Author Affiliations: Division of Gastroenterology, Department of Internal Medicine, Hallym University College of Medicine, Dongtan Sacred Heart Hospital, 40 Seokwoo-dong, Hwasung, Kyungki-Do 445-170, Republic of Korea (Byun HW, Hong EM, Park SH, Koh DH, Choi MH, Jang HJ, Kae SH and Lee J)

 

Corresponding Author: Jin Lee, MD, Division of Gastroenterology, Department of Internal Medicine, Hallym University College of Medicine, Dongtan Sacred Heart Hospital, 40 Seokwoo-dong, Hwasung, Kyungki-Do 445-170, Republic of Korea (Tel: 82-31-8086-2015; Fax: 82-31-8086-2029; Email: jinlee@medimail.co.kr)

 

© 2014, Hepatobiliary Pancreat Dis Int. All rights reserved.

doi: 10.1016/S1499-3872(14)60009-6

 

 

Contributors: LJ proposed the study. BHW and LJ performed research, wrote the first draft and analyzed the data. All authors contributed to the design and interpretation of the study and to further drafts. LJ is the guarantor.

Funding: None.

Ethical approval: Not needed.

Competing interest: No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

 

 

BACKGROUND: Statins are suggested to preserve gallbladder function by suppressing pro-inflammatory cytokines and preventing cholesterol accumulation in gallbladder epithelial cells. They also affect cross-talk among the nuclear hormone receptors that regulate cholesterol-bile acid metabolism in the nuclei of hepatocytes. However, there is controversy over whether or how statins change the expression of peroxisome proliferator-activated receptor (PPAR)α, PPARγ, liver X receptor α (LXRα), farnesoid X receptor (FXR), ABCG5, ABCG8, and 7α-hydroxylase (CYP7A1) which are directly involved in the cholesterol saturation index in bile.

 

METHODS: Human Hep3B cells were cultured on dishes. MTT assays were performed to determine the appropriate concentrations of reagents to be used. The protein expression of PPARα and PPARγ was measured by Western blotting analysis, and the mRNA expression of LXRα, FXR, ABCG5, ABCG8 and CYP7A1 was estimated by RT-PCR.

 

RESULTS: In cultured Hep3B cells, pravastatin activated PPARα and PPARγ protein expression, induced stronger expression of PPARγ than that of PPARα, increased LXRα mRNA expression, activated ABCG5 and ABCG8 mRNA expression mediated by FXR as well as LXRα, enhanced FXR mRNA expression, and increased CYP7A1 mRNA expression mediated by the PPARγ and LXRα pathways, together or independently.

 

CONCLUSION: Our data suggested that pravastatin prevents cholesterol gallstone diseases via the increase of FXR, LXRα and CYP7A1 in human hepatocytes.

 

(Hepatobiliary Pancreat Dis Int 2014;13:65-73)

 

KEY WORDS: pravastatin; PPARγ; liver X receptor α; farnesoid X receptor; gallstone disease

The cholesterol saturation index (CSI) of bile produced by hepatocytes is a major determinant of cholesterol gallstone formation and cho-lesterolosis in the gallbladder (GB).^{[1, 2]} Representative nuclear hormone receptors regulate cholesterol-bile acid metabolism. These receptors include liver X receptor α (LXRα), farnesoid X receptor (FXR), and peroxisome proliferator-activated receptor (PPAR), and they influence the regulatory targets of one another.^[3-5]

 

LXRα in hepatocytes works as a heterodimer with retinoid X receptor (RXR) to lower the cholesterol concentration in response to oxycholesterol. Activation of LXRα in hepatocytes induces the transcription of 7α-hydroxylase (CYP7A1), the rate-limiting enzyme in hepatic bile acid synthesis, and leads to conversion of cholesterol to bile acid.^[6] In addition, activated LXRα in hepatocytes induces ABCG5/ABCG8 located at the canalicular membrane of cells, and transports excessive intracellular cholesterol into bile. Thus, sustained activation of LXRα-mediated ABCG5/ABCG8 produces supersaturated bile, resulting in the formation of cholesterol gallstones.^[7-10] However, a recent study^[11] revealed that the LXR response element (LXRE) is not conserved in the human CYP7A1 promoter. Accordingly, there is controversy over whether LXR agonist activates CYP7A1 in human hepatocytes.^{[12, 13]}

 

FXR in hepatocytes also works as a heterodimer with RXR to maintain adequate concentrations of bile acid. An increased concentration of bile acid in hepatocytes activates FXR, inducing intracellular CYP7A1 and sterol-12-hydroxylase (CyP8B1) in another pathway of bile acid synthesis, resulting in decreased synthesis of bile acid.^[14-16] Additionally, activated FXR induces bile salt exporter pump (BSEP, ABCB11), which secretes intracellular bile acid into bile, and multi-drug resistance gene 3 (MDR3, ABCB4), which secretes intracellular phospholipids into bile, thereby maintaining the solubility of cholesterol in hepatic bile.^[8] Recently, it was suggested that activation of FXR induces hepatic expression of ABCG5/ABCG8, which may play a more important role in maintaining hepatic cholesterol homeostasis.^{[13, 17]}

 

PPARs, upon heterodimerization with RXR, function as transcriptional regulators of glucose and lipid metabolism.^{[18, 19]} Some studies^{[20, 21]} have indicated that stimulation of PPARα and PPARγ increases LXRα expression, and that a PPAR response element (PPRE) exists in the promoter of LXRα and functions as a PPAR-selective response element. In addition, our previous study^[22] in GB epithelial cells (GBECs) documented that PPARα and PPARγ ligands can block the NF-κB pathway, thus modulating the inflammatory reaction, and that PPARα and PPARγ agonists also induce LXRα-mediated ABCA1 activation. These results suggest that PPAR agonist can preserve GB function. Interestingly, fibrates known as PPARα agonists, contribute to cholesterol gallstone formation because they not only suppress the synthesis of bile acid by PPARα-mediated down regulation of CYP7A1 but also stimulate LXRα-ABCG5/ABCG8, which promotes cholesterol excretion into bile, leading to cholesterol supersaturation of bile.^{[23, 24]} Accordingly, PPARα agonist cannot be used to prevent gallstones, despite the ability of PPARα to preserve GB function. Statin, an hydroxy-methl-glutaryl CoA (HMG-CoA) reductase inhibitor that is safely used for the treatment of dyslipidemia, inhibits the synthesis of cholesterol in the liver. Clinical evidence on the preventive role of statin in gallstone formation is conflicting. Population-based case-control studies, however, recently reported that long-term sustained statin use decreases the risk of gallstone diseases and reduces the risk of surgery for gallstone diseases.^[25,26] Most studies suggest that statin reduces the CSI of bile, or at least does not worsen the biliary lipid composition,^{[27, 28]} despite PPARα induction. We also demonstrated that in GBECs, statins activate PPARα and PPARγ, modulate the inflammatory response by blocking lipopolysaccharide-induced TNF-α production, and eliminate excessive cholesterol via the LXRα/RXR-ABCA1-mediated cholesterol efflux system.^[29]

 

Little is known about the mechanisms of how statin affects the homeostasis of hepatic cholesterol-bile acid metabolism, which is associated with the CSI of bile. The present study was to investigate the effects of statin on various regulatory factors, including PPARα, PPARγ, LXRα, FXR, ABCG5/ABCG8, and CYP7A1, in human hepatocytes.

 

 

Dulbecco's modified Eagle's medium (DMEM) was from Gibco (Grand Island, NY, USA). Fetal bovine serum (FBS) and penicillin/streptomycin were from Hyclone (South Logan, UT, USA). Trypsin/ethylene diamine tetraacetic acid (EDTA), dimethyl sulfoxide (DMSO), 3-[4, 5-dimethylthiazol-2-yl] diphenyl tetrazolium bromide (MTT), 22(R)-hydroxycholesterol, and ursodeoxycholic acid (UDCA) were from Sigma Chemicals (St. Louis, MO, USA). Pravastatin, WY-14643, and troglitazone were from Cayman Chemicals (Ann Arbor, MI, USA). The peroxidase-conjugated anti-rabbit IgG antibody was from Amersham Biosciences (Buckinghamshire, UK). The rabbit polyclonal anti-human β-actin antibody was from Cell Signaling Technology (Danvers, MA, USA). The Western blot detection kit (Visualizer) was from Upstate (Lake Placid, NY, USA). Rabbit polyclonal anti-mouse PPARα antibody (a synthetic peptide corresponding to N-terminal amino acids 1-18 of mouse PPARα (NP_038482.3), which has 100% homology with human PPARα) and rabbit polyclonal PPARγ anti-human antibody were purchased from Abcam (Cambridge, UK).

 

Hep3B cells, a hepatoma cell line, were used for our experiments. Stock cultures were grown on 100-mm dishes in DMEM (including 4.5 g/L glucose) supp­lemented with 10% FBS, 2 mmol/L L-glutamine, 1.5 g/L sodium bicarbonate, 100 IU/mL penicillin, and 100 µg/mL streptomycin. The medium was changed twice a week, and the cells were maintained at 37 �� in an incubator with 5% CO₂. The cells were passaged when confluent (every 5 to 7 days) using trypsin (2.5 g/L) and EDTA (1.0 g/L). For experiments, the cells were grown on 60-mm dishes, and the medium was changed to serum free medium (SFM) containing 0.2% bovine serum albumin (BSA) (Sigma, St. Louis, MO, USA).

 

The loading dose of chemicals or reagents used in the experiments described below was determined by MTT assays as the maximum concentration that did not affect the proliferation of cells for 24 hours. Cells were plated at a density of 5×10⁴ cells/mL in 24-well plates and cultured to 60% confluence in serum-containing regular medium. They were then incubated with or without various concentrations of reagents (pravastatin, WY-14643, and troglitazone) in SFM for 24 hours. MTT (0.5 mg/mL) was then added to each well, and the cells were incubated for another 4 hours at 37 ��. After decanting the medium, 500 µL of DMSO was added to each well. After 10 minutes of constant and gentle shaking, the color intensity (proportional to the number of live cells) was assessed with an ELX800 (Biotek, Winooski, VT, USA) at a 570 nm wavelength.

 

Hep3B cells were cultured to confluence on 60-mm dishes. The cells were then incubated in SFM containing 0.2% BSA with pravastatin (1, 5, 10, 20 µmol/l), WY-14643 (100 µmol/L), troglitazone (10 µmol/L), UDCA (150 µmol/L), or 22(R)-hydroxycholesterol (10 µmol/L) for 24 hours as indicated. The cells were harvested, and RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RT-PCR was performed with the ampiRivert 1-step RT-PCR system according to the manufacturer's instructions (Gendepot, Barker, TX, USA). The sequences of primers are described in Table. The total reaction was performed in 50 µL mixtures containing total RNA (1 µg), 20 pmol primer, RT-PCR enzyme mix (5 U AMV transcriptase, 1.25 U Taq DNA polymerase), and 2× RT-PCR buffer mix (2× reaction buffer, 0.4 mmol/L dNTP, and 3.5 mmol/L MgCl₂). The RT reaction was set at 45 �� for 45 minutes and then at 95 �� for 3 minutes. The PCR conditions were as follows: denaturation at 94 �� for 30 seconds, annealing at 53-58 �� for 30 seconds, and extension at 72 �� for 30 seconds. The reaction was ended with an additional extension at 72 �� for 7 minutes, and then samples were chilled to 4 ��. The mRNA was amplified by 26 cycles for LXRα, 40 cycles for ABCG5 and CYP7A1, 35 cycles for ABCG8, and 25 cycles for FXR. The PCR products were fractionated by electrophoresis on 2% agarose gels containing ethidium bromide.

 

Hep3B cells were cultured to confluence on 60-mm dishes with regular media. The cells were treated with pravastatin (5, 10, 20 µmol/L), WY14643 (100 µmol/L), or troglitazone (10 µmol/L) in SFM containing 0.2% BSA for 24 hours as indicated. They were then washed with PBS and harvested with lysis buffer [50 mmol/L Tris (pH 7.5), 150 mmol/L NaCl, 1 mmol EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 µmol/L phenylmethylsulfonyl fluoride (PMSF), 5 µg/mL aprotinin, and 5 µg/mL leupeptin]. Proteins were quantified by the Bradford assay (Sigma St. Louis, Mo, USA). SDS-PAGE was performed with a 4% stacking gel and 10% resolving gel, followed by transfer to a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). The membranes were blocked overnight at 4 �� in a blocking solution [5% skim milk in Tris-buffer with Tween-20 (TBS-T): 200 mmol/L Tris, 500 mmol/L NaCl, pH 7.5, and 0.05% v/v Tween-20]. The membranes were then incubated with rabbit polyclonal PPARα antibody or rabbit polyclonal PPARγ antibody for 1 hour at room temperature. At the same time, the rabbit polyclonal β-actin antibody was used to detect the β-actin housekeeping protein to normalize the amount of protein. The membranes were washed with TBS-T and incubated with peroxidase-conjugated anti-rabbit IgG for 1 hour at room temperature. The membrane was washed and incubated using a Visualizer Western blot detection kit for 5 minutes. Autoradiography was then performed. The signal intensities for specific bands on the Western blotting were quantified using NIH Image J density analysis software (Version 1.20).

 

Results from each experiment are expressed as the mean±SD of duplicate cultures, and all results described are representative of at least three separate experiments. One-way analysis of variance (ANOVA) for three or more unpaired groups or Student's t test for two unpaired groups was used, and P<0.05 was considered statistically significant.

 

 

The expression of PPARα and PPARγ in response to pravastatin (a hydrophilic HMG-CoA reductase inhibitor) was studied in human Hep3B cells. We used 100 µmol/L WY-14643 as a positive control for PPARα and 10 µmol/L troglitazone as a positive control for PPARγ. PPARα expression was increased significantly by a high concentration of pravastatin (P<0.05, versus no-treatment control), but was increased only slightly by low concentrations, 5 or 10 µmol/L of pravastatin (Fig. 1A). PPARγ expression was increased significantly by low concentrations of pravastatin (P<0.01, versus no-treatment control) as well as by a high concentration (P<0.001, versus no-treatment control) in a concentration dependent manner (Fig. 1B). These findings suggest that pravastatin has a greater effect on the expression of PPARγ than on the expression of PPARα.

 

To assess whether statins stimulate the expression of LXRα mRNA, which is induced by activated PPAR, cultured Hep3B cells were incubated with various concentrations of pravastatin, 10 µmol/L 22(R)-hydroxycholesterol as an LXRα positive control, and no-treatment as a negative control. When the treatment concentration of pravastatin was 5 µmol/L or more, the expression of LXRα mRNA was significantly increased, compared with the no-treatment control (P<0.05), to a level similar to that observed with 22(R)-hydroxycholesterol treatment (Fig. 2).

 

As a bile acid sensor, FXR is important for the regulation of cholesterol homeostasis. In order to evaluate whether statin stimulates the expression of FXR mRNA, cultured Hep3B cells were incubated with various concentrations of pravastatin, 150 µmol/L UDCA as a positive control, and no-treatment as a negative control. Pravastatin increased the expression of FXR mRNA significantly in a concentration dependent manner (P<0.01, Fig. 3).

 

We found that pravastatin induced the expression of PPARα, PPARγ, LXRα, and FXR. Because no LXREs have been identified in the promoters of ABCG5 or ABCG8, whether a potent LXR agonist can induce ABCG5 and ABCG8 in human hepatocytes is controversial.^[11] On the other hand, activation of FXR may induce hepatic expression of ABCG5/ABCG8 through a common FXR response element, which promotes biliary-free cholesterol secretions.^{[13, 17]} Accordingly, RT-PCR for ABCG5/ABCG8 was performed using the same conditions as for LXRα to evaluate whether pravastatin induces the expression of ABCG5 and ABCG8 mRNA. The increased expression of ABCG5 mRNA was significantly dependent on the concentration of pravastatin (Fig. 4A). A high concentration of pravastatin (10 or 20 µmol/L) induced the expression of ABCG8 mRNA, though not as much as 22(R)-hydroxycholesterol (Fig. 4B). We also examined whether FXR induces the expression of ABCG5 and ABCG8 mRNA in Hep3B cells. The expression of ABCG5/ABCG8 mRNA was significantly increased by UDCA, an FXR ligand, as well as by LXRα ligand. However, LXRα stimulation induced a stronger expression of ABCG5 and ABCG8 mRNA, compared with FXR stimulation by UDCA (P<0.05) (Fig. 5).

 

In this study, pravastatin appeared to stimulate the expression of FXR mRNA and LXRα mRNA in cultured Hep3B cells. However, the LXRE is not conserved in the human CYP7A1 promoter,^[11] and LXR agonists repress CYP7A1 in human hepatocytes.^[12] Therefore, we wondered whether pravastatin stimulates the expression of CYP7A1, which is activated by LXRα and inhibited by FXR via negative feedback. To determine this, RT-PCR was performed on Hep3B cells stimulated by pravastatin. As shown in Fig. 6A, pravastatin significantly enhanced the expression of CYP7A1 mRNA in a concentration dependent manner in Hep3B cells. The results in Fig. 6B indicate that activation of CYP7A1 mRNA by pravastatin is mediated by the PPARγ and LXRα pathways, together or independently.

 

 

In this study, statin affected the cross-talk between nuclear hormone receptors, including PPARα/PPARγ, LXRα, and FXR, associated with cholesterol-bile acid-phospholipid balance in hepatic bile, probably contributing to the prevention of cholesterol gallstones. Additionally, the current study documented possible molecular mechanisms by which statin prevents gallstones formation.

 

The present study demonstrated in hepatocytes that statin stimulates PPARα, PPARγ, and LXRα expression, and that PPARγ expression changes much more than PPARα expression, even in low concentrations of pravastatin. Considering that PPARγ agonist blocks the repression of CYP7A1 mRNA^[30] and has a more powerful anti-inflammatory effect than fibrates,^[22] the stronger activation of PPARγ induced by statin may play a preventive role in cholesterol gallstones formation. Some studies have suggested probable mechanisms by which statins activate PPARγ: statins enhance peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) activity by suppressing Akt phosphorylation,^[31] and PPARγ also interacts with PGC-1α.^[32] On the other hand, PGC-1α plays a role in the prevention of cholelithiasis via interaction with the bile acid sensor, FXR,^[33] and PPARγ may prevent cholesterol accumulation in GBECs by LXRα-mediated ABCA1 activation.^[22]

 

ABCG5 and ABCG8 are expressed predominantly on the basolateral (bile-contacting) side of hepatocytes and on the apical (luminal) side of enterocytes in the proximal small intestine of humans and mice. They play a pivotal role in transporting excessive cholesterol from hepatocytes into bile or from enterocytes into the lumen.^[10] Accordingly, overexpression of ABCG5 and ABCG8 in hepatocytes may induce supersaturation of bile, resulting in the formation of gallstones.^[8] LXRα, a nuclear receptor that plays a key role in regulating genes involved in cholesterol trafficking, is required for the increased expression of ABCG5 and ABCG8 in response to cholesterol or oxycholesterol.^{[10, 34]} However, it was reported that a potent LXR agonist or cholesterol treatment failed to induce ABCG5 and ABCG8 in primary human hepatocytes.^[13] In contrast, another study^[35] indicated that the expression of both ABCG5 and ABCG8 increases substantially upon LXR activation in human enterocytes. In addition, even though no LXREs have been identified in the promoters of ABCG5 or ABCG8, the intergenic region was found to act as a bidirectional promoter and be partially responsive to treatment with LXR agonists.^[36] Results from studies investigating whether statin activates the expression of LXRα, ABCA1, or ABCG5/ABCG8 are inconsistent.^[37-41] On the other hand, the hepatic expression of ABCG5/ABCG8 is induced by activation of FXR through a common FXRE, which promotes cholesterol secretions, and the FXR-ABCG5/ABCG8 pathway may play a more important role in maintaining hepatic cholesterol homeostasis than the LXRα-ABCG5/ABCG8 pathway in human hepatocytes.^{[13, 17]} The present study showed that pravastatin stimulated the expression of ABCG5/ABCG8 mRNA in Hep3B cells through the activation of LXRα and FXR. Although there might be differences between cancer cells and normal hepatocytes, the result suggests that statin mediates cholesterol secretion into bile by inducing ABCG5/ABCG8 expression via both LXRα and FXR activation in hepatocytes.

 

Although statin activates PPARα and LXRα-ABCG5/ABCG8, which are associated with supersaturated bile, no studies have reported that statin induces gallstones formation. Thus, we considered other potential reasons for increased cholesterol solubility in bile, including CYP7A1 and FXR participation in bile acid homeostasis. Controversy exists over the effects of statins on FXR expression in hepatocytes.^{[42, 43]} Our study demonstrated that statin enhances FXR expression in a concentration dependent manner in hepatocytes. Enhanced FXR expression induced by pravastatin, along with other possible mechanisms described in the next paragraph, presumably contributes to the maintenance or increase of cholesterol solubility in bile. On the other hand, PGC-1α, PPARγ, and hepatocyte nuclear factor-4α (HNF-4α) mRNAs are induced after a prolonged stimulus such as fasting, and PGC-1α coactivates PPARγ and/or HNF-4α bound to a direct repent-1 (DR-1) element in the FXR promoter, to induce FXR mRNA expression.^[44] In addition, statin induces PGC-1α, PPARγ, and HNF-4α.^[31,45] Taken together, statin probably induces FXR through PGC-1α, PPARγ, and HNF-4α activation.

 

CYP7A1 is mainly regulated at the gene transcription level by bile acid, cholesterol, amino acid taurine, cytokines, hormones, and transcriptional regulators.^[11,46] The LXRE does not exist in the human CYP7A1 promoter,^[11] and some studies^[14,47,48] reported that LXR agonists repress CYP7A1 in human hepatocytes.^[12] Furthermore, FXR inhibits CYP7A1, and PPARα agonist represses CYP7A1.^{[23, 24]} Accordingly, we expected CYP7A1 mRNA levels in Hep3B cells treated with pravastatin to decrease or remain unchanged. However, contrary to our expectations, pravastatin enhanced CYP7A1 expression. Possible mechanisms responsible for the enhanced expression of CYP7A1 by statin are as follows: 1) Although no LXREs have been identified in the promoter of CYP7A1, the intergenic region may partially respond to LXR agonists like ABCG5 and ABCG8.^[36] In this experiment, enhanced expression of CYP7A1 induced by 22(R)-hydroxycholesterol supports this theory. 2) We observed that PPARγ agonist increased the expression of CYP7A1. PGC-1α activates CYP7A1 transcription together with HNF-4α by increasing HNF-4α-mediated transactivation of CYP7A1,^[49] and PPARγ also interacts with PGC-1α.^[44] Therefore, we suggest that the statin-PPARγ-PGC-1α-HNF-4α pathway may be involved. Additional study is needed to substantiate this hypothesis. This result does indicate that statin promotes hepatic bile acid synthesis by inducing CYP7A1 and maintains cholesterol solubility in bile.

 

We used Hep3B cells in this study in view of the technical advantages of hepatocyte culture, because Hep3B and HepG2 hepatoma cell lines have cellular characteristics similar to normal hepatocytes. Many studies on enzymes, receptors, and transporters, such as CYP7A1,^{[42, 46, 50]} LXRα,^{[51, 52]} FXR,^[43] and PPAR,^[53] associated with cholesterol metabolism in hepatocytes have been performed with HepG2 or Hep3B cells. In addition, if functional protein studies or over/under-expression studies for specific gene targets were performed, they would clearly demonstrate the cross-talk between nuclear hormone receptors. However, the primary objectives of the study were to demonstrate the change in the expression of the proteins or mRNAs of specific gene related to lipid solubility in bile, and our previous study in hamsters have already documented that pravastatin contributes to the prevention of gallstones formation.^[54] Moreover, we agree that the nucleation and growth of cholesterol gallstones in humans is a complex phenomenon which is associated with not only cholesterol liver metabolism but also gallbladder parietal factors such as mucins, gallbladder motility, and calcium bilirubinate precipitation.^{[55, 56]} Accordingly, a preventive effect on gallstones disease could be achieved by acting also on the other factors involved in gallstones formation. 

 

A study^[57] indicated that the expression of FXR and its target gene small heterodimer partner (SHP) was strongly down-regulated in human hepatocellular carcinoma cells, which is consistent with a tumor suppressive role of FXR.^[58] Therefore, Hep3B cells treated with pravastatin might show some different response compared with primary hepatocytes in expressional change of FXR mRNA, which may be another limitation of this study. In an aspect, our results showed a possibility that statin may have anti-neoplastic effect because statin enhanced expression of FXR mRNA in Hep3B cells.

 

In conclusion, in human hepatocytes, pravastatin induces the expression of PPARγ, more so than that of PPARα, and activates the expression of FXR as well as LXRα. In addition, pravastatin activates ABCG5/ABCG8 expression, which is mediated by both LXRα and FXR, and increases CYP7A1 expression, which is induced by activation of LXRα and PPARγ, together or independently. We postulate that statin increases cholesterol solubility in bile by simultaneous activation of FXR which promotes bile acid excretion and CYP7A1 which enhances hepatic bile acid synthesis,^[8] although statin also activates LXRα-ABCG5/ABCG8 which excretes cholesterol into bile.^[7-10] We thus suggest that statin-induced increased expression of both FXR and LXRα and enhancement of CYP7A1 expression in human hepatocytes, with the known action of statin to preserve GB function, can play a preventive role in cholesterol gallstones diseases.

 

 

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