低氧对Caco-2细胞P-gp表达及功能的影响

Effects of hypoxia on the expression and function of P-gp in Caco-2 cells

目的:低氧会改变许多药物的口服生物利用度,其中也包括多种P糖蛋白(P-glycoprotein,P-gp)的底物(药物),提示低氧可能影响肠上皮细胞P-gp的功能。Caco-2细胞单层膜模型是当前研究肠上皮P-gp功能的经典模型。本研究通过结合Caco-2细胞单层膜模型与低氧处理,研究低氧对Caco-2细胞P-gp表达和功能的影响,有助于阐明高原低氧环境改变肠上皮药物转运的机制。方法:正常培养的Caco-2细胞在1%的O_(2)浓度条件下分别培养24、48、72 h,提取膜蛋白后,采用蛋白质印迹法检测P-gp的表达水平,选择表达变化最显著的缺氧时间用于后续研究。于transwell小室中培养Caco-2细胞21 d,建立Caco-2细胞单层膜模型后,将其分为常氧对照组与低氧组,常氧对照组以正常条件继续培养72 h,低氧组在1%的O_(2)浓度条件下继续培养72 h,通过跨膜电阻(transepithelial electrical resistance,TEER)、荧光黄表观渗透系数(apparent permeability coefficient,Papp)、碱性磷酸酶(alkaline phosphatase,AKP)活性及透射电镜下观察微绒毛与紧密连接结构,评价Caco-2细胞单层膜的完整性和极化状态。采用双向转运实验检测P-gp特异性底物罗丹明123(rhodamine 123,Rh123)的Papp,计算外排率。将Caco-2细胞在塑料培养瓶中连续培养21 d形成Caco-2细胞单层膜,在1%O_(2)浓度条件下处理72 h后,采用蛋白质印迹法检测膜蛋白中P-gp的表达水平。结果:1%的O_(2)浓度处理下调Caco-2细胞P-gp的表达,其中处理72 h下调最为显著(P<0.01);低氧组Caco-2细胞单层膜TEER大于400Ω·cm^(2),荧光黄Papp小于5×10^(-7)cm/s,肠腔侧与基底侧AKP活性比值大于3。表明Caco-2细胞单层膜模型建立成功,且低氧处理未影响模型的完整性与极化状态。与常氧对照组相比,低氧组Caco-2细胞单层膜Rh123的外排率显著降低(P<0.01);1%的O_(2)浓度培养72 h,下调Caco-2细胞单层膜P-gp的表达(P<0.01)。结论:低氧抑制Caco-2细胞P-gp功能,其可能与P-gp表达水平降低有关。

Objective:Hypoxia can alter the oral bioavailability of drugs,including various substrates(drugs)of P-glycoprotein(P-gp),suggesting that hypoxia may affect the function of P-gp in intestinal epithelial cells.Currently,Caco-2 monolayer model is the classic model for studying the function of intestinal epithelial P-gp.This study combines the Caco-2 monolayer model with hypoxia to investigate the effects of hypoxia on the expression and function of P-gp in Caco-2 cells,which helps to elucidate the mechanism of changes in drug transport on intestinal epithelial cells in high-altitude hypoxia environment.Methods:Normally cultured Caco-2 cells were cultured in 1%oxygen concentration for 24,48,and 72 h,respectively.After the extraction of the membrane proteins,the levels of P-gp were measured by Western blotting.The hypoxia time,with the most significant change of P-gp expression,was selected as the subsequent study condition.After culturing Caco-2 cells in transwell cells for 21 days and establishing a Caco-2 monolayer model,they were divided into a normoxic control group and a hypoxic group.The normoxic control group was continuously cultured in normal condition for 72 h,while the hypoxic group was incubated for 72 h in 1%oxygen concentration.The integrity and polarability of Caco-2 cells monolayer were evaluated by transepithelial electrical resistance(TEER),apparent permeability(Papp)of lucifer yellow,the activity of alkaline phosphatase(AKP),and microvilli morphology and tight junction structure under transmission electron microscope.Then,the Papp of rhodamine 123(Rh123),a kind of P-gp specific substrate,was detected and the efflux rate was calculated.The Caco-2 cell monolayer,culturing at plastic flasks,was incubated for 72 h in 1%oxygen concentration,the expression level of P-gp was detected.Results:P-gp was decreased in Caco-2 cells with 1%oxygen concentration,especially the duration of 72 h(P<0.01).In hypoxic group,the TEER of monolayer was more than 400Ω·cm^(2),the Papp of lucifer yellow was less than 5×10^(−7) cm/s,and the ratio of AKP activity between apical side and basal side was greater than 3.The establishment of Caco-2 monolayer model was successful,and hypoxia treatment did not affect the integrity and polarization state of the model.Compared with the normoxic control group,the efflux rate of Rh123 was significantly reduced in Caco-2 cell monolayer of the hypoxic group(P<0.01).Hypoxia reduced the expression of P-gp in Caco-2 cell monolayer(P<0.01).Conclusion:Hypoxia inhibits P-gp function in Caco-2 cells,which may be related to the decreased P-gp level.