VDR/vitamin D receptor; PXR; FXR; Microbial Metabolite; Endogenous Metabolite

Lithocholic Acid has an IC50 of 0.7 μM and 1.4 μM, respectively, to block FXR activation produced by GW4064 and CDCA [5]. In HepG2 cells, 100 nM GW4064-induced BSEP expression is inhibited by 10-30 μM lithocholic acid over a 24-hour period [5]. Lithocholic Acid (0-500 μM) suppresses neuroblastoma cell growth (BE(2)-m17, SK-n-SH, SK-n-MCIXC, and Lan-1) in a dose-dependent manner[3].

Lithocholic Acid (LCA) as an Effective Cholestasis Inducer: Mechanisms and Modeling Methods
Pathogenic Mechanisms
• As a toxic secondary bile acid, LCA induces intrahepatic cholestasis by altering hepatocyte membrane composition and bile secretion function.
• Its toxic effects include promoting biliary epithelial cell injury, inflammatory cell infiltration, and bile acid metabolism disorders.
• It exacerbates cholestasis by activating nuclear receptor pathways (e.g., FXR/PXR).

Animal Model Construction
Rat Model (Acute Cholestasis)
Strain: Male Wistar rats (250–300 g)
Dosing Protocol:
Dose: 0.2 μmol/100 g
Route: Intravenous injection (dissolved in 7.5% bovine serum albumin + 0.45% saline)
Sacrifice Time: 1 hour post-administration

Mouse Model (Subacute Cholestasis)
Strain: Male ICR mice (5–7 weeks old)
Dosing Protocol:
Dose: 150 mg/kg
Route: Oral gavage (dissolved in corn oil)
Frequency: Twice daily for 5 administrations
Sacrifice Time: 12 hours after the last dose

Evaluation Parameters
Serum Biochemistry:
• Significant increases in total bilirubin (TBIL) and direct bilirubin (DBIL)
• Elevated liver enzyme activities (ALT, AST, ALP)

Histopathology:
• Hepatocyte vacuolar degeneration and bile duct proliferation
• Inflammatory cell infiltration in portal areas
• Bile capillary cholestasis

Functional Changes:
• Transient increase followed by a significant decrease in bile flow
• Disrupted bile acid (TBA) metabolism

Key Considerations
• Strictly control IV concentration to prevent hemolysis.
• Administer oral doses at fixed light-cycle timepoints to minimize circadian rhythm effects.
• Include vehicle control groups (bovine serum albumin/corn oil) to exclude solvent interference.

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When added to the food at a rate of 0.6% for seven days, lithocholic acid raises the levels of TGFB1, TGFBR1, and TGFBR2 mRNA in the liver of male C57BL/6 mice, activates SMAD3, and causes biliary injury [4]. Male C57BL/6 mice given intraperitoneal injections of lithocholic acid (125 mg/kg, twice daily for four days) develop liver damage and have elevated levels of AST, ALT, and ALP [2].

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The cholestatic liver damage was generated by alphanaphthyl isothiocyanate (ANIT), 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) and Lithocholic acid (LCA). The results indicated that the levels of bile acids were commonly increased in plasma of three mouse cholestasis models, while arginine was decreased. The level of plasma glutathione was decreased in ANIT- and LCA-induced intrahepatic PBC and PSC, respectively. But, the liver glutathione was decreased in DDC induced extrahepatic PSC. The level of plasma phospholipids was elevated in ANIT and DDC models, whereas that was depleted in LCA model. And protoporphyrin IX was significantly increased in the liver of DDC model. These metabolomics data could potentially distinguish the metabolic differences of three types of cholestasis, contributing to the understanding of the potential mechanism of cholestatic liver damage. [2]

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Lithocholic acid (LCA) induced expression of TGFB1 and the receptors TGFBR1 and TGFBR2 in the liver. In addition, immunohistochemistry revealed higher TGFβ expression around the portal vein after Lithocholic acid/LCA exposure and diminished SMAD3 phosphorylation in hepatocytes from Smad3-null mice. Serum metabolomics indicated increased bile acids and decreased lysophosphatidylcholine (LPC) after LCA exposure. Interestingly, in Smad3-null mice, the metabolic alteration was attenuated. LCA-induced lysophosphatidylcholine acyltransferase 4 (LPCAT4) and organic solute transporter β (OSTβ) expression were markedly decreased in Smad3-null mice, whereas TGFβ induced LPCAT4 and OSTβ expression in primary mouse hepatocytes. In addition, introduction of SMAD3 enhanced the TGFβ-induced LPCAT4 and OSTβ expression in the human hepatocellular carcinoma cell line HepG2. In conclusion, considering that Smad3-null mice showed attenuated serum ALP activity, a diagnostic indicator of cholangiocyte injury, these results strongly support the view that TGFβ-SMAD3 signaling mediates an alteration in phospholipid and bile acid metabolism following hepatic inflammation with the biliary injury[4].

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Lithocholic acid exposure enhanced TGFβ and the receptors mRNA level in the liver [4]
The influence of Lithocholic acid (LCA) exposure on TGFβ signaling was investigated using C57BL/6 mice treated with the synthetic AIN93G diet (Cont) and 0.6% LCA-supplemented AIN93G diet (LCA) for 7 days. Hepatic TGFB1, TGFBR1, and TGFBR2 mRNA levels increased after LCA exposure, although TGFBR3 mRNA level did not changed in the livers (Fig. 1). These results suggest that LCA exposure stimulates TGFβ signaling in the livers.

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LCA-induced liver injury was alleviated in Smad3-null mice [4]
TGFβ activates SMAD3 via the TGFβ receptors. Thus, to investigate whether SMAD3 was involved in Lithocholic acid/LCA-induced liver injury, Smad3-null mice were treated with control diet and LCA diet for 6 days. After LCA exposure, the liver mass of Smad3-null mice was smaller than that of LCA-treated wild-type mice (Fig. 2A). In addition, LCA-increased serum ALP activities were significantly attenuated in the Smad3-null mice, although serum ALT activities were not changed (Fig. 2B, C). Furthermore, liver histology showed mild features of inflammatory cell infiltration around the portal vein in Smad3-null mice, which was not observed in similarly treated wild-type mice (Fig. 2D). Immunohistochemistry revealed TGFβ protein around the portal vein with lower expression of the TGFβ in Smad3-null mice compared with wild-type mice (Fig. 2E), suggesting lower TGFβ stimulation of the SMAD3 activation in the liver (supplementary Fig. I). In addition, a dramatic attenuation of the SMAD3 phosphorylation signal was observed in the hepatocyte nuclei of Smad3-null mice (Fig. 2E). These results suggest that TGFβ-SMAD3 signaling is associated with the LCA-induced biliary injury and raise the possibility that TGFβ-SMAD3 signaling alters hepatic metabolism.

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Difference between wild-type and Smad3-null mice in the serum metabolome after Lithocholic acid/LCA exposure [4]
To examine serum metabolites, PLS and contribution analyses were performed with UPLC-ESI-QTOFMS negative mode data derived from serum of mice fed Lithocholic acid/LCA or control diet. PLS analysis showed a separation between the LCA-treated wild-type (Fig. 3A) and the LCA-treated Smad3-null group that was further examined with a loadings plot (Fig. 3B). Contribution analysis revealed 10 enhanced and 10 attenuated ions as the top-ranking ions giving rise to the separation. Lysophosphatidylcholine (LPC) and fatty acid fragments were determined as raised ions in Smad3-null mice compared with the wild-type mice (Table 1). The most lowered ions were derived from bile salts (Table 2). After LCA feeding, the serum metabolome of Smad3-null mice was much different from that of the wild-type mice in LPC and bile salts.

Treatment of cells with Lithocholic acid/LCA, hydrogen peroxide or z-DEVD-fmk [3]
Stock solutions of Lithocholic acid/LCA in varying concentrations were first made in 100% dimethyl sulfoxide (DMSO). For treatment of cultured human NB, human BC and rat GL cells, these stock solutions were then diluted to the indicated final concentration of LCA (the final concentration of DMSO was always kept at 1%) in either a 1:1 mixture of DMEM and Ham's F-12 Nutrient Mixture supplemented with 10% FBS (for SK-n-MCIXC and BE(2)-m17 cells), EMEM supplemented with 10% FBS (for SK-n-SH cells), DMEM supplemented with 10% BCS (for Lan-1 cells) or a 1:1 mixture of DMEM and Ham's F-12 Nutrient Mixture supplemented with 10% FBS and 1X anti-mycotic/anti-biotic (for MCF7 and F98 cells). For treatment of human primary neurons with LCA, stock solutions of LCA in varying concentrations made in 100% DMSO were diluted in EMEM containing 0.225% sodium bicarbonate, 1 mM sodium pyruvate, 2 mM L-glutamine, 0.1% dextrose and 5% FBS to the indicated final concentration of LCA (the final concentration of DMSO was always kept at 1%). For treatment of cultured cancer cells or primary neurons with LCA, they were incubated for 48 hours in the presence LCA at the indicated final concentrations; control cells were treated with an empty DMSO vehicle only. For cell treatment with hydrogen peroxide, its 30% stock solution was diluted in sterile H2O and added directly to the cell cultures after 24 h of their pre-treatment with LCA or an empty DMSO vehicle only; cells were then incubated for 24 h. In experiments involving cell treatment with z-DEVD-fmk, this caspase-3 inhibitor was added to a final concentration of 5 μM simultaneously with LCA or an empty DMSO vehicle only.

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Cell viability assays [3]
The number of viable cells in cultures exposed to Lithocholic acid/LCA was measured using the MTT (3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide)-based CellTiter 96 Non-Radioactive Cell Proliferation Assay. In this assay, only viable, metabolically active cells were able to reduce the yellow tetrazolium salt of MTT to form a purple formazan product. This insoluble product was solubilized by the addition of a detergent. The resulting intracellular purple formazan was then detected spectrophotometrically using a 96-well plate reader at a wavelength of 570 nm. The signal was corrected to account for cellular debris using a wavelength of 630 nm. Chromatin in cells exposed to LCA and/or hydrogen peroxide and/or z-DEVD-fmk was visualized with the fluorescent dye Hoechst used at a final concentration of 4 μM in culture media and viewed using fluorescence microscopy. For each cell culture, the percentage of viable non-apoptotic cells carrying intact, non-fragmented nuclei containing non-condensed chromatin was calculated. Dead apoptotic cells carried fragmented nuclei containing condensed chromatin, a hallmark event of apoptotic death.

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Visualization of mitochondria and measurement of the mitochondrial membrane potential by fluorescence microscopy [3]
Mitochondrial morphology of cells treated with Lithocholic acid/LCA was visualized using MitoTracker Red CMXRos used at a concentration of 125 nM in the culture media. Cells were viewed using fluorescence microscopy, and the percentage of cells displaying fragmented mitochondria was calculated. The mitochondrial membrane potential (∆Ψ) was measured using tetramethylrhodamine ethyl ester (TMRE), a cell-permeant, cationic fluorescent dye. The extent of reversible sequestration of TMRE by mitochondria is proportional to the value of ∆Ψ. Cells were incubated with 50 nM TMRE for 20 min and directly viewed using fluorescence microscopy. The percentage of TMRE-positive cells displaying a detectable level of ∆Ψ was calculated.

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Transient Transfection Assay [5]
HepG2 cells were seeded at a density of 3.2 × 104 cells/well of 96-well plates in DMEM containing 10% FBS 24 h prior to transfection. Cells were transfected with transfection mixes in serum-free Opti-MEM I medium using the FuGENE6 transfection reagent according to the manufacturer's instructions. Typically, transfection mixes for each well contained 0.405 μl of FuGENE6, 3 ng of pcDNA3.1-GAL4-hFXR (LBD) expression vector, 3 ng of pcDNA3.1-hRXRα expression construct, and 60 ng of pUAS(5X)-tk-LUC reporter vector and 60 ng of pCMV-lacZ as an internal control for transfection efficiency. Cells were incubated in the transfection mixture for 4 h at 37 °C in an atmosphere of 10% CO2. The cells were then incubated for ∼40–48 h in fresh DMEM containing 5% charcoal stripped FBS with or without various concentrations of ligands. Cell lysates were produced using reporter lysis buffer according to the manufacturer's directions. Luciferase activity in cell extracts was determined using luciferase assay buffer in an ML3000 luminometer. β-Galactosidase activity was determined using β-d-galactopyranoside as described previously. Luciferase activities were normalized to β-galactosidase activities individually for each well. When assayed for antagonist activity of Lithocholic acid/LCA, cells were incubated with increasing concentrations of LCA in the presence of 100 nm GW4064.

Animal/Disease Models: Male mice (C57BL/6)[4].
Doses: 0.6% LCA-supplement diet, with the AIN93G diet as a control
Route of Administration: in diet, for 6 days
Experimental Results: Induced liver injury. Activated TGFβ-SMAD3 signaling. Increased serum ALP activities.

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Animal/Disease Models: Male mice (C57BL/6)[2].
Doses: 125 mg/kg, dissolved in corn oil
Route of Administration: ip, twice a day for four days
Experimental Results: Induced liver injury, generated necrosis and neutrophilic -granulocytic infiltrate (H&E staining). Increased AST, ALT and ALP level.

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Animals studies [2]
Male C57BL/6 mice (6–8 weeks old) were housed under temperature and humidity-controlled conditions with a 12 h light/12 h dark cycle. Thirty mice were randomly divided into six groups: (1) control of ANIT (ANIT-C); (2) ANIT; (3) control of DDC (DDC-C); (4) DDC; (5) control of Lithocholic acid/LCA (LCA-C); (6) LCA. ANIT dissolved in corn oil was given at a single dose of 75 mg/kg by gavage and mice were sacrificed 48 h after treatment (Fang et al., 2017; Tang et al., 2016). The administration of DDC was slightly modified on the previous reports (Dai et al., 2017; Fickert et al., 2007). DDC dissolved in corn oil was given at an oral dose 100 mg/kg for seven consecutive days, and mice were euthanized 24 h after the last dose of DDC. LCA dissolved in corn oil was intraperitoneal administered at dose of 125 mg/kg twice a day for four days consecutively (Beilke et al., 2009; Owen et al., 2010) and sacrificed 24 h after the last dose of LCA. The mice treated with corn oil were used as the control groups. All the mice were sacrificed by CO2 inhalation. Subsequently, mice plasma and liver samples were collected for biochemical assay, histopathology and metabolomic analysis. Biochemical assay included ALT, AST and ALP. Part of liver tissue were stained with hematoxylin and eosin (H&E) for histopathology using the method described previously (Hu et al., 2018) and the others were stored at −80 °C.

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Animals and diets [4]
Male mice (C57BL/6), MAD homolog 3 (Smad3)-null mice, and background-matched wild-type mice were housed in temperature- and light-controlled rooms and given water and pelleted NIH-31 chow ad libitum. For the Lithocholic acid/LCA studies, mice were given 0.6% LCA-supplement diet with the AIN93G diet as a control. Three wild-type and three Smad3-null mice were fed the control diet, and five wild-type and four Smad3-null mice were given the LCA diet.

Metabolism / Metabolites
LITHOCHOLIC ACID (24)C(14) IS CONVERTED BY RAT LIVER HOMOGENATE INTO 3ALPHA-6BETA-DIHYDROXY-5BETA-CHOLANIC ACID, 7SIGMA-HYDROXYLATION OCCURS, HYDROXYLATION CONJUGATION WITH TAURINE & FORMATION OF 3-SULFATE ESTER CAN BE DEMONSTRATED.
LABELED LITHOCHOLATE WAS INJECTED INTO GALLSTONE PATIENTS & HEALTHY VOLUNTEERS, MAJORITY OF RADIOACTIVITY IN BILE (50-60%) WAS PRESENT AS SULFATED CONJUGATES. DEGREE OF SULFATION WAS GREATER FOR GLYCINE THAN TAURINE CONJUGATES, WHICH SUGGESTED PREFERENTIAL SULFATION OF GLYCINE CONJUGATES.
Lithocholic Acid has known human metabolites that include 6alpha-Hydroxylithocholic acid.

Interactions
SKIN TUMOR INHIBITION DECR IN FOLLOWING ORDER OF ACIDS: CHENODEOXYCHOLIC, LITHOCHOLIC, DEOXYCHOLIC, & CHOLIC.
16ALPHA-CYANOPREGNENOLONE (5 MG IP TWICE DAILY FOR 2 DAYS) INCR IN VITRO RAT LIVER MICROSOMAL 6BETA- & 7ALPHA-HYDROXYLATION OF LITHOCHOLIC ACID BY FACTORS OF 2 & 3-4 RESPECTIVELY. THIS MAY ACCOUNT FOR PREVENTION OF LITHOCHOLIC ACID-INDUCED CHOLELITHIASIS.
LITHOCHOLIC ACID (24)C(14) IS CONVERTED BY RAT LIVER HOMOGENATE INTO 3ALPHA-6BETA-DIHYDROXY-5BETA-CHOLANIC ACID. ADDN OF ETHANOL TO ENZYMATIC SYSTEM RESULTS IN INHIBITION OF FORMATION OF 3ALPHA, 6BETA-DIHYDROXY-5BETA-CHOLANIC ACID.
SODIUM LITHOCHOLATE INCR MNNG (N-METHYL-N'-NITRO-N-NITROSOGUANIDINE) INDUCED COLON TUMOR INCIDENCE IN BOTH GERM-FREE & CONVENTIONAL RATS (F344). /SODIUM LITHOCHOLATE/
LCA was also tested as a promoter of N-Nitrobis(2-hydroxypropyl)amine (BHP) induced carcinogenesis. Two groups of 5 to 6-wk-old hamsters (number not stated) were given 500 mg/kg BHP subcutaneously once per week for 5 weeks, and group 3 was given no further treatment; group 4 was given 0.5% LCA in feed for 30 weeks, all animals were autopsied at 35 weeks. There was no difference in food consumption or body weight between these 2 groups. There were no differences in number on liver lesions (group 3: 15/15 hyperplastic nodules, 2/15 hepatocellular carcinoma, 1/15 cholangiocarcinoma; group 4: 22/22 hyperplastic nodules, 3/22 hepatocellular carcinoma, 3/22 cholangiocarcinoma). However, there was a significant difference in the pancreatic tumors: group 3 had 4/15 gross tumors, 5/15 carcinomas, 4/15 adenomas while group 4 had 13/22 gross tumors, 15/22 carcinomas (P<.04) and 2/22 adenomas. Under the conditions of this experiment, LCA was not carcinogenic when administered alone, but was an effective promoter of BHP pancreatic carcinogenesis.

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mouse LD50 oral 3900 mg/kg Progress Report for Contract No. NIH-NCI-E-C-72-3252, Submitted to the National Cancer Institute by Litton Bionetics, Inc., NCI-E-C-72-3252(1973)

[1]. Effect on blood lipids of very high intakes of fiber in diets low in saturated fat and cholesterol. N Engl J Med, 1993. 329(1): p. 21-6.

[2]. Metabolomic analysis of cholestatic liver damage in mice. Food Chem Toxicol. 2018 Jul 14;120:253-260.

[3]. Lithocholic bile acid selectively kills neuroblastoma cells, while sparing normal neuronal cells. Oncotarget, 2011. 2(10): p. 761-82.

[4]. TGF-beta-SMAD3 signaling mediates hepatic bile acid and phospholipid metabolism following lithocholic acid-induced liver injury. J Lipid Res, 2012. 53(12): p. 2698-707.

[5]. Lithocholic acid decreases expression of bile salt export pump through farnesoid X receptor antagonist activity. J Biol Chem. 2002 Aug 30;277(35):31441-7.

Hexagonal leaflets (from alcohols) or prisms (from acetic acid) or white powder. (NTP, 1992)
Lithocholic acid is a monohydroxy-5beta-cholanic acid with a alpha-hydroxy substituent at position 3. It is a bile acid obtained from chenodeoxycholic acid by bacterial action. It has a role as a human metabolite, a mouse metabolite and a geroprotector. It is a bile acid, a monohydroxy-5beta-cholanic acid and a C24-steroid. It is a conjugate acid of a lithocholate.
Lithocholic acid has been reported in Homo sapiens with data available.
A bile acid formed from chenodeoxycholate by bacterial action, usually conjugated with glycine or taurine. It acts as a detergent to solubilize fats for absorption and is itself absorbed. It is used as cholagogue and choleretic.

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As described above that bile ducts obstruction are the pathological feature of PBC and PSC, several bile acids were commonly increased in three chemical-induced cholestatic models, including TCDCA, T-α/β-MCA and TH/UDCA. In addition to the elevated bile acids in three cholestasis models, the level of arginine was decreased. ANIT and DDC models disrupted the lipids metabolism, which resulted in the increase of LPCs and carnitines levels in both plasma and liver. Different from ANIT and LCA models, liver GSH was decreased in DCC-induced liver damage (Fig. S3). DDC model induced the accumulation of PPIX in the liver, while the level of PPIX was decreased in ANIT and LCA models. Considering that PPIX was converted to heme for oxygen transfer in vivo, the decreased PPIX in ANIT and LCA models might be an adaptive response for improvement of cell damage. Taken together, these changes may partly illustrate the differences of three types of cholestatic liver damage, contributing to the understanding of potential mechanism from different types of cholestasis. The data presented herein establish the benefits of metabolomics approach in biomarker discovery for cholestatic liver injury.[2]

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Aging is one of the major risk factors of cancer. The onset of cancer can be postponed by pharmacological and dietary anti-aging interventions. We recently found in yeast cellular models of aging that lithocholic acid (LCA) extends longevity. Here we show that, at concentrations that are not cytotoxic to primary cultures of human neurons, LCA kills the neuroblastoma (NB) cell lines BE(2)-m17, SK-n-SH, SK-n-MCIXC and Lan-1. In BE(2)-m17, SK-n-SH and SK-n-MCIXC cells, the LCA anti-tumor effect is due to apoptotic cell death. In contrast, the LCA-triggered death of Lan-1 cells is not caused by apoptosis. While low concentrations of LCA sensitize BE(2)-m17 and SK-n-MCIXC cells to hydrogen peroxide-induced apoptotic cell death controlled by mitochondria, these LCA concentrations make primary cultures of human neurons resistant to such a form of cell death. LCA kills BE(2)-m17 and SK-n-MCIXC cell lines by triggering not only the intrinsic (mitochondrial) apoptotic cell death pathway driven by mitochondrial outer membrane permeabilization and initiator caspase-9 activation, but also the extrinsic (death receptor) pathway of apoptosis involving activation of the initiator caspase-8. Based on these data, we propose a mechanism underlying a potent and selective anti-tumor effect of LCA in cultured human NB cells. Moreover, our finding that LCA kills cultured human breast cancer and rat glioma cells implies that it has a broad anti-tumor effect on cancer cells derived from different tissues and organisms.[3]

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Bile salt export pump (BSEP) is a major bile acid transporter in the liver. Mutations in BSEP result in progressive intrahepatic cholestasis, a severe liver disease that impairs bile flow and causes irreversible liver damage. BSEP is a target for inhibition and down-regulation by drugs and abnormal bile salt metabolites, and such inhibition and down-regulation may result in bile acid retention and intrahepatic cholestasis. In this study, we quantitatively analyzed the regulation of BSEP expression by FXR ligands in primary human hepatocytes and HepG2 cells. We demonstrate that BSEP expression is dramatically regulated by ligands of the nuclear receptor farnesoid X receptor (FXR). Both the endogenous FXR agonist chenodeoxycholate (CDCA) and synthetic FXR ligand GW4064 effectively increased BSEP mRNA in both cell types. This up-regulation was readily detectable at as early as 3 h, and the ligand potency for BSEP regulation correlates with the intrinsic activity on FXR. These results suggest BSEP as a direct target of FXR and support the recent report that the BSEP promoter is transactivated by FXR. In contrast to CDCA and GW4064, lithocholate (LCA), a hydrophobic bile acid and a potent inducer of cholestasis, strongly decreased BSEP expression. Previous studies did not identify LCA as an FXR antagonist ligand in cells, but we show here that LCA is an FXR antagonist with partial agonist activity in cells. In an in vitro co-activator association assay, LCA decreased CDCA- and GW4064-induced FXR activation with an IC(50) of 1 microm. In HepG2 cells, LCA also effectively antagonized GW4064-enhanced FXR transactivation. These data suggest that the toxic and cholestatic effect of LCA in animals may result from its down-regulation of BSEP through FXR. Taken together, these observations indicate that FXR plays an important role in BSEP gene expression and that FXR ligands may be potential therapeutic drugs for intrahepatic cholestasis.[5]

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LCA was recently identified as a PXR/SXR agonist ligand. PXR is thought to be the second bile acid receptor and plays a critical role in liver detoxification (27, 28). However, there is no evidence supporting the involvement of PXR in BSEP gene regulation. Indeed, the PXR-specific ligand rifampicin did not regulate BSEP expression, suggesting that PXR is not involved in BSEP regulation and further supporting the conclusion that the down-regulation of BSEP by LCA is mediated through the antagonist activity on FXR. BSEP expression is critically important for liver protection. The identification of FXR as an important regulator of BSEP not only provides a molecular mechanism for FXR-mediated BSEP gene regulation but also suggests a potential for FXR ligands as therapeutic drugs for intrahepatic cholestasis and lipid disorders.[5]

C24H40O3

376.57

376.297

C, 76.55; H, 10.71; O, 12.75

434-13-9

Allolithocholic acid;2276-94-0;Isoallolithocholic acid;2276-93-9;Isolithocholic acid;1534-35-6;Lithocholic acid-d4;83701-16-0;Lithocholic acid-d5;52840-06-9

9903

White to off-white solid powder

1.1±0.1 g/cm3

511.0±23.0 °C at 760 mmHg

183-188 °C(lit.)

276.9±19.1 °C

0.0±3.0 mmHg at 25°C

1.528

6.7

2

3

4

27

574

9

C[C@H](CCC(=O)O)[C@H]1CC[C@@H]2[C@@]1(CC[C@H]3[C@H]2CC[C@H]4[C@@]3(CC[C@H](C4)O)C)C

SMEROWZSTRWXGI-HVATVPOCSA-N

InChI=1S/C24H40O3/c1-15(4-9-22(26)27)19-7-8-20-18-6-5-16-14-17(25)10-12-23(16,2)21(18)11-13-24(19,20)3/h15-21,25H,4-14H2,1-3H3,(H,26,27)/t15-,16-,17-,18+,19-,20+,21+,23+,24-/m1/s1

3alpha-Hydroxy-5beta-cholan-24-oic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.52 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (5.52 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: ≥ 1 mg/mL (2.66 mM) (saturation unknown) in 10% EtOH + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 10.0 mg/mL clear EtOH stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 4: ≥ 1 mg/mL (2.66 mM) (saturation unknown) in 10% EtOH + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 10.0 mg/mL clear EtOH stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix well.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 5: ≥ 1 mg/mL (2.66 mM) (saturation unknown) in 10% EtOH + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one),clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 10.0 mg/mL clear EtOH stock solution to 900 μL of corn oil and mix well.

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 (Please use freshly prepared in vivo formulations for optimal results.)