基于VEGF-A通路PLCγ1-PI3K/Akt反馈机制的蒲黄炭化瘀止血炮制机理研究

Carbonized pollen typha has the effect on removing blood stasis and hemostasis, however, the mechanism of the effect and the pollen processing are still unclear. According to the treatment effects of carbonized pollen typha on eliminating stasis and bleeding in traditional Chinese medicine (TCM), we proposed a hypothesis based on the previous studies about that the pollen charcoal worked via VEGF-A pathway by regulating the balance between the PLCγ1-PI3K/Akt negative feedback pathway. PLCγ1 pathway promote the synthesis of cyclooxygenase, prostaglandin and TXA2, contract uterus and vascular smooth muscle to play a hemostatic effect. PI3K/Akt feedback pathway repair and promote the regeneration of the vascular endothelial cells by regulating eNOS, relaxing blood vessels, reducing vWF levels to inhibit platelet aggregation and thrombus formation. This item plans to select biological indicators which can reveal the blood stasis-eliminating and hemostatic effects of carbonized pollen typha based on the TCM principles. We will application biological separation technology to track different processing degree of carbonized pollen in blood hemostasis material-effect relationship, explore the key steps of the activity in vivo and in vitro model of PLCγ1-PI3K/Akt feedback pathway. The dose-activity and time-activity relationships of carbonized pollen will be analyzed, and the mechanism of the effect and the processing will be expounded in the overall, tissue, cellular and molecular levels. These studies will help for rational application in clinical and provide a scientific basis for processing standards.

蒲黄炭具有化瘀止血作用，但现有研究工作未能全面阐释其止血而不留瘀的作用机制和炮制机理等科学问题。基于蒲黄炭临床应用情况和前期工作基础，提出蒲黄炭通过调控VEGF-A信号通路中PLCγ1传导通路及PI3K/Akt负反馈通路的平衡，起到化瘀止血作用的假说：PLCγ1信号通路促进体内环氧化酶、前列腺素合成，收缩子宫和血管平滑肌，促进TXA2合成、激活凝血系统，起到止血作用；PI3K/Akt反馈通路调节eNOS的水平，舒张血管，下调vWF的活性，抑制血小板聚集，减少血管内皮损伤及血栓形成，促进血管的修复。本项目应用生物导向分离技术追踪不同炮制程度蒲黄炭化瘀止血物质-效应相关性，探索蒲黄炭对体内外评价模型中PLCγ1-PI3K/Akt反馈通路关键步骤的影响，分析其量-效、时-效关系，以期在整体、组织、细胞及分子多层次揭示其化瘀止血的作用机制和炮制机理，为蒲黄炭临床合理应用和制定炮制标准提供科学依据。

蒲黄炭是典型的化瘀止血药物，具有显著的止血不留瘀的双向调节特色。本课题运用现代技术从宏观变化、微观结构、化学成分变化及热力学变化等多角度表征炒炭炮制过程；利用近红外光谱结合数学建模以实现对蒲黄炭炮制程度的快速判别；以UPLC-Q-TOF-MS技术探究炮制过程中小分子化合物的变化，运用整合方法（包括代谢组学、网络药理学、体内高通量筛选模型）筛选出6个质量标志物（isorhamnetin-3-O-neohesperidoside，isorhamnetin-3-O-rutinoside，kaempferol-3-O-neohesperidoside，naringenin，quercetin和isorhamnetin）。多糖是具有良好药效活性的天然产物，蒲黄中的均一多糖可通过VEGF通路促进血管生成，发挥化瘀作用。从过程表征、定性分析和定量标准三个方面以及大/小分子两个角度阐明了蒲黄炭化瘀止血的物质基础。从细胞、斑马鱼和大鼠三个层次上考察了蒲黄炭化瘀止血的药效作用，一方面可通过PI3K-AKT通路，促进NO释放，促进血管舒张，抑制血管内皮凋亡，发挥化瘀作用；另一方面，作用于PLCγ1通路，促进前列腺素释放，收缩血管，同时改善血管屏障的完整性发挥止血作用。运用生物信息学和分子生物学技术进一步明确蒲黄炭对VEGF-A信号通路中PLCγ1传导通路及PI3K/Akt负反馈通路平衡具有调节作用。低剂量、短时间的蒲黄炭刺激即可激活PLCγ1通路，实现较好的止血疗效。随着时间、剂量的累加，蒲黄炭可促进PI3K/AKT通路，进而发挥化瘀作用，以实现止血不留瘀的药效。

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