Menaquinone-4 exerts a protective effect against carbon tetrachloride-induced acute liver injury in mice by alleviating ferroptosis
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.
国家科技期刊平台
NSTL
万方数据
维普
