Berberine Regulates OATP1B1 Via FXR/LXRα-dependent Mechanism

Background:Berberine is a quinoline alkaloid.It is reported that berberine can regulate the expression of transporters through nuclear receptors.The combination therapy of statins with berberine can enhance lipid-lowering effect,reduce dosage of statins,and decrease risk of adverse effects.Previous studies have shown that statins are mainly transported into the liver through OATPs to exert lipid-lowering effects,and the expression of OATPs are regulated by nuclear receptors.Therefore,whether berberine can regulate the expression of OATPs through mediating nuclear receptors,and then affecting the uptake of statins,remains to be further studied.Objectives:This study aimed to explore the mechanism of drug-drug interaction in combination of berberine and statins to provide theoretical and scientific basis for deep exploration of berberine in clinical rational drug use and combinational therapy.Methods:1.The effect of berberine on the expression of mRNA and protein of OATP1B1and OATP1B3 and the expression and distribution of FXR and LXRαin HepG2 cells was investigated by RT-qPCR,Western blot and other experimental techniques.2.The effect of berberine on the uptake of rosuvastatin in HepG2 cells was investigated by HPLC-MS/MS experimental technique.3.The effect of berberine on the regulatory pathway of FXR/LXRα-OATP1B1in hFXR or hLXRα-overexpressing HepG2 cells was investigate by using the dual-luciferase reporter gene assay.4.The effect of berberine on the expression of OATP1B1 protein was investigated in hFXR or hLXRα-silenced HepG2 cells by small interfering RNA technique.Results:1.The results of Western blot and RT-qPCR analysis showed that berberine（10⁵0μM）significantly upregulated the expression of OATP1B1 mRNA and protein in HepG2 cells,but had no effect on the expression of OATP1B3.2.The results of HPLC-MS/MS analysis showed that the uptake of rosuvastatin in HepG2 cells was significantly increased by 1.24-fold（5μM）,1.42-fold（10μM）,1.78-fold（25μM）and 1.93-fold（50μM）after treated with berberine at different concentrations for 24 h,and the EC₅₀ value was determained as 19.01±1.21μM.3.The results of the dual-luciferase reporter gene assay showed that 2.5⁵0μM berberine could increase the luciferase activity of OATP1B1 reporter gene in a concentration-dependent manner in HepG2-hFXR-OATP1B1-luc and HepG2-hLXRα-OATP1B1-luc stable reporter cell lines,and the EC₅₀ were 12.19±0.86μM and32.15±2.32μM,respectively;The EC₅₀ of GW4064 and GW3965 were 2.56±0.21μM and 2.37±0.36μM,respectively.4.The results of Western blot analysis demonstrated that both GW4064 and GW3965 could significantly induce the expression of OATP1B1 protein,and the effect was further enhanced after the combination of berberine,but the effect of berberine on the expression of OATP1B1 protein was obviously reversed by the transfection of the corresponding siRNA.5.The results of RT-qPCR and Western blot analysis showed that berberine could signifcantly increase the mRNA and protein levels of FXR,but had no impact on the expression of LXRαin HepG2 cells;By comparing the differences in the expression of FXR and LXRαproteins in cytoplasm and nucleus,it was found that the expression of nucleus FXR and LXRαprotein in HepG2 cells was significantly increased,while the expression of cytoplasmic FXR and LXRαprotein decreased slightly.Conclusion:1.Berberine can increase the expression of OATP1B1 by activating FXR and LXRα,thereby increasing the uptake of rosuvastatin.2.Berberine can increase the expression of FXR and induce the nuclear translocation;Berberine can induce the nuclear translocation of LXRα.