Ma Bo, Wang Jing, Sun Jing, Li Ming, Xu Huibo, Sun Guibo, Sun Xiaobo
Institute of Medicinal Plant Development, Chinese Academy Medical Sciences and Peking Union Medical College 151, Malianwa North Road, Haidian District, Beijing, 100193, PR China.
Jilin Academy of Traditional Chinese Medicine 1745, Works and Peasants Road, Chaoyang District, Changchun City, 130021, PR China.
Int J Clin Exp Pathol. 2014 Apr 15;7(5):1957-66. eCollection 2014.
Rhynchophylline (Rhy) is the major component of Uncaria species, which is used in Chinese traditional medicine for the treatment of central nervous system disorders. However, its oral bioavailability has not been known. This study aims to investigate the intestinal permeability and related mechanisms of Rhy using cultured human epithelial Caco-2 cells. The cytotoxicity of Rhy on Caco-2 cells was evaluated with MTT assay. The effect of Rhy on the integrity of Caco-2 cell monolayer was assayed with transepithelial electrical resistance. The permeability of Rhy across cell monolayer was assayed by measuring Rhy quantity in received side with HPLC. The effect of Rhy on the expression of P-glycoprotein and MDR1 was detected with Western blot and flow cytometry, respectively. In the concentration of Rhy, which did not produce toxicity on cell viability and integrity of Caco-2 cell monolayer, Rhy crossed the monolayer with velocity 2.765.57×10^-6 cm/sec and 10.6815.66×10^-6 cm/sec from apical to basolateral side and from basolateral to apical side, respectively. The permeability of Rhy was increased by verapamil, a P-glycoprotein inhibitor, or rhodamine123, a P-glycoprotein substrate. Rhy revealed an induction effect on P-glycoprotein expression in Caco-2 cells. These results demonstrate the low permeability of Rhy in intro, and suggest that P-glycoprotein may underlie the mechanism.
钩藤碱（Rhy）是钩藤属植物的主要成分，在中国传统医学中用于治疗中枢神经系统疾病。然而，其口服生物利用度尚不清楚。本研究旨在利用培养的人上皮Caco-2细胞研究钩藤碱的肠道通透性及相关机制。采用MTT法评估钩藤碱对Caco-2细胞的细胞毒性。采用跨上皮电阻法检测钩藤碱对Caco-2细胞单层完整性的影响。通过高效液相色谱法测定接收侧钩藤碱的量来检测钩藤碱跨细胞单层的通透性。分别采用蛋白质免疫印迹法和流式细胞术检测钩藤碱对P-糖蛋白和多药耐药蛋白1（MDR1）表达的影响。在钩藤碱浓度对Caco-2细胞活力和单层完整性无毒性的情况下，钩藤碱从顶侧向基底侧和从基底侧向顶侧穿过单层的速度分别为2.765.57×10^-6 cm/秒和10.6815.66×10^-6 cm/秒。P-糖蛋白抑制剂维拉帕米或P-糖蛋白底物罗丹明123可增加钩藤碱的通透性。钩藤碱对Caco-2细胞中P-糖蛋白的表达有诱导作用。这些结果表明钩藤碱在体内的通透性较低，并提示P-糖蛋白可能是其机制基础。
