Figure 1.
Cytotoxicity of berberine (BBR) in three cell lines.
(A–F) The cytotoxicity and lethality of BBR using MTT assay. (A, B) HepG2, (C, D) HeLa. (E, F) SY5Y. *P<0.05, **P<0.01, ***P<0.005, statistical significance of BBR treated groups relative to control groups. Data are presented as mean ± S.D. from six independent experiments (n = 6).
Figure 2.
Transportation of BBR through the cells.
(A–I) Images of the subcellular location of BBR in cells before and after 0.5 µg/ml BBR administration for 1 h. (A–C) HepG2 cells; (D–F) HeLa cells; (G–I) SY5Y cells. The fluorescence of BBR is shown in green, differential interference contrast (DIC) figures represent the scale of cells and scale bar is 10 µm.
Figure 3.
Chromatograms and kinetic behavior of BBR in the three cell lines.
(A–C) HPLC chromatograms. (A) Standard BBR; (B) Blank sample from BBR treated cells; (C) Samples from cells with BBR administration for 12 h. (D) Standard curves of BBR concentration verse AU intensity using HPLC assay. (E–G) Kinetic behavior of BBR in the cells in plot with the administration time in 24 h. (E) HepG2; (F) HeLa; (G) SY5Y cells. (H, I) LC/MS chromatograms of BBR in HepG2 cells after the administration for 24 h. (H) the mass spectrum of BBR; (I) the chromatogram of BBR. Standard BBR was shown in green; the sample was shown in purple color. Data are presented as mean ± S.D. from six independent experiments (n = 6).
Figure 4.
Effect of P-glycoprotein (P-gp) inhibitor on BBR uptake in HepG2, HeLa and SY5Y cells.
Cells were treated with 0.5 µg/ml BBR and 0.3 µmol Zosuquidar, the inhibitor of P-gp activity, for 12 h. The intracellular concentrations of BBR were determined by HPLC assay. Data are presented as mean ± S.D from three independent experiments (n = 3). * v.s. the control, P<0.05. ** v.s. the control, P<0.005, t-test.
Figure 5.
Molecular docking of berberine (BBR) to Homo sapiens P-gp.
(A) Side and (B) top view of the BBR binding pocket of P-gp. (C, D) the overall docking views of BBR in the binding pocket of P-gp. Blue mesh: protein's electron isodensity map around the binding site. Protein are represented as cartoon (transparency = 20%), small molecules and important amino acids (as labeled) are represented as lines (white and green, carbon. red, oxygen. blue, nitrogen). Yellow dash line, hydrogen bond.
Figure 6.
BBR modulates the transcription and expression of P-gp.
(A) mRNA expressions of endogenous P-gp using real-time PCR assay. (B) Western blotting assay showed changes of P-gp protein with BBR treatment. (C) The ratio changes of P-gp versus β-actin. Data are presented as mean ± S.D. from 3 independent experiments (n = 3). The concentration of BBR was 0.5 µg/ml. * v.s. the control, P<0.05; ** v.s. the control, P<0.005, t-test. For panels in (A) and (C), Left, HepG2; Middle, HeLa; Right, SY5Y.
Figure 7.
BBR does not act on P-gp promoter.
(A) Schematic map of P-gp 1 kb promoter fused GFP (P-gp promoter-GFP) construct. Black, P-gp promoter. Green, reading frame of EGFP. (B–D) mRNA expressions of P-gp 1 kb promoter fused GFP in the presence or absence of BBR by real-time PCR assay. (B), HepG2; (C), HeLa; (D), SY5Y. (E) Western blotting assay showed changes of P-gp 1 kb promoter fused GFP protein with BBR treatment. (F–H) The ratio changes of GFP versus β-actin. Data are presented as mean ± S.D. from 3 independent experiments (n = 3). “n.s.” means no statistical significance.