冰片促灯盏乙素透体外血-视网膜内屏障的作用研究

Study on the Effect of Borneol on Scutellarin Permeabilization in vitro Blood-Retinal Barrier

目的 观察不同比例冰片对灯盏乙素在体外血-视网膜内屏障的促透作用。方法 完成高效液相色谱-串联质谱联用(HPLC-MS/MS)检测灯盏乙素药物浓度的方法学开发和验证;体外培养大鼠视网膜血管内皮细胞,并用Transwell建立视网膜内皮细胞屏障模型;在体外血-视网膜内屏障模型的上室中给予含有不同比例冰片的灯盏溶液,分时段提取透屏障后的HBSS液,采用HPLC-MS/MS检测透屏障的下室HBSS液中灯盏乙素浓度。结果 (1)自建HPLC-MS/MS检测灯盏乙素的方法特异性好,准确度、灵敏度高,各项方法学验证均符合2015版《中国药典》第四部中《9012生物样品定量分析方法验证指导原则》的要求。(2)建立体外血-视网膜内屏障模型成功,在体外内皮细胞屏障模型中,上室灯盏乙素浓度为100μg·mL^(-1),在2 h后,上室中未添加冰片的下室灯盏乙素药物透过浓度为8.82μg·mL^(-1),上室添加冰片分别为60μg·mL^(-1)、120μg·mL^(-1)、240μg·mL^(-1)、480μg·mL^(-1)的下室中的灯盏乙素药物浓度分别为9.50μg·mL^(-1)、9.57μg·mL^(-1)、11.30μg·mL^(-1)、12μg·mL^(-1)。在冰片浓度为480μg·mL^(-1)时,下室中灯盏乙素的能达到最大浓度为12μg·mL^(-1)。结论 (1)灯盏乙素在HBSS中的含量检测方法学验证成功;(2)冰片可能具备促进灯盏乙素透体外血-视网膜内屏障模型的作用;(3)在上室的冰片药物浓度达到480μg·mL^(-1)时,本研究灯盏乙素透过体外血-视网膜内屏障模型达到最大药物浓度。

Objective To observe the permeability promoting effect of breviscapine on blood-retinal barrier in vitro with different proportions of borneol. Methods The development and validation of HPLC-MS/MS method for the determination of breviscapine concentration was completed. Rat retinal vascular endothelial cells were cultured in vitro and the retinal endothelial cell barrier model was established by Transwell. Breviscapine solution containing different proportions of borneol was given in the upper chamber of the in vitro blood-retinal barrier model. HBSS solution behind the barrier was extracted at different stages, and the concentration of breviscapine in the lower chamber HBSS solution of the barrier was detected by HPLC-MS/MS. Results(1)The self-built HPLC-MS/MS method for the determination of brecapycin showed good specificity, high accuracy and sensitivity, and all the validation methods were in line with the requirements of Guiding Principles for The Validation of Quantitative Analysis Methods of Biological Samples in The Fourth Part of Chinese Pharmacopoeia(2015 edition).(2) The in vitro blood-retinal barrier model was successfully established. In the in vitro endothelial cell barrier model, the concentration of upper compartment breviscapine was 100 μg·mL^(-1), and after 2 h, the permeation concentration of lower compartment breviscapine without borneol was 8.82 μg·mL^(-1). The concentrations of breviscapine in upper chamber were 9.50 μg·mL^(-1), 9.57 μg·mL^(-1), 11.30 μg·mL^(-1)and 12 μg·mL^(-1)when borneol was 60 μg·mL^(-1), 120 μg·mL^(-1), 240 μg·mL^(-1)and 480 μg·mL^(-1), respectively. When borneol concentration was 480 μg/mL, the maximum concentration of breviscapine in the lower chamber was 12 μg·mL^(-1).Conclusion(1)The determination method of breviscapine in HBSS was verified successfully.(2)Borneol may have the effect of promoting breviscapine to penetrate the blood-intraretinal barrier model.(3)When the concentration of borneol in the upper chamber reached 480 μg·mL^(-1), the maximum concentration of breviscapine reached through the in vitro blood-intraretinal barrier model.