Liang Jingjing, Ran Yuanyuan, Hu Changbin, Zhou Jie, Ye Lin, Su Wei, Liu Zongjian, Xi Jianing
Beijing Rehabilitation Hospital, Capital Medical University, Beijing, China.
School of Materials Science and Engineering, Beijing Institute of Technology, Beijing, China.
Int Immunopharmacol. 2025 Jan 27;146:113931. doi: 10.1016/j.intimp.2024.113931. Epub 2024 Dec 28.
Pulmonary fibrosis (PF) is a chronic, progressive, and irreversible lung interstitial disease of unknown etiology with a fatal outcome. M2 macrophages have been recognized to play a significant role in PF pathogenesis. The role of protein hypoxia-inducible factor 1-α (HIF-1α) in M2 macrophage polarization in PF is largely unknown. This study aimed to investigate the role of macrophage HIF-1α in the regulation of PF.
PF was induced in C57BL/6 mice by the intratracheal injection of bleomycin (BLM), and small hairpin RNA (shRNA) lentiviral construct specifically targeting HIF-1α were designed for in vitro and in vivo experiments. In the in vitro experiment, bone marrow-derived macrophages (BMDMs) were used to explore molecular mechanism analysis. In the in vivo experiment, mice were administered BLM intratracheally on day 0, treated with shRNA on day 7, and sacrificed on day 21. Histopathological techniques (H&E and Masson's trichrome staining) were used to evaluate PF severity. Western blot, immunofluorescence, quantitative real-time PCR, and flow cytometry were performed to explore the underlying mechanisms.
HIF-1α was upregulated and macrophages polarized toward M2 phenotype in BLM-induced mouse pulmonary fibrosis models. By constructing HIF-1α knockdown shRNA lentiviral construct, we found that the knockdown of HIF-1α in macrophages significantly suppressed M2-type polarization in vitro, hence alleviating fibrosis in lung epithelial cells. Further results revealed that HIF-1α in macrophages promoted M2-type polarization by mediating the signal transducer and activator of transcription 6 (STAT6) arginine methylation. Meanwhile, its arginine methylation modification site is at position Arg27. Further experiments indicated that the regulation of STAT6 arginine methylation by HIF-1α mainly depended on the protein arginine methyltransferase 1 (PRMT1). Finally, animal experiments demonstrated that Knockdown of HIF-1α, PRMT1, and STAT6 relieved the BLM-induced pulmonary fibrosis of mice.
HIF-1α may act as a novel factor to promote macrophage of the M2 program. Therapeutic approaches to target macrophage HIF-1α may act as a new therapeutic strategy to combat PF in the future.
肺纤维化（PF）是一种病因不明的慢性、进行性且不可逆的肺间质疾病，预后不良。M2巨噬细胞在PF发病机制中发挥重要作用。蛋白缺氧诱导因子1-α（HIF-1α）在PF中M2巨噬细胞极化中的作用在很大程度上尚不清楚。本研究旨在探讨巨噬细胞HIF-1α在PF调控中的作用。
通过气管内注射博来霉素（BLM）诱导C57BL/6小鼠发生PF，并设计特异性靶向HIF-1α的小发夹RNA（shRNA）慢病毒构建体用于体外和体内实验。在体外实验中，使用骨髓来源的巨噬细胞（BMDM）进行分子机制分析。在体内实验中，小鼠在第0天经气管内给予BLM，在第7天用shRNA处理，并在第21天处死。采用组织病理学技术（苏木精-伊红染色和Masson三色染色）评估PF严重程度。进行蛋白质免疫印迹、免疫荧光、定量实时聚合酶链反应和流式细胞术以探究潜在机制。
在BLM诱导的小鼠肺纤维化模型中，HIF-1α上调且巨噬细胞向M2表型极化。通过构建HIF-1α敲低的shRNA慢病毒构建体，我们发现巨噬细胞中HIF-1α的敲低在体外显著抑制M2型极化，从而减轻肺上皮细胞中的纤维化。进一步结果显示，巨噬细胞中的HIF-1α通过介导信号转导和转录激活因子6（STAT6）的精氨酸甲基化促进M2型极化。同时，其精氨酸甲基化修饰位点位于第27位精氨酸。进一步实验表明，HIF-1α对STAT6精氨酸甲基化的调控主要依赖于蛋白精氨酸甲基转移酶1（PRMT1）。最后，动物实验表明，敲低HIF-1α、PRMT1和STAT6可减轻BLM诱导的小鼠肺纤维化。
HIF-1α可能作为促进M2程序巨噬细胞的新因子。靶向巨噬细胞HIF-1α的治疗方法可能成为未来对抗PF的新治疗策略。
