Lyu Xinyi, Shi Jiahao, Liu Qi, Jiang Mingjun, Liu Xilian, Li Yulan, Ding Shuqin, Dai Xianpeng
The Second Affiliated Hospital, Department of Vascular Surgery, Hengyang Medical School, University of South China, Hengyang, China.
The Second Affiliated Hospital, Department of Endocrinology and Metabolism, Hengyang Medical School, University of South China, Hengyang, China.
Front Immunol. 2025 Mar 21;16:1560589. doi: 10.3389/fimmu.2025.1560589. eCollection 2025.
Long term high-dose erythropoietin (EPO) had been reported inducing the formation of abdominal aortic aneurysm (AAA) in mice. When using this model, we found that EPO treated mice showed significant splenomegaly. This is an interesting phenomenon, and its mechanism has not been reported. Therefore, this study aims to explore its mechanism.
C57BL/6 mice were given intraperitoneal injection of recombinant human EPO at 10000 IU/kg/day, and the control mice were treated with normal saline (vehicle). After 3 weeks, the spleens were harvested. Pathological changes in histology were observed using Hematoxylin and Eosin (H&E) staining. The differential expression genes (DEGs) were identified using RNA sequencing (RNA-Seq), verified with the real-time quantitative polymerase chain reaction (RT-qPCR). The functional-enrichment analysis including Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Reactome enrichment analysis were performed to reveal the functional characteristics and related biological pathways of DEGs. Immunohistofluorescence (IHF) and flow cytometry (FCM) were used to detect immune cell subsets and proliferation markers.
EPO treatment resulted in splenomegaly, spleen microstructure disorder, splenic corpuscular atrophy, indistinct germinal center, and unclear boundary between white and red pulp structures. RNA-Seq showed that EPO treatment suppressed gene expression associated with immune responses, while promoted cell cycle and DNA replication. IHF and FCM validated that, at the cellular level, T, B, M1 cells were significantly reduced, and M2 cells were significantly decreased after EPO treatment. The proliferation analysis showed that the portion of EDU or Ki-67cells consisted of granulocytes and macrophages, and after EPO treatment, only macrophages showed a significant increase in their number and proportion, while granulocytes did not show a significant response to EPO stimulation.
Long term high-dose EPO treatment may lead to splenomegaly and immunosuppression of the local immune microenvironment in mice. The mechanism may be related to the increased anti-inflammatory and immunomodulatory functions caused by M2 cells. The study provides, for the first time, the transcriptomic characteristics and immunological of the spleens of EPO treated mice, providing a new perspective for the study of the effects of EPO on mice.
长期大剂量促红细胞生成素（EPO）已被报道可诱导小鼠腹主动脉瘤（AAA）形成。在使用该模型时，我们发现经EPO处理的小鼠出现显著的脾肿大。这是一个有趣的现象，其机制尚未见报道。因此，本研究旨在探讨其机制。
将C57BL/6小鼠按10000 IU/kg/天的剂量腹腔注射重组人促红细胞生成素，对照小鼠用生理盐水（溶剂）处理。3周后，采集脾脏。用苏木精和伊红（H&E）染色观察组织学病理变化。使用RNA测序（RNA-Seq）鉴定差异表达基因（DEG），并用实时定量聚合酶链反应（RT-qPCR）进行验证。进行包括基因本体论（GO）、京都基因与基因组百科全书（KEGG）和Reactome富集分析在内的功能富集分析，以揭示DEG的功能特征和相关生物学途径。采用免疫组织荧光（IHF）和流式细胞术（FCM）检测免疫细胞亚群和增殖标志物。
EPO处理导致脾肿大、脾脏微观结构紊乱、脾小体萎缩、生发中心不明显、白髓和红髓结构边界不清。RNA-Seq显示，EPO处理抑制了与免疫反应相关的基因表达，同时促进了细胞周期和DNA复制。IHF和FCM验证，在细胞水平上，EPO处理后T、B、M1细胞显著减少，M2细胞显著减少。增殖分析表明，EDU或Ki-67细胞部分由粒细胞和巨噬细胞组成，EPO处理后，只有巨噬细胞的数量和比例显著增加，而粒细胞对EPO刺激未表现出显著反应。
长期大剂量EPO处理可能导致小鼠脾肿大和局部免疫微环境免疫抑制。其机制可能与M2细胞引起的抗炎和免疫调节功能增强有关。本研究首次提供了EPO处理小鼠脾脏的转录组学特征和免疫学特征，为研究EPO对小鼠的影响提供了新的视角。
