Inhibition of P-glycoprotein by HIV protease inhibitors increases intracellular accumulation of berberine in murine and human macrophages

Abstract

Background: HIV protease inhibitor (PI)-induced inflammatory response in macrophages is a major risk factor for cardiovascular diseases. We have previously reported that berberine (BBR), a traditional herbal medicine, prevents HIV PI-induced inflammatory response through inhibiting endoplasmic reticulum (ER) stress in macrophages. We also found that HIV PIs significantly increased the intracellular concentrations of BBR in macrophages. However, the underlying mechanisms of HIV PI-induced BBR accumulation are unknown. This study examined the role of P-glycoprotein (P-gp) in HIV PI-mediated accumulation of BBR in macrophages.

Methodology and principal findings: Cultured mouse RAW264.7 macrophages, human THP-1-derived macrophages, Wild type MDCK (MDCK/WT) and human P-gp transfected (MDCK/P-gp) cells were used in this study. The intracellular concentration of BBR was determined by HPLC. The activity of P-gp was assessed by measuring digoxin and rhodamine 123 (Rh123) efflux. The interaction between P-gp and BBR or HIV PIs was predicated by Glide docking using Schrodinger program. The results indicate that P-gp contributed to the efflux of BBR in macrophages. HIV PIs significantly increased BBR concentrations in macrophages; however, BBR did not alter cellular HIV PI concentrations. Although HIV PIs did not affect P-gp expression, P-gp transport activities were significantly inhibited in HIV PI-treated macrophages. Furthermore, the molecular docking study suggests that both HIV PIs and BBR fit the binding pocket of P-gp, and HIV PIs may compete with BBR to bind P-gp.

Conclusion and significance: HIV PIs increase the concentration of BBR by modulating the transport activity of P-gp in macrophages. Understanding the cellular mechanisms of potential drug-drug interactions is critical prior to applying successful combinational therapy in the clinic.

Figure 1. Effect of HIV PIs on BBR uptake in RAW264.7 mouse macrophages.

Cells were treated with BBR (5 µM) with or without individual HIV PIs (AMPV, RITV, or LOPV, 15 µM) for 5 min, 15 min, 1 h, 4 h, 12 h or 24 h. The intracellular concentrations of BBR and HIV PIs were determined by HPLC analysis as described in “Methods” and normalized with total protein amount of viable cells. Data are means ± S.D. of three sets of samples. A) Time course of BBR uptake without or with individual HIV PIs; B) Time course of AMPV uptake without or with BBR; C) Time course of RITV uptake without or with BBR; D) Time course of LOPV without or with BBR.

Figure 2. Effect of HIV PIs on BBR uptake in macrophages.

A. Intracellular BBR accumulation after exposure to AMPV, RITV, LOPV (5, 15 and 25 µM) for 4 h in RAW 264.7 macrophages. B. Intracellular BBR accumulation after exposure to AMPV, RITV, LOPV (2.5, 5 and 10 µM) for 72 h in RAW 264.7 macrophages. Cells were pretreated with different concentrations of individual HIV PIs for 72 h, then treated with BBR for 4 h. C. Intracellular BBR accumulation after exposure to AMPV, RITV, LOPV (5, 15 and 25 µM) for 4 h in THP-1 macrophages. D. Intracellular BBR accumulation after exposure to AMPV, RITV, LOPV (2.5, 5 and 10 µM) for 72 h in THP-1 macrophages. Cells were pretreated with different concentrations of individual HIV PIs for 72 h, then treated with BBR for 4 h. The intracellular cellular BBR amount was determined by HPLC analysis as described in “Methods” and normalized with total protein amount of viable cells. Data are means ± S.D. of three sets of samples. * p<0.05, statistical significance of HIV PI-treated group relative to vehicle control group.

Figure 3. Effect of P-gp and MRP inhibitors on BBR uptake in RAW264.7 macrophages.

RAW264.7 macrophages were treated with BBR (5 µM) in the absence or presence of P-gp inhibitors, verapamil (100 µM) and haloperidol (50 µM), or MRP inhibitors, MK571 (10 µM) and bromosulfalein (80 µM) for 4 h. The intracellular concentrations of BBR were determined as described in “Methods”. Data are means ± S.D. of three sets of samples. *p<0.05, statistical significance of transporter inhibitor-treated group relative to BBR group.

Figure 4. Effect of HIV PIs on P-gp transporter activity in RAW264.7 macrophages.

A. Intracellular Rh123 accumulation after exposure to AMPV, RITV, LOPV (5, 15 and 25 µM), and verapamil (100 µM) for 4 h. B. Intracellular digoxin accumulation after exposure to AMPV, RITV, LOPV (5, 15 and 25 µM), and verapamil (100 µM) for 4 h. Data are means ± S.D. of three sets of samples. * p<0.05, statistical significance of HIV PI-treated group relative to vehicle control group.

Figure 5. Influence of HIV PIs on BBR uptake in MDCK cells.

Wild-type MDCK and P-gp-transfected MDCK cells were treated with BBR (5 µM) in the presence of different amount of individual HIV PIs (5, 15, and 25 µM) or verapamil (100 µM) for 4 h, respectively. The intracellular BBR amount was measured by HPLC analysis as described in “Methods” and normalized with total protein amount of viable cells. Data are means ± S.D. of three sets of samples. * p<0.05 statistical significance of HIV PI-treated group relative to BBR group. A. Intracellular BBR concentration after exposure to AMPV, RITV, LOPV, and verapamil for 4 h in MDCK/WT and MDCK/P-gp cells. B. Relative changes of intracellular BBR concentration compared to basal levels after treatment with HIV PIs and verapamil for 4 h in MDCK/WT and MDCK/P-gp cells.

Figure 6. Molecular docking of BBR and HIV PIs to mouse P-gp.

A and B: side and top view of the drug binding pocket of P-gp, respectively. Most of the surface of binding pocket colored green is hydrophobic and aromatic residues. C–F: The overall docking views of BBR, AMPV, RITV, and LOPV in the binding pocket.