Microbial Metabolite; Human Endogenous Metabolite; Caspase TNF-α

Taurochenodeoxycholic acid (TUDCA) induces dissociation of CD34+ HSCs from stromal cells by decreasing adhesion molecule expression. Through the activation of Akt and ERK, it promotes bone marrow stem cell mobilization, differentiation into endothelial progenitor cells (EPCs), and enhancement of EPC proliferation, invasion, and tube formation[1]. By activating the caspase cascade in macrophages, TCDCA causes the apoptosis process, which may involve the PKC/JNK signaling pathway[2].

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TCDCA/Taurochenodeoxycholic acid induced NR8383 cells apoptosis [2]
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 [2]
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 [2]
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 [2]
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).

TUDCA has neuroprotective effects in neuronal cultures and beneficial effects on ischemia reperfusion in animal models, decreasing infarct area and inflammation via attenuation of endoplasmic reticulum (ER) stress. Organic anion transporter (OATP) 2, OATP8, and the Na+–taurocholate cotransporting polypeptide (NTCP) allow TUDCA to enter target cells. By inducing the expression of MAP kinase phosphatase 1 (MKP1), TUDCA prevents neointimal hyperplasia by encouraging the death of smooth muscle cells. TUDCA also lowers ER stress, which protects the hepatocytes and helps to reestablish glucose homeostasis. In vivo neovascularization is accelerated by TUDCA[1]. TCDCA can significantly reduce the pulmonary coefficient in the model mice at doses of 0.05 and 0.1g/kg. TCDCA can extremely significantly reduce the expression levels of TNF- and TIMP-2 in pulmonary tissues in pulmonary fibrosis mice (P>0.01), the expression level of MMP-9 can extremely significantly increase (P>0.01), and MMP2 is unaffected by TCDCA at dosages of 0.05 and 0.1g/kg or greater. TCDCA can extremely significantly increase the expression level of MMP-9, while it has no significant effects on MMP2. TCDCA therefore inhibits the development of pulmonary fibrosis in mice[3].

Flow cytometry analysis [2]
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 [2]
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 [2]
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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Although serum bile acid concentrations are approximately 10 µM in healthy subjects, the crosstalk between the biliary system and vascular repair has never been investigated. In this study, tauroursodeoxycholic acid (TUDCA) induced dissociation of CD34(+) hematopoietic stem cells (HSCs) from stromal cells by reducing adhesion molecule expression. TUDCA increased CD34(+) /Sca1(+) progenitors in mice peripheral blood (PB), and CD34(+) , CD31(+) , and c-kit(+) progenitors in human PB. In addition, TUDCA increased differentiation of CD34(+) HSCs into EPC lineage cells via Akt activation. EPC invasion was increased by TUDCA, which was mediated by fibroblast activating protein via Akt activation. Interestingly, TUDCA induced integration of EPCs into human aortic endothelial cells (HAECs) by increasing adhesion molecule expression. In the mouse hind limb ischemia model, TUDCA promoted blood perfusion by enhancing angiogenesis through recruitment of Flk-1(+) /CD34(+) and Sca-1(+) /c-kit(+) progenitors into damaged tissue. In GFP(+) bone marrow-transplanted hind limb ischemia, TUDCA induced recruitment of GFP(+) /c-kit(+) progenitors to the ischemic area, resulting in an increased blood perfusion ratio. Histological analysis suggested that GFP(+) progenitors mobilized from bone marrow, integrated into blood vessels, and differentiated into VEGFR(+) cells. In addition, TUDCA decreased cellular senescence by reducing levels of p53, p21, and reactive oxygen species and increased nitric oxide. Transplantation of TUDCA-primed senescent EPCs in hind limb ischemia significantly improved blood vessel regeneration, as compared with senescent EPCs. Our results suggested that TUDCA promoted neovascularization by enhancing the mobilization of stem/progenitor cells from bone marrow, their differentiation into EPCs, and their integration with preexisting endothelial cells[1].

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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 [2].

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[3].

[1]. Stem Cells . 2015 Mar;33(3):792-805.

[2]. Eur J Pharmacol . 2016 Sep 5:786:109-115.

[3]. Pak J Pharm Sci . 2013 Jul;26(4):761-5.

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.

C26H45NO6S

499.7036

499.296

516-35-8

Taurochenodeoxycholic acid sodium;6009-98-9;Taurochenodeoxycholic acid-d4 sodium;2410279-85-3

387316

White to off-white solid

1.2±0.1 g/cm3

1.552

2.1

4

6

7

34

858

10

S(C([H])([H])C([H])([H])N([H])C(C([H])([H])C([H])([H])[C@@]([H])(C([H])([H])[H])[C@@]1([H])C([H])([H])C([H])([H])[C@@]2([H])[C@]3([H])[C@@]([H])(C([H])([H])C4([H])C([H])([H])[C@@]([H])(C([H])([H])C([H])([H])[C@]4(C([H])([H])[H])[C@]3([H])C([H])([H])C([H])([H])[C@@]21C([H])([H])[H])O[H])O[H])=O)(=O)(=O)O[H]

BHTRKEVKTKCXOH-BJLOMENOSA-N

InChI=1S/C26H45NO6S/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)/t16-,17+,18-,19-,20+,21+,22-,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]ethanesulfonic acid

Taurochenodeoxycholic acid; TAUROCHENODEOXYCHOLIC ACID; TCDCA; 516-35-8; Taurochenodeoxycholate; Taurochenodesoxycholic acid; Chenyltaurine; Chenodeoxycholyltaurine; Taurine chenodeoxycholate; 12-Deoxycholyltaurine; 12-Deoxycholyltaurine

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: ~99 mg/mL(~198.1 mM)
Water: ~99 mg/mL(~198.1 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.16 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 (4.16 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 (4.16 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 4: 100 mg/mL (200.12 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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