TNFα-reliant FSP1 up-regulation promotes intervertebral disc degeneration via caspase 3-dependent apoptosis

Abstract

Intervertebral disc degeneration (IDD) is a common chronic inflammatory degenerative disease that causes lower back pain. However, the underlying mechanisms of IDD remain unclear. Ferroptosis suppressor protein 1 (FSP1) is a newly identified suppressor for ferroptosis. This study aims to investigate the role of FSP1 in IDD. Nucleus pulposus (NP) tissues in humans were collected and NP cells from rats were isolated to detect FSP1 expression pattern. The relationship between FSP1-mediated ferroptosis and apoptosis was identified using FSP1 inhibitor iFSP1. RNA sequencing was utilized to seek downstream molecules and related signaling pathways. Moreover, both exogenous recombinant FSP1 protein and endogenous small interfering RNA were implemented in this study to clarify the role of FSP1 in tumor necrosis factor-alpha (TNFα)-mediated NP cell apoptosis. Ultimately, the underlying mechanisms of FSP1-related signaling pathway in IDD were uncovered both in vitro and in vivo. As a result, FSP1 was up-regulated in human degenerative NP tissues and after TNFα stimulation. FSP1 inhibition by iFSP1 fails to trigger ferroptosis in NP cells while inhibiting TNFα-mediated apoptosis. Further experiments demonstrated that FSP1 was closely related to TNFα-reliant caspase 3 activation and mitochondrial damage. However, the exogenous addition of recombinant protein FSP1 does not induce cell death or intensify the efficacy of TNFα. Mechanically, FSP1 is involved in TNFα-mediated NF-κB signaling activation to accelerate the development of IDD. This study demonstrated that FSP1 promotes IDD through TNFα-reliant NF-κB signaling activation and caspase 3-dependent apoptosis. These findings suggested a novel therapeutic target for the treatment of IDD.

Keywords: Caspase 3; FSP1; Intervertebraldisc degeneration; NF-κB; TNFα; iFSP1.

Figure 1

FSP1 is up-regulated during IDD and responsive to TNFα. (A) Representative MRI-T2 images of the lumbar spine from IDD patients with Pfirrmann grade II or grade V respectively. The lower panels show pictures of the indicated segment at a high magnification. (B) Hematoxylin and eosin (H&E) staining on human NP tissue in two groups. Scale bar, 100 μm. (C, D) Western blot of FSP1 expression in two groups and its quantification. (E) Immunohistochemistry staining of FSP1 in two groups. Scale bar, 100 μm. (F) Immunofluorescence staining of FSP1 in rat NP cells with or without TNFα stimulation. Scale bar, 20 μm. (G, H) Western blot of FSP1 expression in rat NP cells with or without TNFα stimulation and its quantification. FSP1, ferroptosis suppressor protein 1; IDD, intervertebral disc degeneration; TNFα, tumor necrosis factor alpha; NP, nucleus pulposus. ∗P < 0.05, ∗∗P < 0.01.

Figure 2

Inhibition of FSP1 fails to induce ferroptosis. (A–C) Rat NP cells were treated with or without TNFα and RNA sequencing was performed. The differentially expressed genes were presented in the volcano plot. Inflammatory response and TNF signaling pathway were activated on GSEA. (D) FSP1 was up-regulated after TNFα stimulation. (E, F) Transcriptional levels of FSP1 and GPX4 in normal NP cells. (G) Cell viability of NP cells with different concentrations of iFSP1 treatment. (H) Cell morphology after treatment with different doses of iFSP1 (0, 1, 2, 5, 10, and 20 μM). Scale bar, 200 μm. (I, J) Cell apoptosis of NP cells after treatment with different concentrations of iFSP1 (0, 1, 2, 5, 10, and 20 μM) and quantification of apoptotic cells was performed by flow cytometry. (K, L) Live-death staining in NP cells with stimulation by different doses of iFSP1 and death rate (%) calculation. Scale bar, 200 μm. (M) Cell morphology of transmission electron microscope in control and iFSP1 treated groups. Low, 5 μm; high, 1 μm. (N, O) Western blot of FSP1 and GPX4 expression in rat NP cells with stimulation by different doses of iFSP1 and their quantification. (P) Schematic diagram of the role of FSP1 and GPX4 in fighting against ferroptosis in NP cells. FSP1, ferroptosis suppressor protein 1; NP, nucleus pulposus; TNFα, tumor necrosis factor alpha; GPX4, glutathione peroxidase 4. ∗∗P < 0.01, ∗∗∗P < 0.001.

Figure 3

FSP1-evoked apoptosis by TNFα is related to caspase 3 activation and mitochondrial damage. (A, B) TNFα stimulated rat NP cells were co-cultured with or without iFSP1 and RNA sequencing was performed. The differentially expressed genes were presented in the volcano plot and the TNF signaling pathway was inhibited on GSEA after iFSP1 treatment. (C) The heat map revealed no significance of ferroptotic genes between the two groups, while apoptotic genes containing caspase 3 and caspase 4 were both down-regulated. (D, E) Western blot analysis of FSP1, OPA1, Drp1, Mfn1, Mfn2, Bax, Bcl2, pro-caspase 3, and cleaved caspase 3 in TNFα stimulated rat NP cells with or without iFSP1 treatment and their quantification. (F) Transmission electron microscope of TNFα stimulated rat NP cells with or without iFSP1 treatment. Low, 5 μm; high, 1 μm. (G) JC-1 staining of NP cells of indicated three groups. Scale bar, 20 μm. (H) Caspase-3 GreenNuc™ staining and caspase-3 immunofluorescence in TNFα stimulated rat NP cells with or without iFSP1 treatment. Scale bar, 100 μm. (I) Annexin V/PI staining of three groups and apoptotic rate (%) quantification. FSP1, ferroptosis suppressor protein 1; NP, nucleus pulposus; PI, propidium iodide; TNFα, tumor necrosis factor alpha; ns, not significance. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Figure 4

Fsp1 knockdown reduces cellular reactivity to TNFα and dampens the apoptotic phenotype. (A) Cell morphology in normal NP cells or after siFSP1 transfection. (B, C) Western blot analysis of FSP1 and GPX4 after three times of siFSP1 transfection, and their quantification. (D, E) Western blot analysis of FSP1 in TNFα stimulated NP cells with or without siFSP1 transfection, and its quantification. (F, G) Western blot analysis of Bax, Bcl2, pro-caspase 3, and cleaved caspase 3 in TNFα stimulated NP cells with or without siFSP1 transfection, and their quantification. (H) Caspase-3 GreenNuc™ staining in TNFα stimulated rat NP cells with or without siFSP1 transfection. Scale bar, 100 μm. (I) Annexin V/PI staining in TNFα stimulated rat NP cells with or without siFSP1 transfection, and apoptotic rate (%) quantification. (J, K) Western blot analysis of OPA1, Drp1, Mfn1, and Mfn2 in TNFα stimulated NP cells with or without siFSP1 transfection, and their quantification. (L) Transmission electron microscope of TNFα stimulated rat NP cells with or without siFSP1 transfection. Low, 5 μm; high, 1 μm. (M) JC-1 staining of NP cells of indicated two groups. Scale bar, 20 μm. FSP1, ferroptosis suppressor protein 1; PI, propidium iodide; TNFα, tumor necrosis factor alpha; GPX4, glutathione peroxidase 4; ns, not significance. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Figure 5

Exogenous FSP1 is incapable of triggering cell death and unable to potentiate TNFα-derived cell demise. (A) Cell morphology of NP cells after treatment of different recombinant FSP1 concentrations (0, 10, 20, 50, 100, and 200 ng/mL). Scale bar, 200 μm. (B) Cell viability of NP cells after treatment of different recombinant FSP1 concentrations. (C, D) Annexin V/PI staining in rat NP cells with addition of different doses of FSP1, and apoptotic rate (%) quantification. (E, F) Live-death staining in NP cells with stimulation by different doses of FSP1, as well as death rate (%) calculation. Scale bar, 100 μm. (G) Cell morphology of TNFα (20 ng/mL) stimulated NP cells after treatment of different recombinant FSP1 concentrations (0, 20, 50, 100, and 200 ng/mL). Scale bar, 200 μm. (H) Cell viability of TNFα stimulated NP cells after treatment of different recombinant FSP1 concentrations. (I, J) Annexin V/PI staining in TNFα stimulated rat NP cells with addition of different doses of FSP1, and apoptotic rate (%) quantification. (K, L) Live-death staining of TNFα stimulated NP cells with stimulation by different doses of FSP1 and death rate (%) calculation. Scale bar, 100 μm. FSP1, ferroptosis suppressor protein 1; PI, propidium iodide; TNFα, tumor necrosis factor alpha; ns, not significance.

Figure 6

Inhibition of FSP1 ameliorates intervertebral disc degeneration in rats. (A) A needle puncture rat model was established and radiological tests were performed on time-line. (B) A sketch map of the novel method for calculation of DHI. (C, D) MRI of rat tails on three indicated groups and MRI index (%) calculation by ImageJ. (E, F) X-ray of rat tails of three indicated groups and DHI calculation. (G, H) Histological investigations comprising hematoxylin and eosin (H&E) staining, Safranin O staining, Masson staining, and Alcian Blue staining were performed in three groups. Scale bar, 1000 μm. (I, J) Immunohistochemistry of FSP1 and caspase 3 of three groups. Scale bar, 200 μm in low magnification and 20 μm in high magnification. FSP1, ferroptosis suppressor protein 1; MRI, magnetic resonance imaging; DHI, disc height index. ∗P < 0.05, ∗∗∗P < 0.001.

Figure 7

FSP1 is a downstream of TNFα and mediates NF-κB signaling activation. (A) TNFα stimulated rat NP cells were co-cultured with or without iFSP1 and RNA sequencing was performed. The differentially expressed genes of NF-κB signaling were shown in heat maps. (B) Independent or conjoint genes in TNF signaling and NF-κB signaling. (C) NF-κB signaling pathway was inhibited on GSEA after iFSP1 treatment. (D) Western blot of p-p65 and p-IκBα in TNFα stimulated NP cells after treatment of different iFSP1 concentrations. (E) Immunofluorescence staining of p65 in TNFα stimulated NP cells in the existence or absence of iFSP1. Scale bar, 29.34 μm. (F) Immunofluorescence staining of p65 and p-IκBα in rat intervertebral disc tissues of three groups. Scale bar, 200 μm. FSP1, ferroptosis suppressor protein 1; TNFα, tumor necrosis factor alpha.

Figure 8

The schematic depicting a proposed model for the function of ferroptosis suppressor protein 1 (FSP1) in intervertebral disc degeneration.