基于SphK2/S1P/S1PR1信号途径研究地榆“补血助止血”功效内涵

As is well-known, Chinese herb Sanguisorba officinalis L. (SOL) excels at cooling blood, hemostasis, detoxification and healing sore. However, according to Ben Cao Xin Bian, the hemostatic efficacy of SOL is related to blood-tonic efficacy. The efficacy connotation of SOL-mediated “Blood Nourishment Assisting in Hemostasis” is unclear. Our previous research showed that ingredients of SOL could increase the peripheral level of complete blood cells. The subsequent research found that saponins of SOL were capable of promoting the proliferation of bone marrow cells through upregulation of TPO receptors expression. Ellagic acids of SOL could facilitate proliferation and differentiation of colony-forming unit-megakaryocytes, of which the molecular mechanism involved enhancive phosphorylated Akt and transcription factor GATA-1 expression, without binding TPO receptors. But, ellagic acids were of high affinity for S1PR1 by molecular docking approach. So, it is speculated that besides TPO/c-Mpl, SphK2/S1P/S1PR1 may be another mechanism that participates in SOL ingredients-induced thrombopoiesis. Therefore, we put forward scientific hypothesis such as “The efficacy of Sanguisorba Officinalis L.-mediated blood nourishment assisting in hemostasis is related to SphK2/S1P/S1PR1 signaling pathway.” In this study, we will investigate: ①the effect of SOL decoction on hematopoietic and hemostatic function in blood-deficient mice; ②the effect of SOL decoction on hematopoietic and hemostatic function in gene-knockout mice; ③the effect of SOL decoction and SOL-containing serum on proliferation and differentiation in gene-knockout HEL cells along with regulatory mechanism. We tend to elucidate the efficacy connotation of SOL-mediated “Blood Nourishment Assisting in Hemostasis” with a new perspective and provide new ideas for novel platelet-promoting drugs.

中药地榆以凉血止血、解毒敛疮见长，然《本草新编》记载其止血功效与“补血”相关，但其功效内涵不明。前期研究发现，地榆成分可升高外周全血细胞水平。其中，皂苷类物质通过上调TPO受体表达以促进骨髓细胞增殖；鞣花酸类物质通过提高Akt磷酸化水平和GATA-1表达以促进巨核系祖细胞增殖和分化。计算机模拟分子对接发现，鞣花酸类成分与S1PR1具有显著结合力。由此推测，除TPO/c-Mpl外，SphK2/S1P/S1PR1是地榆“补血助止血”的另一可能机制。由此我们提出科学假说：“地榆补血助止血功效与SphK2/S1P/S1PR1信号途径有关”。本项目拟研究：①地榆水煎液对血虚模型小鼠造血和止血功能的作用；②地榆水煎液对基因敲除小鼠造血和止血功能的作用；③地榆水煎液和含药血清对基因敲除巨核细胞增殖和分化的作用和调控机制。以新视角阐释地榆“补血助止血”功效内涵，也为发现新型促血小板生成药物提供新思路。

背景：.中药地榆以凉血止血、解毒敛疮见长，然《本草新编》记载其止血功效与“补血”相关，但其功效内涵不明。前期研究发现，地榆成分可升高外周全血细胞水平。其中，皂苷类物质通过上调TPO受体表达以促进骨髓细胞增殖；鞣花酸类物质通过提高Akt磷酸化水平和GATA-1表达以促进巨核系祖细胞增殖和分化。计算机模拟分子对接发现，鞣花酸类成分与S1PR1具有显著结合力。由此推测，除TPO/c-Mpl外，尚存在其他分子信号机制参与地榆“补血助止血”功效发挥。基于此研究地榆“补血助止血”功效内涵。.主要研究内容：.（1）中药地榆水煎液（水提取物干燥品）制备及指纹图谱鉴定。.（2）基于X射线辐照小鼠模型与c-mpl基因敲除小鼠模型研究地榆水煎液促血小板生成效应及其止血功能。.（3）基于X射线辐照小鼠模型研究地榆水煎液促血小板生成分子机制。.（4）基于细胞模型研究地榆水提取物干燥品促巨核细胞分化效应与分子机制。.（5）地榆单体成分分离与鉴定。.（6）地榆活性成分促巨核细胞分化/血小板生成效应与分子机制研究。.重要结果：.（1）地榆水煎液显著提高辐照小鼠外周血血小板计数以及骨髓巨核细胞数目，并显著抑制骨髓细胞凋亡。.（2）地榆水煎液有恢复c-mpl-/-小鼠外周血血小板计数的作用趋势。.（3）上调MAPK信号是地榆水煎液促巨核细胞分化/血小板生成的主要分子机制。.（4）地榆水提取物干燥品显著促进K562细胞的巨核分化，并提高K562细胞MEK和ERK的磷酸化水平。.（5）地榆活性成分TMEA和DMAG均能显著促进HEL细胞的巨核分化以及提高辐照小鼠外周血血小板计数；作用机制与分别激活PI3K/Akt/mTOR和HIF-1β/ERK1/2/NF-E2分子信号有关。.科学意义：.本研究为揭示地榆“补血助止血”功效的科学内涵提供实验依据，为扩大地榆临床应用奠定理论基础。地榆活性成分的探索为寻找促血小板生成的新靶点药物提供新思路。