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Pathological mechanism of ferroptosis in a rat model of α‑naphthyl isothiocyanate‑induced chronic cholestasis

  • Authors:
    • Zhen Guo
    • Jiaxuan Wang
    • Yiwen Wang
    • Xinzhu Liu
    • Yubing Xia
    • Ping Liu
    • Li Qi
    • Jia Liu
    • Xiaoning Wang
  • View Affiliations / Copyright

    Affiliations: Luzhou Key Laboratory of Research and Development of Medical Institution Preparations and Large‑Scale Health Products, The Affiliated Traditional Chinese Medicine Hospital, Southwest Medical University, Luzhou, Sichuan 646000, P.R. China, Institute of Interdisciplinary Science, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, P.R. China
    Copyright: © Guo et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 92
    |
    Published online on: January 14, 2026
       https://doi.org/10.3892/mmr.2026.13802
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Abstract

Ferroptosis is an iron‑dependent form of cell death associated with liver pathologies. However, its role in chronic cholestasis remains to be fully elucidated. The present study therefore investigated the pathological mechanism of ferroptosis in a rat model of α‑naphthyl isothiocyanate (ANIT)‑induced chronic cholestasis and evaluated the therapeutic potential of the iron chelator deferoxamine (DFO). Wistar rats were used to establish a chronic cholestasis model via ANIT administration, with a subset of animals receiving DFO treatment. Wistar rats that were subjected to chronic ANIT exposure were found to develop severe liver injury, characterized by impaired function, inflammation and fibrosis. In addition, pronounced iron deposition and hallmark features of ferroptosis, including elevated lipid peroxidation, depleted glutathione, and aberrant expression of acyl‑CoA synthetase long‑chain family member 4 and cyclooxygenase 2, were observed. Ultrastructural analysis revealed distinctive mitochondrial abnormalities consistent with ferroptosis. Mechanistically, these changes appeared to be mediated by suppression of the Kelch‑like ECH‑associated protein 1/nuclear factor erythroid 2‑related factor 2/heme oxygenase 1 antioxidant pathway and dysregulation of key iron metabolism proteins, including transferrin receptor 1 and ferroportin 1. Intervention with DFO markedly ameliorated the cholestatic injury, reduced iron overload and lipid peroxidation, mitigated mitochondrial damage, and normalized the expression of key proteins involved in ferroptosis, antioxidant defense and iron homeostasis. Taken together, these findings suggested that ferroptosis may be a key pathological mechanism in chronic cholestasis, driven by the concurrent disruption of antioxidant and iron metabolic capacities in hepatocytes. Therefore, targeting iron overload may be a promising therapeutic strategy for cholestasis.

View Figures

Figure 1

Schematic diagram of the experiment.
ANIT, α-naphthyl isothiocyanate; DFO, deferoxamine.

Figure 2

Liver function of rats with chronic
cholestasis is impaired and histopathological analysis of liver
tissue reveals inflammation. The control group was fed a chow diet,
and the ANIT and DFO-treated groups were intragastrically
administered ANIT olive oil solution with or without DFO
(n=8/group). (A) TBA, (B) ALP, (C) TBIL, (D) DBIL, (E) GGT, (F)
LDL, (G) TCH, (H) TG, (I) ALT and (J) AST were measured.
**P<0.01, ***P<0.001. (K) Hematoxylin and eosin staining
(scale bar, 200 µm). Arrows indicate bile duct hyperplasia and
inflammatory cell infiltration in the portal area. ANIT, α-naphthyl
isothiocyanate; DFO, deferoxamine; TBA, total bile acid; ALP,
alkaline phosphatase; TBIL, total bilirubin; DBIL, direct
bilirubin; GGT, γ-glutamyl transferase; LDL, low-density
lipoprotein; TCH, total cholesterol; TG, triglyceride; ALT, alanine
aminotransferase; AST, aspartate aminotransferase.

Figure 3

Intrahepatic collagen fiber
deposition is increased in rats with chronic cholestasis. Control
group was fed a chow diet, whereas ANIT and DFO-treated groups were
intragastrically administered ANIT olive oil solution with or
without DFO (n=8/group). (A) Sirius red staining (scale bar, 200 µ
m) and (B) collagen-positive area in the liver. (C) HYP levels in
the liver. (D) Changes in α-SMA protein expression. The blots shown
are representative of three independent experiments. The dotted
line indicates that the lanes were non-adjacent on the original
gel. (E) Relative expression levels of α-SMA. Results Data are
presented as the mean ± SD. P-values were determined by one-way
ANOVA. *P<0.05, ***P<0.001. ANIT, α-naphthyl isothiocyanate;
DFO, deferoxamine; HYP, hydroxyproline; α-SMA, α-smooth muscle
actin.

Figure 4

Free iron ions are aberrantly
increased in serum and liver samples from rats with chronic
cholestasis. Control group was fed a chow diet, whereas ANIT and
DFO-treated groups were intragastrically administered ANIT olive
oil solution with or without DFO (n=8/group). (A) Serum iron, (B)
liver iron and (C) liver Fe2+ levels were detected. (D)
Liver Prussian blue Fe3+ positive area. Data are
presented as the mean ± SD. P-values were determined by one-way
ANOVA. *P<0.05, **P<0.01, ***P<0.001. (E) Prussian blue
staining (scale bar, 200 µ m). ANIT, α-naphthyl isothiocyanate;
DFO, deferoxamine.

Figure 5

Levels of lipid peroxidation and
ferroptosis markers are increased in the liver of rats with chronic
cholestasis and the liver ultrastructure is affected in a
DFO-reversible manner. Control group was fed a chow diet, whereas
ANIT and DFO-treated groups were intragastrically administered ANIT
olive oil solution with or without DFO (n=8/group). Levels of (A)
MDA, (B) GSH and (C) GSH-PX were detected. (D) Changes in
ferroptosis marker protein expression. The blots shown are
representative of three independent experiments. A dotted line
indicates that the lanes were non-adjacent on the original gel. All
target protein bands and their corresponding loading control bands
shown side-by-side were derived from the same membrane. Relative
expression levels of (E) ASCL4 and (F) COX2. Data are presented as
the mean ± SD. P-values were determined by one-way ANOVA.
*P<0.05, **P<0.01, ***P<0.001. (G) Transmission electron
microscope sections, blue arrows indicate mitochondrial cristae,
yellow arrows indicate outer mitochondrial membrane and green
arrows point to mitochondria (scale bars, 5 and 2 µ m). DFO,
deferoxamine; ANIT, α-naphthyl isothiocyanate; MDA,
malondialdehyde; GSH, glutathione; GSH-PX, glutathione peroxidase;
ASCL4, acyl-CoA synthetase long-chain family member 4; COX2,
cyclooxygenase 2.

Figure 6

Abnormal expression of antioxidant
and iron metabolism-related proteins in liver samples of rats with
chronic cholestasis. Control group was fed a chow diet, whereas the
ANIT and DFO-treated groups were intragastrically administered ANIT
olive oil solution with or without DFO (n=8/group). (A) Changes in
ferroptosis antioxidant-related protein expression. The blots shown
are representative of three independent experiments. A dotted line
indicates that the lanes were non-adjacent on the original gel. All
target protein bands and their corresponding loading control bands
shown side-by-side were derived from the same membrane. Relative
expression levels of (B) Nrf2, (C) Keap1, (D) XCT, (E) HO-1 and (F)
GPX4. (G) Changes in iron metabolism-related protein expression.
The blots shown are representative of three independent
experiments. A dotted line indicates that the lanes were
non-adjacent on the original gel. All target protein bands and
their corresponding loading control bands shown side-by-side were
derived from the same membrane. Relative expression levels of (H)
TFR1, (I) FPN1, (J) DMT1, (K) Steap3 and (L) FTH1. Data are
presented as the mean ± SD. P-values were determined by one-way
ANOVA. *P<0.05, **P<0.01, ***P<0.001. ANIT, α-naphthyl
isothiocyanate; DFO, deferoxamine; Nrf2, nuclear factor
erythroid-2-related factor 2; Keap1, Kelch-like ECH-associated
protein 1, XCT, cystine/glutamate transporter; HO-1, heme oxygenase
1; GPX4, glutathione peroxidase 4; TFR1, transferrin receptor 1;
FPN1, ferroportin 1; DMT1, divalent metal transporter 1; Steap3,
six-transmembrane epithelial antigen of the prostate 3; FTH1,
ferritin heavy chain 1.

Figure 7

Diagram of the mechanism of
ferroptosis. ASCL4, acyl-CoA synthetase long-chain family member 4;
COX2, cyclooxygenase 2; DMT1, divalent metal transporter 1; FPN,
ferroportin; GSSG, oxidized glutathione; GPX4, glutathione
peroxidase 4; GSH, glutathione; HO-1, heme oxygenase 1; Keap1,
Kelch-like ECH-associated protein 1; LIP, labile iron pool; LPCAT3,
lysophosphatidylcholine acyltransferase 3; NCOA4, nuclear receptor
coactivator 4; NOX1, nicotinamide adenine dinucleotide phosphate
oxidase 1; NQO1, NAD(P)H quinone dehydrogenase 1; Nrf2, nuclear
factor erythroid-2-related factor 2; PL-PUFA-OOH,
phospholipid-polyunsaturated fatty acid hydroperoxide; SLC7A11,
solute carrier family 7 member 11; Steap3, six-transmembrane
epithelial antigen of the prostate 3; TFR1, transferrin receptor
1.
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Copy and paste a formatted citation
Spandidos Publications style
Guo Z, Wang J, Wang Y, Liu X, Xia Y, Liu P, Qi L, Liu J and Wang X: <p>Pathological mechanism of ferroptosis in a rat model of &alpha;‑naphthyl isothiocyanate‑induced chronic cholestasis</p>. Mol Med Rep 33: 92, 2026.
APA
Guo, Z., Wang, J., Wang, Y., Liu, X., Xia, Y., Liu, P. ... Wang, X. (2026). <p>Pathological mechanism of ferroptosis in a rat model of &alpha;‑naphthyl isothiocyanate‑induced chronic cholestasis</p>. Molecular Medicine Reports, 33, 92. https://doi.org/10.3892/mmr.2026.13802
MLA
Guo, Z., Wang, J., Wang, Y., Liu, X., Xia, Y., Liu, P., Qi, L., Liu, J., Wang, X."<p>Pathological mechanism of ferroptosis in a rat model of &alpha;‑naphthyl isothiocyanate‑induced chronic cholestasis</p>". Molecular Medicine Reports 33.3 (2026): 92.
Chicago
Guo, Z., Wang, J., Wang, Y., Liu, X., Xia, Y., Liu, P., Qi, L., Liu, J., Wang, X."<p>Pathological mechanism of ferroptosis in a rat model of &alpha;‑naphthyl isothiocyanate‑induced chronic cholestasis</p>". Molecular Medicine Reports 33, no. 3 (2026): 92. https://doi.org/10.3892/mmr.2026.13802
Copy and paste a formatted citation
x
Spandidos Publications style
Guo Z, Wang J, Wang Y, Liu X, Xia Y, Liu P, Qi L, Liu J and Wang X: <p>Pathological mechanism of ferroptosis in a rat model of &alpha;‑naphthyl isothiocyanate‑induced chronic cholestasis</p>. Mol Med Rep 33: 92, 2026.
APA
Guo, Z., Wang, J., Wang, Y., Liu, X., Xia, Y., Liu, P. ... Wang, X. (2026). <p>Pathological mechanism of ferroptosis in a rat model of &alpha;‑naphthyl isothiocyanate‑induced chronic cholestasis</p>. Molecular Medicine Reports, 33, 92. https://doi.org/10.3892/mmr.2026.13802
MLA
Guo, Z., Wang, J., Wang, Y., Liu, X., Xia, Y., Liu, P., Qi, L., Liu, J., Wang, X."<p>Pathological mechanism of ferroptosis in a rat model of &alpha;‑naphthyl isothiocyanate‑induced chronic cholestasis</p>". Molecular Medicine Reports 33.3 (2026): 92.
Chicago
Guo, Z., Wang, J., Wang, Y., Liu, X., Xia, Y., Liu, P., Qi, L., Liu, J., Wang, X."<p>Pathological mechanism of ferroptosis in a rat model of &alpha;‑naphthyl isothiocyanate‑induced chronic cholestasis</p>". Molecular Medicine Reports 33, no. 3 (2026): 92. https://doi.org/10.3892/mmr.2026.13802
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