Bai Zhi-Ru, Fei Hong-Qiang, Li Na, Cao Liang, Zhang Chen-Feng, Wang Tuan-Jie, Ding Gang, Wang Zhen-Zhong, Xiao Wei
Jiangsu Kanion Pharmaceutical Co., Ltd., Lianyungang 222001, China.
State Key Laboratory of New-tech for Traditional Chinese Medicine Pharmaceutical Process, Lianyungang 222001, China.
Zhongguo Zhong Yao Za Zhi. 2016 Feb;41(3):509-513. doi: 10.4268/cjcmm20160325.
Prostaglandin (PG) E2 is an active substance in pathological and physiological mechanisms, such as inflammation and pain. The in vitro high-throughput assay for screening the inhibitors of reducing PEG2 production is a useful method for finding out antiphlogistic and analgesic candidates. The assay was based on LPS-induced PGE2 production model using a homogeneous time-resolved fluorescence(HTRF) PGE2 testing kit combined with liquid handling automation and detection instruments. The critical steps, including the cell density optimization and IC50 values determination of a positive compound, were taken to verify the stability and sensibility of the assay. Low intra-plate, inter-plate and day-to-day variability were observed in this 384-well, high-throughput format assay. Totally 5 121 samples were selected from the company's traditional Chinese medicine(TCM) material base library and used to screen PGE2 inhibitors. In this model, the cell plating density was 2 000 cells for each well; the average IC₅₀ value for positive compounds was (7.3±0.1) μmol; the Z' factor for test plates was more than 0.5 and averaged at 0.7. Among the 5 121 samples, 228 components exhibited a PGE2 production prohibition rate of more than 50%, and 23 components exhibited more than 80%. This model reached the expected standards in data stability and accuracy, indicating the reliability and authenticity of the screening results. The automated screening system was introduced to make the model fast and efficient, with a average daily screening amount exceeding 14 000 data points and provide a new model for discovering new anti-inflammatory and analgesic drug and quickly screening effective constituents of TCM in the early stage.
前列腺素（PG）E2是炎症和疼痛等病理生理机制中的一种活性物质。用于筛选降低PEG2生成抑制剂的体外高通量检测方法是寻找抗炎和镇痛候选药物的有效方法。该检测基于脂多糖诱导的PGE2生成模型，使用均相时间分辨荧光（HTRF）PGE2检测试剂盒，并结合液体处理自动化和检测仪器。采取了包括细胞密度优化和阳性化合物IC50值测定等关键步骤，以验证该检测的稳定性和灵敏度。在这种384孔高通量检测中观察到板内、板间和日间变异较低。从公司的中药原料库中总共挑选了5121个样品用于筛选PGE2抑制剂。在该模型中，每孔细胞接种密度为2000个细胞；阳性化合物的平均IC₅₀值为（7.3±0.1）μmol；测试板的Z'因子大于0.5，平均为0.7。在5121个样品中，228种成分的PGE2生成抑制率超过50%，23种成分的抑制率超过80%。该模型在数据稳定性和准确性方面达到了预期标准，表明筛选结果的可靠性和真实性。引入了自动筛选系统，使该模型快速高效，平均每日筛选量超过14000个数据点，并为发现新的抗炎和镇痛药物以及在早期快速筛选中药有效成分提供了新的模型。
