| Objective:β-adrenergic receptors are important targets for drug discovery. We have developed a new β-Adrenergic receptor/cell membrane chromatography-offline-ultra-performance liquid chromatography/mass spectrometry nethod for screening active ingredients from traditional Chinese medicines. In addition, biological effects of active component were also investigated in order to search for a new type of β1AR antagonist. It will be a useful method for drug discovery as a leading compound resource.Methods:In this study, the pEGFP-N1plasmid containing β1AR cDNA was transfected into the CHO-S cells and positive clones were selected by G418of900μg·ml-1after2to3weeks. The expression level of β1-AR in CHO-S cells was assessed by flow cytometry, con-focal microscope and Western blotting using rabbit anti-β1AR antibody. CHO-S cells with high β1-AR expression levels were used to prepare cell membrane stationary phase in a β1-AR/CMC model. The retention fractions were separated and identified by UPLC/MS system. The screening results found that isoimperatorin from Rhizoma et Radix Notopterygii was the targeted component which could act on (31AR in similar manner of metoprolol as a control drug. Cell viability was determined via a colorimetric method using the CCK-8assay. β1-AR/CHO-S cells were seeded in6-well plates and cultured in metoprolol (10μM) and active components for24h. The cells were cultured in DMEM containing ISO (50μmmol·L-1) for12h and then prepared to measure levels of cAMP and PKA using ELISA. β1-AR/CHO-S cells were cultured in metoprolol(10μM) and active components for24h and then incubated ISO (50μmol·L-1) for12h. Apoptotic cells were detected by Annexin V and propidium odide (PI) double labeling following the manufacturer’s protocols using flow cytometry.Results:After3weeks of selection, the positive cells were chosen for the identification. Comparison of the native CHO-S cells, transfected CHO-S cells displayed a fusion protein band of β1-AR and EGFP and the level of endogenous (β1-AR was very low. The expression of β1-AR protein in the transfected CHO-S cell and native CHO-S cells were also detected by con-focal microscope and flow cytometry. The result showed that the expression of β1AR in the transfected CHO-S was96.31±8.7%and β1AR protein can express on the cell membrane. The results indicated that the transfected CHO-S can highly expresses β1-AR membrane protein, which is applied to improvement of the specificity and selectivity of CMC model. The recognition, separation and identification ability of the β1AR/CMC-offline-UPLC/MS method was assessed using Nitrendipine and metoprolol standard solutions. The chromatographic system was able to selectively recognize the components acting on the β1-AR/CHO-S cell membrane. The β1AR/CMC-offline-UPLC/MS method was applied for screening active components from Radix Angelicae Biserratae extract. The retention fraction was separately collected, evaporated and injected into UPLC/MS system for separation and identification. From UV, MS and previously reported data, the retention fraction was confirmed as isoimperatorin (IMP). To examine the toxicity of the drug, β1-AR/CHO-S cells were treated with IMP (0.1-20μM) and incubated for24h. IMP did not influence viability in cultured cells up to5μM. In contrast, in all groups treated with IMP and metoprolol, concentrations of cAMP and PKA were lower than in the ISO group (P<0.01), suggesting that treatment with these drugs regulated the high levels of cAMP and PKA induced by the excessive activity of β1-AR. Also IMP (5μM) and metoprolol significantly (P<0.01) decreased cell apoptosis compared to ISO group.Conclusions:In summary, we established a β1AR/CMC-offline-UPLC/MS system for screening β1-AR antagonists from a complex system. The use of the β1-AR/CMC model increased the identification selectivity and sensitivity to active components on the target receptors. This method should be useful for drug discovery using natural medicinal herbs as leading compounds. |
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