Zhejiang Key TCM Laboratory for Chinese Resource Innovation and Transformation, Jinhua Academy, School of Pharmaceutical Sciences, The Third Affiliated Hospital, Academy of Chinese Medical Science, Zhejiang Chinese Medical University, Hangzhou, Zhejiang, 310053, China.
Department of Oncology, Chongqing Traditional Chinese Medicine Hospital, Chongqing, 400021, China.
J Ethnopharmacol. 2023 Sep 15;313:116533. doi: 10.1016/j.jep.2023.116533. Epub 2023 Apr 24.
Myelosuppression, also known as bone marrow suppression (BMS), is a pathological phenomenon of the decrease in the production of blood cells and further lead to immune homeostasis disorder. Astragalus mongholicus Bunge (AM, checked with The World Flora Online, http://www.worldfloraonline.org, updated on January 30, 2023) is a traditional Chinese medicine with efficacy of tonifying Qi and strengthening body immunity in thousands of years of clinical practice in China. Astragaloside IV (AS-IV) is a major active ingredient of AM, which plays an important role in regulating immune system through different ways.
This study was aimed to investigate the protective effect and mechanism of AS-IV on macrophages in vitro and cyclophosphamide (CTX)-induced immunosuppressive mice in vivo, and to provide experimental basis for the prevention and treatment of AS-IV in myelosuppression.
Based on network pharmacology and molecular docking technology, the core targets and signaling pathways of saponins of AM against myelosuppression were screened. And then, the immunoregulatory effect of AS-IV on RAW264.7 cells was investigated by cellular immune activity and cellular secretion analysis in vitro. In this way, the effects of AS-IV on the main potential targets of HIF-1α/NF-κB signaling pathway were analyzed by qRT-PCR and Western blot methods. Furthermore, comprehensive analysis of the effects of AS-IV against CTX-induced mice were conducted on the basis of immune organs indices analysis, histopathological analysis, hematological analysis, natural killer cell activity analysis and spleen lymphocyte transformation activity analysis. In order to further verify the relationship between active ingredients and action targets, drug inhibitor experiments were finally conducted.
AS-IV, as a potential anti-myelosuppressive compound, was screened by systematic pharmacological methods to act on target genes including HIF1A and RELA together with the HIF-1α/NF-κB signaling pathway. Further studies by molecular docking technology showed that AS-IV had good binding activity with HIF1A, RELA, TNF, IL6, IL1B and other core targets. Besides, cellular and animal experiments validation results showed that AS-IV could enhance the migration and phagocytosis of RAW264.7 cells, and protect the immune organs such as spleen and thymus together with bone tissues from damage. By this means, immune cell function including spleen natural killer cell and lymphocyte transformation activity were also enhanced. In addition, white blood cells, red blood cells, hemoglobin, platelets and bone marrow cells were also significantly improved in the suppressed bone marrow microenvironment (BMM). In kinetic experiments, the secretion of cytokines such as TNF-α, IL-6 and IL-1β were increased, and IL-10, TGF-β1 were decreased. The key regulatory proteins such as HIF-1α, NF-κB, PHD3 in HIF-1α/NF-κB signaling pathway were also regulated in the results of upregulated expression of HIF-1α, p-NF-κB p65 and PHD3 at the protein or mRNA level. Finally, the inhibition experiment results suggested that AS-IV could significantly improve protein response in immunity and inflammation such as HIF-1α, NF-κB and PHD3.
AS-IV could significantly relieve CTX-induced immunosuppressive and might improve the immune activity of macrophages by activating HIF-1α/NF-κB signaling pathway, and provide a reliable basis for the clinical application of AS-IV as a potentially valuable regulator of BMM.
骨髓抑制，又称骨髓抑制（BMS），是血细胞生成减少的病理现象，进一步导致免疫稳态紊乱。蒙古黄芪（AM，经《世界植物在线》[http://www.worldfloraonline.org]核对，更新于 2023 年 1 月 30 日）是一种在中国数千年临床实践中具有补气、增强身体免疫力功效的中药。黄芪甲苷（AS-IV）是 AM 的主要活性成分之一，通过不同途径在调节免疫系统中发挥重要作用。
本研究旨在探讨 AS-IV 对体外巨噬细胞和环磷酰胺（CTX）诱导的免疫抑制小鼠的保护作用及其机制，为 AS-IV 防治骨髓抑制提供实验依据。
基于网络药理学和分子对接技术，筛选 AM 皂苷类化合物防治骨髓抑制的核心靶标和信号通路。然后，通过细胞免疫活性和细胞分泌分析，在体外研究 AS-IV 对 RAW264.7 细胞的免疫调节作用。通过 qRT-PCR 和 Western blot 方法，分析 AS-IV 对 HIF-1α/NF-κB 信号通路主要潜在靶标蛋白的影响。此外，基于免疫器官指数分析、组织病理学分析、血液学分析、自然杀伤细胞活性分析和脾淋巴细胞转化活性分析，综合评价 AS-IV 对 CTX 诱导的小鼠的作用。为了进一步验证活性成分与作用靶点之间的关系，最后进行了药物抑制剂实验。
AS-IV 作为一种潜在的抗骨髓抑制化合物，通过系统药理学方法筛选出与 HIF-1α/NF-κB 信号通路相关的靶基因，包括 HIF1A 和 RELA。进一步的分子对接技术研究表明，AS-IV 与 HIF1A、RELA、TNF、IL6、IL1B 等核心靶标具有良好的结合活性。此外，细胞和动物实验验证结果表明，AS-IV 可增强 RAW264.7 细胞的迁移和吞噬作用，保护脾、胸腺等免疫器官和骨骼免受损伤。通过这种方式，还增强了脾自然杀伤细胞和淋巴细胞转化活性等免疫细胞功能。此外，在骨髓微环境（BMM）中，白细胞、红细胞、血红蛋白、血小板和骨髓细胞也得到了显著改善。在动力学实验中，TNF-α、IL-6 和 IL-1β 等细胞因子的分泌增加，而 IL-10、TGF-β1 则减少。HIF-1α/NF-κB 信号通路中的关键调节蛋白，如 HIF-1α、p-NF-κB p65 和 PHD3，在蛋白或 mRNA 水平上也表现出上调表达。最后，抑制实验结果表明，AS-IV 可显著改善 HIF-1α、NF-κB 和 PHD3 等免疫和炎症相关蛋白的反应。
AS-IV 可显著缓解 CTX 诱导的免疫抑制，并通过激活 HIF-1α/NF-κB 信号通路，提高巨噬细胞的免疫活性，为 AS-IV 作为一种有价值的 BMM 调节剂的临床应用提供可靠依据。
