梓醇扶正制毒配伍从SLC7A11/GPX4通路抑制铁死亡减轻雷公藤甲素肝毒性

doi:10.12122/j.issn.1673-4254.2025.04.16

摘要/Abstract

摘要：

目的研究梓醇扶正制毒配伍对雷公藤甲素所致肝损伤的减毒作用及其铁死亡相关机制。方法通过动物实验筛选雷公藤甲素所致小鼠肝毒性的最佳剂量，再将20只C57BL/6J小鼠分为空白组、雷公藤甲素组（0.6 mg/kg）、梓醇组（4.5 mg/kg）和两药配伍组（0.6 mg/kg+4.5 mg/kg），5只/组。灌胃给药13 d后，检测小鼠肝组织病理、肝功能指标、亚细胞结构、脂质过氧化指标、亚铁离子沉积和铁死亡相关蛋白。在肝HL7702细胞中，采用铁死亡抑制剂Fer-1（1 μmol/L）抑制铁死亡后，检测肝功能、Fe²⁺浓度和脂质过氧化；再将肝HL7702细胞分为空白组、梓醇组（80 μg/L）、雷公藤甲素组（20 μg/L）和两药配伍组（80 μg/L+20 μg/L），检测肝功能指标、Fe²⁺浓度、脂质过氧化、ROS水平和铁死亡相关蛋白，进一步验证铁死亡在梓醇减轻雷公藤甲素肝毒性中的作用。结果梓醇可减轻雷公藤甲素所致小鼠肝组织病理损伤并降低ALT、AST和LDH（P<0.01），同时逆转雷公藤甲素所致的Fe²⁺浓度和MDA的升高、GP_X的降低（P<0.05）。Fer-1抑制铁死亡逆转了雷公藤甲素所致肝HL7702细胞ALT、AST和LDH的升高（P<0.01）。Western blotting和qRT-PCR结果显示，梓醇能逆转雷公藤甲素所致的铁死亡相关SLC7A11、FTH1、GPX4蛋白和基因表达（P<0.05）。结论梓醇通过SLC7A11/GPX4通路抑制过度铁死亡减轻雷公藤甲素的肝毒性。

关键词: 梓醇, 雷公藤甲素, 肝毒性, 铁死亡

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

张林落, 李长青, 皇玲玲, 周学平, 娄媛媛. 梓醇扶正制毒配伍从SLC7A11/GPX4通路抑制铁死亡减轻雷公藤甲素肝毒性[J]. 南方医科大学学报, 2025, 45(4): 810-818.

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.

图/表 6

表1 qRT-PCR引物序列

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

图1 TP导致C57BL/6J小鼠肝损伤

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.

图2 CAT能减轻TP所致C57BL/6J小鼠肝损伤

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.

图3 CAT能改善TP所致C57雌性小鼠的铁死亡

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.

图4 抑制铁死亡能改善雷公藤甲素所致肝HL7702细胞的损伤

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

图5 CAT能抑制铁死亡改善TP所致肝HL7702细胞的损伤

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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