铁调素调节巨噬细胞极化发挥抗纤维化作用的研究  

Anti-fibrotic Role of Hepcidin through Modulation of Macrophage Polarization

作　　者：荣子毓 刘莹莹 王晓凡 黄龙 程玮 范旭[1] 王雪 刘琳[1] 王萍[1] 刘天会[1] 丛敏[1] RONG Ziyu;LIU Yingying;WANG Xiaofan;HUANG Long;CHENG Wei;FAN Xu;WANG Xue;LIU Lin;WANG Ping;LIU Tianhui;CONG Min(Liver Research Center,Beijing Friendship Hospital,Capital Medical University,State Key Laboratory of Digestive Health and National Clinical Research Center of Digestive Disease,Beijing 100050,China)

机构地区：[1]首都医科大学附属北京友谊医院肝病中心、消化健康全国重点实验室暨国家消化系统疾病临床医学研究中心,北京100050

出　　处：《中国生物工程杂志》2025年第2期13-24,共12页China Biotechnology

基　　金：国家自然科学基金(82170614);王宝恩肝纤维化基金(CFHPC2021042)资助项目。

摘　　要：目的:探究铁调素(hepcidin)在肝纤维化过程中发挥的抗纤维化作用及其调控的主要靶细胞。方法:构建含有hepcidin的重组腺相关病毒AAV-hepcidin,肝组织切片Masson染色和实时荧光定量PCR(qPCR)评估过表达hepcidin对四氯化碳肝纤维化模型的保护作用。利用qPCR、免疫印迹及免疫荧光标记检测膜铁转运蛋白(ferroportin,FPN)在小鼠原代肝星状细胞(p-HSCs)和巨噬细胞RAW 264.7中的表达差异。检测含有hepcidin的条件培养基对转化生长因子-β1(transforming growth factor-β1,TGF-β1)刺激下p-HSCs活化的作用,检测柠檬酸铁铵(FAC)对p-HSCs活化及铁死亡的影响,以及hepcidin对RAW 264.7极化的影响。结果:冰冻切片和酶联免疫吸附试验(ELISA)结果显示,hepcidin在小鼠肝脏内实现高表达。肝组织Masson染色和qPCR结果显示,发生肝纤维化时,过表达hepcidin的小鼠肝组织纤维化程度较对照组减轻。FPN在RAW 264.7中的表达量高于其在p-HSCs的表达量。经含有hepcidin的条件培养基和TGF-β1处理后,q-PCR和蛋白质印迹法结果显示,hepcidin对p-HSCs的活化无明显抑制作用。经FAC刺激后,FAC对p-HSCs的活化及铁死亡相关指标无明显作用。含有hepcidin的条件培养基和脂多糖(LPS)处理RAW 264.7后,炎症相关基因及蛋白质的表达水平均较对照组下降,说明hepcidin可以有效抑制巨噬细胞向M1型极化。结论:Hepcidin在肝纤维化进展过程中起保护作用,其主要通过抑制小鼠肝巨噬细胞向M1型转化发挥抗纤维化作用。Objective:This study aims to investigate the anti-fibrotic effects of hepcidin during the progression of liver fibrosis and to identify the primary cellular targets involved.Methods:To ensure stable,high levels of hepcidin expression in vivo,a recombinant adeno-associated virus carrying the hepcidin gene(AAV-hepcidin)was constructed and administered to mice by tail vein injection.The transduction efficiency of AAV-hepcidin was verified by detecting green fluorescence in frozen liver tissue sections,while serum hepcidin levels were quantified by enzyme-linked immunosorbent assay(ELISA)six weeks post-injection.To evaluate the anti-fibrotic potential of hepcidin,liver fibrosis was induced in mice by repeated intraperitoneal injections of carbon tetrachloride(CCl4),a known hepatotoxin.Fibrosis progression was assessed by Masson’s trichrome staining,which reveals collagen deposition in liver sections,and quantitative real-time PCR(qPCR)analysis of liver tissue to measure fibrosis-related gene expression levels.Differential expression of the membrane iron transporter ferroportin(FPN)in primary hepatic stellate cells(p-HSCs)and RAW 264.7 macrophages was assessed using qPCR,Western blotting,and immunofluorescence staining to determine the target-specific effects of hepcidin.Additionally,the influence of hepcidin-conditioned medium on p-HSCs activation under transforming growth factor-β1(TGF-β1)stimulation and the effects of ferric ammonium citrate(FAC)on markers of ferroptosis and p-HSCs activation were investigated.To evaluate hepcidin’s impact on immune modulation,the effect of hepcidin-conditioned medium on RAW 264.7 macrophage polarization under lipopolysaccharide(LPS)stimulation was examined by analyzing the mRNA and protein expression levels of inflammation-related markers by qPCR and Western blotting.Results:Frozen sections and ELISA results confirmed stable and high hepcidin expression in mouse liver.Compared to controls,hepcidin overexpression was associated with reduced liver fibrosis as evidenced by Mass

关 键 词：肝纤维化 肝星状细胞 巨噬细胞 铁调素 膜铁转运蛋白 

分 类 号：Q593.1[生物学—生物化学]