The Mechanism of Linggui Zhugan Decoction (苓桂术甘汤) in Inhibiting Ferroptosis for Treating Nonalcoholic Steatohepatitis via the Hepcidin-Ferroportin Signaling Pathway
Author：TIAN Tian1, FAN Lirong2,3, LI Lan1, WU Rui1, GUO Xuan1, GAO Zhiyuan1, SU Xiuhai1,4, ZHANG Junling5
Unit：1.Cangzhou Integrated Traditional Chinese and Western Medicine Hospital, Cangzhou Hebei 061001, China; 2.Botou City Traditional Chinese Medicine Hospital, Cangzhou Hebei 062150, China; 3.Fanjilong Renowned Elder Chinese Medicine Doctor Expert Studiol, Cangzhou Hebei 062150, China; 4.Su Xiuhai Renowned Elder Chinese Medicine Expert Studio, Cangzhou Hebei 061001, China; 5.Caofeidian Vocational and Technical College, Tangshan Hebei 063200, China
Quote：引用：田甜，范立荣，李兰，武锐，郭煊，高志远，苏秀海，张俊玲.基于Hepcidin-Ferroportin信号通路研究苓桂术甘汤抑制铁死亡治疗非酒精性脂肪性肝炎的机制[J].中医药导报,2026,32(2):24-31.
DOI：10.13862/j.cn43-1446/r.2026.02.005
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Abstract：
Objective: To investigate the therapeutic
effect of Linggui Zhugan decoction (LGZG) on mice with nonalcoholic
steatohepatitis (NASH) and its impact on ferroptosis mediated by the
Hepcidin-Ferroportin signaling pathway. Methods: Totally 65 mice were randomly
divided into a normal group (n=10) and a modeling group (n=55). Mice in the
normal group were fed a standard diet, while a NASH mouse model was established
in the modeling group using a methionine and choline-deficient diet. The 50
successfully modeled mice were then randomly assigned to model group, silibinin
(SFJB) group, low-dose LGZG group, medium-dose LGZG group, and high-dose LGZG
group, 10 rats in each group. Each group received the corresponding drug at
different doses via gavage. After six weeks, body weight and liver weight were
measured, and the liver index was calculated. Serum levels of alanine
aminotransferase (ALT) and aspartate aminotransferase (AST), as well as liver
levels of total cholesterol (TC) and triglycerides (TG), were detected. Liver
histopathological changes were assessed by hematoxylin-eosin (HE) staining, and
the NAFLD activity score (NAS) was evaluated. Frozen liver sections were
stained with Oil Red O to observe lipid distribution, and the average optical
density (AOD) was calculated to assess hepatic lipid accumulation. Levels of
total iron (Fe), reactive oxygen species (ROS), 4-hydroxynonenal (4-HNE), and
malondialdehyde (MDA) in liver tissue
were measured. The expression levels of acyl-CoA synthetase long-chain family
member 4 (ACSL4) mRNA, transferrin (Trf) mRNA, iron-responsive element-binding
protein 2 (IREB2) mRNA, ferritin heavy chain (Fth) mRNA, hepcidin (Hamp), and
ferroportin (Slc40a1) mRNA in mouse liver were detected by qPCR. The protein
expression levels of ACSL4, Transferrin, IREB2, Fth, Hepcidin, and Ferroportin
in mouse liver were detected by Western blotting. Results: Compared with the
normal group, the model group showed significantly lower body weight and a
higher liver index (P<0.01). Compared with the model group, the SFJB,
medium-dose LGZG, and high-dose LGZG groups showed significantly higher body
weight and lower liver index (P<0.01). Compared with the normal group, the
model group showed significantly higher serum ALT and AST levels and hepatic TC
and TG levels (P<0.01). Compared with the model group, the SFJB, medium-dose
LGZG, and high-dose LGZG groups showed significantly lower serum ALT and AST
levels and hepatic TC and TG levels (P<0.05 or P<0.01). HE and Oil Red O
staining showed clear liver structure and orderly hepatic plates in the normal
group, while the model group exhibited obvious hepatocyte cord derangement and
significant steatosis. Compared with the model group, liver injury was
alleviated, lipid droplets were reduced, and hepatocyte structure was more
intact in the low-dose LGZG group. Liver structure was markedly improved,
hepatocyte arrangement tended to be normal, and steatosis was significantly
reduced in the SFJB, medium-dose LGZG, and high-dose LGZG groups. Compared with
the normal group, the model group showed significantly higher NAS and AOD
(P<0.01). Compared with the
model group, the SFJB,
medium-dose LGZG, and high-dose LGZG groups showed significantly lower NAS and
AOD (P<0.05 or P<0.01). Compared with the normal group, the model group
showed significantly higher hepatic Fe, ROS, 4-HNE, and MDA levels (P<0.01).
Compared with the model group, the SFJB, medium-dose LGZG, and high-dose LGZG
groups showed significantly lower hepatic Fe, ROS, 4-HNE, and MDA levels
(P<0.01). Compared with the normal group, the model group showed
significantly higher relative expression levels of ACSL4, Transferrin, IREB2,
and Hepcidin, but significantly lower levels of Fth and Ferroportin, in the
liver (P<0.01). Compared with the model group, the SFJB, medium-dose LGZG,
and high-dose LGZG groups showed significantly lower relative expression levels
of ACSL4, Transferrin and IREB2, but significantly higher level of Fth, in the
liver (P<0.05 or P<0.01). Compared with the model group, the SFJB,
low-dose LGZG, medium-dose LGZG, and high-dose LGZG groups showed significantly
lower relative expression level of Hepcidin and higher level of Ferroportin in
the liver (P<0.05 or P<0.01). Compared with the normal group, the model
group showed significantly higher relative expression levels of ACSL4 mRNA, Trf
mRNA, IREB2 mRNA and Hamp mRNA, but significantly lower levels of Fth mRNA and
Slc40a1 mRNA, in the liver (P<0.01). Compared with the model group, the
SFJB, medium-dose LGZG, and high-dose LGZG groups showed significantly lower
relative expression levels of ACSL4 mRNA, Trf mRNA, IREB2 mRNA and Hamp mRNA,
but significantly higher level of Fth mRNA and Slc40a1 mRNA, in the liver
(P<0.05 or P<0.01). Conclusion: Linggui Zhugan decoction has a
therapeutic effect on NASH mice and it can alleviate hepatic lipid
peroxidation. Its mechanism may be related to regulating the
Hepcidin-Ferroportin signaling pathway to ameliorate ferroptosis.
Key words：nonalcoholic steatohepatitis; Linggui Zhugan decoction; ferroptosis; lipid peroxidation; Hepcidin-Ferroportin pathway; mice
摘要：
目的：研究苓桂术甘汤（LGZG）对非酒精性脂肪性肝炎（NASH）小鼠的治疗效果及其对铁调素（Hepcidin）-铁转运蛋白（Ferroportin）信号通路介导的铁死亡的影响。方法：将65只小鼠随机分为正常组（10只）和造模组（55只）。正常组小鼠常规饲料喂养，造模组小鼠通过蛋氨酸和胆碱缺乏饮食诱导建立NASH小鼠模型。将50只造模成功小鼠随机分为模型组、水飞蓟宾（SFJB）组、LGZG低剂量组、LGZG中剂量组、LGZG高剂量组，每组10只。各组灌胃不同剂量的相应药物。6周后称量各组小鼠体质量、肝脏质量，并计算肝指数；检测血清丙氨酸氨基转移酶（ALT）、天冬氨酸氨基转移酶（AST）水平及肝组织中总胆固醇（TC）、甘油三酯（TG）水平；肝组织HE染色评估肝脏病变情况，并评定非酒精性脂肪性肝病（NAFLD）活动度评分（NAS）；冰冻组织油红O染色观察脂肪在肝组织中的分布情况，并计算平均光密度（AOD）评估小鼠肝组织脂质堆积水平；检测肝组织总铁（Fe）、活性氧（ROS）、4-羟基壬烯醛（4-HNE）、丙二醛（MDA）水平；采用qPCR检测小鼠肝组织酰基辅酶A合成酶长链家族成员4（ACSL4） mRNA、转铁蛋白（Trf） mRNA、铁反应元件结合蛋白2（IREB2） mRNA、铁蛋白重链（Fth） mRNA、铁调素（Hamp） mRNA、铁转运蛋白（Slc40a1）mRNA表达水平；采用Western blotting法检测小鼠肝组织ACSL4、铁传递蛋白（Transferin）、IREB2、Fth、Hepcidin、Ferroportin蛋白表达水平。结果：模型组小鼠体质量低于正常组（P<0.01），肝指数高于正常组（P<0.01）；SFJB组、LGZG中剂量组、LGZG高剂量组小鼠体质量高于模型组（P<0.01），肝指数低于模型组（P<0.01）。模型组小鼠血清ALT、AST水平及肝脏TC、TG水平高于正常组（P<0.01）；SFJB组、LGZG中剂量组、LGZG高剂量组小鼠血清ALT、AST水平及肝脏TC、TG水平均低于模型组（P<0.05或P<0.01）。HE和油红O染色结果显示，正常组小鼠肝脏结构清晰，肝板排列有序；模型组小鼠肝脏出现明显的肝细胞索排列异常，存在显著的脂肪变性特征；与模型组相比，LGZG低剂量组小鼠肝组织损伤有所减轻，脂滴数量减少，肝细胞结构较为完整；SFJB组、LGZG中剂量组和LGZG高剂量组小鼠肝组织结构明显改善，肝细胞排列趋于正常，脂肪变性程度显著减轻。模型组小鼠NAS、AOD高于正常组（P<0.01）；SFJB组、LGZG中剂量组、LGZG高剂量组小鼠NAS、AOD均低于模型组（P<0.05或P<0.01）。模型组小鼠肝组织Fe、ROS、4-HNE、MDA水平高于正常组（P<0.01）；SFJB组、LGZG中剂量组、LGZG高剂量组小鼠肝组织Fe、ROS、4-HNE、MDA水平低于模型组（P<0.01）。模型组小鼠肝脏ACSL4、Transferrin、IREB2、Hepcidin蛋白相对表达量高于正常组（P<0.01），Fth、Ferroportin蛋白相对表达量低于正常组（P<0.01）；SFJB组、LGZG中剂量组、LGZG高剂量组小鼠肝组织ACSL4、Transferrin、IREB2蛋白相对表达量均低于模型组，Fth蛋白相对表达量均高于模型组，差异均有统计学意义（P<0.05或P<0.01）；SFJB组、LGZG低剂量组、LGZG中剂量组及LGZG高剂量组小鼠肝组织Hepcidin蛋白相对表达量均低于模型组，Ferroportin蛋白相对表达量均高于模型组，差异均有统计学意义（P<0.05或P<0.01）。模型组小鼠肝脏ACSL4 mRNA、Trf mRNA、IREB2 mRNA、Hamp mRNA相对表达量高于正常组（P<0.01），Fth mRNA、Slc40a1 mRNA相对表达量低于正常组（P<0.01）；SFJB组、LGZG中剂量组、LGZG高剂量组小鼠肝组织ACSL4 mRNA、Trf mRNA、IREB2 mRNA、Hamp mRNA相对表达量均低于模型组，Fth
mRNA、Slc40a1 mRNA相对表达量均高于模型组，差异均有统计学意义（P<0.05或P<0.01）。结论：苓桂术甘汤对NASH小鼠具有治疗作用，可改善NASH小鼠肝脏脂质过氧化，其作用机制可能与调控Hepcidin-Ferroportin信号通路来改善铁死亡有关。
关键词：非酒精性脂肪性肝炎;苓桂术甘汤；铁死亡；脂质过氧化；Hepcidin-Ferroportin通路；小鼠
Release time：2026-03-05
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