MBD2缺陷通过抑制M2巨噬细胞极化减轻CCl诱导的肝纤维化。

MBD2 deficiency attenuates CCl-induced hepatic fibrosis by inhibiting M2 macrophage polarization.

作者信息

Yu Wen, Li Ruihua, Wang Yixian, Wang Ya-Nan, Huang Yaxuan, Zhang Yanxiang, Nie Hao, Liu Lian, Chen Hui, Zheng Bing, Gong Quan

机构信息

Department of Laboratory Medicine, the First Affiliated Hospital of Yangtze University, Jingzhou, Hubei, China; Department of Immunology, School of Medicine, Yangtze University, Jingzhou, China; Department of Clinical Laboratory, The eighth Hospital of Wuhan, Wuhan, Hubei, China.

Department of Laboratory Medicine, the First Affiliated Hospital of Yangtze University, Jingzhou, Hubei, China; Department of Immunology, School of Medicine, Yangtze University, Jingzhou, China.

出版信息

Int Immunopharmacol. 2025 Aug 6;164:115306. doi: 10.1016/j.intimp.2025.115306.

DOI:10.1016/j.intimp.2025.115306

PMID:40773897

Abstract

PURPOSE

Methyl-CpG-binding domain protein 2 (MBD2) serves as a pivotal reader of DNA methylation and has been linked to fibrosis in the kidney and lung. However, its role and the underlying mechanisms in liver fibrosis remain unclear.

MATERIALS AND METHODS

Hepatic fibrosis (HF) was induced in C57BL/6 mice using carbon tetrachloride (CCl). Liver tissues were examined for MBD2 and α-smooth muscle actin (α-SMA) expression at 1, 3, and 5 weeks post-induction. Immunofluorescence analysis was used to detect MBD2 and the macrophage marker F4/80 in liver tissues. To dissect the role of MBD2 in the pathogenesis of HF, a macrophage-specific MBD2 knockout (KO) mouse and their littermates were challenged with CCl for 5 weeks. Serum levels of alanine transaminase (ALT), aspartate transaminase (AST), and lactate dehydrogenase (LDH) were measured. Liver tissue sections were stained with hematoxylin and eosin (H&E), Masson's trichrome, and Sirius red for histopathological examination. Expression of fibrosis-specific proteins, including hydroxyproline (Hyp), α-SMA, and collagen type I (col-1), was assessed. Expression of M2 macrophage markers arginase-1 (Arg-1), YM1, and found in inflammatory zone 1 (FIZZ1), as well as the M1 macrophage marker inducible nitric oxide synthase (iNOS), was evaluated. For in vitro analysis, peritoneal macrophages (PMs) and bone marrow-derived macrophages (BMDMs) from MBD2 KO mice and control littermates were extracted. Macrophage polarization was induced using interleukin-4 (IL-4) to assess the expression of Arg-1, YM1, and FIZZ1.

RESULTS

MBD2 protein expression was found to be significantly increased during HF progression. MBD2 deficiency in macrophages led to reduced serum ALT and AST levels, decreased hepatic tissue damage, lower hepatic Hyp content, and reduced expression of α-SMA and col-1 compared to the control group. Expression of M2 macrophage markers Arg-1, YM1, and FIZZ1 in liver tissues was lower in the MBD2-deficient group, consistent with the results of in vitro macrophage polarization assays. However, there was no significant difference in iNOS expression.

CONCLUSION

The study demonstrates that macrophage-specific MBD2 deletion inhibits M2 macrophage polarization and ameliorates CCl-induced HF in mice.

摘要

目的

甲基化CpG结合域蛋白2（MBD2）是DNA甲基化的关键识别蛋白，与肾脏和肺部纤维化有关。然而，其在肝纤维化中的作用及潜在机制尚不清楚。

材料与方法

使用四氯化碳（CCl）诱导C57BL/6小鼠发生肝纤维化（HF）。在诱导后1、3和5周检查肝组织中MBD2和α平滑肌肌动蛋白（α-SMA）的表达。采用免疫荧光分析检测肝组织中MBD2和巨噬细胞标志物F4/80。为了剖析MBD2在HF发病机制中的作用，用CCl对巨噬细胞特异性MBD2基因敲除（KO）小鼠及其同窝小鼠进行攻击5周。检测血清丙氨酸转氨酶（ALT）、天冬氨酸转氨酶（AST）和乳酸脱氢酶（LDH）水平。肝组织切片用苏木精和伊红（H&E）、Masson三色染色法和天狼星红染色进行组织病理学检查。评估纤维化特异性蛋白的表达，包括羟脯氨酸（Hyp）、α-SMA和I型胶原（col-1）。评估M2巨噬细胞标志物精氨酸酶-1（Arg-1）、Ym1和炎症区1发现的蛋白（FIZZ1）以及M1巨噬细胞标志物诱导型一氧化氮合酶（iNOS）的表达。为了进行体外分析，提取来自MBD2基因敲除小鼠和对照同窝小鼠的腹腔巨噬细胞（PMs）和骨髓来源的巨噬细胞（BMDMs）。使用白细胞介素-4（IL-4）诱导巨噬细胞极化，以评估Arg-1、Ym1和FIZZ1的表达。

结果

发现MBD2蛋白表达在HF进展过程中显著增加。与对照组相比，巨噬细胞中MBD2缺乏导致血清ALT和AST水平降低、肝组织损伤减轻、肝Hyp含量降低以及α-SMA和col-1表达减少。MBD2缺陷组肝组织中M2巨噬细胞标志物Arg-1、Ym1和FIZZ1的表达较低，这与体外巨噬细胞极化试验的结果一致。然而，iNOS表达没有显著差异。