Hirsutine, a novel megakaryopoiesis inducer, promotes thrombopoiesis via MEK/ERK/FOG1/TAL1 signaling

Abstract

BackgroundThrombocytopenia (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. PurposeThis study aimed to identify more effective active compounds for TP therapy. MethodsLiquid 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. ResultsWe 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. ConclusionIn 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.