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Ferroptosis inhibitor liproxstatin-1 alleviates metabolic dysfunction-associated fatty liver disease in mice: potential involvement of PANoptosis

Abstract

Ferroptosis is a new form of regulated cell death characterized by excessive iron accumulation and uncontrollable lipid peroxidation. The role of ferroptosis in metabolic dysfunction-associated fatty liver disease (MAFLD) is not fully elucidated. In this study we compared the therapeutic effects of ferroptosis inhibitor liproxstatin-1 (LPT1) and iron chelator deferiprone (DFP) in MAFLD mouse models. This model was established in mice by feeding a high-fat diet with 30% fructose in water (HFHF) for 16 weeks. The mice then received LPT1 (10 mg·kg−1·d−1, ip) or DFP (100 mg·kg−1·d−1, ig) for another 2 weeks. We showed that both LPT1 and DFP treatment blocked the ferroptosis markers ACSL4 and ALOX15 in MAFLD mice. Furthermore, LPT1 treatment significantly reduced the liver levels of triglycerides and cholesterol, lipid peroxidation markers 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA), and ameliorated the expression of lipid synthesis/oxidation genes (Pparα, Scd1, Fasn, Hmgcr and Cpt1a), insulin resistance, mitochondrial ROS content and liver fibrosis. Importantly, LPT1 treatment potently inhibited hepatic apoptosis (Bax/Bcl-xL ratio and TUNEL+ cell number), pyroptosis (cleavages of Caspase-1 and GSDMD) and necroptosis (phosphorylation of MLKL). Moreover, LPT1 treatment markedly inhibited cleavages of PANoptosis-related caspase-8 and caspase-6 in MAFLD mouse liver. In an in vitro MAFLD model, treatment with LPT1 (100 nM) prevented cultured hepatocyte against cell death induced by pro-PANoptosis molecules (TNF-α, LPS and nigericin) upon lipid stress. On the contrary, DFP treatment only mildly attenuated hepatic inflammation but failed to alleviate lipid deposition, insulin resistance, apoptosis, pyroptosis and necroptosis in MAFLD mice. We conclude that ferroptosis inhibitor LPT1 protects against steatosis and steatohepatitis in MAFLD mice, which may involve regulation of PANoptosis, a coordinated cell death pathway that involves apoptosis, pyroptosis and necroptosis. These results suggest a potential link between ferroptosis and PANoptosis.

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Fig. 1: The inhibitor LPT1 blocks ferroptosis without disrupting iron accumulation in a mouse MAFLD model.
Fig. 2: The ferroptosis inhibitor LPT1 decreases steatosis and mitochondrial ROS in a mouse MAFLD model.
Fig. 3: The ferroptosis inhibitor LPT1 mitigates liver injury and fibrosis in a mouse MAFLD models.
Fig. 4: The ferroptosis inhibitor LPT1 suppresses steatohepatitis and ‘PANoptosis’ in a mouse MAFLD models.
Fig. 5: The ferroptosis inhibitor LPT1 inhibits cleavage of PANoptosis-related Caspase-6 and Caspase-8.
Fig. 6: The ferroptosis inhibitor LPT1 directly blocks PANoptosis in hepatocytes upon lipid stress.
Fig. 7: An iron chelator DFP reduces ferroptosis but does not ameliorate steatosis.
Fig. 8: An iron chelator DFP alleviates steatohepatitis.
Fig. 9: An iron chelator DFP does not block PANoptosis.

References

  1. Eslam M, Sanyal AJ, George J, International Consensus P. MAFLD: a consensus-driven proposed nomenclature for metabolic associated fatty liver disease. Gastroenterology. 2020;158:1999–2014.e1.

    Article  CAS  PubMed  Google Scholar 

  2. Eslam M, George J. MAFLD: a holistic view to redefining fatty liver disease. J Hepatol. 2021;74:983–5.

    Article  PubMed  Google Scholar 

  3. Younossi ZM, Rinella ME, Sanyal AJ, Harrison SA, Brunt EM, Goodman Z, et al. From NAFLD to MAFLD: implications of a premature change in terminology. Hepatology. 2021;73:1194–8.

    Article  PubMed  Google Scholar 

  4. Paik JM, Golabi P, Younossi Y, Mishra A, Younossi ZM. Changes in the global burden of chronic liver diseases from 2012 to 2017: the growing impact of NAFLD. Hepatology. 2020;72:1605–16.

    Article  PubMed  Google Scholar 

  5. Friedman SL, Neuschwander-Tetri BA, Rinella M, Sanyal AJ. Mechanisms of NAFLD development and therapeutic strategies. Nat Med. 2018;24:908–22.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Trauner M, Fuchs CD. Novel therapeutic targets for cholestatic and fatty liver disease. Gut. 2022;71:194–209.

    Article  CAS  PubMed  Google Scholar 

  7. Loomba R, Friedman SL, Shulman GI. Mechanisms and disease consequences of nonalcoholic fatty liver disease. Cell. 2021;184:2537–64.

    Article  CAS  PubMed  Google Scholar 

  8. Dixon SJ, Lemberg KM, Lamprecht MR, Skouta R, Zaitsev EM, Gleason CE, et al. Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell. 2012;149:1060–72.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Yang WS, SriRamaratnam R, Welsch ME, Shimada K, Skouta R, Viswanathan VS, et al. Regulation of ferroptotic cancer cell death by GPX4. Cell. 2014;156:317–31.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Friedmann Angeli JP, Schneider M, Proneth B, Tyurina YY, Tyurin VA, Hammond VJ, et al. Inactivation of the ferroptosis regulator Gpx4 triggers acute renal failure in mice. Nat Cell Biol. 2014;16:1180–91.

    Article  CAS  PubMed  Google Scholar 

  11. Ingold I, Berndt C, Schmitt S, Doll S, Poschmann G, Buday K, et al. Selenium utilization by GPX4 is required to prevent hydroperoxide-induced ferroptosis. Cell. 2018;172:409–22.e21.

    Article  CAS  PubMed  Google Scholar 

  12. Doll S, Proneth B, Tyurina YY, Panzilius E, Kobayashi S, Ingold I, et al. ACSL4 dictates ferroptosis sensitivity by shaping cellular lipid composition. Nat Chem Biol. 2017;13:91–8.

    Article  CAS  PubMed  Google Scholar 

  13. Chu B, Kon N, Chen D, Li T, Liu T, Jiang L, et al. ALOX12 is required for p53-mediated tumour suppression through a distinct ferroptosis pathway. Nat Cell Biol. 2019;21:579–91.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Bersuker K, Hendricks JM, Li Z, Magtanong L, Ford B, Tang PH, et al. The CoQ oxidoreductase FSP1 acts parallel to GPX4 to inhibit ferroptosis. Nature. 2019;575:688–92.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Doll S, Freitas FP, Shah R, Aldrovandi M, da Silva MC, Ingold I, et al. FSP1 is a glutathione-independent ferroptosis suppressor. Nature. 2019;575:693–8.

    Article  CAS  PubMed  Google Scholar 

  16. Lee H, Zandkarimi F, Zhang Y, Meena JK, Kim J, Zhuang L, et al. Energy-stress-mediated AMPK activation inhibits ferroptosis. Nat Cell Biol. 2020;22:225–34.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Mao C, Liu X, Zhang Y, Lei G, Yan Y, Lee H, et al. DHODH-mediated ferroptosis defence is a targetable vulnerability in cancer. Nature. 2021;593:586–90.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Qi J, Kim JW, Zhou Z, Lim CW, Kim B. Ferroptosis affects the progression of nonalcoholic steatohepatitis via the modulation of lipid peroxidation-mediated cell death in mice. Am J Pathol. 2020;190:68–81.

    Article  CAS  PubMed  Google Scholar 

  19. Li X, Wang TX, Huang X, Li Y, Sun T, Zang S, et al. Targeting ferroptosis alleviates methionine-choline deficient (MCD)-diet induced NASH by suppressing liver lipotoxicity. Liver Int. 2020;40:1378–94.

    Article  CAS  PubMed  Google Scholar 

  20. Tsurusaki S, Tsuchiya Y, Koumura T, Nakasone M, Sakamoto T, Matsuoka M, et al. Hepatic ferroptosis plays an important role as the trigger for initiating inflammation in nonalcoholic steatohepatitis. Cell Death Dis. 2019;10:449.

    Article  PubMed  PubMed Central  Google Scholar 

  21. Nevzorova YA, Boyer-Diaz Z, Cubero FJ, Gracia-Sancho J. Animal models for liver disease - A practical approach for translational research. J Hepatol. 2020;73:423–40.

    Article  PubMed  Google Scholar 

  22. Im YR, Hunter H, de Gracia Hahn D, Duret A, Cheah Q, Dong J, et al. A systematic review of animal models of nafld finds high-fat, high-fructose diets most closely resemble human NAFLD. Hepatology. 2021;74:1884–901.

    Article  CAS  PubMed  Google Scholar 

  23. Lee S, Karki R, Wang Y, Nguyen LN, Kalathur RC, Kanneganti TD. AIM2 forms a complex with pyrin and ZBP1 to drive PANoptosis and host defence. Nature. 2021;597:415–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Lei G, Zhang Y, Koppula P, Liu X, Zhang J, Lin SH, et al. The role of ferroptosis in ionizing radiation-induced cell death and tumor suppression. Cell Res. 2020;30:146–62.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Rao SS, Portbury SD, Lago L, McColl G, Finkelstein DI, Bush AI, et al. The iron chelator deferiprone improves the phenotype in a mouse model of tauopathy. J Alzheimers Dis. 2020;77:753–71.

    Article  CAS  PubMed  Google Scholar 

  26. Li DJ, Tong J, Li YH, Meng HB, Ji QX, Zhang GY, et al. Melatonin safeguards against fatty liver by antagonizing TRAFs-mediated ASK1 deubiquitination and stabilization in a beta-arrestin-1 dependent manner. J Pineal Res. 2019;67:e12611.

    Article  CAS  PubMed  Google Scholar 

  27. Li DJ, Liu J, Hua X, Fu H, Huang F, Fei YB, et al. Nicotinic acetylcholine receptor alpha7 subunit improves energy homeostasis and inhibits inflammation in nonalcoholic fatty liver disease. Metabolism. 2018;79:52–63.

    Article  CAS  PubMed  Google Scholar 

  28. Tong J, Li D, Meng H, Sun D, Lan X, Ni M, et al. Targeting a novel inducible GPX4 alternative isoform to alleviate ferroptosis and treat metabolic-associated fatty liver disease. Acta Pharm Sin B. 2022;12:3650–66.

  29. Chi C, Fu H, Li YH, Zhang GY, Zeng FY, Ji QX, et al. Exerkine fibronectin type-III domain-containing protein 5/irisin-enriched extracellular vesicles delay vascular ageing by increasing SIRT6 stability. Eur Heart J. 2022;ehac431. https://doi.org/10.1093/eurheartj/ehac431.

  30. Li DJ, Sun SJ, Fu JT, Ouyang SX, Zhao QJ, Su L, et al. NAD(+)-boosting therapy alleviates nonalcoholic fatty liver disease via stimulating a novel exerkine Fndc5/irisin. Theranostics. 2021;11:4381–402.

    Article  PubMed  PubMed Central  Google Scholar 

  31. Zilka O, Shah R, Li B, Friedmann Angeli JP, Griesser M, Conrad M, et al. On the mechanism of cytoprotection by Ferrostatin-1 and Liproxstatin-1 and the role of lipid peroxidation in ferroptotic cell death. ACS Cent Sci. 2017;3:232–43.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Ursini F, Maiorino M. Lipid peroxidation and ferroptosis: the role of GSH and GPx4. Free Radic Biol Med. 2020;152:175–85.

    Article  CAS  PubMed  Google Scholar 

  33. Hadian K, Stockwell BR. SnapShot: ferroptosis. Cell. 2020;181:1188–e1.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Wang J, Liu W, Li JC, Li M, Li B, Zhu R. Hepcidin downregulation correlates with disease aggressiveness and immune infiltration in liver cancers. Front Oncol. 2021;11:714756.

    Article  PubMed  PubMed Central  Google Scholar 

  35. Jeong SH, Kim HB, Kim MC, Lee JM, Lee JH, Kim JH, et al. Hippo-mediated suppression of IRS2/AKT signaling prevents hepatic steatosis and liver cancer. J Clin Invest. 2018;128:1010–25.

    Article  PubMed  PubMed Central  Google Scholar 

  36. Luna-Vargas MPA, Chipuk JE. Physiological and pharmacological control of BAK, BAX, and Beyond. Trends Cell Biol. 2016;26:906–17.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Murphy JM, Czabotar PE, Hildebrand JM, Lucet IS, Zhang JG, Alvarez-Diaz S, et al. The pseudokinase MLKL mediates necroptosis via a molecular switch mechanism. Immunity. 2013;39:443–53.

    Article  CAS  PubMed  Google Scholar 

  38. Shi J, Zhao Y, Wang K, Shi X, Wang Y, Huang H, et al. Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death. Nature. 2015;526:660–5.

    Article  CAS  PubMed  Google Scholar 

  39. Xu B, Jiang M, Chu Y, Wang W, Chen D, Li X, et al. Gasdermin D plays a key role as a pyroptosis executor of non-alcoholic steatohepatitis in humans and mice. J Hepatol. 2018;68:773–82.

    Article  CAS  PubMed  Google Scholar 

  40. Samir P, Malireddi RKS, Kanneganti TD. The PANoptosome: a deadly protein complex driving pyroptosis, apoptosis, and necroptosis (PANoptosis). Front Cell Infect Microbiol. 2020;10:238.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Fritsch M, Gunther SD, Schwarzer R, Albert MC, Schorn F, Werthenbach JP, et al. Caspase-8 is the molecular switch for apoptosis, necroptosis and pyroptosis. Nature. 2019;575:683–7.

    Article  CAS  PubMed  Google Scholar 

  42. Zheng M, Karki R, Vogel P, Kanneganti TD. Caspase-6 is a key regulator of innate immunity, inflammasome activation, and host defense. Cell. 2020;181:674–87. e13.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Li X, Zhang M, Huang X, Liang W, Li G, Lu X, et al. Ubiquitination of RIPK1 regulates its activation mediated by TNFR1 and TLRs signaling in distinct manners. Nat Commun. 2020;11:6364.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Jiang X, Stockwell BR, Conrad M. Ferroptosis: mechanisms, biology and role in disease. Nat Rev Mol Cell Biol. 2021;22:266–82.

    Article  PubMed  PubMed Central  Google Scholar 

  45. Zilka O, Poon JF, Pratt DA. Radical-trapping antioxidant activity of copper and nickel bis(thiosemicarbazone) complexes underlies their potency as inhibitors of ferroptotic cell death. J Am Chem Soc. 2021;143:19043–57.

    Article  CAS  PubMed  Google Scholar 

  46. Shah R, Margison K, Pratt DA. The potency of diarylamine radical-trapping antioxidants as inhibitors of ferroptosis underscores the role of autoxidation in the mechanism of cell death. ACS Chem Biol. 2017;12:2538–45.

    Article  CAS  PubMed  Google Scholar 

  47. Krainz T, Gaschler MM, Lim C, Sacher JR, Stockwell BR, Wipf P. A mitochondrial-targeted nitroxide is a potent inhibitor of ferroptosis. ACS Cent Sci. 2016;2:653–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Tonnus W, Meyer C, Steinebach C, Belavgeni A, von Massenhausen A, Gonzalez NZ, et al. Dysfunction of the key ferroptosis-surveilling systems hypersensitizes mice to tubular necrosis during acute kidney injury. Nat Commun. 2021;12:4402.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Tarangelo A, Magtanong L, Bieging-Rolett KT, Li Y, Ye J, Attardi LD, et al. p53 suppresses metabolic stress-induced ferroptosis in cancer cells. Cell Rep. 2018;22:569–75.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Xue H, Chen D, Zhong YK, Zhou ZD, Fang SX, Li MY, et al. Deferoxamine ameliorates hepatosteatosis via several mechanisms in ob/ob mice. Ann N Y Acad Sci. 2016;1375:52–65.

    Article  CAS  PubMed  Google Scholar 

  51. Anderson N, Borlak J. Molecular mechanisms and therapeutic targets in steatosis and steatohepatitis. Pharmacol Rev. 2008;60:311–57.

    Article  CAS  PubMed  Google Scholar 

  52. Fontana L, Zhao E, Amir M, Dong H, Tanaka K, Czaja MJ. Aging promotes the development of diet-induced murine steatohepatitis but not steatosis. Hepatology. 2013;57:995–1004.

    Article  CAS  PubMed  Google Scholar 

  53. Jadhav S, Protchenko O, Li F, Baratz E, Shakoury-Elizeh M, Maschek A, et al. Mitochondrial dysfunction in mouse livers depleted of iron chaperone PCBP1. Free Radic Biol Med. 2021;175:18–27.

    Article  CAS  PubMed  Google Scholar 

  54. Zhu Z, Zhang Y, Huang X, Can L, Zhao X, Wang Y, et al. Thymosin beta 4 alleviates non-alcoholic fatty liver by inhibiting ferroptosis via up-regulation of GPX4. Eur J Pharmacol. 2021;908:174351.

    Article  CAS  PubMed  Google Scholar 

  55. Wang X, Ma B, Wen X, You H, Sheng C, Bu L, et al. Bone morphogenetic protein 4 alleviates nonalcoholic steatohepatitis by inhibiting hepatic ferroptosis. Cell Death Discov. 2022;8:234.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Funding

This work was supported by the grants from National Natural Science Foundation (82073915, 82274030, 81971306, and 81773719), National Key Research and Development Program Stem Cell and Translational Research Key Projects (2018YFA0108301), Shanghai Talent Development Fund (2020091), Shanghai Science and Technology Commission Grants (21XD1424900, 2020-JMRH1-KJ12, 21S11901200 and 19140904700), Shanghai Shuguang Program (19SG32) and Shanghai Rising Stars of Medical Talent Development Program-Clinical Pharmacist Project [SHWRS(2020)_087].

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Contributions

JT: Writing-original draft, Methodology, Investigation. XTL: Methodology, Formal analysis, Investigation. ZZ: Formal analysis and Investigation. YL: Formal analysis, Investigation, Writing – review & editing. DYS: Formal analysis and Writing – review & editing. XJW: Investigation and Writing – review & editing. SXOY: Investigation and Writing – review & editing. CLZ: Resources (Synthesis of LPT1). FMS: Formal analysis, Resources and Writing – review & editing. PW: Conceptualization, Writing – original draft, Supervision, Funding acquisition, Writing-review & editing. DJL: Conceptualization, Writing – original draft, Supervision, Funding acquisition, Writing – review & editing.

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Correspondence to Pei Wang or Dong-jie Li.

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Tong, J., Lan, Xt., Zhang, Z. et al. Ferroptosis inhibitor liproxstatin-1 alleviates metabolic dysfunction-associated fatty liver disease in mice: potential involvement of PANoptosis. Acta Pharmacol Sin (2022). https://doi.org/10.1038/s41401-022-01010-5

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  • DOI: https://doi.org/10.1038/s41401-022-01010-5

Keywords

  • ferroptosis
  • PANoptosis
  • MAFLD
  • liver disease

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