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. 2021 Mar;16(3):561-566.
doi: 10.4103/1673-5374.293157.

Liproxstatin-1 is an effective inhibitor of oligodendrocyte ferroptosis induced by inhibition of glutathione peroxidase 4

Bao-You Fan  1 , Yi-Lin Pang  1 , Wen-Xiang Li  1 , Chen-Xi Zhao  1 , Yan Zhang  1 , Xu Wang  1 , Guang-Zhi Ning  1 , Xiao-Hong Kong  2 , Chang Liu  2 , Xue Yao  1 , Shi-Qing Feng  1
Affiliations

Liproxstatin-1 is an effective inhibitor of oligodendrocyte ferroptosis induced by inhibition of glutathione peroxidase 4

Bao-You Fan et al. Neural Regen Res. 2021 Mar.

Abstract

Our previous studies showed that ferroptosis plays an important role in the acute and subacute stages of spinal cord injury. High intracellular iron levels and low glutathione levels make oligodendrocytes vulnerable to cell death after central nervous system trauma. In this study, we established an oligodendrocyte (OLN-93 cell line) model of ferroptosis induced by RSL-3, an inhibitor of glutathione peroxidase 4 (GPX4). RSL-3 significantly increased intracellular concentrations of reactive oxygen species and malondialdehyde. RSL-3 also inhibited the main anti-ferroptosis pathway, i.e., SLC7A11/glutathione/glutathione peroxidase 4 (xCT/GSH/GPX4), and downregulated acyl-coenzyme A synthetase long chain family member 4. Furthermore, we evaluated the ability of several compounds to rescue oligodendrocytes from ferroptosis. Liproxstatin-1 was more potent than edaravone or deferoxamine. Liproxstatin-1 not only inhibited mitochondrial lipid peroxidation, but also restored the expression of GSH, GPX4 and ferroptosis suppressor protein 1. These findings suggest that GPX4 inhibition induces ferroptosis in oligodendrocytes, and that liproxstatin-1 is a potent inhibitor of ferroptosis. Therefore, liproxstatin-1 may be a promising drug for the treatment of central nervous system diseases.

Keywords: cell death; central nervous system; factor; ferroptosis; oligodendrocyte; oxidation; pathway; repair; spinal cord injury.

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Conflict of interest statement

None

Figures

Figure 1
Figure 1
RSL-3 induces the death of OLN93 oligodendrocytes. (A) OLN93 oligodendrocytes express Oligo2, which is mainly nuclear. The fluorescent indicator is Alexa Fluor 488 for Oligo2 (green). (B) GPX4 is mainly expressed in the nucleus of OLN93 oligodendrocytes. The fluorescent indicator is Cy3 for GPX4 (red). (C) After incubation with RSL-3 for 24 hours, the viability of OLN93 oligodendrocytes was decreased in a dose-dependent manner (CC50 of RSL-3 was 7.89 μM). The cell viability was assessed by MTT (n= 5). (D) Cell death was confirmed by PI and Hoechst 33342 staining for 24 hours (n= 3). The concentration of RSL-3 was 7.89 μM. (E, F) ROS accumulated following treatment with RSL-3 (7.89 μM) in OLN93 oligodendrocytes at 24 hours (n= 3). Scale bars: 100 μm. Data are expressed as the mean ± SD. ** P< 0.01 (one-way analysis of variance followed by post hoc Tukey’s test). CC50: Cytotoxicity concentration 50%; DAPI: 4′,6-diamidino-2-phenylindole; DMSO: dimethyl sulfoxide; GPX4: glutathione peroxidase 4; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Oligo2: oligodendrocyte transcription factor 2; PBS: phosphate-buffered saline; PI: propidium iodide; ROS: reactive oxygen species.
Figure 2
Figure 2
The expression of ferroptosis-associated pathway proteins in oligodendrocytes detected by western blot assay. (A–F) The expression of GPX4 (A, B), xCT (C, D) and ACSL4 (E, F). The expression levels (standardized based on the GAPDH level first and then calculated relative to the DMSO group) of xCT, GPX4 and ACSL4 were decreased in the RSL-3 (7.89 μM) group compared with the DMSO group. Data are expressed as the mean ± SD (n = 3). *P < 0.05, **P < 0.01 (one-way analysis of variance followed by Tukey’s post hoc test). ACSL4: Acyl-coenzyme A synthetase long chain family member 4; DMSO: dimethyl sulfoxide; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; GPX4: glutathione peroxidase 4; PBS: phosphate-buffered saline; xCT: SLC7A11.
Figure 3
Figure 3
The inhibitory effects of Lipro-1, edaravone and deferoxamine on ferroptosis of OLN93 oligodendrocytes at 24 hours. (A–C) The EC50 of Lipro-1 (A), edaravone (B) and deferoxamine (C) against ferroptosis in OLN93 oligodendrocytes (n = 5). (D) Characteristics and EC50 of Lipro-1, edaravone and deferoxamine. (E, F) Death of OLN93 oligodendrocytes treated with RSL-3 (7.89 μM) and Lipro-1 (1 μM) (n = 3). Scale bars: 100 μm. Data are expressed as the mean ± SD. ****P < 0.0001 (one-way analysis of variance followed by Tukey’s post hoc test). DMSO: Dimethyl sulfoxide; EC50: mean effective concentration; Hoechst: Hoechst 33342; Lipro-1: liproxstatin-1; PBS: phosphate-buffered saline; PI: propidium iodide.
Figure 4
Figure 4
Mechanism of Lipro-1-mediated inhibition of ferroptosis in OLN93 oligodendrocytes. To observe the maximal effect of Lipro-1, the dose of Lipro-1 was 1 μM, and the dose of RSL-3 was 7.89 μM. (A) MDA levels. (B) Mitochondrial lipid peroxidation. Lipro-1 suppressed mitochondrial lipid peroxidation. (C) GSH levels. (D, E) Relative expression of GPX4. (F) Number of FSP1-positive cells. Lipro-1 restored FSP1-positive cells. The fluorescent indicator is Alexa Fluor 488 for FSP1 (green). Scale bars: 100 μm. Data are expressed as the mean ± SD (n = 3). **P < 0.01, ***P < 0.001, ****P < 0.0001 (one-way analysis of variance followed by Tukey’s post hoc test). DAPI: 4′,6-Diamidino-2-phenylindole; DMSO: dimethyl sulfoxide; FSP1: ferroptosis suppressor protein 1; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; GPX4: glutathione peroxidase 4; GSH: glutathione; Lipro-1: liproxstatin-1; MDA: malondialdehyde; MitoPeDPP: 3-[4-(perylenylphenylphosphino)phenoxy]propyltriphenylphosphonium iodide; PBS: phosphate-buffered saline.
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References

    1. Askari VR, Shafiee-Nick R. Promising neuroprotective effects of β-caryophyllene against LPS-induced oligodendrocyte toxicity: A mechanistic study. Biochem Pharmacol. 2019;159:154–171. - PubMed
    1. Bersuker K, Hendricks JM, Li Z, Magtanong L, Ford B, Tang PH, Roberts MA, Tong B, Maimone TJ, Zoncu R, Bassik MC, Nomura DK, Dixon SJ, Olzmann JA. The CoQ oxidoreductase FSP1 acts parallel to GPX4 to inhibit ferroptosis. Nature. 2019;575:688–692. - PMC - PubMed
    1. Brigelius-Flohé R, Maiorino M. Glutathione peroxidases. Biochim Biophys Acta. 2013;1830:3289–3303. - PubMed
    1. Chen L, Hambright WS, Na R, Ran Q. Ablation of the ferroptosis inhibitor glutathione peroxidase 4 in neurons results in rapid motor neuron degeneration and paralysis. J Biol Chem. 2015;290:28097–28106. - PMC - PubMed
    1. Chu J, Liu CX, Song R, Li QL. Ferrostatin-1 protects HT-22 cells from oxidative toxicity. Neural Regen Res. 2020;15:528–536. - PMC - PubMed