基于细胞代谢动力学原理的高灵敏性中药抗菌活性评价体系研究

It has been proved that the traditional Chinese medicine has curative effect on bacterial infection disease. But the current chemical research of antibacterial drugs screening shows low sensitivity when used in Chinese medicine screening. We, from the whole view of Chinese medicine and based on the "mass conservation" cell metabolism dynamics principle, propose a hypothesis that "the change of the flow rate of material consumption in the process of bacteria metabolism will highly reflect the influence of Chinese medicine to the growth and breeding of bacteria and the release of related toxin. In order to verify the hypothesis, our group, based on a essay with a various time measurement of ATP concentration of the glucose (carbon source)in Escherichia coli and Staphylococcus aureus with Andrographis paniculata as carrier, has established a dynamic model using the determination of concentration of 20 types of amino acids (nitrogen source) with amino acid analysis and the determination of bacterial counts and the internal and external toxin quantification. In addition, the “substance-mechanism-effect” traditional Chinese medicine sensitive antibacterial activity evaluation system has been initially established through the comparison of direction and strength change of power system model parameters w/ or w/o Andrographis paniculata, the determination of the interference and effectiveness of traditional Chinese medicine on the pathogen life cycle, meanwhile, by the evaluation of the sensitivity difference of parametric model on the antibacterial effect magnification and the determination of antibacterial rate and traditional absolute weight of virulence factors. The study has important application value on the establishment of new methods and perspective for the expansion of the traditional Chinese medicine antibacterial screening.

中药对细菌感染性疾病疗效确切。但目前承袭化学药物抗菌筛选思路的研究方法用于中药时，灵敏度较低。申请人从中医药整体观出发，基于“物料守恒”的细胞代谢动力学原理，提出“细菌新陈代谢过程中物质流消耗速率的改变，可从整体上高灵敏地反应中药对细菌生长繁殖及相关毒素释放的影响”这一研究假说。为验证假说，项目组以工作基础较好的穿心莲为载体，连续测定大肠埃希菌和金色葡萄球菌胞体中葡萄糖(碳源)产物ATP浓度，以及培养基和胞体内中20种氨基酸（氮源）浓度，同时进行细菌计数和内外毒素定量测定。最终建立相关动力学模型，通过比较模型参数在有无穿心莲时的改变方向及强度，明确药物对致病菌生命周期干扰环节和效能，评价模型参数对抗菌作用的放大倍率与传统抑菌率和毒力因子绝对量测定的灵敏度差异性，初步建立“物质－机制－疗效”相结合的中药高灵敏抗菌活性评价体系。该研究对拓展中药抗菌筛选方法建立的新方向和新角度具有重要应用价值。

本项目围绕“物料守恒”的细胞代谢动力学原理，以细菌新陈代谢过程中物质流消耗速率、细菌生长繁殖及相关毒素释放的动力学过程为重点测量对象，测定了穿心莲内酯对大肠埃希菌（Escherichia coli，E.coli）、金黄色葡萄球菌（Staphyloccocus aureus Rosenbach，S.aureus）、铜绿假单胞菌（Pseudomonas aerμginosa，P.aeruginosa）标准株和野生株PAO1共四种细菌的增殖动力学，测定了穿心莲内酯对金黄色葡萄球菌胞内外20种氨基酸和葡萄糖消耗的动力学及对己糖激酶活性的影响，测定了穿心莲内酯对大肠埃希菌内毒素释放的动力学、测定了穿心莲内酯对铜绿假单胞菌PAO1 QS系统核心基因lasR、rhlR、 pvdQ表达的动力学过程。在以上数据的基础上，采用生长干预模型，建立了细菌增殖动力学、葡萄糖消耗动力学的数学模型，进行了参数拟合，比较模型参数在有无穿心莲内酯时的改变方向及强度，证实了动力学定量系统可灵敏的量化筛选相关抗菌中药，通过这一方法也明确了穿心莲内酯具有较弱的影响细菌代谢的作用。鉴于以上动力学模型参数定量表明穿心莲内酯对细菌直接作用较弱，因此进一步开展了其与阿奇霉素合用对细菌感染性肺炎的药效动力学研究，建立了整合两单用与合用的统一模型。通过比较发现，穿心莲内酯对感染性肺炎具有较显著的改善作用，其对整体动物实验的作用显著强于对细菌的直接抑制作用。目前正在开展溃疡性结肠炎模型的疾病动力学模型，以待完善后评定穿心莲内酯合用化学抗菌药的动力学参数改变。项目组也探索性的构建携带GFP基因的自发荧光大肠埃希菌，采用激光共聚焦技术观测24h内穿心莲内酯对细菌增殖能力的变化。通过以上测定与模型建立，以模型参数为核心，初步建立了“物质－机制－疗效”相结合的中药高灵敏抗菌活性评价体系。该研究对拓展中药抗菌筛选方法建立的新方向和新角度具有应用价值。