2024, 40(1): 121-128.
DOI: 10.12449/JCH240121
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.
YE L, ZHAO F, HUANG QQ, et al. Menaquinone-4exerts a protective effect against carbon tetrachloride-induced acute liver injury in mice by alleviating ferroptosis[J]. J Clin Hepatol, 2024, 40(1): 121-128.. doi: 10.12449/JCH240121.