Gandou Fumu Decoction improves liver steatosis by inhibiting hepatocyte ferroptosis in mice with Wilson's disease through the GPX4/ACSL4/ALOX15 signaling pathway

doi:10.12122/j.issn.1673-4254.2025.07.13

Abstract

Abstract:

Objective To explore the mechanism of Gandou Fumu Decoction (GDFMD) for improving Wilson's disease (WD) in tx-J mice. Methods With 6 syngeneic wild-type mice as the control group, 30 tx-J mice were randomized into WD model group, low-, medium- and high-dose GDFMD treatment groups, and Fer-1 treatment group. Saline (in control and model groups) and GDFMD (3.48, 6.96 or 13.92 g/kg) were administered by gavage, and Fer-1 was injected intraperitoneally once daily for 14 days. Oil red and HE staining were used to observe lipid deposition and pathological conditions in the liver tissue; ALT, AST, albumin, AKP levels were determined to assess liver function of the mice. Western blotting and RT-qPCR were used to detect hepatic protein and mRNA expressions of GPX4, ACSL4, ALOX15, FTH1, FLT, TFR1, FAS, SCD1, and ACOX1, and Fe²⁺, MDA, ROS, SOD, GSH and 4-HNE levels were analyzed to assess oxidative stress. Results The mouse models of WD showed obvious fatty degeneration in the liver tissue significantly increased serum levels of ALT, AST and AKP, decreased albumin level, increased Fe²⁺, MDA, ROS, 4-HNE levels, decreased SOD and GSH levels (P<0.05), lowered protein expressions of ACOX1, GPX4, FTH1, FLT, FAS, and SCD1, and increased protein contents of TFR1, ACSL4 and ALOX15 in the liver. Treatment with GDFMD and Fer-1 improved liver histopathology and liver function of the mouse models, decreased the levels of Fe²⁺, MDA and ROS, increased SOD and GSH levels, and reversed the changes in hepatic protein expressions. Conclusion GDFMD improves liver steatosis in mouse models of WD possibly by inhibiting hepatocyte ferroptosis through the GPX4/ACSL4/ALOX15 signaling pathway.

Key words: Gandou Fumu Decoction, wilson's diseast, ferroptosis, steatosis of the liver

Mengying ZHANG, Chenling ZHAO, Liwei TIAN, Guofang YU, Wenming YANG, Ting DONG. Gandou Fumu Decoction improves liver steatosis by inhibiting hepatocyte ferroptosis in mice with Wilson's disease through the GPX4/ACSL4/ALOX15 signaling pathway[J]. Journal of Southern Medical University, 2025, 45(7): 1471-1478.

Figures/Tables 8

Tab.1 Primer sequences for RT-qPCR

Gene	Sequence (5'to3')
GPX4	CGCCAAAGTCCTAGGAAACG
GPX4	ATGCACACGAAACCCCTGTA
ACSL4	GACAGGCCAGTGTGAACGTA
ACSL4	TCAGCCCATATCCCTGACCA
ALOX15	CGGCGACCAGTATCTCTGAC
ALOX15	TTCCAGGAGTTTCGAACCCG
FTH1	GGAGCATGCCGAGAAACTGA
FTH1	GTCATCACGGTCTGGTTTCTTT
FTL	GATCGGGATGACGTGGCTC
FTL	TTGAGATGGCTTCTGCACAT
TFR1	GTGGAGTCTCCCGAGGGTTA
TFR1	TGGGCATTTGCAACCTTTTCT
ACOX1	TTTGTGGAACCTGTTGGCCT
ACOX1	TCGAAGATGAGTTCCGTGGC
SCD1	ACAGCCTGTTCGTTAGCACC
SCD1	TATCCATAGAGATGCGCGGC
FAS	GTCCTGCCTCTGGTGCTTG
FAS	AGCAAAATGGGCCTCCTTGA
GAPDH	GTGTTCCTACCCCCAATGTG
GAPDH	GTCATTGAGAGCAATGCCAG

Fig.1 Effects of Gandou Fumu Decoction (GDFMD) on liver histopathology and serum biochemical indicators of liver function in tx-J mouse models of Wilson's disease (WD). a: HE staining showing histopathological changes in the liver of the mice. b-e: Comparison of serum levels of ALT, AST, AKP, and albumin (ALB) among the groups (Mean±SD, n=6). A: Blank group; B: Model group; C: Low-dose GDFMD group; D: Medium-dose GDFMD group; E: High-dose GDFMD group; F: Fer-1 group (the same group names are used consistently in all the following figures). *P<0.05, **P<0.01 vs group A; #P<0.05,##P<0.01 vs group B.

Fig.2 Histopathological changes in the liver tissues of tx-J mice in different groups (Oil red staining).

Fig.3 Expression levels of ACOX1, SCD1 and FAS protein in the liver tissue of the mice in each group (Mean±SD, n=6). a: Results of Western blotting of the proteins. b-d: Relative expressions of ACOX1, SCD1 and FAS proteins. *P<0.05 vs group A; #P<0.05 vs group B.

Fig.4 Expression levels of ACOX1 (a), SCD1 (b) and FAS (c) mRNA in the liver tissues of the mice (Mean±SD, n=6). **P<0.01 vs group A; #P<0.05 vs group B.

Fig.5 Levels of Fe2+ (a), Cu2+ (b), MDA (c), ROS (d), SOD (e), GSH (f) and 4-HNE (g) in the liver tissues of the tx-J mice (Mean±SD, n=6). **P<0.01 vs group A; #P<0.05, ##P<0.01 vs group B.

Fig.6 Relative expression levels of FTH1, FLT, TFR1, GPX4, ACSL4, and ALOX15 proteins in the liver tissues of the mice in each group. a: Protein bands detected by Western blotting. b-g: Relative expressions of GPX4, ACSL4, ALOX15, TFR1, FLT, and FTH1 proteins (Mean±SD, n=6) . *P<0.05 vs group A; #P<0.05, ##P<0.01 vs group B.

Fig.7 Relative mRNA expression levels of FTH1, FLT, TFR1, GPX4, ACSL4 and ALOX15 in the liver tissues of the mice in each group. a-f: Relative mRNA expression levels of GPX4, ACSL4, ALOX15, TFR1, FLT and FTH1 (Mean±SD, n=6). **P<0.01 vs group A; #P<0.05, ##P<0.01 vs group B.

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