R/S-普萘洛尔的手性吸收动力学研究

R/S- propranolol Chiral Absorption Kinetics

目的:采用蛋白质组学研究策略,研究R/S-普萘洛尔(Propranolol,PRO)的手性吸收动力学特征,建立其差异蛋白质图谱。方法:体外培养Caco-2细胞并使之在培养瓶底部融合达80%以上;将含160μmol/L的R-PRO和S-PRO的细胞培养液分别加入到培养瓶中,在培养箱中静置培养4 h;吸弃药液,用刮除法收集培养瓶中的Caco-2细胞,细胞全蛋白提取液裂解并收集Caco-2细胞全蛋白,Bradford法蛋白定量;取100μg全蛋白进行双向电泳,建立R-PRO和S-PRO的差异蛋白质图谱;对差异蛋白进行质谱鉴定和生物信息学检索,根据蛋白功能探讨R/S-普萘洛尔的手性吸收动力学特征。结果:R-PRO和S-PRO的蛋白质图谱差异不明显,对其中的明显差异蛋白质点进行质谱检测,成功鉴定到8个蛋白,分别为:GRP78、GRP75、HSP7C、HSP71、Cytokeratin 8、PIP5K、Ran、Prohibition,经生物物信息学检索,这些蛋白的功能主要涉及细胞的应急反应、能量代谢、生长繁殖等,与药物的主动吸收无直接相关。结论:成功建立了R-PRO和S-PRO的差异蛋白质图谱;R-PRO和S-PRO在蛋白质组学方面虽然存在差异,但这些蛋白与药物的主动吸收无直接相关,推测两药在体外的吸收转运无手性差异,与其它方法的研究结果一致。此外,研究中发现的差异蛋白提示,R-PRO和S-PRO在对细胞的新陈代谢方面有差异,可能与两者在体内的系统清除率的差异相关,这有待进一步研究证实。

Objective： To explore the proteomics strategy to research R/S-propranolol （PRO） chiral absorption kinetics, and to establish their differences in protein profiles. Methods： Human colon cancer 2 （Caco-2）cells were cultured in vitro and fusion more than 80%; Add the cell culture medium containing 160 mol / L R-PRO and S-PRO cell to the flask in incubator for 4h; Aspirate liquid, scrape and collect the culture flasks of Caco-2 cells, extract protein from the treated cells and apply the Bradford method for protein quantification, 100 g protein were used for two-dimensional electrophoresis, to establish the differences in protein profiles in the R-PRO and the S-PRO, differentially expressed proteins were identified by mass spectrometry and bioinformatics, and determined their function in the chiral R/S-PRO absorption kinetics. Results： R-PRO and S-PRO protein profiles had no significant difference in mass spectrometry, of the significant differential protein spots, we successfully identified 8 proteins, respectively： GRP78, GRP75, HSP7C, HSP71, Cytokeratin 8, PIP5K, Ran, Prohibition. Through biophysical information retrieval, the function of these proteins was mainly related to the emergency response of the cells, energy metabolism, growth and reproduction, not directly related to the active absorption. Conclusions： The differences in protein profiles in the R-PRO and the S-PRO were successfully established; R-PRO and S-PRO in proteomics, although there are differences, but these proteins are not directly related to active absorption which implies that the two drugs have no ehiral feature in the absorption kinetics. The finding of the study is consistent with other reports. In addition, these differentially expressed proteins, according to their function, suggesting that R-PRO and S-PRO might have differences in cell metabolism and associate with the difference of two drugs in the system clear rate in vivo, which needs to be confirmed by further studies.