基于TRP/凝血级联通路融合生物信息学的姜炭温经止血作用机制的研究

Ginger charcoal is a classic medicine of warming channel for arresting bleeding. The last NSFC task initially explained the processing mechanism of ginger charcoal from its chemical components difference in vitro and PK-PD correlation. However, the mechanism of warming channel for arresting bleeding of ginger charcoal is not yet clear. The proteomics experiments found that the differential proteins mainly participate in the regulation of the TRP/coagulation cascade pathway after administrating the ginger charcoal, and it suggested that ginger charcoal may activate the relevant target protein of TRP/coagulation cascade and thus regulate the disorder of the energy metabolism and the coagulation system, and exert its effect of warming channel for arresting bleeding. Therefore, the project will study the regulate the TRP/coagulation cascade pathway for deficiency-cold and bleed by crude and processed ginger charcoal with multidisciplinary techniques, such as analytical chemistry, molecular biology and bioinformatics. And combining the information of key target proteins of the deficiency-cold and bleed and chemical constituents, construct the interactions between the medicinal components and targets with molecular docking techniques. At the same time it will be verified experimentally on the molecular and animal experiment level. Through the above statements, the mechanism of warming channel for arresting bleeding of ginger charcoal will be explained, revealing its multi-component and multi-target effects. Furthermore, it will illuminate the processing mechanism of ginger charcoal and the scientific connotation of carbonizing drug characteristic. And also providing a reference for the study of hemostatic mechanism and processing mechanism of similar carbon drugs.

姜炭是经典的温经止血药，上一个基金项目从体外化学成分变化和PK-PD相关性的角度初步阐释姜炭炮制机理。但姜炭温经止血作用机制尚不清楚，课题组前期蛋白质组学实验结果发现，姜炭给药后差异蛋白主要参与TRP/凝血级联通路的调控，提示姜炭可能通过激活TRP/凝血级联通路相关靶点蛋白，调节虚寒性出血证引起的能量代谢和凝血系统等紊乱而发挥温经止血作用。故本项目拟以TRP/凝血级联通路为切入点，融合分析化学、分子生物学和生物信息学等多学科技术，研究姜炭炮制前后对虚寒性出血证TRP/凝血级联通路相关靶点蛋白的调控表达；结合虚寒性出血证的关键靶点蛋白和化学成分信息，采用分子对接技术构建药效成分（群）-靶点相互作用关系，并从分子和整体动物水平进行实验验证；阐明姜炭温经止血的作用机制，揭示其多成分多靶点作用规律；进一步完善姜炭炮制机理、炒炭存性和一物两性的科学内涵。为同类炭药止血作用机制和炮制机理研究提供借鉴。

姜炭是经典的温经止血药，上一个基金项目从体外化学成分变化和PK-PD相关性角度阐释姜炭炮制机理，但其作用机制尚不清楚。本项目首先进行虚寒性出血证大鼠（DCHS）和脑出血斑马鱼的药效学实验，结果显示姜炭具有较好的温经止血作用。蛋白质组学结果显示姜炭通过调控TRP/凝血级联通路中TRPV1、TRPA1、Fermt3、ACTB、Talin、αIIbβ3、FⅩⅢb、Kng、PLC-β和纤维蛋白原等靶点达到温经止血作用。定量代谢组学结果显示姜炭通过调节血清中甘氨酸、丝氨酸和苏氨酸代谢、淀粉和蔗糖代谢、半乳糖和花生四烯酸代谢等途径，调节尿液中戊糖、磷酸、谷胱甘肽、氨基酸代谢等途径调节体内能量代谢发挥温经止血作用。同时建立液质定量测定大鼠体液中药效生物标志物肌酐、4-羟基苯甲酸、邻苯二甲酸二丁酯的整体药效学评价方法。采用液质鉴定干姜姜炭中57个成分，确定25个差异化合物。采用网络药理学方法建立差异化合物与DCHS 蛋白靶点的成分-靶点网络，结果显示Dehydro-6-gingerol，6-gingerol，8-Gingerol，6-gingediol-3-acetate，6-gingediol，6-Paradol，10-Gingerol等通过调控凝血级联通路中PROCR，PLAT，F2，FGG，PLAU，F10，F7，F11，MAPK1，GP1BA，FGG等蛋白达到温经止血作用。姜炭动物和细胞实验结合体外凝血实验结果发现姜炭能促进TNF-α、TF、FXII、FXa、FIIa、ITGB3、ICAM-1、PAI-1，TRPA1、TRPV1蛋白表达，降低AT-3、t-PA/PAI-1的表达而达到温经止血作用。活性成分姜酮、6-paradol和二乙酰-6-姜辣素通过促进TNF-α、FⅩa、FⅫ、FVII、ICAM-1、VWF和ITGB3等一个或多个靶点的表达而起到止血作用。利用机器视觉、近红外、电子鼻、色差计等数字智能化手段对姜炭不同炮制程度饮片图像颜色、气味和近红外特征进行数字化分析，结合化学计量学可准确定性判别和成分含量预测。该研究阐明姜炭对DCHS温经止血的作用机制和炒炭存性炮制机理，为同类炭药止血作用机制和炮制机理研究提供借鉴。