四烯甲萘醌（MK-4）对CCl<sub>4</sub>诱导的急性肝损伤小鼠模型的保护作用分析

叶露; 赵凡; 黄倩倩; 张佳怡; 王建青

doi:10.12449/JCH240121

四烯甲萘醌（MK-4）对CCl₄诱导的急性肝损伤小鼠模型的保护作用分析

DOI: 10.12449/JCH240121

叶露¹,
赵凡¹,
黄倩倩^{2, 3},
张佳怡¹,
王建青^{1, 2, 3, , ,}

1.
安徽医科大学药学院，合肥 230032
2.
安徽医科大学第一附属医院药学部，合肥 230012
3.
安徽省公共卫生临床中心，合肥 230012

基金项目:

国家自然科学基金 (82073566);

安徽省高校优秀青年人才支持计划资助项目 (gxyq2019014);

临床药学与药理学共建项目（2020） ;

安徽省公共卫生临床中心安徽医科大学第一附属医院北区科研培育基金资助项目 (2023YKJ14);

安徽省公共卫生临床中心安徽医科大学第一附属医院北区科研培育基金资助项目 (2023YKJ06);

安徽省公共卫生临床中心安徽医科大学第一附属医院北区科研培育基金资助项目 (2023YKJ11)

伦理学声明：本研究方案于2022年5月23日经由安徽医科大学动物伦理委员会审批，批号：202200523，符合实验室动物管理与使用准则。

利益冲突声明：本文不存在任何利益冲突。

作者贡献声明：叶露、赵凡、张佳怡负责实验；叶露负责查阅文章，撰写论文；黄倩倩、王建青负责修改文章并最后定稿。

详细信息

通信作者:
王建青， jianqingwang81@126.com （ORCID： 0000-0002-7935-9520）

计量
- 文章访问数: 229
- HTML全文浏览量: 124
- PDF下载量: 34
- 被引次数: 0
出版历程
- 收稿日期: 2023-03-30
- 录用日期: 2023-05-29
- 出版日期: 2024-01-23

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

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）

摘要

摘要: 目的探究四烯甲萘醌（MK-4）通过减轻铁死亡对四氯化碳（CCl₄）诱导的急性肝损伤（ALI）小鼠的保护作用及其机制。方法选用8周龄成年ICR雄鼠，适应性饲养后，分为Control组、MK-4组、CCl₄模型组（6、12、24 h）、MK-4+CCl₄组（6、12、24 h），每组6只。Control组腹腔注射同等剂量玉米油；MK-4组腹腔注射40 mg/kg的MK-4溶液，1 h后腹腔注射同等剂量玉米油；MK-4+CCl₄组（6、12、24 h）先腹腔注射40 mg/kg的MK-4溶液，1 h后和CCl₄模型组（6、12、24 h）同时腹腔注射0.3 mL/kg CCl₄溶液，分别在6、12、24 h进行取材。通过HE染色观察小鼠肝脏的病理变化；普鲁士蓝染色检测肝组织中铁聚集；生化仪检测小鼠血清中AST和ALT水平；试剂盒检测肝匀浆中组织铁含量、氧化应激指标丙二醛（MDA）、谷胱甘肽（GSH）水平；RT-PCR检测铁死亡标志基因长链脂酰辅酶A合成酶4（ACSL4）、前列腺素内过氧化物酶2（PTGS2）和谷胱甘肽过氧化物酶4（GPX4）以及铁代谢相关基因铁调素调节蛋白（HJV）、转铁蛋白受体1（TFR1）和膜转铁蛋白（FPN）的表达，Western Blot检测GPX4的蛋白表达水平。计量资料多组间比较采用单因素方差分析，进一步两两比较采用LSD-t检验。结果在时效研究中，与Control组相比，CCl₄模型组（12、24 h）的肝质量系数和血清ALT、AST水平明显上升（P值均<0.05），且通过HE染色结果也反映出，肝损伤程度随着时间延长逐渐加重。同时，MK-4+CCl₄（6、12、24 h）组和CCl₄模型组（6、12、24 h）相比时，MK-4+CCl₄（12 h）组的肝质量系数和血清ALT、AST水平下降（P值均<0.05），且肝组织的坏死面积减少，因此在后续研究中采用12 h的小鼠各组织样本进行检测。与Control组相比，CCl₄组MDA上升，GSH下降，差异均有统计学意义（P值均<0.05）；与CCl₄组相比，MK-4+CCl₄组MDA下降，GSH上升，差异均有统计学意义（P值均<0.05）。铁死亡关键指标ASCL4、PTGS2，与Control组相比，CCl₄组显著上升，GPX4则显著下降，差异均有统计学意义（P值均<0.05）；MK-4+CCl₄组较CCl₄组而言，ASCL4、PTGS2的mRNA水平下降，GPX4的mRNA水平上升，差异均有统计学意义（P值均<0.05）。蛋白质免疫印迹的结果显示，与Control组相比，CCl₄组蛋白表达水平下降，MK-4+CCl₄组较CCl₄组相比，蛋白表达水平上升，差异均有统计学意义（P值均<0.05）。普鲁士蓝染色结果显示：与Control组相比，CCl₄组铁聚集明显上升，MK-4干预后，MK-4+CCl₄组较CCl₄组铁聚集点下降。同时在对小鼠肝脏铁代谢基因的检测中发现，与Control组相比，CCl₄组铁含量显著上升，FPN、HJV的mRNA水平下降，TFR1的mRNA水平上升，差异均有统计学意义（P值均<0.05）；进行MK-4进行保护后，铁含量下降，FPN、HJV的mRNA水平上升，TFR1的mRNA水平下降，差异均有统计学意义（P值均<0.05）。结论 MK-4提前干预可减轻CCl₄诱导的小鼠ALI，其机制可能主要是通过抑制铁死亡并改善了小鼠肝脏铁代谢相关基因的表达。
- 肝功能衰竭，急性 /
- 铁死亡 /
- 甲萘醌4 /
- 小鼠，近交ICR
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

HTML全文

图 1 各组小鼠肝脏组织病理变化HE

注： a，正常对照组；b，CCl₄（6 h）组；c，CCl₄（12 h）组；d，CCl₄（24 h）组；e，MK-4组；f，MK-4+CCl₄（6 h）组；g，MK-4+CCl₄（12 h）组；h，MK-4+CCl₄（24 h）组。

Figure 1. Pathological changes of liver tissue in each group

下载: 全尺寸图片幻灯片

图 2 各组小鼠肝脏组织MDA及GSH水平

Figure 2. MDA and GSH levels in liver tissue of mice in each group

下载: 全尺寸图片幻灯片

图 3 各组小鼠肝脏组织铁死亡相关基因的表达

Figure 3. Expression of genes related to ferroptosis in liver tissue of mice in each group

下载: 全尺寸图片幻灯片

图 4 Western Blot检测各组小鼠肝脏组织中GPX4蛋白水平

Figure 4. The levels of GPX4 protein in liver tissues of mice in each group were detected by Western Blot

下载: 全尺寸图片幻灯片

图 5 各组小鼠肝脏组织铁聚集变化（普鲁士蓝染色，×40）

注： a，Control组；b，CCl₄组；c，MK-4组；d，MK-4+CCl₄组。箭头所指为铁聚集颗粒。

Figure 5. Iron accumulation in liver tissue of mice in each group Prussian dyeing（×40）

下载: 全尺寸图片幻灯片

图 6 各组小鼠肝脏组织铁含量以及铁代谢相关基因的表达

Figure 6. Iron content and iron metabolism related gene expression in liver tissue of mice in each group

下载: 全尺寸图片幻灯片

表 1 基因的引物序列

Table 1. The primer sequence of the gene

序号	基因名	序列（5′-3′）
1	18S	F：GTAACCCGTTGAACCCCATT
1	18S	R：CCATCCAATCGGTAGTAGCG
2	ACSL4	F：CTTCCTCTTAAGGCCGGGAC
2	ACSL4	R：TGCCATAGCGTTTTTCTTAGATTT
3	PTGS2	F： GGGCTCAGCCAGGCAGCAAAT
3	PTGS2	R：GCACTGTGTTTGGGGTGGGCT
4	GPX4	F：GCAACCAGTTTGGGAGGCAGGA
4	GPX4	R：CCTCCATGGGACCATAGCGCTTC
5	FPN	F：ATGGCTACCGCTGGTGTGGC
5	FPN	R：CACCTTGCCATCGTTGCGGC
6	HJV	F：ATGGGCCAGTCCCCTAGTC
6	HJV	R：GACGAGACATACTCGGCATTG
7	TFR1	F：CGAACTGGCTGGTCATCT
7	TFR1	R：TGTCTCTTGTCTCTACATTCCT

下载: 导出CSV

表 2 各组小鼠肝质量系数比较

Table 2. Liver weight coefficient of mice in each group

组别	动物数（只）	肝质量系数
Control组	6	0.049±0.003
MK-4组	6	0.046±0.002
CCl₄（6 h）组	6	0.054±0.003
CCl₄（12 h）组	6	0.056±0.003^1）
CCl₄（24 h）组	6	0.053±0.002^1）
MK-4+CCl₄（6 h）组	6	0.051±0.002
MK-4+CCl₄（12 h）组	6	0.052±0.003^2）
MK-4+CCl₄（24 h）组	6	0.055±0.002
F值		8.088
P值		<0.01
注：与Control组比较，1）P<0.05；与CCl₄（12 h）组比较，2）P<0.05。

下载: 导出CSV

表 3 各组小鼠血清中 ALT、AST 水平

Table 3. Serum ALT and AST levels of mice in each group

组别	动物数（只）	ALT（U/L）	AST（U/L）
Control组	6	42.920±2.676	134.480±21.603
MK-4组	6	51.200±10.232	140.000±25.140
CCl₄（6 h）组	6	231.540±73.919^1）	245.500±29.844^1）
CCl₄（12 h）组	6	9 285.600±1 990.930^1）	7 611.000±2 429.269^1）
CCl₄（24 h）组	6	31 123.750±3 635.768^1）	18 536.000±7 222.513^1）
MK-4+CCl₄（6 h）组	6	215.700±48.565	242.850±25.378
MK-4+CCl₄（12 h）组	6	5 053.000±380.391^2）	3 258.000±1280.555^2）
MK-4+CCl₄（24 h）组	6	29 431.250±2 538.912	11 605.000±6 190.560
F值		275.144	13.426
P值		<0.001	<0.01
注：与Control组比较，1）P<0.01；与CCl₄（12 h）组比较，2）P<0.05。

下载: 导出CSV

参考文献(18)

[1]	SHRESTHA DB, BUDHATHOKI P, SEDHAI YR, et al. N-acetyl cysteine versus standard of care for non-acetaminophen induced acute liver injury: A systematic review and meta-analysis[J]. Ann Hepatol, 2021, 24: 100340. DOI: 10.1016/j.aohep.2021.100340.
[2]	BHAKUNI GS, BEDI O, BARIWAL J, et al. Animal models of hepatotoxicity[J]. Inflamm Res, 2016, 65( 1): 13- 24. DOI: 10.1007/s00011-015-0883-0.
[3]	LIANG DG, MINIKES AM, JIANG XJ. Ferroptosis at the intersection of lipid metabolism and cellular signaling[J]. Mol Cell, 2022, 82( 12): 2215- 2227. DOI: 10.1016/j.molcel.2022.03.022.
[4]	JIANG XJ, STOCKWELL BR, CONRAD M. Ferroptosis: Mechanisms, biology and role in disease[J]. Nat Rev Mol Cell Biol, 2021, 22( 4): 266- 282. DOI: 10.1038/s41580-020-00324-8.
[5]	LIN FY, CHEN WY, ZHOU JH, et al. Mesenchymal stem cells protect against ferroptosis via exosome-mediated stabilization of SLC7A11 in acute liver injury[J]. Cell Death Dis, 2022, 13( 3): 271. DOI: 10.1038/s41419-022-04708-w.
[6]	VERVOORT LM, RONDEN JE, THIJSSEN HH. The potent antioxidant activity of the vitamin K cycle in microsomal lipid peroxidation[J]. Biochem Pharmacol, 1997, 54( 8): 871- 876. DOI: 10.1016/s0006-2952(97)00254-2.
[7]	LI JR, LIN JC, WANG H, et al. Novel role of vitamin k in preventing oxidative injury to developing oligodendrocytes and neurons[J]. J Neurosci, 2003, 23( 13): 5816- 5826. DOI: 10.1523/JNEUROSCI.23-13-05816.2003.
[8]	MISHIMA E, ITO J, WU ZJ, et al. A non-canonical vitamin K cycle is a potent ferroptosis suppressor[J]. Nature, 2022, 608( 7924): 778- 783. DOI: 10.1038/s41586-022-05022-3.
[9]	DIXON SJ, LEMBERG KM, LAMPRECHT MR, et al. Ferroptosis: An iron-dependent form of nonapoptotic cell death[J]. Cell, 2012, 149( 5): 1060- 1072. DOI: 10.1016/j.cell.2012.03.042.
[10]	QIAN MY, LIU MY, TAO CL, et al. Research progress of macrophages death[J]. Chin J Immun, 2023, 39( 1): 204- 210. DOI: 10.3969/j.issn.1000-484X.2023.01.034. 钱敏雅, 刘满宇, 陶晨璐, 等. 巨噬细胞死亡方式的研究进展[J]. 中国免疫学杂志, 2023, 39( 1): 204- 210. DOI: 10.3969/j.issn.1000-484X.2023.01.034.
[11]	SAMRA YA, HAMED MF, EL-SHEAKH AR. Hepatoprotective effect of allicin against acetaminophen-induced liver injury: Role of inflammasome pathway, apoptosis, and liver regeneration[J]. J Biochem Mol Toxicol, 2020, 34( 5): e22470. DOI: 10.1002/jbt.22470.
[12]	SATO T, SCHURGERS LJ, UENISHI K. Comparison of menaquinone-4 and menaquinone-7 bioavailability in healthy women[J]. Nutr J, 2012, 11: 93. DOI: 10.1186/1475-2891-11-93.
[13]	YANG WS, SRIRAMARATNAM R, WELSCH ME, et al. Regulation of ferroptotic cancer cell death by GPX4[J]. Cell, 2014, 156( 1-2): 317- 331. DOI: 10.1016/j.cell.2013.12.010.
[14]	STOCKWELL BR, ANGELI JPF, BAYIR H, et al. Ferroptosis: A regulated cell death nexus linking metabolism, redox biology, and disease[J]. Cell, 2017, 171( 2): 273- 285. DOI: 10.1016/j.cell.2017.09.021.
[15]	PIETRANGELO A. Genetics, genetic testing, and management of hemochromatosis: 15 years since hepcidin[J]. Gastroenterology, 2015, 149( 5): 1240- 1251.e4. DOI: 10.1053/j.gastro.2015.06.045.
[16]	ZHANG L, LIAO YQ, XIA QC, et al. Ferroptosis regulatory signaling pathway and its research progress in related diseases[J]. Chin J Clin Pharmacol Ther, 2022, 27( 2): 227- 234. DOI: 10.12092/j.issn.1009-2501.2022.02.015. 张亮, 廖勇群, 夏秦川, 等. 铁死亡调控信号通路以及在相关疾病中的研究进展[J]. 中国临床药理学与治疗学, 2022, 27( 2): 227- 234. DOI: 10.12092/j.issn.1009-2501.2022.02.015.
[17]	YANG WS, STOCKWELL BR. Ferroptosis: Death by lipid peroxidation[J]. Trends Cell Biol, 2016, 26( 3): 165- 176. DOI: 10.1016/j.tcb.2015.10.014.
[18]	FENG H, STOCKWELL BR. Unsolved mysteries: How does lipid peroxidation cause ferroptosis[J]. PLoS Biol, 2018, 16( 5): e2006203. DOI: 10.1371/journal.pbio.2006203.