茵陈蒿汤对自身免疫性肝炎小鼠肝细胞铁死亡的抑制作用及其机制分析

李竹蓉; 陈晨; 郭地; 吕思学; 武嘉文; 杨娜; 刘杨

doi:10.12449/JCH240311

茵陈蒿汤对自身免疫性肝炎小鼠肝细胞铁死亡的抑制作用及其机制分析

DOI: 10.12449/JCH240311

山西中医药大学基础医学院，山西晋中 030619

基金项目:

山西省科学技术厅山西省重点国别科技合作项目 (202104041101013);

山西中医药大学科技创新团队项目 (2022TD2003);

中西医结合防治风湿免疫病山西省科技创新人才重点团队 (202204051002033);

山西中医药大学学科建设项目 (2023XKJS-03);

山西省自然科学基金 (202203021222272)

伦理学声明：本研究方案于2021年9月29日经由山西中医药大学实验动物伦理委员会审批，批号：2019LL41。

利益冲突声明：本文不存在任何利益冲突。

作者贡献声明：李竹蓉负责设计论文框架，起草论文及研究过程的实施；陈晨、郭地负责实验操作，数据收集；吕思学、武嘉文负责统计学分析；杨娜、刘杨负责论文修改；刘杨负责拟定写作思路，指导撰写文章并最后定稿。

详细信息

通信作者:
刘杨， liuyang1980@sxtcm.edu.cn （ORCID： 0000-0002-6627-5002）

计量
- 文章访问数: 425
- HTML全文浏览量: 117
- PDF下载量: 46
- 被引次数: 0
出版历程
- 收稿日期: 2023-06-09
- 录用日期: 2023-08-15
- 出版日期: 2024-03-20

Role and mechanism of action of Yinchenhao Decoction in inhibiting ferroptosis of hepatocytes in mice with autoimmune hepatitis

School of Basic Medical Sciences，Shanxi University of Chinese Medicine，Jinzhong，Shanxi 030619，China

Research funding:

Shanxi Provincial Key Country Science and Technology Cooperation Project Shanxi Provincial Science and Technology Department (202104041101013);

Science and Technology Innovation Team Project of Shanxi University of Traditional Chinese Medicine (2022TD2003);

Key Team of Science and Technology Innovation Talents of Shanxi Province for Prevention and Treatment of Rheumatological and Immunological diseases by Integrated Chinese and Western Medicine (202204051002033);

Discipline Construction Project of Shanxi University of Traditional Chinese Medicine (2023XKJS-03);

Natural Science Foundation Project of Shanxi Province (202203021222272)

More Information

Corresponding author: LIU Yang， liuyang1980@sxtcm.edu.cn （ORCID： 0000-0002-6627-5002）

摘要

摘要: 目的探讨茵陈蒿汤对自身免疫性肝炎小鼠肝细胞铁死亡的抑制作用及机制。方法选取SPF级雌性C57BL/6小鼠18只，采用随机数字表法分为正常组、模型组、治疗组，每组6只。模型组和治疗组采用刀豆蛋白A（Con A）尾静脉注射制备自身免疫性肝炎小鼠模型，正常组注射生理盐水。治疗组于造模前14天，给予茵陈蒿汤（4.68 g生药/kg）预防性灌胃治疗，末次灌胃给药后注射Con A。分别检测ALT、AST、IFN-γ、TNF-α、铁离子、GSH、ROS、ATP、MDA水平，计算肝脏指数、脾脏指数，并观察GPX4、SLC7A11的表达；比较各组小鼠肝脏病理组织变化。正态分布的计量资料3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。结果与正常组比较，模型组肝脏指数、脾脏指数、ALT、AST、IFN-γ、TNF-α、铁离子、ROS、MDA水平均升高（P值均<0.05），GSH、ATP含量及GPX4、SLC7A11蛋白表达水平降低（P值均<0.05）。与模型组比较，治疗组肝脏指数、脾脏指数、ALT、AST、IFN-γ、TNF-α、铁离子、ROS、MDA水平均降低（P值均<0.05），GSH、ATP含量及GPX4、SLC7A11蛋白表达水平升高（P值均<0.01）。HE染色结果显示，与正常组比较，模型组小鼠肝脏出现大面积肝细胞变性、坏死、门管区炎细胞聚集，治疗组处理的小鼠肝脏坏死和炎性浸润程度较模型组有所减轻。结论 Con A诱导肝组织损伤可能与铁死亡有关。茵陈蒿汤可以增加SLC7A11/GPX4蛋白的表达水平进而抵抗Con A诱导的肝细胞铁死亡。
- 肝炎，自身免疫性 /
- 茵陈蒿汤 /
- 铁死亡 /
- 谷胱甘肽过氧化酶 /
- 小鼠，近交C57BL
Abstract: Objective To investigate the role and mechanism of action of Yinchenhao Decoction in inhibiting ferroptosis of hepatocytes in mice with autoimmune hepatitis. Methods A total of 18 specific pathogen-free female C57BL/6 mice were selected and divided into normal group， model group， and treatment group using a random number table， with 6 mice in each group. The mice in the model group and the treatment group were injected with concanavalin A （Con A） via the caudal vein to establish a mouse model of autoimmune hepatitis， and those in the normal group were injected with normal saline. The mice in the treatment group were given prophylactic treatment with Yinchenhao Decoction （4.68 g crude drug/kg） by gavage at 14 days before modeling， and Con A was injected after the last gavage. The levels of alanine aminotransferase （ALT）， aspartate aminotransferase （AST）， interferon gamma （IFN-γ）， tumor necrosis factor-α （TNF-α）， iron ion， glutathione （GSH）， reactive oxygen species （ROS）， adenosine triphosphate （ATP）， and malondialdehyde （MDA） were measured； liver index and spleen index were calculated； the expression levels of GPX4 and SLC7A11 were measured； liver histopathological changes were compared between groups. A one-way analysis of variance was used for comparison of normally distributed continuous data between three groups， and the least significant difference t-test was used for further comparison between two groups. Results Compared with the normal group， the model group had significant increases in liver index， spleen index， ALT， AST， IFN-γ， TNF-α， iron ion， ROS and MDA （all P<0.05） and significant reductions in the content of GSH and ATP and the protein expression levels of GPX4 and SLC7A11 （all P<0.05）. Compared with the model group， the treatment group had significant reductions in liver index， spleen index， ALT， AST， IFN-γ， TNF-α， iron ion， ROS and MDA （all P<0.05） and significant increases in the content of GSH and ATP and the protein expression levels of GPX4 and SLC7A11 （all P<0.05）. HE staining showed that compared with the normal group， the model group showed massive hepatocyte degeneration and necrosis and inflammatory cell aggregation at the portal area， and compared with the model group， the treatment group had alleviation of liver necrosis and inflammatory infiltration. Conclusion Liver injury induced by Con A may be associated with ferroptosis. Yinchenhao Decoction can increase the protein expression levels of SLC7A11 and GPX4 protein and thus inhibit ferroptosis of hepatocytes induced by Con A.
- Hepatitis， Autoimmune /
- Yinchenhao Decoction /
- Ferroptosis /
- Glutathione Peroxidase /
- Mice， Inbred C57BL

HTML全文

图 1 茵陈蒿汤对小鼠肝组织形态学的影响（HE染色）

注：黑色箭头所示为淋巴细胞浸润程度。

Figure 1. Effect of Yinchenhao decoction on liver morphology of mice （HE staining）

下载: 全尺寸图片幻灯片

图 2 茵陈蒿汤对小鼠肝组织中GPX4和SLC7A11蛋白表达的影响

Figure 2. Effect of Yinchenhao decoction on GPX4 and SLC7A11 protein expression in liver tissue of mice

下载: 全尺寸图片幻灯片

表 1 茵陈蒿汤对小鼠肝/脾指数、血清ALT和AST水平的影响

Table 1. Effect of Yinchenhao decoction on liver and spleen index， serum ALT and AST levels in mice

组别	动物数（只）	肝脏指数（mg/g）	脾脏指数（mg/g）	ALT（U/L）	AST（U/L）
正常组	6	50.09±1.60	3.51±0.31	63.51±12.29	106.56±3.50
模型组	6	62.84±4.38^1）	6.74±0.69^1）	307.31±22.95^1）	231.85±9.95^1）
治疗组	6	50.64±1.63^2）	3.63±0.67^2）	93.87±17.64^2）	108.28±11.61^2）
F值		38.33	59.31	321.40	377.50
P值		<0.01	<0.01	<0.01	<0.01
注：与正常组比较，1）P<0.01；与模型组比较，2）P<0.01。

下载: 导出CSV

表 2 茵陈蒿汤对小鼠肝组织中ROS产生、铁离子、MDA、GSH、ATP水平的影响

Table 2. Effects of Yinchenhao decoction on ROS production， Fe， MDA， GSH and ATP levels in liver tissue of mice

组别	动物数（只）	ROS（AU）	铁离子（μmol/kg）	MDA（nmol/mg）	GSH（μmol/g）	ATP（nmol/g）
正常组	6	6.00±1.75	137.97±16.91	1.14±0.13	17.91±3.20	0.30±0.04
模型组	6	19.09±1.63^1）	867.09±161.88^1）	1.82±0.21^1）	2.09±0.37^1）	0.18±0.02^1）
治疗组	6	7.85±2.48^2）	303.93±135.25^2）	1.41±0.06^2）	34.10±7.44^2）	0.65±0.11^2）
F值		76.32	58.70	31.49	70.11	69.92
P值		<0.01	<0.01	<0.01	<0.01	<0.05
注：与正常组比较，1）P<0.05；与模型组比较，2）P<0.01。

下载: 导出CSV

表 3 茵陈蒿汤对Con A诱导肝损伤小鼠血清IFN-γ、TNF-α的影响

Table 3. Effects of Yinchenhao decoction on serum IFN-γ and TNF-α production in mice with Con A induced liver injury

组别	动物数（只）	IFN-γ（pg/mL）	TNF-α（pg/mL）
正常组	6	356.08±19.93	309.19±13.32
模型组	6	405.57±49.80^1）	357.44±25.72^1）
治疗组	6	357.13±8.92^2）	318.84±8.16^2）
F值		4.87	12.95
P值		<0.05	<0.01
注：与正常组比较，1）P<0.05；与模型组比较，2）P<0.05。

下载: 导出CSV

参考文献(32)

[1]	TL J, MIQUEL R, ZACHOU K, et al. Features and outcome of AIH patients without elevation of IgG[J]. JHEP Rep, 2020, 2( 3): 100094. DOI: 10.1016/j.jhepr.2020.100094.
[2]	MIELI-VERGANI G, VERGANI D, CZAJA AJ, et al. Autoimmune hepatitis[J]. Nat Rev Dis Primers, 2018, 4: 18017. DOI: 10.1038/nrdp.2018.17.
[3]	PAPE S, SNIJDERS RJALM, GEVERS TJG, et al. Systematic review of response criteria and endpoints in autoimmune hepatitis by the International Autoimmune Hepatitis Group[J]. J Hepatol, 2022, 76( 4): 841- 849. DOI: 10.1016/j.jhep.2021.12.041.
[4]	European Association for the Study of the Liver. EASL clinical practice guidelines: Autoimmune hepatitis[J]. J Hepatol, 2015, 63( 4): 971- 1004. DOI: 10.1016/j.jhep.2015.06.030.
[5]	LV T, LI M, ZENG N, et al. Systematic review and meta-analysis on the incidence and prevalence of autoimmune hepatitis in Asian, European, and American population[J]. J Gastroenterol Hepatol, 2019, 34( 10): 1676- 1684. DOI: 10.1111/jgh.14746.
[6]	TRIVEDI PJ, HIRSCHFIELD GM. Recent advances in clinical practice: Epidemiology of autoimmune liver diseases[J]. Gut, 2021, 70( 10): 1989- 2003. DOI: 10.1136/gutjnl-2020-322362.
[7]	ZHANG KH, LI JC, SHI Z, et al. Ginsenosides regulates innate immunity to affect immune microenvironment of AIH through hippo-YAP/TAZ signaling pathway[J]. Front Immunol, 2022, 13: 851560. DOI: 10.3389/fimmu.2022.851560.
[8]	CHEN SQ. Treating of autoimmune hepatitis with removing heat and dampness of Prof. ZHOU zhongying[J]. J Pract Tradit Chin Intern Med, 2013, 27( 1): 16, 18. DOI: 10.3969/j.issn.1671-7813.2013.01(s).08. 陈四清. 周仲瑛教授清热化湿治疗免疫性肝炎[J]. 实用中医内科杂志, 2013, 27( 1): 16, 18. DOI: 10.3969/j.issn.1671-7813.2013.01(s).08.
[9]	LI S, SHI MJ, XIAO HM, et al. Data mining for diagnosis and treatment law of autoimmune hepatitis based on Chinese medical records[J]. J Tradit Chin Med, 2017, 58( 14): 1237- 1240. DOI: 10.13288/j.11-2166/r.2017.14.017. 黎胜, 施梅姐, 萧焕明, 等. 自身免疫性肝炎中医医案诊治规律数据挖掘[J]. 中医杂志, 2017, 58( 14): 1237- 1240. DOI: 10.13288/j.11-2166/r.2017.14.017.
[10]	ZHU LJ, CHEN DZ, ZHU Y, et al. GPX4-regulated ferroptosis mediates S100-induced experimental autoimmune hepatitis associated with the Nrf2/HO-1 signaling pathway[J]. Oxid Med Cell Longev, 2021, 2021: 6551069. DOI: 10.1155/2021/6551069.
[11]	CHEN X, KANG R, KROEMER G, et al. Ferroptosis in infection, inflammation, and immunity[J]. J Exp Med, 2021, 218( 6): e20210518. DOI: 10.1084/jem.20210518.
[12]	LEE H, ZANDKARIMI F, ZHANG YL, et al. Energy-stress-mediated AMPK activation inhibits ferroptosis[J]. Nat Cell Biol, 2020, 22( 2): 225- 234. DOI: 10.1038/s41556-020-0461-8.
[13]	WANG KC, WU WR, JIANG XW, et al. Multi-omics analysis reveals the protection of gasdermin D in concanavalin A-induced autoimmune hepatitis[J]. Microbiol Spectr, 2022, 10( 5): e0171722. DOI: 10.1128/spectrum.01717-22.
[14]	LI J, ZUO ZY. Formulas of Chinese Medicine[M]. Fifth Edition. Beijing: China Press of Traditional Chinese Medicine, 2021: 237- 238. 李冀, 左铮云. 方剂学[M]. 第五版. 北京: 中国中医药出版社, 2021: 237- 238.
[15]	SHEN H, HUANG YY, CHEN T, et al. Optimization of the water extraction for Yinchenhao Decoction by orthogonal experimental design[J]. China Pharm, 2020, 29( 11): 14- 17. DOI: 10.3969/j.issn.1006-4931.2020.11.004. 沈晗, 黄玉宇, 陈汀, 等. 正交试验设计优选茵陈蒿汤水煎煮工艺[J]. 中国药业, 2020, 29( 11): 14- 17. DOI: 10.3969/j.issn.1006-4931.2020.11.004.
[16]	LU Y, SUN FF, ZENG Z, et al. Research advances on autoimmune hepatitis[J/CD]. Chin J Liver Dis(Electronic Version), 2022, 14( 4): 1- 9. DOI: 10.3969/j.issn.1674-7380.2022.04.001. 路遥, 孙芳芳, 曾湛, 等. 自身免疫性肝炎研究进展[J/CD]. 中国肝脏病杂志(电子版), 2022, 14( 4): 1- 9. DOI: 10.3969/j.issn.1674-7380.2022.04.001.
[17]	MACK CL, ADAMS D, ASSIS DN, et al. Diagnosis and management of autoimmune hepatitis in adults and children: 2019 practice guidance and guidelines from the American association for the study of liver diseases[J]. Hepatology, 2020, 72( 2): 671- 722. DOI: 10.1002/hep.31065.
[18]	LOHSE AW, SEBODE M, BHATHAL PS, et al. Consensus recommendations for histological criteria of autoimmune hepatitis from the International AIH Pathology Group: Results of a workshop on AIH histology hosted by the European Reference Network on Hepatological Diseases and the European Society of Pathology: Results of a workshop on AIH histology hosted by the European Reference Network on Hepatological Diseases and the European Society of Pathology[J]. Liver Int, 2022, 42( 5): 1058- 1069. DOI: 10.1111/liv.15217.
[19]	Chinese Society of Hepatology, Chinese Medical Association. Guidelines on the diagnosis and management of autoimmune hepatitis(2021)[J]. J Clin Hepatol, 2022, 38( 1): 42- 49. 中华医学会肝病学分会. 自身免疫性肝炎诊断和治疗指南(2021)[J]. 临床肝胆病杂志, 2022, 38( 1): 42- 49.
[20]	WANG LY, LIU YC, DU TT, et al. ATF3 promotes erastin-induced ferroptosis by suppressing system Xc[J]. Cell Death Differ, 2020, 27( 2): 662- 675. DOI: 10.1038/s41418-019-0380-z.
[21]	GUO YF, SUN FX, LI XL, et al. Clinical application of Yinchenhao Decoction in hepatobiliary disease[J]. J Liaoning Univ Tradit Chin Med, 2020, 22( 3): 57- 60. DOI: 10.13194/j.issn.1673-842x.2020.03.016. 郭雨菲, 孙凤霞, 李晓玲, 等. 茵陈蒿汤在肝胆疾病中临床应用[J]. 辽宁中医药大学学报, 2020, 22( 3): 57- 60. DOI: 10.13194/j.issn.1673-842x.2020.03.016.
[22]	ZHANG JY, LIU XK, WU JR, et al. A bioinformatics investigation into the pharmacological mechanisms of the effect of the Yinchenhao Decoction on hepatitis C based on network pharmacology[J]. BMC Complement Med Ther, 2020, 20( 1): 50. DOI: 10.1186/s12906-020-2823-y.
[23]	LI DH, WU HW, ZHANG SJ, et al. New progress in pharmacological action of Yinchenhao Decoction[J]. Gansu Sci Technol, 2021, 37( 11): 151- 154. DOI: 10.3969/j.issn.1000-0952.2021.11.045. 李东辉, 吴红伟, 张淑娟, 等. 茵陈蒿汤药理作用研究新进展[J]. 甘肃科技, 2021, 37( 11): 151- 154. DOI: 10.3969/j.issn.1000-0952.2021.11.045.
[24]	YUAN HS, PRATTE J, GIARDINA C. Ferroptosis and its potential as a therapeutic target[J]. Biochem Pharmacol, 2021, 186: 114486. DOI: 10.1016/j.bcp.2021.114486.
[25]	XIONG H, ZHANG AH, GUO YJ, et al. A clinical and animal experiment integrated platform for small-molecule screening reveals potential targets of bioactive compounds from a herbal prescription based on the therapeutic efficacy of Yinchenhao Tang for jaundice syndrome[J]. Engineering, 2021, 7( 9): 213- 238.
[26]	YAO YQ, CAO Q, WANG X, et al. Identification of constituents in vitro and blood-absorbed ingredients of protective effect on acute liver injury from Yin Chen Hao Decoction based on UPLC-QTOF/MS[J]. Acta Pharm Sin, 2023, 58( 5): 1173- 1180. DOI: 10.16438/j.0513-4870.2022-1139. 姚一青, 曹奇, 王璇, 等. 基于UPLC-QTOF/MS的茵陈蒿汤体外成分鉴定和急性肝损伤防护作用入血成分研究[J]. 药学学报, 2023, 58( 5): 1173- 1180. DOI: 10.16438/j.0513-4870.2022-1139.
[27]	CHEN YT, LUO GM, ZHOU L, et al. Discussion on the mechanism of Yinchenhao Decoction in treating intrahepatic cholestasis of pregnancy based on network pharmacology-molecular docking[J]. Mod Tradit Chin Med Mater Med World Sci Technol, 2021, 23( 5): 1588- 1597. DOI: 10.11842/wst.20210105007. 陈云婷, 罗光明, 周丽, 等. 基于网络药理学-分子对接探讨茵陈蒿汤治疗妊娠期肝内胆汁淤积症的作用机制[J]. 世界科学技术-中医药现代化, 2021, 23( 5): 1588- 1597. DOI: 10.11842/wst.20210105007.
[28]	SHI XH, MANG J, XU ZX. Research progress in cell death modes of cerebral ischemia-reperfusion injury[J]. J Jilin Univ:Med Edit, 2022, 48( 6): 1635- 1643. DOI: 10.13481/j.1671-587X.20220633. 石晓花, 莽靖, 徐忠信. 脑缺血再灌注损伤细胞死亡模式的研究进展[J]. 吉林大学学报(医学版), 2022, 48( 6): 1635- 1643. DOI: 10.13481/j.1671-587X.20220633.
[29]	YUAN Y, ZHAI YY, CHEN JJ, et al. Kaempferol ameliorates oxygen-glucose deprivation/reoxygenation-induced neuronal ferroptosis by activating Nrf2/SLC7A11/GPX4 axis[J]. Biomolecules, 2021, 11( 7): 923. DOI: 10.3390/biom11070923.
[30]	YANG WC, WANG YX, ZHANG CG, et al. Maresin1 protect against ferroptosis-induced liver injury through ROS inhibition and Nrf2/HO-1/GPX4 activation[J]. Front Pharmacol, 2022, 13: 865689. DOI: 10.3389/fphar.2022.865689.
[31]	LEI XG, ZHU JH, CHENG WH, et al. Paradoxical roles of antioxidant enzymes: Basic mechanisms and health implications[J]. Physiol Rev, 2016, 96( 1): 307- 364. DOI: 10.1152/physrev.00010.2014.
[32]	TANG HM, TANG HL. Cell recovery by reversal of ferroptosis[J]. Biol Open, 2019, 8( 6): bio043182. DOI: 10.1242/bio.043182.