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.

Keywords: MAFLD; PANoptosis; ferroptosis; liver disease.

Fig. 1. The inhibitor LPT1 blocks ferroptosis without disrupting iron accumulation in a mouse MAFLD model.

a Schematic representation of experimental design illustrating the treatment protocol with liproxstatin-1 (LPT1) in a high-fat high-fructose diet (HFHF)-induced MAFLD mouse model. C57BL/6 J mice were divided into four groups: Chow, Chow+LPT1, HFHF and HFHF + LPT1 groups. MAFLD was induced by HFHF diet for 16 weeks. LPT1 was given intraperitoneally (10 mg·kg⁻¹·d⁻¹) for two weeks. b Effect of LPT1 treatment on body weight and liver weight. c, d Fe²⁺ levels and GSH levels in liver tissues of four groups of mice. e, f Representative immunoblot and quantitative analysis of GPX4 protein level in liver tissues of four groups of mice. GAPDH as loading control. n = 4 biologically independent experiments. g–i Representative immunoblot and quantitative analysis of ACSL4 and ALOX15 protein levels in liver tissues of four groups of mice. GAPDH as loading control. n = 4 biologically independent experiments. j The mRNA level of Hamp in liver tissues of four groups of mice. n = 6 biologically independent experiments. k Immunoblotting analysis of 4-hydroxynonenal (4-HNE)-adducted protein levels in liver tissues of four groups of mice. n = 6 biologically independent experiments. l Malondialdehyde (MDA) levels in liver tissues of four groups of mice. n  = 6 biologically independent experiments. The data were presented as Means ± SEM and analyzed by One way-ANOVA followed by Tukey’s post hoc test. *P < 0.05, **P < 0.01 vs Chow; ^#P < 0.05, ^##P < 0.01 vs HFHF. NS no significance.

Fig. 2. The ferroptosis inhibitor LPT1 decreases steatosis and mitochondrial ROS in a mouse MAFLD model.

a Oil red O staining showing the lipid droplets (red) in liver tissues of four groups of mice. b Liver triglycerides levels in liver tissues of four groups of mice. n = 6 biologically independent experiments. c Liver cholesterol levels in liver tissues of four groups of mice. n = 6 biologically independent experiments. d–g Representative immunoblot analysis of p-Akt, Akt, p-IRS1, IRS-1 and PPARα in liver tissues from four groups of mice. GAPDH was used as loading control. h–l The mRNA levels of lipid oxidation gene Pparα and lipid synthesis genes including Scd1, Fasn, Hmgcr and Cpt1a in liver tissues of four groups of mice. n = 4–6 biologically independent experiments. m Mitochondrial ROS content measured by MitoSox Dye (red) in liver tissues of four groups of mice. Hoechst 33342 (blue) was used to label nuclei. n = 9 biologically independent experiments. The data were presented as Means ± SEM and analyzed by One way-ANOVA followed by Tukey’s post hoc test. *P < 0.05, **P < 0.01 vs Chow; ^#P < 0.05, ^##P < 0.01 vs HFHF. NS no significance.

Fig. 3. The ferroptosis inhibitor LPT1 mitigates liver injury and fibrosis in a mouse MAFLD models.

a, b Serum ALT and AST levels in four groups of mice. c–f The mRNA levels of pro-fibrotic genes α-SMA, Tgf-β, Timp-1 and Col1α1 in liver tissues from four groups of mice. n  = 4 biologically independent experiments. g Immunohistochemistry staining of α-SMA and quantitative analysis in liver tissues from four groups of mice. Scale bars, 100 μm. h Masson’s trichrome staining showing the collagen fibers (blue) in liver tissues from four groups of mice. Scale bars, 100 μm. i, j Serum ALT and AST levels in four groups of mice fed with MCD for 4 weeks. LPT1 was given intraperitoneally (10 mg·kg⁻¹·d⁻¹) for two weeks. n = 6 per group. k MAFLD activity score calculated based on H&E staining in liver from four groups mice fed with MCD or normal chow diet. Scale bars, 100 μm. n = 6 per group. l Masson’s trichrome staining showing the liver fibrosis from four groups of mice fed with MCD or normal chow diet. Scale bars, 100 μm. n = 6 per group. The data were presented as Means ± SEM and analyzed by One way-ANOVA followed by Tukey’s post hoc test. **P < 0.01 vs Chow; ^#P < 0.05. NS no significance.

Fig. 4. The ferroptosis inhibitor LPT1 suppresses steatohepatitis and ‘PANoptosis’ in a mouse MAFLD models.

a, b The mRNA level of pro-inflammatory factor TNF-α and IL-1β in liver tissues from four groups of mice. n = 6 biologically independent experiments. c TUNEL staining showing apoptotic cells in liver tissues from four groups of mice. The arrows show TUNEL positive cells (brown). Scale bars, 100 μm. d–f Representative immunoblot images and quantitative analysis on phosphorylated MLKL (p-MLKL), total MLKL, Bax (a pro-apoptotic protein) and Bcl-xL (an anti-apoptotic protein) protein levels in liver tissues from four groups of mice. GAPDH as loading control. g The enzymatic activity of pyroptosis executor Caspase-1 in liver tissues from four groups of mice. n = 5 biologically independent experiments. h The mRNA level of pyroptosis executor Gsdmd in liver tissues from four groups of mice. n = 4 biologically independent experiments. i–k Representative immunoblot images and quantitative analysis on the cleavage of Caspase-1 and GSDMD in liver tissues from four groups of mice. GAPDH as loading control. l–n The mRNA level of IL-6, IL-1β and TNF-α in liver tissues from four groups of mice fed with MCD or normal chow diet for 4 weeks. n = 6 biologically independent experiments. The data were presented as Means ± SEM and analyzed by One way-ANOVA followed by Tukey’s post hoc test. *P < 0.05, **P < 0.01 vs Chow; ^#P < 0.05, ^##P < 0.01. NS no significance.

Fig. 5. The ferroptosis inhibitor LPT1 inhibits cleavage of PANoptosis-related Caspase-6 and Caspase-8.

a Representative immunoblot analysis of Caspase-6 and Caspase-8 levels in liver tissues from four groups of mice. GAPDH as loading control. b Immunohistochemistry staining showing the effects of LPT1 treatment on cleaved Caspase-6 (p18 subunit) in MAFLD mice liver tissues. Scale bars, 100 μm. n = 18 biologically independent experiments. c Immunohistochemistry staining showing the effects of LPT1 treatment on cleaved Caspase-8 (p18 subunit) in MAFLD mice liver tissues. Scale bars, 100 μm. n = 18 biologically independent experiments. The data were presented as Means ± SEM and analyzed by One way-ANOVA followed by Tukey’s post hoc test. **P < 0.01 vs Chow; ^#P < 0.05 vs HFHF. NS no significance.

Fig. 6. The ferroptosis inhibitor LPT1 directly blocks PANoptosis in hepatocytes upon lipid stress.

a The effects of ferroptosis inhibitor LPT1 on cell viability and LDH release in an in vitro MAFLD model. AML12 cells were cultured in DME/F-12 medium and treated with PANoptosis-inducing molecules (TLN), a combination of TNF-α (100 mg/mL), LPS (50 ng/mL) and Nigericin (20 mmol/L), in the presence of PA stimuli (300 nmol/L). The LPT1 was added into the PA stimuli culture medium to reach the final concentration at 100 nmol/L. Cell viability and LDH release were measured using commercial kits. b The apoptosis and necroptosis induced by PANoptosis-inducing molecules TLN were measured by flow cytometry analysis with YO-PRO-1/PI staining. c The mRNA levels of Scd1 and Fasn in AML12 cells treated by PANoptosis-inducing molecules TLN in the presence of PA. d Oil red O staining showing the lipid content in AML12 cells treated by PANoptosis-inducing molecules TLN in the presence of PA. The data were presented as Means ± SEM and analyzed by One way-ANOVA followed by Tukey’s post hoc test. n = 6 per group. *P < 0.05, **P < 0.01 vs Control; ^#P < 0.05, ^##P < 0.01. NS no significance.

Fig. 7. An iron chelator DFP reduces ferroptosis but does not ameliorate steatosis.

a Schematic representation of experimental design illustrating the treatment protocol with deferiprone (DFP) in a HFHF-induced MAFLD mouse model. C57BL/6 J mice were divided into three groups: Chow, HFHF and HFHF + DFP groups. MAFLD was induced by HFHF diet for 16 weeks. Mice in HFHF + DFP group were given DFP by oral gavage (100 mg·kg⁻¹·d⁻¹) for two weeks. b The body and liver weight in three groups of mice. c The Fe²⁺ levels in liver tissues in three groups of mice. d The mRNA level of Hamp in liver tissues in three groups of mice. n = 6 biologically independent experiments. e Immunohistochemistry staining and quantitative analyses of ferroptosis markers (ACSL4, ALOX15, Ferritin, Transferrin and GPX4) in liver tissues in three groups of mice. f The GSH levels in liver tissues in three groups of mice. g Oil red O staining showing the lipid droplets (red) in liver tissues in three groups of mice. h The mRNA levels of Pparα, Fasn, Hmgcr and Cpt1a in liver tissues in three groups of mice. n  = 4 biologically independent experiments. i Liver triglycerides and cholesterol contents in three groups of mice. j Representative immunoblot analysis of p-Akt, Akt, p-IRS1 and IRS-1 in liver tissues from three groups of mice. GAPDH was used as a loading control. The data were presented as Means ± SEM and analyzed by One way-ANOVA followed by Tukey’s post hoc test. *P < 0.05, **P < 0.01 vs Chow; ^#P < 0.05, ^##P < 0.01 vs HFHF. NS no significance.

Fig. 8. An iron chelator DFP alleviates steatohepatitis.

a Mitochondrial ROS level was determined by flow cytometry with MitoSOX dye. b The MDA levels in liver tissues in three groups of mice. c Representative immunoblot images and quantitative analysis of 4-HNE protein level in liver tissues in three groups of mice. GAPDH as loading control. n = 3 biologically independent experiments. d Immunohistochemistry staining of TNF-α in liver tissues in three groups of mice. Scale bars, 100 μm. n = 8 biologically independent experiments. e Immunohistochemistry staining of α-SMA, Masson’s trichrome staining and Sirius Red staining (red under light microscopy [LM]; orange/green under polarized microscopy [PM]) in liver tissues in three groups of mice. Scale bars, 100 μm. f ELISA of IL-6, IL-1β and IL-18 levels in liver tissues of three groups of mice. The data were presented as Means ± SEM and analyzed by One way-ANOVA followed by Tukey’s post hoc. *P < 0.05, **P < 0.01 vs Chow; ^#P < 0.05, ^##P < 0.01 vs HFHF. NS no significance.

Fig. 9. An iron chelator DFP does not block PANoptosis.

a Representative immunoblot images and quantitative analyses of p-MLKL, MLKL, GSDMD and Caspase-6 in liver tissues in three groups of mice. GAPDH as loading control. n = 3 biologically independent experiments. b Serum ALT and AST levels in three groups of mice which were induced by HFHF diet for 16 weeks. DFP was given by oral gavage (100 mg·kg⁻¹·d⁻¹) for two weeks. c A schematic representation depicting the different impacts of ferroptosis inhibitor (radical trapping antioxidant) and iron chelator on steatosis and steatohepatitis in MAFLD condition. Ferroptosis inhibitor not only inhibits ferroptosis, but also blocks apoptosis, pyroptosis and necroptosis (PANoptosis). By contrary, iron chelator does not affect PANoptosis process. The data were presented as Means ± SEM and analyzed by One way-ANOVA followed by Tukey’s post hoc. **P < 0.01 vs Chow. NS no significance.