小檗碱、巴马汀和药根碱在大鼠原代肝细胞中的摄取转运特征

Uptake Characterizations of Berberine,Palmatine and Jateorhizine in Rat Primary Hepatocytes

目的通过大鼠原代肝细胞模型,明确小檗碱、巴马汀和药根碱的肝细胞摄取特征。方法 Seglen两步法分离大鼠原代肝细胞并悬浮培养,应用已知底物瑞舒伐他汀评价转运体的功能,将药物在4和37℃分别与肝细胞孵育不同时间,UPLC-MS/MS测定肝细胞摄取量,考察温度和时间对药物摄取的影响,并计算转运参数。通过浓度依赖的摄取实验,计算得到K_m、V_(max)和主动摄取率等参数。在肝细胞模型上,将转运体的特异抑制剂与小檗碱、巴马汀和药根碱共孵育,观察阳性抑制剂对受试药物转运的影响。结果小檗碱、巴马汀和药根碱在37℃条件下的摄取量随着底物浓度的增高而增加,4℃条件下摄取量较低且变化很小,3种生物碱进入肝细胞的CL_(active)/CL_(uptake)分别为85.26%、74.90%和57.74%。阳性抑制剂布洛芬、地高辛、甘草酸和盐酸哌唑嗪能显著降低小檗碱和巴马汀的摄取,甘草酸和盐酸哌唑嗪能减少药根碱的摄取。结论小檗碱、巴马汀和药根碱进入肝细胞均以主动转运为主,Oatp1a1、Oatp1a4、Oatp1b2和Oct1可能介导了小檗碱和巴马汀的肝脏摄取,Oatp1b2和Oct1参与了药根碱的主动转运。

OBJECTIVE To evaluate the uptake characterizations of berberine, palmatine and j ateorhizine through hepatic up- take model with rat primary hepatocytes. METHODS Rat hepatoeytes were isolated by Seglen＇s two-step method and cultured in sus- pension. The activity of transporters was assessed using rosuvastatin which is known as the substrate of the transporters. The tested drugs were incubated with rat hepatocytes at 4 or 37 ℃ for various periods of time and the drugs were determined using UPLC-MS/MS method to calculate the uptake amounts. The effect of cultural time and temperature on the hepatic uptake of these compounds were as- sessed. The kinetic parameters such as Kin, Vmax and CLsctive/CLuptake were calculated from concentration dependent uptake experiments. The effects of positive inhibitors on the uptake of berberine, palmatine and jateorhizine were also surveyed by pre-ineubation of the in- hibitors with the rat hepatocytes, followed by co-incubation with berberine, palmatine and jateorhizine. RESULTS The hepatic up- take of berberine, palmatine and jateorhizine increased along with the increase of concentration at 37 ℃ while the change of hepatic up- take was very little at 4 ℃. The value of CLactive/CLuptakewas 85.26%, 74. 90% and 57.74% for berberine, palmatine and jateorhiz- inc. Ibuprofen, digoxin, glycyrrhizin and prazosin which were the known inhibitors of transporters could remarkably reduce the hepatic uptake of berberine and palmatine, respectively. Glycyrrhizin and prazosin could reduce the uptake of jateorhizine. CONCLUSION The transport of berberine, palmatine and jateorhizine into hepatocytes is mainly through an active process. Berberine and palmatine are the substrates of Oatplal, Oatpla4, Oatplb2 and Oct1, and the uptake of jateorhizine might be associated with Oatplb2and Oct1.