Endogenous Metabolite; Microbial Metabolite

Cholestasis represents pathophysiologic syndromes defined as impaired bile flow from the liver. As an outcome, bile acids accumulate and promote hepatocyte injury, followed by liver cirrhosis and liver failure. Glycochenodeoxycholic acid (GCDCA) is relatively toxic and highly concentrated in bile and serum after cholestasis. However, the mechanism underlying GCDCA-induced hepatotoxicity remains unclear. In this study, we found that GCDCA inhibits autophagosome formation and impairs lysosomal function by inhibiting lysosomal proteolysis and increasing lysosomal pH, contributing to defects in autophagic clearance and subsequently leading to the death of L02 human hepatocyte cells. Notably, through tandem mass tag (TMT)-based quantitative proteomic analysis and database searches, 313 differentially expressed proteins were identified, of which 71 were increased and 242 were decreased in the GCDCA group compared with those in the control group. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that GCDCA suppressed the signaling pathway of transcription factor E3 (TFE3), which was the most closely associated with autophagic flux impairment. In contrast, GCDCA-inhibited lysosomal function and autophagic flux were efficiently attenuated by TFE3 overexpression. Specifically, the decreased expression of TFE3 was closely related to the disruption of reactive oxygen species (ROS) homeostasis, which could be prevented by inhibiting intracellular ROS with N-acetyl cysteine (NAC). In summary, our study is the first to demonstrate that manipulation of ROS/TFE3 signaling may be a therapeutic approach for antagonizing GCDCA-induced hepatotoxicity. [1]

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Methods: TGF-β mRNA expression was documented in bile duct epithelial cells exposed to varying concentrations of the toxic bile acid; glycochenodeoxycholic acid (GCDCA) ± PC. Results: In these experiments, as well as in co-culture experiments where bile duct epithelial cells were cultured with peripheral blood mononuclear cells and myofibroblasts, TGF-β mRNA expression remained unaltered in the presence or absence of PC. Moreover, collagen type Iα1 mRNA expression by myofibroblasts also remained unaltered.[2]

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The effect of Glycochenodeoxycholic acid/GCDC-induced apoptosis on PKC activity and PKC's role in GCDCA-induced hepatocyte apoptosis is unclear. The specific aims of this study were to determine if GCDC-induced apoptosis changed intracellular PKC activity and if modulation of PKC activity affected GCDC-induced hepatocyte apoptosis. Apoptosis was induced in isolated hepatocytes using GCDC. PKC activity was measured and specific PKC and calpain inhibitors were used to study the effects of PKC and calpain modulation on GCDC-induced apoptosis. After 4 h exposure, 50 microM GCDC induced apoptosis in 42% of hepatocytes. Intracellular PKC activity decreased to 44% of controls 2 h after exposure of hepatocytes to GCDC (p < 0.001). Pre-incubation of hepatocytes with the calpain protease inhibitor restored PKC activity in GCDC exposed hepatocytes to 91 +/- 5% of control cells. Pre-incubation of hepatocytes with a calpain inhibitor decreased GCDC-induced apoptosis as did pre-incubation with the PKC activating phorbol ester, PMA. The combination of calpain inhibition and PMA further reduced GCDC-induced apoptosis but caused low level hepatic apoptosis. Inhibition of PKC with chelerythrine also substantially reduced GCDC-induced hepatocyte apoptosis. GCDC-induced apoptosis is associated with decreases in total cellular PKC activity, which appear to be dependent on intracellular calpain-like protease activity. The combination of protease inhibition and phorbol ester pretreatment preserved total cellular PKC activity and decreased GCDC-induced apoptosis but induced low level apoptosis in the absence of GCDC exposure. PKC inhibition also decreased GCDC-induced hepatocyte apoptosis highlighting the complex interactions of PKC and proteases during GCDC-induced apoptosis. [3]

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Glycochenodeoxycholic acid (GCDC), a component of bile acid (BA), has been reported to induce necrosis in primary human hepatocytes. In the present work, we investigated the function of GCDC in HCC chemoresistance. We found that GCDC promoted chemoresistance in HCC cells by down-regulating and up-regulating the expression of apoptotic and anti-apoptotic genes, respectively. Furthermore, GCDC induced the EMT phenotype and stemness in HCC cells and activated the STAT3 signaling pathway. These findings reveal that GCDC promotes chemoresistance in HCC by inducing stemness via the STAT3 pathway and could be a potential target in HCC chemotherapy [4].

We then assessed whether direct injection of these selected bile acids into the ventricle of the brain could also suppress the HPA axis in vivo. The suppressive effects of bile acids on the HPA axis were restricted to the conjugated bile acids TCA and Glycochenodeoxycholic acid (GCDA), whereas the unconjugated bile acids CA, DCA, and CDCA had no significant effect on circulating corticosterone levels 6 hours after injection (Figure 3A). In parallel, hypothalamic CRH mRNA expression and circulating CRH protein levels were decreased by TCA and Glycochenodeoxycholic acid (GCDA) injection (Figure 3, B and C), whereas the bile acids CA, DCA, and CDCA had no significant effect (data not shown).[5]

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To assess whether the in vivo effect of bile acids was also dependent upon ASBT, we injected an ASBT Vivo-Morpholino or mismatched control sequence into the third ventricle. This significantly suppressed the translation of ASBT protein in the hypothalamic neurons as demonstrated by immunofluorescence (Figure 5A). Direct injection of Glycochenodeoxycholic acid (GCDA) into the third ventricle significantly suppressed the hypothalamic CRH mRNA expression, (Figure 5B) protein content (Figure 5C) and circulating corticosterone levels (Figure 5D), an effect that was attenuated or reversed by ASBT Vivo-Morpholino injection.[5]

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We then assessed whether the ability of bile acids to activate GR is also relevant in vivo. Rats were injected with GR Vivo-Morpholino (or mismatched control sequence) into the lateral ventricle, and the resulting expression of GR in the hypothalamus was assessed by immunofluorescence. As expected, GR immunoreactivity was significantly suppressed after central GR Vivo-Morpholino injection compared with the mismatched control as assessed by immunofluorescence (Figure 7A) and immunoblotting (Figure 7B). Furthermore, subsequent injection of Glycochenodeoxycholic acid (GCDA) into the third ventricle significantly suppressed the CRH mRNA expression (Figure 7C), protein content (Figure 7D), and circulating corticosterone levels (Figure 7E), an effect that was attenuated after GR Vivo-Morpholino injection.[5]

GR luciferase assay [5]
In addition, the GR transcriptional activity was assessed in hypothalamic neurons using a luciferase reporter construct coupled to a promoter region containing the glucocorticoid response element (GRE) consensus sequence following procedures described previously. Neurons were plated onto 96-well plates at a density of 10 000 cells/well and allowed to adhere overnight. Cells were then transfected with the GRE-luciferase reporter construct (0.1-μg DNA/well) with 0.28 μL of TransIT-LT1 transfection reagent overnight at 37°C. After this time, the cells were stimulated with Glycochenodeoxycholic acid (GCDA) or TCA (10μM) and were assayed for luciferase activity using the luciferase assay kit 24 hours after stimulation. Treatments were done at least in quadruplicate, and results are expressed as the degree of change of luciferase activity per microgram of protein.

Cell culture [1]
L02 human normal liver cells were cultured in 1640 medium (HyClone) supplemented with 10% heat-inactivated FCS and 1% (v/v) penicillin/streptomycin (Sigma, St Louis, MO, USA) in a 5% CO2 humidified atmosphere at 37 °C. At 80% confluence, the cells were treated with GCDCA at different concentrations (50, 75, or 100 μM) for 6 h or with 100 μM GCDCA for various periods (0, 1, 3, or 6 h), as described in our previous study (Chen et al., 2013, 2015; Xu et al., 2012). GCDCA was dissolved in sterile phosphate-buffered saline (PBS) to produce a 100 mM stock solution and then used to produce a serial dilution with cell culture medium before application.

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Cell sample preparation and bile acid detection [1]
L02 cells(5 × 106 cells) were treated with 100 μMGCDCA for 6 h, and the cell samples were homogenized on ice in 500 μl of a mixture of chloroform, methanol and water (1:2.5:1, v/v/v). The samples were then centrifuged at 13,000 rpm for 10 min at 4 °C, and a 150-μl aliquot of the supernatant was transferred to an LC sampling vial containing an IS (10 μl L-4-chloro-phenylalanine in water, 5 μg/mL). The deposit was rehomogenized with 500 μl of methanol, and a 150-μl aliquot of supernatant was added to the same vial for drying prior to reconstitution with acetonitrile/H2O (6:4, v/v) to a final volume of 500 μl. After reconstituted with mobile phase, the extract as well as the bile acid reference standards were analyzed with a Waters ACQUITY ultra performance liquid chromatography coupled with a Waters XEVO TQ-S mass spectrometer with an ESI source. The entire UPLC–MS/MS system was controlled by MassLynx 4.1 software. All chromatographic separations were performed with an ACQUITY BEH C18 column (1.7 μm, 100 mm × 2.1 mm internal dimensions) and the injection volume was 5 μL. UPLC-MS raw data obtained with negative mode were analyzed using TargetLynx applications manager version 4.1 to obtain calibration equations and the quantitative concentration of each bile acid in the samples. Lysate samples were measured in ng/well and scaled per mg protein as measured using the Pierce BCA™ Protein Assay Kit.

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Cell death assay [1]
L02 cells were plated in 6-well plates (5 × 105 cells per well). After being treated with GCDCA, the cells were detached with 300 μl of a trypsin EDTA solution. The suspension of detached cells was centrifuged at 300g for 5 min. Then, the pellet was combined with 800 μl of trypan blue solution and dispersed. After staining for 3 min, the cells were counted using an automated cell counter (Bio-Rad, TC10). The dead cells were stained blue. The cell mortality (%) is expressed as the percentage of dead cells/total cells (Chang et al., 2011).

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Cell Culture [2]
KMBC cells were cultured in DMEM supplemented with 110 mg/L sodium pyruvate, 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin. LX-2 cells were cultured in DMEM/F12 supplemented with 0.1 mmol/L non-essential amino acid, 2 mmol/L glutamine, 110 mg/L sodium pyruvate, 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin. All the cells were incubated at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. In experiments, where cells were exposed to GCDCA, the duration of exposure was 24 h.

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Co-culture of KMBC and LX-2 Cells [2]
KMBC and LX-2 cells were cultured in co-culture plates with inserts containing a 3.0-um porous membrane at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. For each plate, 8 × 106 KMBC cells were cultured in the upper chamber and 8 × 106 LX-2 cells in the lower chamber. Culture medium was removed one day before treatment and cells were incubated in the medium described above. The indicated concentrations of GCDCA were added into the upper chamber bath solution for 24 h prior to cell harvesting.

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Co-culture of KMBC Peripheral Blood Mononuclear Cells (PBMC) and LX-2 Cells [2]
KMBC, PBMC (derived from healthy donors) and LX-2 cells were cultured in co-culture plates with inserts containing a 3.0-um porous membrane at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. For each well, 5 × 106 KMBC and 3 × 106 PBMC cells were cultured in the upper chamber and 8 × 106 LX-2 cells in the lower chamber. Culture medium was removed one day before GCDCA exposure and the cells incubated in the medium described above.

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Cell proliferation and cytotoxicity assay [4]
Chemotherapy-induced cell death was determined by cell counting kit-8 assay. Huh7 and LM3 cells were seeded in a 96-well plate at a density of 8 × 103 cells/well and incubated with GCDCA and treated with chemotherapeutic drugs (5-FU and cisplatin) for 24 h and 48 h, respectively. Next, the cells were washed with phosphate-buffered saline (PBS), and cell counting kit-8 (CCK-8) solution (1/10 the volume of media) was added for 1 h. The cell viability was detected at 450 nm using a microplate reader.

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Apoptosis assay [4]
A total of 1 × 105 cells were seeded in a 6-well plate and treated with GCDCA and chemotherapeutic drugs for 24 h and 48 h, respectively. Next, the cells were washed with PBS and resuspended in the PBS and stained with Annexin V and propidium iodide (PI) according to manufacturer’s instructions. Flow cytometry was used to analyze the proportion of apoptotic cells.

Male Sprague Dawley rats (150–175 g) were maintained in a temperature-controlled environment (20°C–22°C) with a 12-hour light, 12-hour dark cycle. Unless otherwise indicated, animals had free access to drinking water and standard rat chow. Rats were fed a diet containing 2% cholestyramine or the control diet AIN-93G for 3 days before either BDL or sham surgeries. Tissue and serum were collected 3 days after surgery between the hours of 8 and 9 am to minimize the circadian variations in glucocorticoid levels. In a separate experiment, rats were injected with 20 pmol of the bile acids cholic acid (CA), deoxycholic acid (DCA), chenodeoxycholic acid (CDCA), Glycochenodeoxycholic acid (GCDA), or TCA in the third ventricle (0 mm medial/lateral, −1.8 mm anterior/posterior, +4.5 mm dorsal/ventral) and serum and tissue were collected 6 hours later. In parallel, rats were infused with 1 mg/kg · d of Vivo-Morpholino sequences into the lateral ventricle at the coordinates (−1.3 mm medial/lateral, −0.2 mm anterior/posterior, +3.5 mm dorsal/ventral) using the brain infusion kits coupled to subcutaneous implanted minipumps for 3 days before the single Glycochenodeoxycholic acid (GCDA) or TCA injection following the method described above. The degree by which the target gene expression was suppressed by Vivo-Morpholino infusion was evaluated by immunofluorescence and immunoblotting as previously described [5].

[1]. Glycochenodeoxycholic acid sodium salt impairs transcription factor E3 -dependent autophagy-lysosome machinery by disrupting reactive oxygen species homeostasis in L02 cells. Toxicol Lett. 2020 Oct 1;331:11-21.

[2]. Glycochenodeoxycholic acid sodium salt Does Not Increase Transforming Growth Factor-Beta Expression in Bile Duct Epithelial Cells or Collagen Synthesis in Myofibroblasts. J Clin Exp Hepatol. 2017 Dec;7(4):316-320.

[3]. Glycochenodeoxycholic acid (GCDC) induced hepatocyte apoptosis is associated with early modulation of intracellular PKC activity. Mol Cell Biochem. 2000 Apr;207(1-2):19-27.

[4]. Glycochenodeoxycholic acid sodium salt induces stemness and chemoresistance via the STAT3 signaling pathway in hepatocellular carcinoma cells. Aging (Albany NY). 2020 Aug 3;12(15):15546-15555.

[5]. Suppression of the HPA Axis During Cholestasis Can Be Attributed to Hypothalamic Bile Acid Signaling. Mol Endocrinol. 2015 Dec;29(12):1720-30.

Glycochenodeoxycholic acid is a bile acid conjugate of glycine, with the bile acid component being 3α,7α-dihydroxy-5β-cholan-24-acyl. It is a human metabolite functionally related to chenodeoxycholic acid and is the conjugated acid of Glycochenodeoxycholic acid salt. Data on Glycochenodeoxycholic acid in Homo sapiens has been reported. It is a bile acid salt formed in the liver from chenodeoxycholic acid and glycine, usually existing as a sodium salt. It acts as a surfactant, dissolving fats to promote absorption, and is also absorbed itself. It has choleretic effects. It is functionally related to chenodeoxycholic acid. It is the conjugate acid of Glycochenodeoxycholic acid.
Glycochenodeoxycholic acid has been reported to exist in Homo sapiens, and relevant data are available.
It is a bile acid formed in the liver from chenodeoxycholic acid and glycine, usually existing as a sodium salt. It acts as a detergent, dissolving fats to facilitate absorption, and is also absorbed itself. It has choleretic and choleretic effects. Recent experimental evidence suggests that dysregulation of hepatic autophagy flux plays a crucial role in the pathogenesis of extrahepatic cholestasis. Impaired autophagy promotes bile acid-induced liver injury and the accumulation of ubiquitinated proteins, while activated autophagy can protect the liver from cholestasis-induced damage (Gao et al., 2014; Kim et al., 2018). Furthermore, Khambu B et al. reported that hepatic autophagy defects impair farnesoid X receptor function and lead to cholestatic injury (Khambu et al., 2019). Consistent with these findings, our results confirm that GCDCA inhibits autophagosome formation and impairs lysosomal function by inhibiting lysosomal proteolysis and increasing lysosomal pH, thereby leading to in vitro autophagic clearance defects. bHLH-leucine zipper protein TFE3, belonging to the MiTF/TFE family, is a key regulator of autophagy and lysosomal biosynthesis and promotes overall cellular degradation (Fan et al., 2018). TFE3 is gradually emerging as a global regulator of cell survival and energy metabolism, with its mechanisms of action including promoting lysosomal gene expression and participating in newly discovered targets such as oxidative metabolism and oxidative stress responses (Pi et al., 2019a; Wang et al., 2019). In recent years, other MiTF/TFE proteins, such as melanocyte-inducible transcription factor (MITF), transcription factor EB (TFEB), and transcription factor EC (TFEC), as major regulators of autophagy and lysosomal biosynthesis, have also been found to be key factors in human disease pathology (Martina et al., 2014). In this study, GCDCA treatment inhibited TFE3 expression and suppressed the activity of the TFE3 reporter gene, thereby reducing the expression of autophagy-related genes. MITF, TFEB, and TFE3 expression patterns were widespread and detected in multiple cell types, while TFEC expression was limited to myeloid cells (Slade and Pulinilkunnil, 2017). Based on these results, we investigated whether other MiT/TFE proteins are involved in the role of GCDCA in L02 cells. Consistent with proteomics analysis, TFE3 mRNA expression was significantly reduced after 6 hours of exposure to different concentrations of GCDCA, while MITF or TFEB levels showed no significant change (Figure S6). These results confirm the important role of TFE3 in GCDCA-mediated autophagy. However, the mechanism of GCDCA-mediated inhibition of TFE3 expression and activity remains unclear. Recent studies have shown that ROS accumulation is associated with TFE3 activation during breast cancer or melanoma cell invasion and migration (Deng et al., 2018; Tan et al., 2018). ROS may play an important role in the inhibition of TFE3 in GCDCA-treated L02 cells. We found that GCDCA induces ROS production in a dose-dependent manner, while NAC pretreatment eliminated this effect. Furthermore, L02 cells incubated with NAC for 2 hours prior to GCDCA treatment showed suppressed ROS production, thus eliminating the effect of GCDCA on the TFE3 pathway. This result contradicts previous findings (Deng et al., 2018; Tan et al., 2018). We propose two possible explanations. First, our results were obtained in a normal cell line rather than a cancer cell line. Second, our study only tested very limited conditions. The exact relationship between ROS and TFE3 may depend on the cell model, and further elucidation of its mechanistic details is needed. In summary, our data suggest that the ROS/TFE3 signaling pathway may serve as a therapeutic target for developing novel therapies to prevent liver injury in patients with extrahepatic cholestasis (Figure 7). Despite these findings, this study has some limitations that deserve emphasis. First, we used only one cell line to evaluate the mechanism of GCDCA-induced hepatotoxicity; future studies will investigate other hepatocyte lines and/or primary hepatocytes. Second, we only investigated GCDCA and not other toxic bile acids. Most importantly, our results were derived from cultured cells, so caution should be exercised when extrapolating in vitro culture results to human patient populations. Further improvements are expected to overcome the current system's limitations in future work, including animal studies and rigorous clinical trials. [1] Despite these findings, there are some limitations worth emphasizing. First, the cell lines used were bile duct epithelial cells and myofibroblasts, rather than primary cells derived from human liver. Whether these cell lines are less sensitive to the toxic effects of GCDCA and the stimulation of profibrotic cytokines than primary cells remains to be determined. Second, this study only examined GCDCA and not other toxic bile acids. Third, the concentration range of GCDCA was referenced to previously reported concentrations in human blood. It is unclear whether higher concentrations of GCDCA exist in human bile, particularly in the bile of patients with primary sclerosing cholangitis (PSC). Fourth, co-culture experiments physically isolate bile duct epithelial cells and peripheral blood mononuclear cells from myofibroblasts, thereby preventing cell-cell contact and potential intercellular communication. Fifth, a longer period of cell exposure to GCDCA might be required to induce bile duct epithelial cell damage. However, rapid biochemical and histological changes in an acute bile duct ligation model negate this possibility. Sixth, it is noteworthy that PBMCs are composed of peripheral blood mononuclear cells, not tissue macrophages. Perhaps certain additional characteristics of the latter cell population are crucial for the expression and release of pro-fibrotic cytokines in this context. Finally, we did not explore whether restoring bile PC concentrations to normal levels could have a beneficial effect on the course of PSC through pathways other than protecting bile duct epithelial cells from toxic bile acid damage. In conclusion, the results of this study do not support the hypothesis that PC deficiency leads to toxic bile acid damage to bile duct epithelial cells, subsequently activating adjacent myofibroblasts, resulting in the exacerbation of fibrosis observed in PSC. Therefore, restoring low PC levels in bile from primary sclerosing cholangitis (PSC) to normal levels does not appear to be an effective treatment for PSC. [2] Studies have shown that the JAK/STAT3 signaling pathway contributes to cancer cell survival and chemotherapeutic resistance. In addition, it can promote the development of cancer stem cell (CSC)-like features. For example, the STAT3 pathway can induce the expression of the CSC marker Nanog in hepatocellular carcinoma initiating cells. Similarly, members of the SOCS and PTPN family have been shown to negatively regulate the JAK/STAT signaling pathway. We found that in hepatocellular carcinoma (HCC) cells, GCDC activates the STAT3 signaling pathway by inhibiting the expression of multiple negative regulators of STAT3 signaling, including SOCS2, SOCS5, PTPN1, and PTPN11. In hepatocellular carcinoma (HCC) cells, glycocholic acid (GCDA)-induced resistance was suppressed when STAT3 expression was inhibited using siRNA. These results suggest that the STAT3 signaling pathway is involved in GCDA-induced chemotherapeutic resistance in HCC cells. In summary, our results indicate that GCDA treatment enhances chemotherapeutic resistance in HCC cells by inducing CSC-like features and EMT phenotypes and by activating the STAT3 signaling pathway by inhibiting the expression of SOCS2, SOCS5, PTPN1 and PTPN1. Therefore, GCDA may be a potential target for the prognosis and treatment of HCC. [3] Studies have shown that the hypothalamus-pituitary-adrenal (HPA) axis is suppressed during cholestatic liver injury. In addition, we have demonstrated that in cholestasis models, serum bile acids can leak into the brain across the blood-brain barrier, leading to an increase in hypothalamic bile acid content. Therefore, this study aimed to investigate the effect of bile acid signaling on the hypothalamus-pituitary-adrenal (HPA) axis. Data showed that the HPA axis was suppressed during cholestatic liver injury, particularly with decreased circulating corticosterone levels and decreased expression of hypothalamic corticotropin-releasing hormone (CRH), which was alleviated by the bile acid sequestrant cholestyramine. Secondly, in vitro experiments showed that treatment of hypothalamic neurons with various bile acids could inhibit the expression and secretion of CRH. However, in vivo experiments showed that the HPA axis was suppressed only after intracerebral injection of taurocholic acid or glycocholic acid (GCDA), while this phenomenon was not observed with other bile acids studied. In addition, we confirmed that taurocholic acid and glycocholic acid (GCDA) activated glucocorticoid receptors after uptake via apical sodium-dependent bile acid transporters, thereby affecting the expression of hypothalamic corticotropin-releasing hormone (CRH). Combined with previous studies, our data support the hypothesis that bile acids enter the brain during cholestatic liver injury, are transported to neurons via apical sodium-dependent bile acid transporters, and activate glucocorticoid receptors to suppress the hypothalamic-pituitary-adrenal (HPA) axis. These data also support the broader hypothesis that bile acids may act as central regulators of hypothalamic peptides that may be altered during liver disease. [5]

C₂₆H₄₃NO₅

449.62

449.314

640-79-9

Glycoursodeoxycholic acid;64480-66-6;Glycochenodeoxycholic acid sodium salt;16564-43-5;Glycochenodeoxycholic acid-d4;1201918-16-2

12544

White to off-white solid powder

4.434

4

5

6

32

727

10

O([H])[C@]1([H])C([H])([H])[C@]2([H])C([H])([H])[C@@]([H])(C([H])([H])C([H])([H])[C@]2(C([H])([H])[H])[C@@]2([H])C([H])([H])C([H])([H])[C@]3(C([H])([H])[H])[C@@]([H])([C@]([H])(C([H])([H])[H])C([H])([H])C([H])([H])C(N([H])C([H])([H])C(=O)O[H])=O)C([H])([H])C([H])([H])[C@@]3([H])[C@@]21[H])O[H]

GHCZAUBVMUEKKP-GYPHWSFCSA-N

InChI=1S/C26H43NO5/c1-15(4-7-22(30)27-14-23(31)32)18-5-6-19-24-20(9-11-26(18,19)3)25(2)10-8-17(28)12-16(25)13-21(24)29/h15-21,24,28-29H,4-14H2,1-3H3,(H,27,30)(H,31,32)/t15-,16+,17-,18-,19+,20+,21-,24+,25+,26-/m1/s1

2-[[(4R)-4-[(3R,5S,7R,8R,9S,10S,13R,14S,17R)-3,7-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanoyl]amino]acetic acid

ChenodeoxycholylglycineLithocholylglycine; GLYCOCHENODEOXYCHOLIC ACID; 640-79-9; Glycochenodeoxycholate; Chenodeoxycholylglycine; Glycine chenodeoxycholate; GCDCA; 12-Deoxycholylglycine; 12-Desoxycholylglycine; Lithocholylglycine, Glycochenodeoxycholate,Glycochenodeoxycholate,Glycine chenodeoxycholate,Glycine chenodeoxycholate, GCDCA,GCDCA Chenodeoxycholylglycine

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)

DMSO : ~100 mg/mL (~222.41 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.56 mM) (saturation unknown) in 10% DMSO + 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 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL 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 2: ≥ 2.5 mg/mL (5.56 mM) (saturation unknown) 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 25.0 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 3: ≥ 2.5 mg/mL (5.56 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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