, 1362: 344156. doi: 10.1016/j.aca.2025.344156.
27. Dong S, Li X, Xu G, et al. Quercetin attenuates the symptoms of osteoarthritis in vitro and in vivo by suppressing ferroptosis via activation of AMPK/Nrf2/Gpx4 signaling. Mol Med Rep, 2025, 31(3): 60. doi: 10.3892/mmr.2024.13425.
28. Zhang T, Zhang Y, Xie J, et al. Ferroptosis in neurodegenerative diseases: mechanisms and therapeutic potential of stem cell derivatives. Front Cell Dev Biol, 2025, 13: 1577382. doi: 10.3389/fcell.2025.1577382.

Chinese Journal of Reparative and Reconstructive Surgery

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

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