Catalpol reduces liver toxicity of triptolide in mice by inhibiting hepatocyte ferroptosis through the SLC7A11/GPX4 pathway: testing the Fuzheng Zhidu theory for detoxification

doi:10.12122/j.issn.1673-4254.2025.04.16

Abstract

Abstract:

Objective To investigate the protective effect of catalpol against triptolide-induced liver injury and explore its mechanism to test the Fuzheng Zhidu theory for detoxification. Methods C57BL/6J mice were randomized into blank control group, catalpol group, triptolide group and triptolide+catalpol group. After 13 days of treatment with the agents by gavage, the mice were examined for liver tissue pathology, liver function, hepatocyte subcellular structure, lipid peroxidation, ferrous ion deposition and expressions of ferroptosis-related proteins in the liver. In a liver cell line HL7702, the effect of catalpol or the ferroptosis inhibitor Fer-1 on triptolide-induced cytotoxicity was tested by examining cell functions, Fe²⁺ concentration, lipid peroxidation, ROS level and the ferroptosis-related proteins. Results In C57BL/6J mice, catalpol significantly alleviated triptolide-induced hepatic injury, lowered the levels of ALT, AST and LDH, and reversed the elevation of Fe²⁺ concentration and MDA level and the reduction of GP_Xlevel. In HL7702 cells, inhibition of ferroptosis by Fer-1 significantly reversed triptolide-induced elevation of ALT, AST and LDH levels. Western blotting and qRT-PCR demonstrated that catalpol reversed abnormalities in expressions of SLC7A11, FTH1 and GPX4 at both the mRNA and protein levels in triptolide-treated HL7702 cells. Conclusion The combined use of catalpol can reduce the hepatotoxicity of triptolide in mice by inhibiting excessive hepatocyte ferroptosis through the SLC7A11/GPX4 pathway.

Key words: catalpol, triptolide, hepatotoxicity, ferroptosis

Linluo ZHANG, Changqing LI, Lingling HUANG, Xueping ZHOU, Yuanyuan LOU. Catalpol reduces liver toxicity of triptolide in mice by inhibiting hepatocyte ferroptosis through the SLC7A11/GPX4 pathway: testing the Fuzheng Zhidu theory for detoxification[J]. Journal of Southern Medical University, 2025, 45(4): 810-818.

Figures/Tables 6

Tab.1 Primers used for qRT-PCR

Gene

Forward primer (5'-3')

Reverse primer (5'-3')

SLC7A11

FTH1

GPX4

GAPDH

GGCAGTTGCTGGGCTGATTTA

CATCAACCGCCAGATCAACCT

TGAAGATCCAACCCAAGGGC

GGAAGCTTGTCATCAATGGAAATC

GATGACGAAGCCAATCCCTGT

GCAAAGTTCTTCAAAGCCACATC

GACGGTGTCCAAACTTGGTG

TGATGACCCTTTTGGCTCCC

Fig.1 Triptolide-induced liver injury in C57BL/6J mice. A: Body weight changes of triptolide-treated mice. B: Liver index (%) changes. C: changes in ALT, AST and LDH levels. D: HE staining of the liver tissues. E: Oil Red O staining of the liver tissue. L, M and H: Low-, moderate-, and high-dose triptolide groups with daily dose of 0.3, 0.6 and 0.9 mg/kg, respectively. Data are presented as Mean±SD (n=5). *P<0.05, **P<0.01, ***P<0.001 vs Control group.

Fig.2 Catalpol alleviated triptolide-induced liver injury in C57BL/6J mice. A: Body weight changes. B: Liver function index (%). C: ALT, AST and LDH levels. D: HE staining results. E: Oil Red O staining results. *P<0.05, ***P<0.001 vs Control group. #P<0.05, ##P<0.01 vs TP group.

Fig.3 Catalpol alleviates triptolide-induced ferroptosis in C57BL/6J mice. A: Subcellular structure of mouse liver tissue (Original magnification: ×10000). B: PB staining (×100). C: MDA and GPX levels. D: mRNA levels of SLC7A11, FTH1 and GPX4 detected by qRT-PCR. E: Expressions of SLC7A11, FTH1 and GPX4 proteins detected by Western blotting. *P<0.05, **P<0.01, ***P<0.001 vs Control group. #P<0.05, ##P<0.01, ###P<0.001 vs TP group.

Fig.4 Inhibition of ferroptosis by Fer-1alleviates triptolide-induced injury in HL7702 cells. A: Cell viability changes (%). B: ALT, AST and LDH levels. C: Fe2+ concentration. D: MDA and GPX levels. E: Cell viability (%). F: Cell viability (%). G: ALT, AST and LDH indicators. H: Fe2+ concentration. I: MDA and GPX indicators. J: PB staining. L: Low-dose triptolide group (5 μg/L). M: Medium-dose triptolide group (20 μg/L). H: High-dose triptolide group (80 μg/L). *P<0.05, **P<0.01, ***P<0.001 vs Control group; #P<0.05, ##P<0.01 vs TP group

Fig.5 Catalpol reduces triptolide-induced HL7702 cell damage by inhibiting ferroptosis. A: Cell viability (%). B: Cell viability (%). C: Cell viability (%). D: ALT, AST and LDH indicators. E: Fe2+ concentration. F: MDA and GPX. G: The ratio of ROS (%). H: Immunohistochemistry of SLC7A11, FTH1 and GPX4. *P<0.05, **P<0.01, ***P<0.001 vs Control group. #P<0.05, ##P<0.01, ###P<0.001 vs TP group.

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