Caco-2 cell line was donated by college of pharmacy, Anhui University of Chinese Medicine; Fetal bovine serum (FBS), Dulbecco's modified Eagle's medium (DMEM), Non-essential amino acids (NEAA), 0.25% trypsin/1 m M EDTA, antibiotic-antimycotic mixture (10,000 U/ml penicillin, 10,000 l g/ml streptomycin) were purchased from Thermo Scientific Co. (Hudson, NH, USA); Hank's Balanced Salt Solution(HBSS), Dimethyl sulfoxide (DMSO), Rhodamine-123 (Rho-123) were purchased from Beijing Solarbio Science & Technology Co., Ltd; Verapamil (VER) were purchased from Sigma Chemical Co. (St. Louis, MO, United States); Geniposide is a white powder with 98% purity (determined by UPLC); All other chemicals used in this study were of analytical grade from commercial sources. Anti-P Glycoprotein antibody [EPR 10364-57] ab170904, Anti-P Glycoprotein antibody [UIC2] (Phycoerythrin) ab93590 was purchased from Abcam(Cambridge, United Kingdom). Chromatographic separation was performed on a C18 column, Luna C18 100A, 250 × 4.60 mm by using the Agilent 1260 HPLC system (Agilent Technologies, CA,USA).
Cell culture and establishment of transwell model
The Caco-2 cell line was cultured in a DMEM medium containing 10% FBS, 1% non-essential amino acids, 1% L-glutamic acid and 1% Penicillin-Streptomycin Solution(100×) at 37 ˚C in a 5% CO2 incubator. Caco-2 cells are in the shape of paving stone-like and grow in clusters. The cells were 30–60 generations for this experiment, which were in logarithmic growth phase.
The cells were seeded onto polycarbonate membrane (transwell inserts) at a density of 1 × 105/well for 21 d to establish the transport model in vitro. Cell suspension was added to the apical (AP) of transwell chamber, and medium was added to the basolateral (BL). The medium was changed once every two days after inoculation, and every day after a week. The cells were cultured for 21 to 23 d to form a cell monolayer. Cell morphology, transepithelial electrical resistance (TEER) and cell polarity were used to determine the integrity of cell monolayer for subsequent transport experiments.
Evaluation of transwell model
The polycarbonate membrane at the bottom of the chamber was cut off and fixed in 2.5% glutaraldehyde overnight. After washing with PBS, cells were immobilized in 1% osmium acid, dehydrated and embedded in ethanol and propylene oxide. After immersion, the cells were transferred to a special embedding board and incubated in oven. The slice was sliced to a thickness of 70 nm. After being stained, the images were observed and recorded under transmission electron microscope.
Transepithelial Electrical Resistance
Caco-2 cells were cultured in transwell chamber and measured on the 3, 6, 9, 12, 15, 18 and 21 d. Before the measurement, the electrode was immersed in 70% ethanol for 15 minutes, and air-dried. During the measurement, the short electrode was inserted into the apical and the long electrode into the basolateral. Note that the electrodes should be inserted vertically and that the ends of the electrodes should not touch the bottom of the transwell chamber. Each chamber was measured three times at random, and the results were recorded as Rt. The results of blank chamber were recorded as R0.The TEER value is calculated according to the formula: TEER=(Rt-R0) × S (S is the effective membrane area of the cell. Generally, TEER values largrer than 500Ω·cm2 can be regarded as single layer compact and complete . The higher the TEER value, the denser the monolayer is, generally not more than 1000 Ω·cm2.
When cells grew in transwell chamber for 4, 7, 15 and 21 d, the cell monolayer was gently washed with PBS and incubated at 37 ˚C for 30 min. Alkaline phosphatase (AKP) activity of AP and BL side was measured by kit. The OD value was measured by a microplate reader at 520 nm. AKP activity = A (measurement) / A (standard) × standard phenol content (0.005 mg) × 100 mL/0.05 mL .
In order to select the appropriate concentration, the toxicity of the test substances at different concentrations to Caco-2 cells was determined by IC50 and MTT assay. Cells were seeded in 96-well plates and incubated overnight after cell counting (104 cells/well). Different concentrations of GE (100, 200, 300, 400, 500, 600, 700, 800, 900 and 1000 µg/mL) were added to the experimental group and incubated at 37 ˚C for 24 hours. Thereafter, 10 µL MTT was added to each well and incubated at 37 ˚C for 4 h. After incubation period, the cell medium was removed and the formed formazan crystals dissolved in 100% DMSO. The 96-well plate was shaken for 15 min at room temperature in the dark and then the absorbance was measured at 520 nm. The IC50 value was calculated by Graphpad Prism software (version 6.0).
In order to determine the concentration of GE, Caco-2 cells were incubated with a series of GE concentrations (1,2,5,10,25,50,100,200 µg/mL) at 37 ˚C with different durations (6,12,24,48 h). The survival rate of Caco-2 cells at different concentrations of GE was examined. The cell survival rate was calculated by the following formula: Cell viability = (OD the xperimental group - OD the blank group) / (OD the negative control group - OD the blank group) × 100%.
HPLC analytical methods
Chromatographic separation was performed on a C18 column (Luna C18 100A, 250 × 4.60 mm) by the Agilent 1260 HPLC system (Agilent Technologies, CA,USA). The GE sample was analyzed by acetonitrile-water (15:85) at 25 ˚C and flow rate was 1.0 mL/min. The injection volume was 10 µL and detection wavelength was 238 nm. For digoxin samples, the mobile phase was acetonitrile-water (35:65) and detection at 220 nm.
Bidirectional transport experiments
Caco-2 cells formed integrated and tight monolayers after 21d differentiation, which used for transport experiments. In the AP-BL transport experiment (the absorptive direction), 0.5 mL drug solutions of GE (10, 25, 50, 100 µg/mL) or in the presence of verapamil (100 µM) were added to the apical chamber (donor), 1.5 mL blank HBSS was added to the basolateral chamber (receiver), and then samples (200 µL) were collected from the basolateral chamber at different time points, 30, 60, 90, 120,150, and 180 min. In the BL-AP transport experiment (the secretory direction), 1.5 mL GE solutions (10, 25, 50, 100 µg/mL) or in the presence of verapamil (100 µM) were added to the basolateral chamber (donor), 0.5 mL blank HBSS was added to the apical chamber (receiver), and then samples (200 µL) were collected from the apical chamber at different time points.
For digoxin assay, bidirectional transport experiments in the presence or absence of verapamil (100 µM) or GE (25, 50, 100 µg/mL) was performed by digoxin (10 µM), as mentioned above. The samples were centrifuged at high speed (16 000 rpm, 20 min). The content of GE was analyzed by HPLC.
The apparent permeability coefficient (Papp) was calculated by formula (1), and the efflux ratio (ER) was calculated by formula (2). Papp = (dQ/dt) / (A × C0) (1), where dQ/dt is the transmission rate of GE per unit time, A is the surface area of Caco-2 monolayer (0.03 cm2), and C0 is the initial drug concentration on the donor side. ER = Papp(BL-AP)/ Papp(AP-BL) (2) .
Flow cytometry analysis
The efflux function of P-gp was performed by rho-123 accumulation and efflux using flow cytometry. The transport activity of P-gp has two phases: accumulation phase and efflux phase. In the accumulation phase, verapamil was used as a P-gp inhibitor to block P-gp activity and rho-123 accumulated in cells. In the efflux phase, the P-gp inhibitor was removed and the high glucose medium was added as an energy substance to re-establish the P-gp energy-dependent efflux phase, and rho-123 flows out of the cell. Accumulation phase: after digestion of the cell suspension, Caco-2 cells were counted at 5 × 105 cells/mL, centrifuged (1000 rpm, 5 min) and suspended in medium containing 10% FBS, rho-123 (5 µM) and verapamil (100 µM). The fluorescent substrates accumulated in the cells durning incubation at 37 ˚C for 1 h. Efflux phase: thereafter, it was centrifuged (4 ˚C, 1000 rpm, 5 min) and washed twice with ice-cold PBS containing 10% FBS. The cells were resuspended in high glucose medium containing various concentrations of GE and incubated at 37 ˚C for 45 min. After rho-123 effluxed, it was washed twice with ice-cold PBS containing 10% FBS and resuspended in ice-cold PBS for analysis of flow cytometry.
The expression of P-gp on Caco-2 cell membrane was also analyzed by flow cytometry. Caco-2 cells were inoculated into six-well plates with 5 × 105 cells/mL, and fused into monolayer cells. Verapamil (100 µM) and GE (25, 50, 100 µg/mL) were administered separately and cultured for 24 h. Subsequently, the cells were washed twice with PBS and digested with 0.25% trypsin/1 mM EDTA to obtain cell suspension. After centrifugation (1000 rpm, 5 min), the cells were suspended in PBS buffer containing 10% FBS, P-gp antibody [UIC2] (Phycoerythrin). Then, the cells were incubated at 37 ˚C for 30 min in the dark, suspended in ice-cold PBS and stored on ice until analysis of flow cytometry.
The accumulation of rho-123 in Caco-2 cells was detected by immunofluorescence to evaluate the efflux function of P-gp. Caco-2 cells were plated in six-well plates at 5 × 105 cells/mL. The cells were then exposed to rho-123 (5 µM) and verapamil (100 µM) for 1 h. After the accumulation period, the cells were washed twice with PBS and were administered with high glucose medium containing different concentrations of GE, followed by incubation at 37˚C for 1 h. Then, the cells was washed twice with PBS and observed in a fluorescence microscope.
Caco-2 cells were inoculated into six-well plates with 5 × 105 cells/mL. After cell fusion, the cells were cultured in medium containing or without GE ( 25, 50, 100 µg/mL) and verapamil (100 µM) for 24 h. The cells were fully lysed by 300 µL lysate (RIPA lysate mixed with PMSF protease inhibitor 100:1 mixed) per pore. The cells were scraped and crushed by ultrasound. The supernatant was centrifuged at 12000 rpm for 20 min and stored at -80 ˚C until analysis. BCA protein quantitative kit was used to determine protein concentration and ensured the same sample concentration. The proteins were separated by SDS-PAGE and transferred to PVDF membrane by electrophoresis. After that, it was blocked with 5% skim milk for 2 hours at room temperature, and the primary antibody (1:2500) was incubated at 4 ˚C overnight after washing with TBST. Finally, the secondary antibody was incubated for 2 h and detected by the ECL luminescent liquid. Band density was measured using Image J analytical software (National Institutes of Health, Bethesda, MD). The relative expression levels of target proteins are shown by the density ratio to GAPDH internal control or to unphosphorylated total protein in the same sample.