Mechanism by which Angelica sinensis polysaccharide regulates bone marrow hematopoietic microenvironment for aplastic anemia

doi:10.12307/2024.739

Abstract

Abstract:

BACKGROUND: How to improve the hematopoietic microenvironment is a hot topic in the treatment of aplastic anemia.

OBJECTIVE: To explore the action mechanism of Angelica sinensis polysaccharide in the treatment of aplastic anemia by combining GEO sequencing analysis, network pharmacology, and experimental validation.

METHODS: Aplastic anemia-related differentially expressed genes were obtained from GEO database, and gene ontology and gene set enrichment analysis were performed. The active components and targets of Angelica sinensis polysaccharide were obtained by combining the literature with PubChem, SwissTargetPrediction, and PharmMapper databases. After the intersection targets were taken, STRING and Cytoscape were used to construct protein-protein interaction network, and gene ontology and Kyoto encyclopedia of genes and genomes enrichment analysis was performed. Mouse model of aplastic anemia was established, and the effect and action mechanism of Angelica sinensis polysaccharide on aplastic anemia were verified by blood cell analyzer, flow cytometry, ELISA, immunohistochemistry, immunofluorescence, and western blot assay.
RESULTS AND CONCLUSION: (1) A total of 834 differentially expressed genes were screened, which were involved in biological processes such as cell development, hematopoiesis, and myeloid cell differentiation. (2) 347 targets related to Angelica sinensis polysaccharide were retrieved and 77 potential therapeutic genes were screened. Among them, the degree values of angiogenic, apoptotic, and immune-related factors such as VEGFA, EGLN1, Bcl-2, interferon-γ, interleukin-2, interleukin-4, and interleukin-6 were significant. (3) KEGG pathway enrichment analysis revealed that the therapeutic targets were mainly enriched in Th17 cell differentiation, NK-related cytotoxicity, cell adhesion factors such as interferon-γ, interleukin-2, and interleukin-4 related signaling pathways. (4) Animal experiments showed that Angelica sinensis polysaccharide significantly improved bone marrow haematopoiesis, increased peripheral blood cell, and bone marrow single nucleated cell counts, and improved the survival rate of mice. Compared with the model group, mice in the Angelica sinensis polysaccharide group showed a significant decrease in the ratio of Th1/Th2 cells (P < 0.01), a decrease in the expression level of interferon-γ (P < 0.01), an increase in the level of interleukin-4 (P < 0.05), a significant increase in the level of VEGFA (P < 0.01), a significant decrease in EGLN1 (P < 0.01), a significant decrease in apoptosis rate of bone marrow single nucleated cells and reactive oxygen species level (P < 0.01), and a significant increase in cleaved Caspase-3 protein expression (P < 0.01), and a significant decrease in Bcl-2/Bax ratio (P < 0.01). (5) These findings show that Angelica sinensis polysaccharide can improve hematopoiesis of aplastic anemia mice by regulating aberrant T-cell subsets and promoting angiogenesis to improve hematopoietic microenvironment, and inhibiting apoptosis of bone marrow mononuclear cells.

Key words: Angelica sinensis polysaccharide, aplastic anemia, Th1/Th2, bone marrow hematopoietic microenvironment, cell apoptosis

CLC Number:

Fu Jiaqi, Chen Xiubao, Cui Xing, Chen Zetao . Mechanism by which Angelica sinensis polysaccharide regulates bone marrow hematopoietic microenvironment for aplastic anemia[J]. Chinese Journal of Tissue Engineering Research, 2025, 29(1): 44-51.

Figures/Tables 2

2.1 网络药理学分析结果 2.1.1 再生障碍性贫血差异表达基因的获取与分析选取GSE3807数据集进行差异表达基因筛选，包含6个再生障碍性贫血患者和2个健康志愿者的骨髓CD3+T细胞测序数据。R语言分析后获得834个差异表达基因(P < 0.05，|logFC| > 2)，并绘制差异表达基因火山图，见图1A。对差异表达基因进行GO和GSEA富集分析，选取P < 0.01前20的GO功能富集结果进行展示，见图1B，差异表达基因参与造血、髓系细胞分化等生物学过程，激活了免疫受体活性、细胞因子受体活性、生长因子活性等分子功能。随后GSEA富集分析显示参与了凋亡、IFN-γ、IL-6-STAT3等相关通路，见图1C-E。 2.1.2 当归多糖靶点网络构建当归多糖属于植物多糖，根据其结构特点分为同聚多糖和杂多糖，目前所提取的多糖以杂多糖为主，见图2A，主要由半乳糖、半乳糖醛酸、葡萄糖、阿拉伯糖和鼠李糖5种单糖组成[12]，通过Pubchem获得单糖结构，借助数据库获得当归多糖潜在靶点347个，通过STRING数据库并导入Cytoscape获得蛋白互作网络图，见图2B。 2.1.3 当归多糖-再生障碍性贫血靶点网络构建及功能分析将当归多糖与再生障碍性贫血靶点取交集后得到77个潜在治疗靶点，见图3A，随后将潜在靶点导入STRING平台获得蛋白相互作用网络，并在Cytoscape软件中进行拓扑分析，结果显示VEGFA、EGLN1、BCL2、IFN-γ、IL-2、IL-4、IL-6等血管生成、凋亡及免疫相关因子度值显著，见图3B，其中VEGFA表达在再生障碍性贫血中呈现显著下调，EGLN1表达则显著上调，提示当归多糖可能通过这些靶点干预再生障碍性贫血。为进一步探讨当归多糖治疗再生障碍性贫血的机制，对潜在靶点进行GO和KEGG富集分析，设置P < 0.05。GO富集的生物学过程主要涉及造血、炎症反应、细胞生长的正向调节等，见图3C。KEGG通路富集分析表明，潜在治疗靶点富集于多个通路，主要包括Th17细胞分化、NK相关细胞毒性、细胞黏附因子等IFN-γ、IL-2、IL-4相关信号通路，见图3D。 2.2 实验验证结果 2.2.1 当归多糖对小鼠造血功能及存活率的影响与对照组相比，模型组、当归多糖组的外周血细胞数量、骨髓单个核细胞数量和造血组织增生度均明显减少(P < 0.01)，证明造模成功，见图4A-E。与模型组相比，应用当归多糖干预后各项指标均得到一定的恢复(P < 0.01)，小鼠存活率显著提高，见图4F，表明当归多糖对再生障碍性贫血患者的造血功能具有改善作用。"

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