Berberine Promotes OATP1B1 Expression and Rosuvastatin Uptake by Inducing Nuclear Translocation of FXR and LXRα

Berberine, a quinoline alkaloid, can be used in combination with statins to enhance hypolipidemic effects and reduce the dose and side effects of statins. The hypolipidemic effects of statins in the liver are mainly regulated by organic anion transporting polypeptides (OATPs), and the expression of OATPs is regulated by nuclear receptors. Berberine has been reported to affect nuclear receptors. However, whether berberine affects the uptake of statins by regulating nuclear receptor-mediated expression of OATPs remains to be determined. The aim of this study was to investigate the effects of berberine on the expression of OATP1B1 in HepG2 and explore the underlying mechanism. In HepG2 cells, 10–50 μM berberine significantly increased the uptake of rosuvastatin by inducing the expression of OATP1B1 mRNA and protein. Dual-Luciferase reporter assay showed that luciferase activity of hFXR and hLXRα activated OATP1B1 promoter was increased by 2.5–50 μM berberine in a concentration-dependent manner, with half-maximal effective concentration (EC50) of 12.19 ± 0.86 and 32.15 ± 2.32 μM, respectively. In addition, after silencing FXR or LXRα by small interfering RNA (siRNA), berberine-induced OATP1B1 expression was significantly attenuated. Western blot analysis of FXR and LXRα protein levels in the cytoplasm and nucleus of HepG2 cells after treatment with berberine showed that berberine induced nuclear translocation and activation of FXR and LXRα. In conclusion, berberine-induced nuclear translocation of FXR and LXRα could activate OATP1B1 promoter, resulting in enhanced expression of OATP1B1 and increased uptake of rosuvastatin.


INTRODUCTION
Statins have been widely used as lipid-lowering drugs because they are inhibitors for hydroxyl methylglutaryl coenzyme A (HMG-CoA) reductase. The combination therapy of statins with bile acid sequestrants, niacin, or ezetimibine have significantly improved efficacy in the treatment of hyperlipidemia (Knapp et al., 2001;Wolfe et al., 2001;Robinson and Davidson, 2006;Huijgen et al., 2010). However, combination therapy have the possibility to increase adverse effects such as muscle toxicity and myopathy, which may be associated with drug interactions mediated by organic anion transporting polypeptide (OATP) transporters (Staffa et al., 2002;Shitara and Sugiyama, 2006;Kitamura et al., 2008;Kunze et al., 2014).
Pregnane X receptor (PXR), constitutive androgen receptor (CAR), farnesoid X receptor (FXR), and liver X receptor a (LXRa) are members of constitutive and ligand-activated nuclear receptor superfamily and play a crucial role in regulating target genes involved in drug metabolism and transport (Urquhart et al., 2007;Staudinger et al., 2013). FXR and LXRa have been characterized as transcriptional factors which regulate the expression of OATP1B1 (Meyer Zu Schwabedissen et al., 2010). Rifampicin significantly increases the expression of OATP1B1 protein and mRNA in hepatocytes by activating PXR (Jigorel et al., 2006).
Berberine is a compound isolated from traditional Chinese medicines and exerts a variety of pharmacological effects such as anti-diabetes, immunoregulation, anti-hypertension, antiarrhythmia, and lipid-lowering (Lau et al., 2001;Kong et al., 2004;Kong et al., 2009;Gu et al., 2015;Montes et al., 2019;Neag et al., 2018;Belwal et al., 2020;). In addition, berberine regulates metabolic enzymes and transporters to affect the absorption, distribution and metabolism of endogenous and exogenous substances in vivo. Berberine increased the blood concentration of cyclosporine A in renal transplanted patients by inhibiting CYP3A4 (Wu et al., 2005). Moreover, berberine can regulate the absorption and metabolism of dextromethorphan, losartan, and midazolam in healthy human by suppressing the activity of CYP2D6, 2C9, and 3A4 (Guo et al., 2012). Berberine-activated LXRa increased the expression of ABCA1 transporter and reduced the accumulation of low-density lipoprotein cholesterol (LDL-c) in macrophages to prevent the formation of foam cells (Lee et al., 2010). Berberine-disrupted STAT5 signaling promoted Ntcp/NTCP expression, resulting in enhanced bile acid uptake (Bu et al., 2017). In addition, berberine-activated RXRa/FXR and RXRa/LXR heterodimers enhanced luciferase activity of FXRE and LXRE (Ruan et al., 2017). Recently, combination of statins and berberine has been clinically used to treat hyperlipidemia. Kong et al. demonstrated that combined use of simvastatin and berberine significantly reduced LDL-c levels in rats and hyperlipidemia patients and adverse effects compared to monotherapy (Kong et al., 2008). The combination of berberine and simvastatin remarkably attenuated adverse effects such as rhabdomyolysis and improved the efficacy and safety of treatment (Bei-Bei et al., 2009;Li et al., 2019). Therefore, we speculated that berberine could induce the expression of OATP1B1 transporter through nuclear receptors and boosts the uptake of statins by hepatocytes, thereby improving the lipid-lowering efficacy in combination treatment. Notably, rosuvastatin is not metabolized by CYP450 but is transported by OATP (White, 2002). Therefore, in this study we used rosuvastatin to avoid the interference by CYP450 and used HepG2 cell as the model to investigate the effects of berberine on the expression of OATP1B1 and explore the underlying mechanisms.

Cell Culture
The human liver carcinoma cell line HepG2 was provided by Novo Biotechnology (Shanghai, China) and cultured in Dulbecco's modified Eagle medium (DMEM, Solarbio Co., Ltd, Beijing, China) supplemented with 10% fetal bovine serum (FBS, Biological Industries, Israel) as described previously (Zhong et al., 2018). Cells were cultured to 70%-80% confluency and then treated with the chemicals for 24 h, and cells treated with 0.1% DMSO (generally considered noncytotoxic) were used as the blank control.

Western Blot Analysis
Cells were lysed with RIPA buffer (Applygen Gene Technology Co., Ltd. Beijing, China), and the nuclear and cytoplasmic proteins were separated and extracted using a nuclear and cytoplasmic extraction kit (Boster, Wuhan, China) according to the manufacturer's instructions. Protein concentrations were quantified with BCA protein assay kit (Vazyme Biotech, Nanjing, China). Proteins (20 mg/sample) were separated using 10% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes. Subsequently, the membranes were blocked for 2 h with 5% skim milk and then incubated overnight at 4°C with primary antibodies. The membranes were washed in TBS and then incubated with horseradish peroxidase-conjugated antirabbit or anti-rat IgG antibody (Santa Cruz, CA, USA) for 1 h at room temperature. GAPDH and Lamin B1 were used as loading controls. The bands were detected using a Bio-Rad ChemiDoc XRS imaging system (Bio-Rad Laboratories).

Rosuvastatin Uptake Assay
Rosuvastatin uptake assay in HepG 2 cells was performed as previously described (Li et al., 2012). Briefly, the cells were seeded at 2 × 10 5 /well into 24-well plates and cultured for 24 h, and then treated with a medium containing berberine or a blank control (0.1% DMSO) at 37°C for 24 h. In the uptake experiments, cells were washed three times with HBS-HEPES (99:1) uptake buffer at 37°C, and then the cells were incubated for 10 min in uptake buffer containing 20 mM rosuvastatin. After the incubation, the buffer was quickly aspirated, the cells were washed three times with ice-cold HBSS-HEPES buffer, and repeatedly thawed three times at −80°C and room temperature. Finally, 100 ml cell lysate was spiked with 20 ml IS (10 ng/ml atorvastatin), and 200 ml methanol was added. The mixture was then vortexed for 1 min and centrifuged at 10,000g for 10 min, with an aliquot (10 µl) automatically injected into the LC-MS/ MS system for analysis, and protein content was determined by BCA method. Three independent experiments were performed in triplicates.
Samples were ionized utilizing an electrospray-ionization probe in the positive-ion mode, and quantification was performed using the multiple-reaction monitoring (MRM) method, with the precursor-to-product transition being m/z 482.3!258.2 for rosuvastatin and m/z 559.2!440.0 for atorvastatin (IS). Nitrogen was used as the curtain and auxiliary gas, and air was used as the nebulizer gas under the following conditions: curtain gas, 40 psi; ion-spray voltage, 5500 V; nebulizer gas, 50 psi; auxiliary gas, 50 psi; and turbo temperature, 500°C. The collision energy (CE) was 45 V for rosuvastatin and 28V for atorvastatin, and the declustering potential (DP) was 118 V for rosuvastatin and 100 V for atorvastatin.

Statistical Analysis
The data from three independent experiment were presented as mean ± standard deviation (mean ± SD), and one-way ANOVA was used to determine the differences among the groups using GraphPad Prism 5.0. p < 0.05 indicated that the differences were significant.

Effect of Berberine on OATP1B1 Expression in HepG2 Cells
To investigate the effects of berberine on the expression of OATP1B1, HepG2 cells were treated with a series of concentrations of berberine (5, 10, 25, and 50 mM) for 24 h, or treated with 25 mM berberine for a series of time (6, 12, 24, and 48 h). Real-time PCR showed that berberine significantly upregulated OATP1B1 mRNA levels in a concentration and time-dependent manner ( Figures 1A, B). Western blot analysis showed that 10-50 mM berberine enhanced the expression of OATP1B1 protein in a concentration-dependent manner after 24-h treatment ( Figures 1C, D).

Berberine Increased Rosuvastatin Uptake in HepG 2 Cells
To investigate the effects of berberine on OATP1B1 transport function, the time-and concentration-dependent uptake assays were conducted, and uptake kinetic parameters were examined in a series concentration or time point (data not shown). The HPLC-MS-MS method for the determination of rosuvastatin was validated with selectivity, precision, accuracy, extract recovery, and matrix effect (Supplementary Figure 1). The uptake of rosuvastatin was linear with time over the first 10 min, and the Km (Michaelis constant) was determined to be 21.50 ± 1.77 mM, thus we performed the specific rosuvastatin uptake test under the condition of 20 mM and 10 min. After treatment with increasing concentrations (2, 5, 10, 25, and 50 mM) of berberine for 24 h, the uptake of rosuvastatin was increased 1.24-fold (5 mM berberinetreated), 1.42-fold (10 mM berberine-treated), 1.78-fold (25 mM berberine-treated), and 1.93-fold (50 mM berberine-treated)  compared to control, respectively ( Figure 2A). The half-maximal effective concentration (EC 50 ) value was measured to be 19.01 ± 1.21 mM ( Figure 2B).

Berberine Enhanced FXR and LXRa Mediated Activation of OATP1B1 Promoter
HepG2 cells were treated with 10 mM of nuclear receptor ligands including rifampicin (PXR ligand), CITCO (CAR ligand), GW4064 (FXR ligand), or GW3965 (LXRa ligand) for 24 h. PCR and Western blot analysis showed that only GW3965 (a specific LXRa agonist) and GW4064 (a specific FXR agonist) markedly upregulated the expression of OATP1B1 mRNA and protein ( Figures 3A, B).
Next we performed dual luciferase reporter assay to investigate the potency of berberine on activating transcriptional activity of LXRa and FXR on OATP1B1 promoter. As shown in Figure 4A, luciferase activity significantly increased after treatment with 25 mM berberine or/and 10 mM corresponding ligands. Meanwhile, the EC 50 value was 12.19 ± 0.86 mM in HepG2-hFXR-OATP1B1-luc cells ( Figure 4B), and the EC 50 value was 32.15 ± 2.32 mM in HepG2-hLXRa-OATP1B1-luc cells ( Figure 4C), while the EC 50 in the two cell lines were 2.56 ± 0.21 and 2.37 ± 0.36 mM, respectively, after GW4064 and GW3965 treatment. These data suggest that berberine could improve hFXR or hLXRa-mediated activation of OATP1B1 luciferase activity in a concentrationdependent manner,

Berberine Enhanced FXR and LXRa Induced Expression of OATP1B1 Protein and Transport Function
To further investigate the potency of LXRa and FXR activated by berberine on regulating OATP1B1 expression, HepG2 cell models with silenced or induced FXR and LXRa activities were  constructed. First, Western blot analysis confirmed that transfection of siRNA-hFXR or siRNA-hLXRa into HepG2 cells significantly reduced FXR and LXRa protein levels compared to control group ( Figures 5A, C). Subsequently, HepG2 cells were treated with berberine, GW4064, GW3965, or/and FXR/LXRa siRNAs. As shown in Figures 5B, D, the induction of OATP1B1 expression by GW4064 and GW3965 was significantly increased by berberine, while FXR or LXRa siRNA eliminated the upregulation of OATP1B1 by berberine. Furthermore, berberine, GW4064 and GW3965 significantly increased the uptake of rosuvastatin by OATP1B1, but knockdown of FXR or LXRa significantly reduced berberine stimulated rosuvastatin uptake by OATP1B1 ( Figure 5E). These results indicate that FXR and LXRa participate in the upregulation of OATP1B1 expression by berberine.

Berberine Induced Nuclear Translocation of FXR and LXRa
Previous studies reported that nuclear receptors such as FXR and LXRa regulate the expression of target genes after ligandinduced nuclear translocation (Lee et al., 2010;Xu et al., 2016). Therefore, we investigated the effect of berberine on the expression and distribution of FXR and LXRa in HepG2 cells. PCR analysis showed that berberine significantly increased FXR mRNA expression in a concentration-dependent manner ( Figure 6A). Consistently, Western blot analysis showed that berberine upregulated the expression of FXR protein in HepG2 cells, except that the induction of FXR protein by berberine was attenuated slightly at the highest dose of 50 mM ( Figure 6B). However, berberine had no significantly effect on the expression of LXRa at both mRNA and protein levels ( Figures 6D, E). Subsequently, cytoplasmic proteins and nuclear proteins were isolated and Western blot analysis showed that nuclear FXR and LXRa protein levels increased significantly after treatment with berberine for 24 h, while cytoplasmic FXR and LXRa protein levels decreased slightly ( Figures 6C, F). These results indicate that berberine could promote nuclear translocation of FXR and LXRa.

DISCUSSION
In this study, we provide the first evidence that berberine enhanced FXR and LXRa mediated upregulation of OATP1B1 expression, resulting in enhanced uptake of the substrate rosuvastatin in HepG2 cells, which may be responsible for improved lipid-lowering efficacy in combination with statins. Berberine is a new type of hypolipidemic drug widely used with favorable clinical efficacy and safety (Kong et al., 2004;Kong et al., 2009). Studies have reported that the combination of berberine with simvastatin can significantly enhance the h y p o l i p i d e m i c e ffi c a c y i n p a t i e n t s a n d r a t s w i t h hyperlipidemia, reduce the dose of statins, and lower the risk of adverse reactions (Bei-Bei et al., 2009;Kong et al., 2009;Li et al., 2019). Notably, we found that rosuvastatin had rapid uptake in HepG2 cells, this may be explained by that rosuvastatin is mainly transported by OATP1B1 and OATP1B3, which are highly expressed in the liver (Hagenbuch and Meier, 2003) Furthermore, we showed that berberine upregulated the expression of OATP1B1 transporter at both mRNA and protein levels in HepG2 cells, which could promote rosuvastatin uptake in HepG2 cells.
The fold induction of luciferase activity was determined using a Dual-Luciferase reporter assay system. DMSO (0.1%) was used as the negative control. Data are expressed as mean ± SD of triplicate independent experiments, ***P < 0.001 compared to the control.
can act on OATP1B1 promoter to regulate the expression (Meyer Zu Schwabedissen et al., 2010). Our study demonstrated that berberine regulated the expression of OATP1B1 by activating FXR and LXRa. This finding was confirmed by Dual-Luciferase reporter assay. The results indicated that berberine had stronger induction on hFXR-mediated transcriptional activation of OATP1B1 than on hLXRa, but the effect was weaker than classical agonists GW4064 and GW3965. However, the combination of berberine and the corresponding agonists can further improve the expression of target proteins. Silencing FXR or LXRa by siRNA dramatically diminished the upregulation of OATP1B1expression by berberine. These results confirmed that FXR and LXRa mediate the effects of berberine on the upregulation of OATP1B1 expression. However, the underlying mechanism by which berberine activates FXR and LXRa in HepG2 cells is unclear. LXRa and . The nuclear and cytoplasmic FXR (C) and LXRa (F) protein levels were quantified by Western blot. The determination of mRNA and protein were normalized to GAPDH, nuclear and cytoplasmic protein was normalized to Lamin B1 and GAPDH, respectively. DMSO (0.1%) was used as the negative control. Data are expressed as mean ± SD of triplicate independent experiments,*P < 0.05, **P < 0.01, ***P < 0.001 compared to the control. FXR have been identified as critical nuclear receptors which bind to the promoters of target genes to regulate transcriptional activity of downstream target genes after ligand-activated nuclear translocation (Meyer Zu Schwabedissen et al., 2010;Xu et al., 2016;Zhou et al., 2016). In this study we found that berberine promoted nuclear translocation and activation of FXR and LXRa, similar to other Chinese herbal medicines such as ginkgolide b (Zhou et al., 2016) and dihydroartemisinin (Xu et al., 2016). In addition, berberine significantly increased the expression of FXR protein and mRNA at 10 and 25 mM, but only moderately upregulated the expression of FXR protein at 50 mM, while the mRNA expression was still significantly upregulated, suggesting that FXR may be subjected to a series of posttranscriptional regulations, such as phosphorylation, acetylation, and glycosylation (Chang, 2009;Sugatani et al., 2014). Although statins are known as HMG-CoA reductase inhibitors, previous study suggested that berberine inhibited HMG-CoA reductase activity via increased phosphorylation of HMG-CoA reductase, leading to reduced hepatic cholesterol level (Wu et al., 2011). In addition, recent reports showed that berberine could inhibit lipogenesis by targeting sterol regulatory element-binding protein (SREBP) related signaling (Yunxin et al., 2019;Zhu et al., 2019). Therefore, furthers studies are needed to demonstrate that berberine and statins in combination can inhibit SREBP signaling and HMG-Co reductase activity to achieve enhanced hypolipidemic effects.
In summary, our results suggest that berberine upregulates the expression of OATP1B1 in HepG2 cells by inducing nuclear translocation of FXR and LXRa, which then activate the expression of OATP1B1 and increase the uptake of rosuvastatin.

DATA AVAILABILITY STATEMENT
All datasets generated for this study are included in the article/ Supplementary Material.

AUTHOR CONTRIBUTIONS
YX, HZ, and CX participated in study design. ML, DZ, and WD conducted the experiments and analyzed the data. JW and SH contributed to the writing of the manuscript.