Bile acid derivative; Microbial Metabolite

In healthy individuals, Taurodeoxychloic Acid has a median sodium concentration of 33.9 nM [1]. With an IC50 of 170 μM, tauodeoxycholic acid inhibits the binding of N-3H-methylscopolamine to M3 muscarine uptake [1]. Tauodeoxycholate (0.05–1.00 mM; 1-6) promotes the growth of intestinal epithelial cells [2]. The test for c-cell proliferation is increased when tauodeoxycholate (0.05–1.00 mM; 24 h) causes a considerable increase in the S phase concentration and a significant decrease in the G1 phase concentration of the cell cycle [2].

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IEC-6 or Caco-2 cells were treated with varying concentrations of Taurodeoxychloic Acid (.05 to 1 mmol/L) and proliferation determined. Apoptosis was measured by use of DNA fragmentation assay and nuclear staining. Cell phase was determined with propidium iodide flow cytometry. C-myc expression was determined by Northern and Western blot analysis, and c-myc function was inhibited by specific c-myc antisense.
Results: There was no change in cell structure. Apoptosis was not induced. Six days after exposure to Taurodeoxychloic Acid, IEC-6 cell proliferation was significantly increased. Flow cytometry showed a significant increase in S-phase concentration and a significant decrease in G1-phase concentration of the cell cycle. Taurodeoxycholate also increased c-myc protein and mRNA expression, and inhibition of c-myc function prevented taurodeoxycholate-induced cell proliferation.
Conclusions: Exposure to physiological concentrations of the bile salt Taurodeoxychloic Acid increases intestinal epithelial cell proliferation. This effect is at least partially mediated through a c-myc-dependent mechanism. Bile salts can have a beneficial effect on the intestinal mucosa [1].

Mice with proven sepsis who received tauodeoxycholate (0.5 mg/kg; injection; once) were protected [1], but not TGR5 KO mice.

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TDCA/Taurodeoxychloic Acid confers protection to mice with sepsis [1]
When we infused Taurodeoxychloic Acid/TDCA i.v. (0.5 mg/kg) at 30 min or 24 h after LPS injection, 80 and 50% of the mice survived, respectively (Figure 1A, p < 0.05). The TDCA dose of 0.4 mg/kg was sufficient to obtain this effect (Figure S1A). In addition, 70% of the mice survived when we infused TDCA at 2 h after cecal ligation and puncture (CLP, Figure 1B, p < 0.05). The plasma Cmax (= 502 ng/ml) of TDCA after i.v. infusion (1 mg/kg) was approximately 1/1,000 of the 50% hemolytic concentration (420 μg/ml) previously reported (32) (Figure S1B) and approximately 1/1,000 less than the cytotoxic dose in vitro (33). TDCA did not protect TGR5 KO mice under sepsis (Figure S1C).

TDCA/Taurodeoxychloic Acid decreased liver and kidney damage in septic mice (Figures 1C,D and Figure S2). H&E staining of the kidney showed that the LPS-injected mice exhibited marked vacuolar degeneration of the tubules (arrows in Figure 1C). TDCA infusion almost completely ameliorated LPS-induced kidney lesions (Figure 1C). The mucopolysaccharides on the basement membranes of the glomerular capillary loops and the tubular epithelium of the kidney were also stained with PAS (arrows in Figure 1C, right column). The loss of the brush border was remarkable in the LPS + PBS group (an arrow) and was significantly recovered by TDCA infusion (an arrow in Figure 1C). TDCA infusion normalized kidney function, liver function and hypotension from 4 h after LPS injection (Figures 1D,E, Figure S2). The production of cytokines, such as TNF-α, MCP-1, IL-6, and IL-1β, was also significantly inhibited by TDCA in both the LPS injection and CLP models (Figures 1F–H, Figure S3).

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The phenotype of CD11B+Gr1hi cells increased by Taurodeoxychloic Acid/TDCA [1]
As previously reported (34), mice with sepsis exhibited reduced splenocyte numbers at 48 h after LPS injection and 72 h after CLP (Figure 2A, Figure S4). However, the total numbers of splenocytes were significantly increased in the LPS + TDCA/Taurodeoxychloic Acid group at 48 h after LPS injection and 72 h in the CLP + TDCA group. CD11b+Gr1+ cells increased both in the LPS + TDCA group and CLP + TDCA group (Figures 2B,C, Figures S4, S5). TDCA treatment did not increase the number of T cells or CD11c+ cells (Figure S6). There were no significant changes in the number of CD4+FoxP3+ Treg cells or the expression of CTLA4 on these cells (Figure S7).

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Bile acids (BAs) control metabolism and inflammation by interacting with several receptors. Here, we report that intravenous infusion of taurodeoxycholate (TDCA) decreases serum pro-inflammatory cytokines, normalizes hypotension, protects against renal injury, and prolongs mouse survival during sepsis. Taurodeoxychloic Acid/TDCA increases the number of granulocytic myeloid-derived suppressor cells (MDSCLT) distinctive from MDSCs obtained without TDCA treatment (MDSCL) in the spleen of septic mice. FACS-sorted MDSCLT cells suppress T-cell proliferation and confer protection against sepsis when adoptively transferred better than MDSCL. Proteogenomic analysis indicated that TDCA controls chromatin silencing, alternative splicing, and translation of the immune proteome of MDSCLT, which increases the expression of anti-inflammatory molecules such as oncostatin, lactoferrin and CD244. TDCA also decreases the expression of pro-inflammatory molecules such as neutrophil elastase. These findings suggest that TDCA globally edits the proteome to increase the number of MDSCLT cells and affect their immune-regulatory functions to resolve systemic inflammation during sepsis.

Cell Proliferation Assay[2]
Cell Types: IEC-6 and caco -2 Cell
Tested Concentrations: 0, 0.05, 0.50, and 1.00 mM
Incubation Duration: 1, 2, 4 Myc IEC-6 cell protein and mRNA expression [2]. and 6-day
Experimental Results: significant stimulation of intestinal epithelial cell proliferation in a dose-dependent manner.

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Cell cycle analysis [2]
Cell Types: IEC-6 cells
Tested Concentrations: 0, 0.05, 0.50, 1.00 mM
Incubation Duration: 24 h
Experimental Results: Cells in S phase increased Dramatically, and cells in G1 phase diminished Dramatically.

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Western Blot Analysis[2]
Cell Types: IEC-6 Cell
Tested Concentrations: 0.5 mM
Incubation Duration: 1 and 6 days
Experimental Results: c-myc protein expression increased Dramatically.

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T-cell proliferation assay [1]
T cells were purified from mouse spleen using MACS with a pan T-cell isolation kit. A total of 2 × 105 normal splenic T cells were stimulated with 1 μg/ml of anti-CD3 and 10 μg/ml of anti-CD28 antibodies in RPMI 1640 medium containing 10% heat-inactivated FBS and 2 mM glutamine. FACS-sorted CD11b+Gr1hi cells from the LPS + PBS group or the LPS + Taurodeoxychloic Acid/TDCA group were mixed with T cells at a final concentration of 4 × 104/well (E:T = 1:5) in a 96-well flat-bottom plate and cultured for 96 h at 37°C in a humidified 5% CO2 atmosphere. T cells cultured without CD11b+Gr1hi cells served as a negative control. The cells were pulsed with 1 μCi [3H] methyl-thymidine for 18 h. The cells were harvested with a Filtermate Harvester, and the isotope incorporation was measured using a MicroBeta Plate Counter. The data are expressed as the counts per minute (cpm) ± standard error of the mean (SEM).

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Adoptive transfer of CD11b+Gr1hi cells [1]
PBS or Taurodeoxychloic Acid/TDCA was infused i.v. via the tail vein at 30 min after LPS injection i.p. into B6 mice. The cells were isolated from the spleen at 24 h after LPS injection. Following incubation of the cells in blocking buffer for 30 min on ice, the cells were stained with a biotin-conjugated anti-mouse Gr1 antibody (clone RB6-8C5), followed by staining with anti-biotin microbeads at 4°C for 15 min. After washing with MACS buffer (0.5% BSA and 2 mM EDTA in PBS), the cells were positively selected using an LS column. The Gr1+ cells presorted with MACS were further stained with mAbs against CD11b (conjugated to PE) and F4/80 (conjugated to FITC) for FACS sorting. The CD11b+Gr1hi F4/80int cells were sorted using a FACSAria. A total of 1 × 105 cells were injected i.v. via the tail vein into B6 mice. The recipient mice were injected i.p. with LPS 24 h prior to adoptive transfer.

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In-solution digestion of cell lysates [1]
The CD11b+Gr1hi cells were FACS-sorted from 2 groups of mice (LPS+PBS and LPS+TDCA/Taurodeoxychloic Acid). The lysates of the FACS-sorted CD11b+Gr1hi splenocytes were prepared using 8 M urea buffer, and the protein concentrations were determined by the BCA assay. Dithiothreitol was added to the lysate (3 mM) and incubated at room temperature for 1 h. The cell lysates were mixed with iodoacetamide (5 mM) and incubated in a dark room for 1 h. One part of the lysates was mixed with 10 parts of 50 mM ammonium bicarbonate and digested with trypsin (1/50 × total protein amount of cell lysate) at 37°C for 16 h. The samples were subsequently desalted using a Macro Spin Column (C-18). The column was activated with 0.1% trifluoroacetic acid (TFA) in 80% acetonitrile in advance and subsequently equilibrated with 0.1% TFA in water (pH < 3.0). The samples were loaded into the column and centrifuged at 1,000 × g for 2 min at room temperature. The column was washed with 0.1% TFA in water, and the peptide fraction was eluted with 0.1% TFA in 80% acetonitrile. The peptide samples were dried with a CentriVap® benchtop vacuum concentrator, and the peptide concentration was determined using a BCA kit.

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Labeling peptides for iTRAQ [1]
Isobaric tags for relative and absolute quantitation (iTRAQ) were used to compare the proteomes of CD11b+GR1hi cells from 2 groups (LPS + PBS and LPS + TDCA/Taurodeoxychloic Acid). One hundred micrograms of peptide from each group were labeled according to the manufacturer's protocol for the iTRAQ reagent kit. Briefly, the peptide samples were reconstituted with 500 mM triethylammonium bicarbonate (TEAB) buffer, sonicated and vortexed. The 4-plex iTRAQ reagent dissolved with ethanol was added to the peptide samples and incubated at room temperature for 1 h. To one part of the sample, 3 parts of 0.05% TFA was added and incubated at room temperature for 30 min. The iTRAQ reagent-labeled peptides were pooled and subsequently concentrated to 300 μl using a CentriVap® benchtop vacuum concentrator. The samples were then mixed with 1 ml of 50 mM triethylammonium bicarbonate (TEAB).

Animal/Disease Models: C57BL/6N mouse, lipopolysaccharide injection model of sepsis [1]
Doses: 0.5 mg/kg
Route of Administration: intravenous (iv) (iv)injection 30 minutes or 24 hrs (hrs (hours)) after LPS injection
Experimental Results:Improved survival rate of septic mice . diminished liver and kidney damage in septic mice. Improves systemic inflammation and normalizes blood pressure in septic mice.

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LPS injection model of sepsis [1]
The survival rate of the female mice was determined after i.p. injection of LPS (20 mg/kg), followed by the i.v. infusion of 200 μl of PBS or Taurodeoxychloic Acid/TDCA for 20 min (0.5 mg/kg, unless otherwise indicated) using a Medfusion 2001 system at 30 min (unless otherwise indicated) after LPS injection. For the protection assay using IL-10 KO mice, 5 mg/kg LPS were injected i.p. For the adoptive transfer experiments, B6 mice were injected i.v. with 100 μl of purified cells. The mice were treated with LPS 24 h prior to adoptive transfer, unless otherwise specified.

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CLP-induced sepsis model [1]
Female B6 mice were anesthetized, and a small abdominal midline incision was made. The cecum was ligated below the ileocecal valve and punctured 3 times using a 23-gauge needle. The abdominal incision was closed with an auto-metal clip. The same procedure was applied to the sham-operated animals, with the exception of the ligation and puncture of the cecum. The mice were subsequently infused with 200 μl of PBS or Taurodeoxychloic Acid/TDCA i.v. at 2 h after CLP.

[1]. Taurodeoxycholate Increases the Number of Myeloid-Derived Suppressor Cells That Ameliorate Sepsis in Mice. Front Immunol. 2018 Sep 18;9:1984.

[2]. Taurodeoxycholate increases intestinal epithelial cell proliferation through c-myc expression. Surgery. 2004 Feb;135(2):215-21.

More than 2 million individuals worldwide suffer from sepsis on an annual basis. Because a plethora of pathogenic signaling pathways are simultaneously activated in septic patients, clinical trials targeting a single inflammatory mediator, coagulation factor or pro-inflammatory signal transducer have not shown significant survival benefits. In contrast to former strategies examining the blockade of pro-inflammatory pathways, targeting intrinsic immune regulatory mechanisms may be more effective for inhibiting the broad spectrum of pathways that are activated in sepsis. For these reasons, in vivo expansion of MDSCLT using a pharmacological dose of Taurodeoxychloic Acid/TDCA may be a plausible approach to inhibit the broad-spectrum pathogenesis exhibited in septic patients.[1]

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This study shows that bile salt Taurodeoxychloic Acid/TDCA at least partially increases intestinal epithelial proliferation by regulating transcription of the proto-oncogene c-myc, which has been shown to play an important regulatory role during intestinal epithelial proliferation. This study further defines physiological roles for bile salts in the intestinal mucosa. Thus TDCA can affect cellular functions not just digestive functions in the intestinal mucosa and these effects occur via mediating expression of cell signaling and transcription factors. Further studies are needed to elucidate the role of bile salts during injury, repair and growth within the intestinal mucosa.[2]

C26H44NO6SNA

521.68500

521.278

C, 59.86; H, 8.50; N, 2.68; Na, 4.41; O, 18.40; S, 6.15

110026-03-4

Taurodeoxycholic acid;516-50-7;Sodium taurodeoxycholate hydrate;207737-97-1;Taurodeoxycholic acid-d4 sodium;2410279-82-0

90478527

White to off-white solid powder

168ºC (dec.)(lit.)

4.526

3

6

7

35

864

10

C[C@H](CCC(=O)NCCS(=O)(=O)[O-])[C@H]1CC[C@@H]2[C@@]1(CC[C@H]3[C@H]2C[C@@H]([C@H]4[C@@]3(CC[C@H](C4)O)C)O)C.[Na+]

VNQXUJQHLHHTRC-WMWRQJSFSA-M

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

sodium;2-[[(4R)-4-[(3R,5R,6S,8S,9S,10R,13R,14S,17R)-3,6-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

Taurodeoxycholic acid sodium hydrate

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 : ~125 mg/mL (~231.61 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (3.85 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 (3.85 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 (3.85 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: 20 mg/mL (37.06 mM) in 0.5% CMC-Na/saline water (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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