铁过载诱导的小鼠肝纤维化过程影响巨噬细胞M2极化

doi:10.12122/j.issn.1673-4254.2025.04.02

南方医科大学学报 ›› 2025, Vol. 45 ›› Issue (4): 684-691.doi: 10.12122/j.issn.1673-4254.2025.04.02

• • 上一篇

铁过载诱导的小鼠肝纤维化过程影响巨噬细胞M2极化

俞佳雯¹^,²(), 周薏², 钱春美³, 穆蓝², 阙任烨¹()

^1.上海中医药大学附属上海市中西医结合医院脾胃病科，上海 200082
^2.上海中医药大学附属市中医医院消化内科，，上海 200071
^3.上海中医药大学附属市中医医院实验中心，上海 200071

收稿日期:2024-11-08 出版日期:2025-04-20 发布日期:2025-04-28
通讯作者: 阙任烨 E-mail:18170389858@163.com;824492@qq.com
作者简介:俞佳雯，在读硕士研究生，E-mail: 18170389858@163.com
基金资助:
国家自然科学基金(82204816);上海市青年科技英才扬帆计划(19YF1445200);上海市卫健委中发办青年基金(2018LQ016);上海中医药大学项目(18LK074)

Effects of liver fibrosis induced by iron overload on M2 polarization of macrophages in mice

Jiawen YU¹^,²(), Yi ZHOU², Chunmei QIAN³, Lan MU², Renye QUE¹()

^1.Department of Spleen and Stomach Diseases, Shanghai Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai 200082, China
^2.Department of Gastroenterology, Shanghai Municipal Hospital of Traditional Chinese Medicine Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai 200071, China
^3.Experimental Center, Shanghai Municipal Hospital of Traditional Chinese Medicine Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai 200071, China

Received:2024-11-08 Online:2025-04-20 Published:2025-04-28
Contact: Renye QUE E-mail:18170389858@163.com;824492@qq.com
Supported by:
National Natural Science Foundation of China(82204816)

摘要/Abstract

摘要：

目的观察铁过载诱导的小鼠肝纤维化过程中肝内巨噬细胞极化的演变过程。方法选取32只6~8周龄C57BL/6小鼠，随机分为对照组（n=8）、模型组（n=24）。模型组每日给予外源性右旋糖酐铁（50 mg/kg）制作慢性铁过载诱导的小鼠肝纤维化模型，在造模第3、5、7周分别处死小鼠，随机分为模型第3、5、7周组，8只/组；对照组给予腹腔注射同等剂量的生理盐水。取小鼠肝脏组织进行Masson染色、Sirius Red染色及免疫组织化学染色观察各组小鼠纤维化程度。取血清进行生化分析及比色法检测ALT、AST等观察肝功能。检测血清铁、铁蛋白、肝脏总铁及肝脏亚铁水平观察小鼠肝脏铁过载情况。免疫荧光双标法检测iNOS⁺/F4/80⁺细胞、CD206⁺/F4/80⁺等细胞观察M1、M2型巨噬细胞比例及分布情况。Western blotting法检测Arg-1、iNOS等蛋白表达。ELISA法检测IL-6、IL-10、TNF-α等蛋白水平。RT-PCR法检测CD206 等蛋白mRNA表达。结果与对照组比较，各模型组小鼠汇管区纤维组织增生逐渐加重，肝小叶结构逐渐破坏，逐见假小叶形成。各模型组小鼠α-SMA、COL-1等蛋白表达呈造模时间依赖升高（P<0.01）。各模型组小鼠血清ALT、AST水平升高，血清铁、铁蛋白、肝脏总铁及亚铁含量均升高（P<0.01）。各模型组小鼠M1极化标志物IL-6、TNF-α、iNOS等蛋白表达升高（P<0.01），以第3周为最高。M2极化标志物IL-10、Arg-1蛋白及CD206 mRNA表达与对照组比较在第3周呈升高趋势（P<0.01），第5周、第7周呈下降趋势（P<0.01）。结论铁过载在各阶段诱导巨噬细胞M1极化增加，在肝纤维化早期可诱导巨噬细胞M2极化增加，而中晚期对M2极化具有负调节的作用。

关键词: 铁过载, 右旋糖酐铁, 肝纤维化, 巨噬细胞极化

Abstract:

Objective To observe the evolution of intrahepatic macrophage polarization in mice with liver fibrosis induced by iron overload. Methods Thirty-two C57BL/6 mice (6-8 weeks) were randomized into control group (n=8) and liver fibrosis model group (n=24) induced by aidly intraperitoneal injection of iron dextran. At the 3rd, 5th, and 7th weeks of modeling, 8 mice in the model group were sacrificed for observing liver fibrosis using Masson, Sirius Red and immunohistochemical staining and detecting serum levels of ALT, AST and the levels of serum iron, ferritin, liver total Fe and ferrous Fe. iNOS⁺/F4/80⁺ cells and CD206⁺/F4/80⁺ cells were detected by double immunofluorescence assay to observe the proportion and distribution of M1 and M2 macrophages. The hepatic expressions of Arg-1, iNOS, IL-6, IL-10, and TNF‑α proteins were detected using Western blotting or ELISA, and the expression of CD206 mRNA was detected using RT-PCR. Results The mice in the model group showed gradual increase of fibrous tissue hyperplasia in the portal area over time, structural destruction of the hepatic lobules and formation of pseudolobules. With the passage of time during modeling, the rat models showed significantly increased hepatic expressions of α-SMA and COL-1, elevated serum levels of ALT, AST, Fe, ferritin, and increased liver total Fe and ferrous Fe levels. The expressions of M1 polarization markers IL-6, TNF‑α, and iNOS all increased with time and reached their peak levels at the 3rd week; The expressions of M2 polarization markers (IL-10 and Arg-1 proteins and CD206 mRNA) significantly increased in the 3rd week and but decreased in the 5th and 7th weeks. Conclusion Iron overload promotes M1 polarization of macrophages in mice. Liver fibrosis in the early stage promotes M2 polarization of macrophages but negatively regulate M2 polarization at later stages.

Key words: iron overload, iron dextran, liver fibrosis, macrophage polarization

俞佳雯, 周薏, 钱春美, 穆蓝, 阙任烨. 铁过载诱导的小鼠肝纤维化过程影响巨噬细胞M2极化[J]. 南方医科大学学报, 2025, 45(4): 684-691.

Jiawen YU, Yi ZHOU, Chunmei QIAN, Lan MU, Renye QUE. Effects of liver fibrosis induced by iron overload on M2 polarization of macrophages in mice[J]. Journal of Southern Medical University, 2025, 45(4): 684-691.

图/表 12

表1 引物序列

Tab.1 Primer sequences for RT-qPCR

Gene	Forward primer	Reverse primer
CD206	AGCTTCATCTTCGGGCCTTTG	GGTGACCACTCCTGCTGCTTTAG
β-actin	TTGTAACCAACTGGGACGATATGG	GATCTTGATCTTCATGGTGCTAGG

图1 右旋糖酐铁诱导的慢性铁过载肝脏病理组织学改变

Fig.1 Histopathological changes of mouse liver with chronic iron overload induced by iron dextran (Original magnification: ×100).

图2 右旋糖酐铁诱导的慢性铁过载肝脏病理组织学改变

Fig.2 Histopathological changes of mouse liver with chronic iron overload induced by iron dextran. **P<0.01 vs Control.

图3 右旋糖酐铁诱导的慢性铁过载肝脏α-SMA及COL-1改变

Fig.3 Changes of α-SMA and COL-1 in mouse liver with chronic iron overload induced by iron dextran (×100).

图4 右旋糖酐铁诱导的慢性铁过载肝脏α-SMA及COL-1改变

Fig.4 Changes of α-SMA and COL-1 in mouse liver with chronic iron overload induced by iron dextran. **P<0.01 vs Control.

图5 右旋糖酐铁诱导的慢性铁过载小鼠肝功能改变

Fig.5 Changes of liver function in chronic iron overload mice induced by iron dextran. **P<0.01 vs Control.

图6 右旋糖酐铁诱导的慢性铁过载血清铁及铁蛋白含量改变

Fig.6 Changes of serum iron and ferritin contents in chronic iron overload induced by iron dextran.**P<0.01 vs Control.

图7 右旋糖酐铁诱导的慢性铁过载肝脏总铁及亚铁含量改变

Fig.7 Changes of total iron and ferrous content in liver with chronic iron overload induced by iron dextran.**P<0.01 vs Control.

图8 右旋糖酐铁诱导的慢性铁过载肝脏巨噬细胞极化标志物改变

Fig.8 Changes of markers of liver macrophage polarization in chronic iron overload induced by iron dextran. A: Changes of M1 polarization markers; B, C: Changes of M2 polarization markers. **P<0.01 vs Control.

图9 右旋糖酐铁诱导的慢性铁过载肝脏巨噬细胞M1极化改变

Fig.9 Changes in liver macrophage M1 polarization induced by chronic iron overload (×100).

图10 右旋糖酐铁诱导的慢性铁过载肝脏巨噬细胞M2极化改变

Fig.10 Changes in M2 polarization of liver macrophages in mice with chronic iron overload (×100).

图11 右旋糖酐铁诱导的慢性铁过载肝脏巨噬细胞M1、M2极化改变

Fig.11 Changes in M1 and M2 polarization of liver macrophages in mice with chronic iron overload. **P<0.01 vs Control.

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铁过载诱导的小鼠肝纤维化过程影响巨噬细胞M2极化

Effects of liver fibrosis induced by iron overload on M2 polarization of macrophages in mice

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