Menaquinone-4 exerts a protective effect against carbon tetrachloride-induced acute liver injury in mice by alleviating ferroptosis

Lu YE; Fan ZHAO; Qianqian HUANG; Jiayi ZHANG; Jianqing WANG

doi:10.12449/JCH240121

Volume 40 Issue 1

Jan. 2024

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Menaquinone-4 exerts a protective effect against carbon tetrachloride-induced acute liver injury in mice by alleviating ferroptosis

DOI: 10.12449/JCH240121

Lu YE¹,
Fan ZHAO¹,
Qianqian HUANG^{2, 3},
Jiayi ZHANG¹,
Jianqing WANG^{1, 2, 3
,
,
,}

1.
Department of Pharmacy，Anhui Medical University，Hefei 230032，China
2.
Department of Pharmacy，The First Affiliated Hospital of Anhui Medical University，Hefei 230012，China
3.
Anhui Public Health Clinical Center，Hefei 230012，China

Research funding:

National Natural Science Foundation of China (82073566);

The Program of Excellent Young Talents in Universities of Anhui Province (gxyq2019014);

Clinical Pharmacy and Pharmacology （2020） ;

Anhui Public Health Clinical Center, Supported by North District Scientific Research and Cultivation Foundation of the First Affiliated Hospital of Anhui Medical University (2023YKJ14);

Anhui Public Health Clinical Center, Supported by North District Scientific Research and Cultivation Foundation of the First Affiliated Hospital of Anhui Medical University (2023YKJ06);

Anhui Public Health Clinical Center, Supported by North District Scientific Research and Cultivation Foundation of the First Affiliated Hospital of Anhui Medical University (2023YKJ11)

More Information

Corresponding author: WANG Jianqing， jianqingwang81@126.com （ORCID： 0000-0002-7935-9520）
Received Date: 2023-03-30
Accepted Date: 2023-05-29
Published Date: 2024-01-23

Abstract

Abstract

Objective To investigate whether menaquinone-4 （MK-4） can exert a protective effect against carbon tetrachloride （CCl₄）-induced acute liver injury （ALI） in mice by alleviating ferroptosis. Methods After adaptive feeding， adult male ICR mice， aged 8 weeks， were divided into Control group， MK-4 group， CCl₄ model group （6-hour， 12-hour， and 24-hour）， and MK-4+CCl₄ group （6-hour， 12-hour， and 24-hour）， with 6 mice in each group. The mice in the Control group were given intraperitoneal injection of an equal dose of corn oil； the mice in the MK-4 group were given intraperitoneal injection of 40 mg/kg MK-4 solution， followed by an equal dose of corn oil after 1 hour； the mice in the MK-4+CCl₄ group （6-hour， 12-hour， and 24-hour） were given intraperitoneal injection of 40 mg/kg MK-4 solution， and after 1 hour， the mice in this group and the CCl₄ model group （6-hour， 12-hour， and 24-hour） were given intraperitoneal injection of 0.3 mL/kg CCl₄ solution， with samples collected at 6， 12， and 24 hours. HE staining was used to observe the pathological changes of mouse liver； Prussian blue staining was used to observe iron accumulation in liver tissue； a biochemical analyzer was used to measure the serum levels of aspartate aminotransferase （AST） and alanine aminotransferase （ALT）； related kits were used to measure the levels of tissue iron content and the oxidative stress indices malondialdehyde （MDA） and glutathione （GSH） in liver homogenate； RT-PCR was used to measure the expression levels of ferroptosis marker genes （acyl-CoA synthetase long-chain family member 4 ［ACSL4］， prostaglandin-endoperoxide synthase 2 ［PTGS2］， and glutathione peroxidase 4 ［GPX4］） and iron metabolism-related genes （hemojuvelin ［HJV］， transferrin receptor 1 ［TFR1］， and ferroportin ［FPN］）， and Western blot was used to measure the protein expression level of GPX4. A one-way analysis of variance was used for comparison of continuous data between multiple groups， and the least significant difference t-test was used for further comparison between two groups. Results In the aging study， compared with the Control group， the CCl₄ model group （6-hour， 12-hour， and 24-hour） had significant increases in liver weight coefficient and the serum levels of ALT and AST （all P<0.05）， and HE staining also showed that liver injury gradually aggravated over time. Meanwhile， compared with the CCl₄ model group （6-hour， 12-hour， and 24-hour）， the MK-4+CCl₄ （12-hour） group had significant reductions in liver weight coefficient and the serum levels of ALT and AST （all P<0.05）， with a reduction in the necrotic area of liver tissue， and therefore， 12-hour mouse tissue samples were used for detection in the following study. Compared with the Control group， the CCl₄ group had a significant increase in MDA and a significant reduction in GSH （both P<0.05）， and compared with the CCl₄ group， the MK-4+CCl₄ group had a significant reduction in MDA and a significant increase in GSH （both P<0.05）. Compared with the Control group， the CCl₄ group had significant increases in the key ferroptosis indices ASCL4 and PTGS2 and a significant reduction in GPX4 （all P<0.05）； compared with the CCl₄ group， the MK-4+CCl₄ group had significant reductions in the mRNA expression levels of ASCL4 and PTGS2 and a significant increase in the mRNA expression level of GPX4 （all P<0.05）. Western blotting showed that compared with the Control group， the CCl₄ group had a significant reduction in the protein expression level of GPX4 （P<0.05）， and compared with the CCl₄ group， the MK-4+CCl₄ group had a significant increase in the protein expression level of GPX4 （P<0.05）. Prussian blue staining showed that compared with the Control group， the CCl₄ group had a significant increase in iron accumulation； after MK-4 intervention， compared with the CCl₄ group， the MK-4+CCl₄ group had a significant reduction in iron accumulation. As for the measurement of iron metabolism genes in mouse liver， compared with the Control group， the CCl₄ group had a significant increase in iron content， significant reductions in the mRNA expression levels of FPN and HJV， and a significant increase in the mRNA expression level of TFR1 （all P<0.05）； after protection with MK-4， there was a significant reduction in iron content， significant increases in the mRNA expression levels of FPN and HJV， and a significant reduction in the mRNA expression level of TFR1 （all P<0.05）. Conclusion MK-4 intervention in advance can alleviate CCl₄-induced ALI in mice， possibly by inhibiting ferroptosis and improving the expression of iron metabolism-related genes in mouse liver.
- Liver Failure， Acute,
- Ferroptosis,
- Menaquinone-4,
- Mice， Inbred ICR

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Menaquinone-4 exerts a protective effect against carbon tetrachloride-induced acute liver injury in mice by alleviating ferroptosis

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Menaquinone-4 exerts a protective effect against carbon tetrachloride-induced acute liver injury in mice by alleviating ferroptosis

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