地榆单体TMEA通过c-kit/PI3K/Akt途径促巨核细胞分化的分子机制

Bleeding caused by thrombocytopenia is a great challenge in clinic for a long time, in which the proliferation and differentiation of megakaryocytes plays a key role in the treatment of thrombocytopenia. Recently, it is reported that the TPO/c-Mpl classic pathway improves the proliferation of megakaryocytes, but have little effect on the differentiation of megakaryocytes and thrombocytopenia. Therefore, the TPO/c-Mpl classic pathway can not explain the regulatory mechanism so far. Our previous studies showed that the TMEA from Sanguisorba officinalis L., an important hemostatic traditional Chinese medicine (TCM), have the significant stimulative effect on the proliferation and differentiation of megakaryocytes. Moreover, TMEA can up-regulate the mRNA expression of c-kit, phosphorylation-level of Akt protein and transcription of GATA-1, rather than the influence of c-Mpl expression. Therefore, we hypothesis that trimethyl ellagic acid promotes the differentiation of megakaryocytes via c-kit/PI3K/Akt pathway instead of TPO/c-Mpl classic pathway. The purpose of this study is aim to investigate the effect of TMEA on the differentiation of megakaryocytes and the associated mechanisms, using the models of c-Mpl-/- (c-Mpl knocked out) HEL cells and c-Mpl-/- mice by multiple techniques including gene knockout, flow cytometry, cellular and molecular biologic methods. This study may help to illuminate the modern medical support for TCM hemostasis theory of Sanguisorba officinalis L. from a new viewpoint, also provide new strategy for the treatment of thrombocytopenia and development of novel original thrombopoietic agents through c-kit/PI3K/Akt pathway.

血小板减少导致的出血长期困扰临床，加速巨核细胞增殖与分化是促血小板生成的关键环节。TPO/c-Mpl通路被认为是调控血小板生成的经典途径，但其激动剂促巨核细胞分化作用不明显且疗效欠佳。课题组前期从临床止血中药地榆中发现三甲基逆没食子酸（TMEA）能显著促进巨核细胞增殖与分化，提高血小板产量；但对TPO受体（c-Mpl）无明显作用，却可上调c-kit表达，促进Akt磷酸化，增强GATA-1转录；提示：TMEA通过c-kit/PI3K/Akt通路而非TPO/c-Mpl途径促巨核细胞分化。为此，本项目拟采用HEL c-Mpl-/-巨核细胞及c-Mpl-/-基因敲除小鼠为模型，应用流式细胞术、分子生物及药理学方法研究地榆单体TMEA促巨核细胞分化的体内、外活性及治疗作用，探讨对巨核细胞的形态、功能及信号通路的调控机制。从新的视角阐释地榆止血功效的科学内涵，也为发现新型促血小板生成药物提供新的思路。

血小板减少导致的出血长期困扰临床，加速巨核细胞增殖与分化是促血小板生成的关键环节。TPO/c-Mpl通路被认为是调控血小板生成的经典途径，课题组前期从临床止血中药地榆中发现三甲基逆没食子酸（TMEA）能显著促进巨核细胞增殖与分化，提高血小板产量，作用过程中发现TMEA可上调c-kit表达，促进Akt磷酸化，增强GATA-1转录。本课题设想TMEA通过c-kit/PI3K/Akt通路促巨核细胞分化。为此，本项目采用HEL c-Mpl-/-巨核细胞及c-Mpl-/-基因敲除小鼠为模型，应用流式细胞术、分子生物及药理学方法研究TMEA促巨核细胞分化的体内外活性及治疗作用。体外实验研究显示：TMEA能够明显浓度依赖的促进HEL和Meg-01细胞体积及多核细胞数目的增多，鬼笔环肽染色显示TMEA显著促进了微管蛋白的胞内表达以及巨核细胞的多倍体化，流式细胞术结果显示TMEA能促进CD41+CD42b+表达，β1-tublin染色显示TMEA干预细胞后细胞质伸出细丝，多倍体的数量增加，核荧光显著增强；透射电镜结果表明TMEA诱导巨核细胞分化成熟并呈现出血小板特征的形态。体内实验结果显示：TMEA能促进血小板减少症小鼠模型的外周血小板水平和白细胞水平升高，对红细胞水平无影响，且能增加肝脏指数，对肝脏具有保护作用；TMEA和TPO对c-Mpl-/-小鼠血小板水平和脏器指数无影响；TMEA不影响WT小鼠血小板水平和脏器指数，但TPO可显著促进WT小鼠血小板水平升高；且TMEA能明显增加辐射损伤小鼠外周血细胞、骨髓有核细胞和脾脏细胞表面抗原CD41与CD61的表达。机制研究结果显示：TMEA可上调转录因子GATA-1，RUNX-1，NF-E2和FLi-1的mRNA表达，且促进核转录因子GATA-1和NF-E2的核转位。TMEA可激活TPOR信号，上调mTOR和ERK信号通路蛋白的表达，及转录因子GATA-1和NF-E2以及β1-tublin的表达，另PI3K和ERK途径抑制剂能分别抑制相关通路蛋白的表达。上述结果提示，TMEA为一种新型结构的TPO通路激动剂，本项目从新的视角阐释地榆止血功效的科学内涵，也为发现新型促血小板生成药物提供新的思路。

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