Department of Pharmacology, School of Pharmacy, Southwest Medical University, Luzhou, China; State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, China.
Department of Pharmacology, School of Pharmacy, Southwest Medical University, Luzhou, China.
Phytomedicine. 2022 Jul 20;102:154150. doi: 10.1016/j.phymed.2022.154150. Epub 2022 May 5.
Thrombocytopenia (TP) remains a challenge in clinical hematology. TP may have serious consequences, such as recurrent skin and mucosal bleeding and increased risk of intracranial and internal organ hemorrhage. However, effective and safe therapeutic drugs for the long-term management of TP are still lacking.
This study aimed to identify more effective active compounds for TP therapy.
Liquid chromatography-mass spectrometry-nuclear magnetic resonance analysis was used to confirm the medicinal species and chemical structure of Hirsutine (HS). The proliferation of HS was examined by Cell Counting Kit (CCK-8) assay on cells lines. The effect of HS on megakaryocyte differentiation was analyzed by evaluating the expression of CD41, CD42b, and DNA ploidy via flow cytometry (FCM). The morphology of megakaryocytes and intermediate cells was observed using an optical microscope. K562 cells were then stained with Giemsa and benzidine. qRT-PCR was used to examine the mRNA expression of GATA-1, GATA-2, FOG-1, TAL-1, RUNX-1, NF-E2, and KLF-1 in K562 cells. Protein levels of the transcription factors were analyzed by western blotting. An MEK inhibitor was used to verify the relationship between the MEK/ERK signaling pathway and CD41/CD42b (FCM), FOG-1, and TAL-1. The Kunming thrombocytopenia mouse model was established by X-ray irradiation (4 Gy) and used to test HS activity and related hematopoietic organ index in vivo. Finally, computer simulations of molecular docking were used to predict the binding energies between HS-MEK and HS-ERK.
We preliminarily identified HS by screening a plant-sourced compound library for natural compounds with megakaryocytic differentiation and maturation (MKD/MKM)-promoting activity. We found that HS not only enhanced MKD/MKM of K562 and Meg01 cells, but also suppressed the decline of peripheral platelet levels in X-ray-induced myelosuppressive mice. In addition, HS promoted MKD via activation of MEK-ERK-FOG1/TAL1 signaling, which may be the key molecular mechanism of HS action in TP treatment. Molecular docking simulations further verified that HS targets the signaling protein MEK with high-affinity.
In this study, we report for the first time that hirsutine boosts MKD/MKM through the MEK/ERK/FOG1/TAL1 signaling pathway and thus represents a promising treatment option for TP.
血小板减少症（TP）仍然是临床血液学的一个挑战。TP 可能会产生严重后果，如皮肤和黏膜反复出血，颅内和内脏出血风险增加。然而，用于 TP 长期管理的有效和安全的治疗药物仍缺乏。
本研究旨在寻找更有效的 TP 治疗活性化合物。
采用液质联用-核磁共振分析方法，对胡芦巴碱（HS）的药用品种和化学结构进行确证。通过细胞计数试剂盒（CCK-8）检测细胞系中 HS 的增殖情况。通过流式细胞术（FCM）评估 CD41、CD42b 和 DNA 倍体，分析 HS 对巨核细胞分化的影响。使用光学显微镜观察巨核细胞和中间细胞的形态。然后用吉姆萨和联苯胺对 K562 细胞进行染色。采用 qRT-PCR 检测 K562 细胞中 GATA-1、GATA-2、FOG-1、TAL-1、RUNX-1、NF-E2 和 KLF-1 的 mRNA 表达。采用 Western blot 分析转录因子的蛋白水平。用 MEK 抑制剂验证 MEK/ERK 信号通路与 CD41/CD42b（FCM）、FOG-1 和 TAL-1 的关系。通过 X 射线照射（4Gy）建立昆明血小板减少症小鼠模型，检测 HS 体内活性及相关造血器官指数。最后，采用计算机模拟分子对接预测 HS-MEK 和 HS-ERK 的结合能。
我们通过筛选具有促进巨核细胞分化和成熟（MKD/MKM）活性的天然化合物的植物源化合物库，初步鉴定出 HS。我们发现 HS 不仅增强了 K562 和 Meg01 细胞的 MKD/MKM，还抑制了 X 射线诱导的骨髓抑制小鼠外周血小板水平的下降。此外，HS 通过激活 MEK-ERK-FOG1/TAL1 信号通路促进 MKD，这可能是 HS 治疗 TP 的关键分子机制。分子对接模拟进一步证实 HS 以高亲和力靶向信号蛋白 MEK。
本研究首次报道胡芦巴碱通过 MEK/ERK/FOG1/TAL1 信号通路促进 MKD/MKM，因此代表了治疗 TP 的一种很有前途的治疗选择。
