Microbial Metabolite; Human Endogenous Metabolite

Taurochenodeoxycholic acid (12-Deoxycholyltaurine) sodium significantly increases the apoptosis rate of NR8383 cells in a concentration-dependent manner. Taurochenodeoxycholic acid sodium, meanwhile, significantly boosts PKC mRNA levels and activities and raises JNK, caspase-3, and caspase-8 mRNA expression levels and activities[1].

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TCDCA/Taurochenodeoxycholic acid induced NR8383 cells apoptosis [1]
We first examined if TCDCA has an apoptotic effect on NR8383 cells. After cells were treated with different concentrations of TCDCA, we used flow cytometry to determine the apoptosis percentage of NR8383 cells. Based on FITC Annexin V and PI double staining, the results demonstrated that TCDCA treatment enhanced the apoptosis rate of NR8383 cells. We found that the apoptosis percentage of NR8383 cells was augmented in the 10 μM TCDCA treatment group, and even higher in the 100 μM TCDCA treatment group. Therefore, TCDCA induced NR8383 cells apoptosis in a concentration-dependent manner (Fig. 1).

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TCDCA/Taurochenodeoxycholic acid effected PKC gene expression and activity [1]
NR8383 cells were treated with different concentrations of TCDCA (100 μM, 10 μM, 1 μM) for 1 h, while control NR8383 cells were incubated in DMEM alone. The gene expression level of PKC was detected by qPCR, and the activity of PKC was observed using Western Blot analysis with anti-PKC alpha and anti-phospho-PKC alpha antibodies. The results revealed that mRNA expression level of PKC was markedly augmented by TCDCA (100 μM, 10 μM and 1 μM) treatments (Fig. 2). Treatment with 100 μM and 1 μM TCDCA remarkably enhanced PKC-α expression level compared with control group (Fig. 3(B)). Besides, phosphorylation of PKC-α was considerably increased by TCDCA (100 μM and 10 μM) treatments (Fig. 3(C)).

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TCDCA/Taurochenodeoxycholic acid effected JNK gene expression and activity [1]
Gö 6983, which is a specific inhibitor of PKC, was used to testify whether the apoptotic process induced by TCDCA via PKC/JNK signaling pathway. For single treatments, NR8383 cells were treated with 100 μM, 10 μM and 1 μM of TCDCA or Gö 6983 (10 μM) for 1 h. For combined-treatments, NR8383 cells were pretreated with Gö 6983 for 1 h before being co-treated with TCDCA (100 μM, 10 μM and 1 μM) for another hour. The gene expression level of JNK was detected by qPCR, and the activity of JNK was observed by Western Blot analysis with anti-JNK1 and anti-phospho-JNK1 antibodies. The results showed that treatments with 100 μM, 10 μM and 1 μM of TCDCA significantly increased JNK mRNA levels. Besides, PKC specific inhibitor markedly reduced the mRNA expression level of JNK compared with control group. Thus, this is supporting a role for PKC as critical signal for the JNK gene expression. While TCDCA (100 Μm, 10 μM and 1 μM)/ Gö 6983 co-treatment markedly increased the mRNA expression level of JNK compared with Gö 6983 single treatment (Fig. 4). JNK expression levels were significantly augmented by TCDCA (100 μM, 10 μM and 1 μM). However Gö 6983 single treatment markedly suppressed JNK protein expression compared with control group (Fig. 5(B)). Furthermore, the expression of phosphorylated JNK was remarkably increased by 100 μM and 10 μM TCDCA. Meanwhile, PKC specific inhibitor significantly prevented JNK phosphorylation expression, suggesting that JNK was a downstream target of PKC activation in NR8383 cells (Fig. 5(C)).

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TCDCA/Taurochenodeoxycholic acid effected caspase-3 and caspase-8 gene expression and activities [1]
The hypothesis that TCDCA induced NR8383 cells apoptosis through activation of JNK was tested using SP600125, which is a specific inhibitor of JNK. JNK performs a catalytic mechanism that activates the caspase cascade. For single treatments, NR8383 cells were treated with 100 μM, 10 μM and 1 μM of TCDCA or SP600125 (10 μM) for 1 h. For combined-treatments, NR8383 cells were pretreated with SP600125 for 1 h before being co-treated with TCDCA (100 μM, 10 μM and 1 μM) for another hour. Caspase-3 and caspase-8 mRNA expression levels were examined using qPCR. We found that TCDCA (100 μM, 10 μM and 1 μM) significantly increased the mRNA expression levels of caspase-3 and caspase-8 in a concentration-dependent manner, while significantly reduced by SP600125 compared with control group. Meanwhile, caspase-3 and caspase-8 mRNA levels were remarkably increased by TCDCA (100 μM, 10 μM and 1 μM)/ SP600125 co-treatments compared with single treatment with SP600125 (Fig. 6).

Taurochenodeoxycholic acid (12-Deoxycholyltaurine; TCDCA; 0.05, 0.1g/kg) sodium lowers the pulmonary coefficient in model mice and lessens the pathological damage to their lungs. It can also lower the expression levels of TNF-α and TIMP-2 in pulmonary tissues in pulmonary fibrosis mice, but it has no appreciable effects on MMP2[2]. Taurochenodeoxycholic acid sodium prevents indomethacin-induced increases in the biliary contents of secondary bile acids and hydrophobicity index and significantly normalizes the clinical inflammatory parameters. It also tends to lessen intestinal inflammation[3]. Taurochenodeoxycholic acid sodium significantly reduces polyarthritis index and paw swelling in AA rats, increases thymus and spleen loss body weight, and corrects radiologic changes. All TCDCA-treated rats have significantly decreased levels of serum and synovium tissue TNF-α, IL-1β, and IL-6 mRNA expression and overproduction[4].

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Intraluminal bacteria, food intake, and bile play important roles in indomethacin-induced small intestinal inflammation in rats. Tauroursodeoxycholic acid (TUDCA) and ursodeoxycholic acid (UDCA) inhibit hydrophobic bile acid-induced damage in various types of cells. We investigated the effects of these bile acids along with the possible influence of other bile acids on this model of inflammation. Clinical and intestinal inflammatory parameters and bile secretion were assessed after 7-day dietary bile acid pretreatments and subsequent indomethacin injections. UDCA significantly enhanced indomethacin-associated reductions in food intake and body weight, increases in gross inflammatory scores and myeloperoxidase activity, and the shortening of small intestinal length. Taurochenodeoxycholic acid (TCDCA) significantly normalized the clinical inflammatory parameters, prevented indomethacin-induced increases in the biliary contents of secondary bile acids and hydrophobicity index, and tended to attenuate the intestinal inflammation. Although elevated biliary levels of muricholic acids and a decreased hydrophobicity index were evident before indomethacin injection in the TCDCA case, these alterations could not explain the TCDCA-mediated protection. Dietary TCDCA attenuates whereas UDCA exacerbates intestinal inflammation in this model. Alterations in the bile composition (increases in UDCA and chenodeoxycholic acid) may explain the observed modification effects.[3]

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Taurochenodeoxycholic acid (TCDCA) is one of the main bioactive substances of animals' bile acid. In this study, we aimed to investigate the anti-arthritic effects and potential mechanism of TCDCA on adjuvant arthritis (AA) in rats. Freund's complete adjuvant (FCA) was used to induce AA in rats. Paw swelling, index of thymus and spleen and body weight growth rate were measured, and polyarthritis index and radiologic changes were observed. The production of TNF-α, IL-1β, IL-6 and IL-10 was detected by ELISA in serum and synoviocytes. mRNA expression of TNF-α, IL-1β, IL-6 and IL-10 was determined by real-time RT-PCR in synovium tissue and synoviocytes. In both prophylactic and therapeutic treatment, TCDCA significantly suppressed paw swelling and polyarthritis index, increased the loss body weight and index of thymus and spleen, and amended radiologic changes in AA rats. The overproduction and mRNA expression of TNF-α, IL-1β and IL-6 were remarkably suppressed in serum and synovium tissue of all TCDCA-treated rats, however, IL-10 was markedly increased in prophylactic treatment. In a definite concentration ranging from 300 μg/mL to 500 μg/mL, TCDCA showed marked inhibition in the overproduction and mRNA expression of TNF-α, IL-1β and IL-6 in synoviocytes in a concentration-dependent manner, but opposite action on IL-10. In conclusion, treatment with TCDCA confers a good anti-adjuvant arthritis activity in rats, which its reparative effects could be mediated via reduction of the protein and mRNA expression of TNF-α, IL-1β and IL-6, and augment of IL-10 in rats [4].

Flow cytometry analysis [1]
FITC Annexin V and Prodium Iodide (PI) binding was used to identify the existence of apoptosis. Cells were treated with different concentrations of TCDCA (100 μM, 10 μM, 1 μM) for 48 h, while control NR8383 cells were incubated in DMEM alone. Then, all steps were performed on the basis of the manufacturer’s protocol. In brief, the NR8383 cells were washed 2–3 times with pre-cooled PBS, centrifuged and resuspended at a concentration of 1×106 cells/ml with 1× Binding Buffer. Then 100 μl of the solution namely 1×105 cells were transferred to 1.5 ml centrifuge tubes and FITC-Annexin V (final concentration 5 μl/100 μl) and PI (final concentration 5 μg/ml) were added. After incubation at 25 °C in the dark for 20 min, apoptosis was instantly detected using a BD FACSAria™ flow cytometer. About 1×104 cells were collected and analyzed with Cell Lab Quanta™ SC Analysis software per sample.

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 RNA isolation and qPCR assays [1]
Quantitative real-time PCR (qPCR) was carried out to analyze the mRNA level of various cytokines. NR8383 cells were added into 24-well plates and cultured overnight for attachment. Thereafter, cells were pre-treated with different inhibitors for 1 h before being co-treated with TCDCA for another hour. The total cellular mRNA was extracted from 24-well plates using Tripre™ RNA reagent. The quality of mRNA was determined by agarose gel electrophoresis and the ratio of OD260/280. Synthesis of cDNA was carried out using PrimeScript™ RT Master Mix kit on the basis of the manufacturer’s protocol. The cDNA was amplified on ViiA™ 7 system using SYBR® Premix Ex Tag™ kit. In brief, a total of 25 μl reaction mixture including 2 μl of cDNA, 12.5 μl of 2×SYBR® Premix Ex Tag™, 1 μl of specific target primers (10 μM) forward and reverse and 8.5 μl of ddH2O. The qPCR thermal cycling settings were 30 s at 95 °C, followed by 39 cycles of 5 s at 95 °C, and 30 s at Tm, and 15 s at 95 °C. The qPCR was carried out with the specific primers (Table 1). All data were calculated based on the comparative Ct formula (Liu et al., 2011a, Liu et al., 2011b) and each sample was normalized by β-actin. Relative mRNA expressions were analyzed according to the Ct values, based on the equation: 2−ΔCt [ΔCt= Ct (PKC, JNK, caspase-3, caspase-8)-Ct (β-actin)]. The melting curves were guaranteed the purity of each reaction.

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 Caspase-3 and caspase-8 activities assays [1]
The enzymatic activities of caspase-3 and caspase-8 were detected using Caspase-Glo® 3/7 and Caspase-Glo® 8 assay kits on the basis of the manufacturer's protocol. In brief, NR8383 cells were added into white 96-well plates at the density of 6000 cells/well in triplicate wells and cultured overnight for attachment. Then cells were pre-treated with JNK inhibitor for 1 h before being co-treated with TCDCA for another hour. Thereafter same volume of Caspase-Glo® reagent was added into each well. Samples were incubated at 25 °C and luminescence was detected after 1 h using a plate-reading luminometer.

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Our former studies have suggested that taurochenodeoxycholic acid (TCDCA) as a signaling molecule shows obvious anti-inflammatory and immune regulation properties. In this research, we tentatively explored the potential effects and the possible mechanism that involve in the apoptotic process in NR8383 cells induced by TCDCA. Using flow cytometry analysis, we evaluated the apoptosis rate. Gene expression levels were determined by qPCR. The expressions of protein kinase C (PKC), Jun N-terminal kinase (JNK) and their phosphorylation were measured by Western Blot. We observed the activities of caspase-3 and caspase-8 with Caspase-Glo® regent. The results demonstrated that TCDCA dramatically improved the apoptosis rate of NR8383 cells in a concentration-dependent manner. In the meantime, PKC mRNA levels and activities were significantly augmented by TCDCA treatments. In addition, JNK, caspase-3 and caspase-8 mRNA expression levels and activities were increased by TCDCA, while they were markedly decreased by specific inhibitors. We conclude that TCDCA contributes to the apoptosis through the activation of the caspase cascade in NR8383 cells, and the PKC/JNK signaling pathway may be involved in this process. These results indicate that TCDCA may be a latent effective pharmaceutical product for apoptosis-related diseases [1].

TCDCA dissociated and depurated [4]
Fresh chicken gall was collected from chicken slaughterhouse, filtered by filter paper, deproteinated by alcohol, depigmented by activated carbon, condensed by rotary evaporator, salted out, extracted, dewatered, after that crude bile acid was obtained. TCDCA was dissociated and depurated from crude bile acid by chromotography techniques and the purity was detected by high performance liquid chromatography and its purity was > 99.5%. Animals and drug treatment [4]
Male Wistar rats, 11–13 weeks old, weighing 160–180 g, were obtained from experimental animal center, academy of military medical sciences in China. All animals were maintained at a controlled temperature (22 ± 2 °C), and a regular light/dark cycle (7:00–19:00 h, light), and all animals had free access to food and water. Animals were divided into six groups of ten each. Group 1 was normal rat (Sham), Group 2 received FCA only, Group 3 and Group 4 received FCA + TCDCA (0.1 g/kg) and FCA + TCDCA (0.2 g/kg), respectively, Groups 3 and 4 were treated beginning from day 0 of injection of FCA, Group 5 and Group 6 received FCA + TCDCA (0.1 g/kg) and FCA + TCDCA (0.2 g/kg), respectively, Group 5 and Group 6 were treated from 14 days after induction. All animals were treated with intragastrical administration and sacrificed after 28 days of induction.

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The present study prepared the pulmonary fibrosis model in mice by using Bleomycin and carry out the investigations on the effects of taurochenodeoxycholic acid (TCDCA) in preventing pulmonary fibrosis in mice. Expression profiles of the bile acid receptors in the lung of mice FXRα and TGR5 were examined, and pulmonary coefficient, pathohistology as well as expression of TNF-α, MMP-2, MMP-9 and TIMP-2 in pulmonary fibrosis mice. The results showed that FXRα and TGR5 simultaneously expressed in the lung of the mice; TCDCA in dosages of 0.05 and 0.1g/kg can extremely significantly decrease the pulmonary coefficient in the model mice (P>0.01), TCDCA in a dosage of 0.2g/kg significantly decreased the pulmonary coefficient in the model mice (P<0.05); TCDCA in dosages of 0.05 and 0.1g/kg significantly reduce the pathological damages on their lungs; TCDCA can extremely significantly decrease the expression levels of TNF-α and TIMP-2 in pulmonary tissues in the pulmonary fibrosis mice (P>0.01), the expression level of MMP-9 extremely significantly increased (P>0.01), while it has no significant effects on MMP2. The results as mentioned above indicated that TCDCA had antagonistic actions on pulmonary fibrosis in mice[2].

[1]. Taurochenodeoxycholic acid induces NR8383 cells apoptosis via PKC/JNK-dependent pathway. Eur J Pharmacol. 2016 Sep 5;786:109-15.

[2]. The effects of taurochenodeoxycholic acid in preventing pulmonary fibrosis in mice. Pak J Pharm Sci. 2013 Jul;26(4):761-5.

[3]. Taurochenodeoxycholic acid ameliorates and ursodeoxycholic acid exacerbates small intestinal inflammation. Am J Physiol. 1997 May;272(5 Pt 1):G1249-57.

[4]. Effects of taurochenodeoxycholic acid on adjuvant arthritis in rats. Int Immunopharmacol. 2011 Dec;11(12):2150-8.

Taurochenodeoxycholic acid is a bile acid taurine conjugate of chenodeoxycholic acid. It has a role as a mouse metabolite and a human metabolite. It is functionally related to a chenodeoxycholic acid. It is a conjugate acid of a taurochenodeoxycholate.
Taurochenodeoxycholic acid is an experimental drug that is normally produced in the liver. Its physiologic function is to emulsify lipids such as cholesterol in the bile. As a medication, taurochenodeoxycholic acid reduces cholesterol formation in the liver, and is likely used as a choleretic to increase the volume of bile secretion from the liver and as a cholagogue to increase bile discharge into the duodenum. It is also being investigated for its role in inflammation and cancer therapy.
Taurochenodeoxycholic acid has been reported in Homo sapiens and Trypanosoma brucei with data available.
A bile salt formed in the liver by conjugation of chenodeoxycholate with taurine, usually as the sodium salt. It acts as detergent to solubilize fats in the small intestine and is itself absorbed. It is used as a cholagogue and choleretic.

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Drug Indication
Taurochenodeoxycholic acid is likely indicated as a choleretic and cholagogue. It is also being investigated for its role in inflammation and cancer therapy.

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Mechanism of Action
Chenodeoxycholic acid is a primary bile acid in the liver that combines with taurine to form the bile acid taurochenodeoxycholic acid. In the bile, taurochenodeoxycholic acid is either a sodium (most) or potassium salt. Taurochenodeoxycholic acid is normally produced in the liver, and its physiologic function as a bile salt is to emulsify lipids such as cholesterol in the bile. As a medication, taurochenodeoxycholic acid reduces cholesterol formation in the liver, and is likely used as a choleretic to increase the volume of bile secretion from the liver and as a cholagogue to increase bile discharge into the duodenum. The mechanism of action of taurochenodeoxycholic acid in inflammation and cancer has yet to be determined.

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Treatment with TCDCA confers a good anti-adjuvant arthritis activity in rats, which its reparative effects could be mediated via reduction of the protein and mRNA expression of TNF-α, IL-1β and IL-6, and augment of IL-10 in rats.[4]

C26H44NNAO6S

521.68

521.278

6009-98-9

Taurochenodeoxycholic acid;516-35-8;Taurochenodeoxycholic acid-d9 sodium;2483832-00-2;Taurochenodeoxycholic acid-d5 sodium;Taurochenodeoxycholic acid-d4-1 sodium;Taurochenodeoxycholic acid-d4 sodium;2410279-85-3

387316

Off-white to light yellow solid

1.164 g/mL at 25 °C(lit.)

215 °C(lit.)

195 °F

n20/D 1.558(lit.)

4.526

4

6

7

34

858

10

C[C@H](CCC(=NCCS(=O)(=O)O)[O-])[C@H]1CC[C@H]2[C@H]3[C@H](CC[C@]12C)[C@@]4(C)CC[C@H](C[C@H]4C[C@H]3O)O.[Na+]

IYPNVUSIMGAJFC-HLEJRKHJSA-M

InChI=1S/C26H45NO6S.Na/c1-16(4-7-23(30)27-12-13-34(31,32)33)19-5-6-20-24-21(9-11-26(19,20)3)25(2)10-8-18(28)14-17(25)15-22(24)29;/h16-22,24,28-29H,4-15H2,1-3H3,(H,27,30)(H,31,32,33);/q;+1/p-1/t16-,17+,18-,19-,20+,21+,22-,24+,25+,26-;/m1./s1

sodium;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]ethanesulfonate

Taurochenodeoxycholic acid sodium salt; P2SD3PHQ3Y; CHENYL TAURINE SODIUM; NSC-681055; Ethanesulfonic acid, 2-[[(3alpha,5beta,7alpha)-3,7-dihydroxy-24-oxocholan-24-yl]amino]-, sodium salt (1:1); TAUROCHENODEOXY CHOLATE SODIUM SALT; TAURINE, N-(3.ALPHA.,7.ALPHA.-DIHYDROXY-5.BETA.-CHOLAN-24-OYL)-, MONOSODIUM SALT; ETHANESULFONIC ACID, 2-(((3.ALPHA.,5.BETA.,7.ALPHA.)-3,7-DIHYDROXY-24-OXOCHOLAN-24-YL)AMINO)-, SODIUM SALT (1:1); ...; 6009-98-9;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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 (~191.7 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (3.99 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 20.8 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: ≥ 2.08 mg/mL (3.99 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 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 3: ≥ 2.08 mg/mL (3.99 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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 (Please use freshly prepared in vivo formulations for optimal results.)