目的 探索淫羊藿苷元治疗血小板减少症的直接作用靶点及作用机制。方法 从BALB/c小鼠股骨中分离得到原代骨髓细胞，利用键合淫羊藿苷元的琼脂糖微球捕获并鉴定靶点蛋白，使用表面等离子共振（surface plasmon resonance，SPR）技术进行靶点确证；采用Western blotting检测淫羊藿苷元对胰岛素受体底物1（insulin receptor substrate 1，IRS1）/蛋白激酶B（protein kinase B，Akt）/哺乳动物雷帕霉素靶蛋白（mammalian target of rapamycin，mTOR）/糖原合成酶激酶3β（glycogen synthase kinase 3β，GSK3β）、Hippo、细胞周期、Janus激酶（Janus kinase，JAK）/信号转导与转录激活子（signal transducer and activator of transcription，STAT）和核因子-κB（nuclear factor-κB，NF-κB）通路相关蛋白的调控作用；通过流式细胞术检测淫羊藿苷元对巨核细胞系分化的影响。结果 共发现20个与造血干细胞增殖分化功能密切相关的靶点蛋白，且淫羊藿苷元可能通过作用于泛素羧基末端酯酶L5（ubiquitin carboxyl-terminal esterase L5，UCHL5）、肿瘤坏死因子α诱导蛋白8（tumor necrosis factor α-induced protein 8，TNFAIP8）、Visfatin靶点，进而激活IRS1/Akt/mTOR/GSK3β和细胞周期通路，并抑制Hippo、JAK/STAT和NF-κB通路，最终促进巨核细胞的分化。结论 淫羊藿苷元通过UCHL5、TNFAIP8、Visfatin等多靶点治疗血小板减少症。

Objective To explore the direct target and mechanism of icaritin on treatment of thrombocytopenia. Methods Primary bone marrow cells were isolated from BALB/c mice. Target proteins were captured and identified by icaritin-binding agarose and further confirmed with surface plasmon resonance (SPR). Western blotting was used to detect the regulation of icaritin on insulin receptor substrate 1 (IRS1)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR)/glycogen synthase kinase 3β (GSK3β), Hippo, cell cycle, Janus kinase (JAK)/signal transducer and activator of transcription (STAT) and nuclear factor-κB (NF-κB) signaling pathways related proteins. The effect of icaritin on differentiation of megakaryocytes was clarified by flow cytometry. Results A total of 20 target proteins associated to proliferation and differentiation of hematopoietic stem cells were found. In addition, icaritin activated IRS1/Akt/mTOR/GSK3β, cell cycle signaling pathways, inhibited Hippo, JAK/STAT and NF-κB signaling pathways through potentially interacting with ubiquitin carboxyl-terminal esterase L5 (UCHL5), tumor necrosis factor α-induced protein 8 (TNFAIP8) and Visfatin to promote the differentiation of megakaryocytes. Conclusion Icaritin treats thrombocytopenia through interacting with multiple target proteins including UCHL5, TNFAIP8, and Visfatin.

R285.5

国家自然科学基金资助项目（81973505）；国家自然科学基金资助项目（81773932）；国家重点研发计划项目（2017YFC1702400，2019YFC1708902）