Inhibitory Effect of Berberine on Broiler P-glycoprotein Expression and Function: In Situ and In Vitro Studies

Abstract

Overcoming P-glycoprotein (P-gp) efflux is a strategy to improve the absorption and pharmacokinetics of its substrate drugs. Berberine inhibits P-gp and thereby increases the bioavailability of the P-gp substrate digoxin in rodents. However, the effects of berberine on P-gp in chickens are still unclear. Here, we studied the role of berberine in modulating broilers P-gp expression and function through both in situ and in vitro models. In addition, molecular docking was applied to analyze the interactions of berberine with P-gp as well as with chicken xenobiotic receptor (CXR). The results showed that the mRNA expression levels of chicken P-gp and CXR decreased in the ileum following exposure to berberine. The absorption rate constant of rhodamine 123 increased after berberine treatment, as detected using an in situ single-pass intestinal perfusion model. Efflux ratios of P-gp substrates (tilmicosin, ciprofloxacin, clindamycin, ampicillin, and enrofloxacin) decreased and the apparent permeability coefficients increased after co-incubation with berberine in MDCK-chAbcb1 cell models. Bidirectional assay results showed that berberine could be transported by chicken P-gp with a transport ratio of 4.20, and this was attenuated by verapamil (an inhibitor of P-gp), which resulted in a ratio of 1.13. Molecular docking revealed that berberine could form favorable interactions with the binding pockets of both CXR and P-gp, with docking scores of -7.8 and -9.5 kcal/mol, respectively. These results indicate that berberine is a substrate of chicken P-gp and down-regulates P-gp expression in chicken tissues, thereby increasing the absorption of P-gp substrates. Our findings suggest that berberine increases the bioavailability of other drugs and that drug-drug interactions should be considered when it is co-administered with other P-gp substrates with narrow therapeutic windows.

Keywords: MDCK-chAbcb1 cell line; P-glycoprotein; berberine; chicken; inhibition.

Figure 1

The effect of berberine on Abcb1 and chicken xenobiotic receptor (CXR) mRNA levels in broilers. (A) Abcb1. (B) CXR. Data are represented as mean ± SEM of three independent experiments. ** p < 0.01.

Figure 2

Mean Rho123 concentrations vs. time curves in the jejunum of broilers after oral administration of berberine for 24 h. Each point represents the mean ± SEM of six broilers. * p < 0.05; ** p < 0.01.

Figure 3

Bi-directional transport of Rho123 across MDCK-chAbcb1 cell monolayer after pre-treatment with berberine for the indicated time. (A) 2 h, Apical to Basolateral (AP→BL) (B) 2 h, Basolateral to Apical (BL→AP) (C) 8 h, Apical to Basolateral (AP→BL). (D) 8 h, Basolateral to Apical (BL→AP). Each point represents as mean ± SEM of three independent experiments.

Figure 4

Bi-directional transport of Rho123 across MDCK-chAbcb1 cell monolayer after pre-treatment with verapamil for 2 h. (A) Apical to Basolateral (AP→BL). (B) Basolateral to Apical (BL→AP). Each point represents as mean ± SEM of three independent experiments.

Figure 5

Bi-directional transport of difference chicken P-gp substrates across MDCK-chAbcb1 cell monolayer after pre-treatment with berberine for 2 h. (A) Tilmicosin. (B) Ciprofloxacin. (C) Clindamycin. (D) Sulfadiazine. (E) Ampicillin. (F) Enrofloxacin. Data are shown as mean ± SEM of three independent experiments. * p < 0.05; ** p < 0.01.

Figure 6

Bi-directional transport of berberine across MDCK and MDCK-chAbcb1 cell monolayers with or without verapamil (A) MDCK cell monolayer without verapamil. (B) MDCK-chAbcb1 cell monolayer without verapamil. (C) MDCK cell monolayer with verapamil. (D) MDCK-chAbcb1 cell monolayer with verapamil. Data are shown as mean ± SEM of three independent experiments. ** p < 0.01.

Figure 7

Final snapshot of the 1 ns molecular dynamics simulation of the CP-gp model build by homology modelling using I-TASSER homology modelling server and embedded in the POPC lipid bilayer. CP-gp is shown in the ribbon representation colored according to secondary structure, while the POPC molecules are shown in the stick representation (hydrogen atoms not shown for clarity).

Figure 8

Molecular docking solution of berberine against CXR target obtained using AutoDock Vina. (A) Berberine (thick grey lines) in the ligand binding domain of CXR (ribbon representation), (B) Ligand interaction diagram of the berberine docking pose (dark green—conventional hydrogen bond; light green—carbon hydrogen bond; dark pink—pi-pi stacked interaction; light pink—pi-alkyl interaction; yellow—pi-sulfur interaction), (C) Berberine in the active sites delineated by the hydrophobic surface and surrounding residues which are labelled and represented as thin grey lines. Hydrogen atoms are not shown for clarity. (D) Dynamic nature of interactions represented by percentage of frames for which interactions was observed during 1 ns of molecular dynamics simulations of the fully solvated system.

Figure 9

Molecular docking solution of berberine against CP-gp target obtained using AutoDock Vina. (A) Berberine (thick grey lines) in the ligand binding domain of CP-gp (ribbon representation), (B) Ligand interaction diagram of the berberine docking pose (dark green—conventional hydrogen bond; light green—carbon hydrogen bond; dark pink—pi-pi stacked interaction; light pink—pi-alkyl interaction; yellow—pi-sulfur interaction), (C) Berberine in the active sites delineated by the hydrophobic surface and surrounding residues which are labelled and represented as thin grey lines. Hydrogen atoms are not shown for clarity. (D) Dynamic nature of interactions represented by percentage of frames for which interactions was observed during 1 ns of molecular dynamics simulations of the fully solvated system.