一种激动剂敏感、快速、简单的基于细胞的信号转导测定法，用于通过亚型 EP1 受体鉴定模拟前列腺素 E2 或 E1 活性的配体：适用于高通量筛选。
An agonist sensitive, quick and simple cell-based signaling assay for determination of ligands mimicking prostaglandin E2 or E1 activity through subtype EP1 receptor: Suitable for high throughput screening.
机构信息
Centre for Experimental Therapeutics and Pharmacoinformatics, Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, University of Houston, Texas 77004, USA.
出版信息
BMC Complement Altern Med. 2011 Feb 7;11:11. doi: 10.1186/1472-6882-11-11.
BACKGROUND
Conventionally the active ingredients in herbal extracts are separated into individual components, by fractionation, desalting, and followed by high-performance liquid chromatography (HPLC). In this study we have tried to directly screen water-soluble fractions of herbs with potential active ingredients before purification or extraction. We propose that the herbal extracts mimicking prostaglandin E(1) (PGE(1)) and E(2) (PGE(2)) can be identified in the water-soluble non-purified fraction. PGE(1) is a potent anti-inflammatory molecule used for treating peripheral vascular diseases while PGE(2) is an inflammatory molecule.
METHODS
We used cell-based assays (CytoFluor multi-well plate reader and fluorescence microscopy) in which a calcium signal was generated by the recombinant EP(1) receptor stably expressed in HEK293 cells (human embryonic kidney). PGE(1) and PGE(2) were tested for their ability to generate a calcium signal. Ninety-six water soluble fractions of Treasures of the east (single Chinese herb dietary supplements) were screened.
RESULTS
After screening, the top ten stimulators were identified. The identified herbs were then desalted and the calcium fluorescent signal reconfirmed using fluorescence microscopy. Among these top ten agonists identified, seven stimulated the calcium signaling (1-40 μM concentration) using fluorescence microscopy.
CONCLUSIONS
Fluorescence microscopy and multi-well plate readers can be used as a target specific method for screening water soluble fractions with active ingredients at a very early stage, before purification. Our future work consists of purifying and separating the active ingredients and repeating fluorescence microscopy. Under ordinary circumstances we would have to purify the compounds first and then test all the extracts from 96 herbs. Conventionally, for screening natural product libraries, the procedure followed is the automated separation of all constituents into individual components using fractionation and high performance liquid chromatography. We, however, demonstrated that the active ingredients of the herbal extracts can be tested before purification using an agonist sensitive, quick and simple cell-based signaling assay for ligands mimicking the agonists, PGE(1) and PGE(2).
背景
传统上，通过分段、脱盐和高效液相色谱（HPLC）等方法，将草药提取物中的有效成分分离成单个成分。在这项研究中，我们试图在纯化或提取之前，直接筛选具有潜在有效成分的草药的水溶性成分。我们提出，可以在水溶性未纯化部分中鉴定出模拟前列腺素 E1（PGE1）和前列腺素 E2（PGE2）的草药提取物。PGE1 是一种用于治疗外周血管疾病的强效抗炎分子，而 PGE2 是一种炎症分子。
方法
我们使用基于细胞的测定法（CytoFluor 多孔板读数器和荧光显微镜），其中通过在 HEK293 细胞（人胚肾）中稳定表达的重组 EP1 受体产生钙信号。测试了 PGE1 和 PGE2 生成钙信号的能力。筛选了 96 种 Treasure of the east（单一中药膳食补充剂）的水溶性成分。
结果
筛选后，确定了前 10 个刺激剂。然后对这些草药进行脱盐处理，并使用荧光显微镜重新确认钙荧光信号。在鉴定出的前 10 个激动剂中，有 7 个在 1-40 μM 浓度下通过荧光显微镜刺激钙信号。
结论
荧光显微镜和多孔板读数器可以作为一种在非常早期阶段筛选具有有效成分的水溶性成分的靶向特异性方法，而无需进行纯化。我们未来的工作包括对活性成分进行纯化和分离，并重复荧光显微镜检测。在通常情况下，我们必须首先纯化化合物，然后测试来自 96 种草药的所有提取物。传统上，对于天然产物文库的筛选，所遵循的程序是使用分段和高效液相色谱法将所有成分自动分离成单个成分。然而，我们证明，使用针对模拟激动剂 PGE1 和 PGE2 的激动剂敏感、快速和简单的基于细胞的配体信号测定法，可以在纯化之前测试草药提取物的有效成分。
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