Hepatic transferrin plays a role in systemic iron homeostasis and liver ferroptosis

Abstract

Although the serum-abundant metal-binding protein transferrin (encoded by the Trf gene) is synthesized primarily in the liver, its function in the liver is largely unknown. Here, we generated hepatocyte-specific Trf knockout mice (Trf-LKO), which are viable and fertile but have impaired erythropoiesis and altered iron metabolism. Moreover, feeding Trf-LKO mice a high-iron diet increased their susceptibility to developing ferroptosis-induced liver fibrosis. Importantly, we found that treating Trf-LKO mice with the ferroptosis inhibitor ferrostatin-1 potently rescued liver fibrosis induced by either high dietary iron or carbon tetrachloride (CCl4) injections. In addition, deleting hepatic Slc39a14 expression in Trf-LKO mice significantly reduced hepatic iron accumulation, thereby reducing ferroptosis-mediated liver fibrosis induced by either a high-iron diet or CCl4 injections. Finally, we found that patients with liver cirrhosis have significantly lower levels of serum transferrin and hepatic transferrin, as well as higher levels of hepatic iron and lipid peroxidation, compared with healthy control subjects. Taken together, these data indicate that hepatic transferrin plays a protective role in maintaining liver function, providing a possible therapeutic target for preventing ferroptosis-induced liver fibrosis.

Graphical abstract

Figure 1.

Trf-LKO mice have impaired iron metabolism. (A) Hepatic Trf protein and serum Trf levels were measured in 8-week-old control (Trf^fl/fl) and Trf-LKO (n = 6-7 mice/group) mice. (B) Serum iron levels, serum NTBI levels, and tissue non-heme iron concentrations were measured in 8-week-old control and Trf-LKO mice (n = 8-10 mice/group). (C) Hepatic Tfr1, Ftl, and Fth mRNA levels were measured in control and Trf-LKO mice using RT-PCR (n = 6 mice/group). (D) Western blot analysis of hepatic Tfr1, ferritin-L (Ftl), and ferritin-H (Fth) proteins in control and Trf-LKO mice (n = 3 mice/group). (E) RT-PCR analysis of hepatic Hamp mRNA in control and Trf-LKO mice (n = 6 mice/group). (F) Perls Prussian blue staining for iron and Fpn1 IHC were performed in liver, spleen, and duodenum sections obtained from control and Trf-LKO mice; the scale bars represent 100 μm. *P < .05, **P <. 01, Student t test. n.s., not significant.

Figure 2.

Loss of hepatic Trf exacerbates liver damage induced by an HID. (A) Non-heme iron was measured in the indicated tissues of control and Trf-LKO mice (n = 4 mice/group) after 8 weeks on an HID. (B) Perls Prussian blue staining of liver sections obtained from HID-fed control and Trf-LKO mice; the scale bars represent 100 μm. (C) Serum ALT levels were measured in control and Trf-LKO mice fed either an SID or an HID (n = 4 mice/group). (D) 4-HNE, Sirius Red, and Masson’s trichrome staining were performed on liver sections obtained from HID-fed control and Trf-LKO mice; the scale bars represent 100 μm. Hepatic MDA, thiol, and SOD activity (E) and hepatic Slc7a11 and Gpx4 mRNA (F) were measured in SID-fed and HID-fed control mice and Trf-LKO mice (n = 4 mice/group). *P < .05, **P < .01, Student t test. n.s., not significant.

Figure 3.

Fer-1 treatment rescues iron overload‒induced ferroptosis in Trf-LKO mice. Hepatic non-heme iron (A), serum ALT (B), hepatic MDA (C), and hepatic thiol (D) levels were measured in HID-fed control mice and Trf-LKO mice treated with saline or Fer-1 (n = 3-6 mice/group). (E) Liver sections obtained from HID-fed control and Trf-LKO mice treated with saline or Fer-1 were stained with 4-HNE, Sirius Red, and Masson’s trichrome; the scale bars represent 100 μm. (F) Quantitative analyses for Sirius red and Masson’s trichrome staining in the indicated groups. (G) Hepatic mRNA levels of the fibrotic genes Col1a1 and PDGF were measured in the indicated groups. *P < .05, **P < .01, one-way analysis of variance with Tukey’s post hoc test. n.s., not significant.

Figure 4.

Loss of hepatic Slc39a14 protects against ferroptosis in Trf-LKO mice. (A) Hepatic non-heme iron was measured in control (Trf^fl/fl), Trf-LKO, and Trf/Slc39a14 double-knockout (DKO) mice fed an SID (n = 4 mice/group). (B) Representative images of Perls Prussian blue‒stained liver sections obtained from SID-fed control, Trf-LKO, and DKO mice; the scale bars represent 100 μm. Hepatic non-heme iron (C), thiol (D), and MDA (E) levels were measured in HID-fed control, Trf-LKO, and DKO mice (n = 4 mice/group). (F) Representative images of liver sections obtained from HID-fed control, Trf-LKO, and DKO mice and stained with Perls Prussian blue, 4-HNE, Sirius Red, and Masson’s trichrome; the scale bars represent 100 μm. *P < .05 and **P < .01, one-way analysis of variance with Tukey’s post hoc test.

Figure 5.

Both inhibiting ferroptosis and deleting hepatic Slc39a14 alleviates CCl₄-induced fibrosis in Trf-LKO mice. Control, Trf-LKO, and DKO mice were treated with vehicle (oil) or CCl₄, and liver sections were obtained and stained with Masson’s trichrome (A), Sirius Red (B), or 4-HNE (C); the scale bars represent 100 μm. Hepatic MDA (D) and non-heme iron (E) were measured in the indicated mice (n = 4-5 mice/group). *P < .05, **P < .01, one-way analysis of variance with Tukey’s post hoc test. n.s., not significant.

Figure 6.

Patients with liver cirrhosis have reduced serum Trf levels. (A) Trf was measured in serum samples obtained from 48 patients with cirrhosis and 52 healthy control subjects. (B-E) The indicated indices of liver fibrosis were measured in the serum of 48 patients with liver cirrhosis and are plotted against serum Trf concentration. The data in panel A were analyzed by using a Student t test; the data in panels B-E were analyzed by using Pearson’s correlation coefficient (r).

Figure 7.

Hepatic Trf, iron, MDA, and SLC39A14 levels in liver biopsy samples obtained from patients with liver cirrhosis and in Trf-LKO mice. Liver biopsy samples were obtained from 20 patients with liver cirrhosis and 20 control subjects who underwent resection for hepatocellular carcinoma. (A,B) Representative images of liver sections stained with Masson’s trichrome, Trf IHC, Perls Prussian blue, MDA IHC, and SLC39A14 IHC. (C) Quantitative analyses of Trf, MDA, and SLC39A14 staining of liver sections obtained from control and cirrhotic biopsy samples. (D) Representative images of murine liver sections stained with Slc39a14 obtained from 12-week-old SID-fed and HID-fed control mice and Trf-LKO mice. The scale bars represent 100 μm. (E) Quantitative analyses of hepatic Slc39a14 staining from 12-week-old SID-fed and HID-fed control mice and Trf-LKO mice. *P < .05, **P < .01, Student t test.