基于TNFα/ERK/NF-E2非经典途径研究地榆酚酸DMAG促进巨核细胞末期分化与成熟的分子机制

血小板减少导致的出血长期困扰临床，治疗的关键是刺激巨核细胞分化与成熟进而促血小板生成。TNFα可正向调控这一过程，但其机制尚未阐明。我们前期研究发现：地榆酚酸（DMAG）为中药地榆促血小板生成的关键成分，核心作用靶点为TNFα，通过上调TNFα表达，浓度依赖地促进巨核细胞分化与成熟，增加胞内微管蛋白表达及ERK磷酸化，触发核内NF-E2高表达，提高小鼠血小板水平，然而DMAG对TPO核心下游通路JAK/STAT无明显影响。由上推测：DMAG通过非依赖TPO的TNFα/ERK/NF-E2通路促巨核细胞分化与成熟。本课题拟以TNFR1-/-巨核细胞及TNFR1-/-小鼠为模型，运用流式细胞术、全荧光小鼠、单细胞测序等证明：①TNFα/ERK为一条新型促血小板生成调控通路；②DMAG通过上调该通路促血小板生成。本课题将从新视角阐释地榆止血功效的科学内涵，有望为发掘新型促血小板生成药物提供新思路。

Bleeding caused by thrombocytopenia is the frequent and serious problem in clinic, while the differentiation and maturation of megakaryocytes play the critical roles to improve the platelet production in the treatment of thrombocytopenia. TNFα/ERK pathway is associated with positive regulation of thrombopoiesis, however, the molecular mechanism has not been fully understood so far. Our previous study found that 3,4'-O-dimethylellagic acid-4-O-α-D-glucoside (DMAG) isolated from Sanguisorba officinalis L. was the most potent compound for the treatment of thrombocytopenia. More interestingly, we found that DMAG did not affect the well-recognized TPO/c-Mpl pathway but remarkabely improve the mRNA expression of TNFα by the qRT-PCR experiments. Furthermore, DMAG could significantly accelerate the differentiation and maturation of megakaryocytes, as well as promote intracellular tubulin expressions and ERK phosphorylations, then induce the consecutively potent expressions of NF-E2 in the megakaryocytic nucleus leading to the terminal platelet production and release. In summary, our findings indicate that DMAG can accelerate the terminal differentiation and maturation of megakaryocytes through the TNFα/ERK/NF-E2 pathway. The present study will use the TNFR1-/- megakaryocytes and TNFR1-/- knockout mice to block the interference of TPO/c-Mpl pathway, then investigate the phenotypes of differentiation and maturation in vitro and in vivo. Meanwhile， we will explore the associated mechanism of DMAG promoting thrombopoiesis by the flow cytometry, full-fluorescent mice, single-cell sequencing and other methods. This research is not only the continuation and development of our previous studies, but also an intensive study of the Sanguisorba officinalis’s hemostatic efficacy, which may make a novel approach for clinical therapeutic strategies.