Fibroblast growth factor 21 attenuates iron overload-induced liver injury and fibrosis by inhibiting ferroptosis

Abstract

Ferroptosis plays a role in several diseases such as iron overload-induced liver diseases. Manipulation of ferroptosis has been explored as a potential therapeutic strategy to treat related diseases. Numerous antioxidants have been identified to control ferroptosis but the cell-autonomous mechanisms responsible for regulating ferroptosis remain elusive. In the present study, we found that iron overload promoted ferroptosis in hepatocytes by excessively inducing HO-1 expression, which contributed to the progression of liver injury and fibrosis, accompanied by the upregulation of the FGF21 protein level in vitro and in vivo. Interestingly, both recombinant FGF21 and Fgf21 overexpression significantly protected against iron overload-induced hepatocytes mitochondria damage, liver injury and fibrosis by inhibiting ferroptosis. In contrast, the loss of FGF21 aggravated iron overload-induced ferroptosis. Notably, FGF21-induced HO-1 inhibition (via the promotion of HO-1 ubiquitination and degradation) and NRF2 activation provide a mechanistic explanation for this phenomenon. Taken together, we identified FGF21 as a novel ferroptosis suppressor. Thus, FGF21 activation may provide an effective strategy for the potential treatment of iron overload-induced ferroptosis-related diseases, such as hereditary haemochromatosis (HH).

Keywords: Ferroptosis; Fibroblast growth factor 21 (FGF21); Lipid peroxidation; Oxidative stress; Ubiquitination.

Fig. 1

Iron overload induces liver damage and fibrosis in mice. 12-week-old C57BL/6J male mice were intraperitoneally injected with Iron dextran (0.1 mg/g body weight per day) or equal volume of PBS for two weeks, serum and liver were collected for analyzing the following indexes. (A) The average body weight change of the control and iron dextran injected mice (n = 8 per group). (B) Liver nonheme iron concentrations. (C) Western blot analysis of iron metabolism relation proteins (TFR1, Fpn and FTH/L) in control and iron dextran injected mice. (D and E) Serum ALT and AST levels in control and iron dextran injected mice (n = 8). (F) The mRNA of hepatic pro-fibrogenic genes (Co11a1, Co13a1, Timp1 and ɑ-SMA) in control and iron-dextran injected mice (n = 8). (G to I) Liver OH^.-, MDA and GSH content in control and iron dextran injected mice (n = 8). (J) Relative Ptgs2 (ferroptosis marker gene) mRNA expression in control and iron dextran injected mice liver (n = 8). (K) Expression of ferroptosis related proteins in control and iron dextran injected mice liver (n = 4).

Fig. 2

Iron overload robustly enhances FGF21 expression in primary hepatocytes and in the liver of mice. Primary hepatocytes were treated with 100 or 1000 μM FAC for 24 h (A to C) Fgf21 mRNA, supernatant FGF21 concentration and FGF21 protein levels were detected in primary hepatocytes following treatment (n = 4 per group). (D) FGF21 protein levels were detected in the liver of FAC and iron dextran injected mice (n = 8). (E) qRT-PCR analysis of iron metabolism and ferroptosis related gene expression in FAC (1000 μM) and/or FGF21 (200 ng/ml) recombinant protein treated primary hepatocytes (n = 4). (F to J) The LIP, ROS, MDA, GSH and CD11-BODIPY levels were detected in FAC (1000 μM) and/or FGF21 (200 ng/ml) recombinant protein treated primary hepatocytes (n = 4). (K) Transmission electron microscopy of primary hepatocytes treated with FAC (1000 μM) and/or FGF21 (200 ng/ml) recombinant protein for 24 h. Single white arrowheads: shrunken mitochondria.

Fig. 3

FGF21 knockdown induces oxidative stress and worsen iron overload induced ferroptosis. Fgf21 was knocked down in primary hepatocytes using Fgf21 siRNA, followed by treatment with or without FAC (1000 μM) for 24 h. (A) qRT-PCR analysis of Fgf21 mRNA expression in primary hepatocytes treated with control siRNA or Fgf21 siRNA. (B to E) Cell viability, LIP, ROS and CD11-BODIPY levels in the cells (n = 4). (F) Iron metabolism and ferroptosis related proteins levels in the cells (n = 3). (G–I) Cell viability, C11-BODIPY levels, and iron metabolism and ferroptosis related proteins expression in above primary hepatocytes treated with vehicle (mock) or 1000uM FAC.

Fig. 4

Exogenous FGF21 rescues FGF21 knockdown-mediated ferroptosis. Fgf21 was knocked down in the primary hepatocytes, followed by mock treatment or treatment with 200 ng/ml recombinant FGF21 for 24 h (A to E) Levels of LIP, ROS, GSH, MDA and C11-BODIPY in the cells (n = 4). (F) Iron metabolism and ferroptosis related proteins expression in the cells (n = 3).

Fig. 5

FGF21 overexpression protects against iron overload-induced liver damage and fibrosis in mice. C57/BL6 male mice were intravenously injected with AdEGFP or AdFGF21-EGFP. Two days later, mice were IP injected with PBS or iron dextran (0.1 mg/g body weight per day) daily for 7 days. Mice were sacrificed on day 7, and serum and liver were harvested. (A) Fgf21 mRNA levels in the liver (n = 10). (B) The body weight graph (n = 10). (C) Hepatic iron concentrations (n = 10). (D and E) Serum ALT and AST levels (n = 10). (F) The mRNA of hepatic pro-fibrogenic genes ɑ-SMA (n = 10). (G and H) Hepatic OH^.- and MDA levels. (I) Iron metabolism and ferroptosis related protein expression in the liver.

Fig. 6

HO-1 activation contributes to iron overload-induced ferroptosis. Primary hepatocytes were transfected with HO-1 plasmid or Ho-1 siRNA for 36 h. (A) Western blot analysis of HO-1 expression in HO-1 over-expression primary hepatocytes. (B to E) Cell viability, LIP, ROS, and CD11-BODIPY in HO-1 over expression primary hepatocytes vs control cells (n = 4). (F) Western blot analysis of HO-1 expression in HO-1 knock down primary hepatocytes. (G to J) Cell viability, LIP, ROS, and C11-BODIPY in Ho-1 knock down primary hepatocytes (n = 4).

Fig. 7

FGF21 attenuates iron overload-induced ferroptosis through promoting HO-1 ubiquitination. (A) Ho-1 mRNA expression levels in the liver of FGF21 over-expression mice (n = 10). (B) Ho-1 mRNA expression levels in primary hepatocytes treated with recombinant FGF21 protein (n = 4). (C) HO-1 protein expression in primary hepatocytes treated with PBS (left) or 200 ng/ml recombinant FGF21 protein (right) for 4 h, followed by 20 μg/ml CHX for 3 h. (D) HEK293T cells were transfected with Flag-tagged ubiquitin cDNA (Ub-Flag) alone or together with HA-tagged Ho-1 cDNA (HO-1-HA) alone or together with FGF21 cDNA or vector plasmid for 36 h. Cell lysates were subjected to immunoprecipitation with anti-HA antibody, and the tag of flag was detected by western blot. (E) Model of proposed mechanism of FGF21 attenuated liver injury and fibrosis induced by iron overload. Iron overload induces hepatocyte ferroptosis, which contributes to liver injury and fibrosis in WT mice (Left panel). Mechanistically, iron overload robustly increases HO-1 expression, which catalyzes the conversion of heme into Fe²⁺, carbon monoxide and biliverdin. Notably, excess Fe²⁺ accumulating in the cells induces ROS generation which could be the direct reason for ferroptosis. In addition, iron overload inhibits the expression of NRF2, which is a master regulator of the antioxidant response. Of particular interest, FGF21 over expression (OE) attenuates liver injury and fibrosis induced by iron overload via suppressing hepatocytes ferroptosis (right panel). FGF21 not only enhances HO-1 ubiquitination and degradation to reduce Fe²⁺ generation in hepatocytes but also activates NRF2 expression to suppress iron overload-induced oxidative damage. Therefore, FGF21 is a novel regulator of ferroptosis and activation of FGF21 may provide an effective strategy for potential treatment in iron-mediated ferroptosis-related disease, such as HH.