Microbial Metabolite; Human Endogenous Metabolite

牛磺鹅脱氧胆酸（12-脱氧胆牛磺酸）钠以浓度依赖性方式显着增加 NR8383 细胞的凋亡率。同时，牛磺鹅去氧胆酸钠显着提高 PKC mRNA 水平和活性，并提高 JNK、caspase-3 和 caspase-8 mRNA 表达水平和活性[1]。

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TCDCA/Taurochenodeoxycholic acid诱导NR8383细胞凋亡[1]
我们首先研究了TCDCA是否对NR8383细胞具有凋亡作用。用不同浓度的TCDCA处理细胞后，我们使用流式细胞术测定NR8383细胞的凋亡百分比。基于FITC-Annexin V和PI双重染色，结果表明TCDCA处理提高了NR8383细胞的凋亡率。我们发现，10μM TCDCA治疗组NR8383细胞的凋亡率增加，100μM TCDCA治疗组甚至更高。因此，TCDCA以浓度依赖的方式诱导NR8383细胞凋亡（图1）。

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TCDCA/Taurochenodeoxycholic acid影响PKC基因表达和活性[1]
NR8383细胞用不同浓度的TCDCA（100μM、10μM、1μM）处理1小时，而对照NR8383电池仅在DMEM中孵育。通过qPCR检测PKC的基因表达水平，并使用抗PKCα和抗磷酸化PKCα抗体的Western Blot分析观察PKC的活性。结果显示，TCDCA（100μM、10μM和1μM）处理显著提高了PKC的mRNA表达水平（图2）。与对照组相比，100μM和1μM TCDCA治疗显著提高了PKC-α的表达水平（图3（B））。此外，TCDCA（100μM和10μM）处理显著增加了PKC-α的磷酸化（图3（C））。

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TCDCA/Taurochenodeoxycholic acid影响JNK基因表达和活性[1]
Gö6983是PKC的特异性抑制剂，用于证明TCDCA是否通过PKC/JNK信号通路诱导凋亡过程。对于单次处理，NR8383细胞用100μM、10μM和1μM的TCDCA或Gö6983（10μM）处理1小时。对于联合处理，NR8383细胞用Gö6982预处理1小时，然后与TCDCA（100μM，10μM，1μM）共处理一小时。通过qPCR检测JNK的基因表达水平，并用抗JNK1和抗磷酸化JNK1抗体通过Western Blot分析观察JNK的活性。结果表明，100μM、10μM和1μM TCDCA处理显著提高了JNK mRNA水平。此外，与对照组相比，PKC特异性抑制剂显著降低了JNK的mRNA表达水平。因此，这支持PKC作为JNK基因表达的关键信号的作用。与Gö6983单次治疗相比，TCDCA（100μm、10μm和1μm）/Gö6983联合治疗显著提高了JNK的mRNA表达水平（图4）。TCDCA（100μM、10μM和1μM）显著增强了JNK的表达水平。然而，与对照组相比，Gö6983单次治疗显著抑制了JNK蛋白的表达（图5（B））。此外，100μM和10μM TCDCA显著增加了磷酸化JNK的表达。同时，PKC特异性抑制剂显著阻止了JNK磷酸化表达，表明JNK是NR8383细胞中PKC激活的下游靶点（图5（C））。

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TCDCA/Taurochenodeoxycholic acid影响胱天蛋白酶-3和胱天蛋白酶-8基因的表达和活性[1]
使用JNK的特异性抑制剂SP600125测试了TCDCA通过激活JNK诱导NR8383细胞凋亡的假设。JNK执行激活胱天蛋白酶级联的催化机制。对于单次处理，NR8383细胞用100μM、10μM和1μM的TCDCA或SP600125（10μM）处理1小时。对于联合处理，NR8383细胞用SP600125预处理1小时，然后与TCDCA（100μM，10μM，1μM）共处理一小时。采用qPCR检测Caspase-3和Caspase-8 mRNA表达水平。我们发现TCDCA（100μM、10μM和1μM）以浓度依赖的方式显著增加了caspase-3和caspase-8的mRNA表达水平，而SP600125与对照组相比显著降低了其表达水平。同时，与SP600125单次治疗相比，TCDCA（100μM、10μM和1μM）/SP600125联合治疗显著提高了半胱氨酸天冬氨酸蛋白酶-3和半胱氨酸天冬氨酰蛋白酶-8的mRNA水平（图6）。

牛磺鹅去氧胆酸（12-Deoxycholyltaurine；TCDCA；0.05、0.1g/kg）钠可降低模型小鼠的肺系数，减轻肺部的病理损伤。还可降低肺纤维化小鼠肺组织中TNF-α、TIMP-2的表达水平，但对MMP2无明显影响[2]。牛磺鹅脱氧胆酸钠可防止吲哚美辛引起的胆汁中次级胆汁酸含量和疏水性指数的增加，并显着使临床炎症参数正常化。它还可以减轻肠道炎症[3]。牛磺鹅脱氧胆酸钠显着降低 AA 大鼠的多发性关节炎指数和爪肿胀，增加胸腺和脾脏重量减轻，并纠正放射学变化。所有接受 TCDCA 治疗的大鼠血清和滑膜组织 TNF-α、IL-1β 和 IL-6 mRNA 表达水平均显着降低，并且产生过量[4]。

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腔内细菌、食物摄入和胆汁在吲哚美辛诱导的大鼠小肠炎症中起着重要作用。牛磺脱氧胆酸（TUDCA）和熊去氧胆酸（UDCA）抑制疏水性胆汁酸诱导的各种细胞损伤。我们研究了这些胆汁酸的影响以及其他胆汁酸对这种炎症模型的可能影响。在7天的饮食胆汁酸预处理和随后注射吲哚美辛后，评估了临床和肠道炎症参数以及胆汁分泌。UDCA显著增强了与吲哚美辛相关的食物摄入量和体重减少、总炎症评分和髓过氧化物酶活性增加以及小肠长度缩短。牛磺酸脱氧胆酸（TCDCA）显著地使临床炎症参数正常化，防止了吲哚美辛诱导的胆汁次生胆汁酸含量和疏水性指数的增加，并倾向于减轻肠道炎症。尽管在TCDCA病例中，注射吲哚美辛之前，胆汁中的盐酸水平明显升高，疏水指数明显降低，但这些变化并不能解释TCDCA介导的保护作用。在该模型中，膳食TCDCA会减弱肠道炎症，而UDCA会加剧肠道炎症。胆汁成分的变化（UDCA和鹅脱氧胆酸的增加）可能解释了观察到的修饰作用。[3]

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牛磺脱氧胆酸（TCDCA）是动物胆汁酸的主要生物活性物质之一。本研究旨在探讨TCDCA对大鼠佐剂性关节炎（AA）的抗关节炎作用及其潜在机制。弗氏完全佐剂（FCA）用于诱导大鼠AA。测量爪肿胀、胸腺和脾脏指数以及体重增长率，并观察多发性关节炎指数和放射学变化。ELISA法检测血清和滑膜细胞中TNF-α、IL-1β、IL-6和IL-10的产生。实时RT-PCR检测滑膜组织和滑膜细胞中TNF-α、IL-1β、IL-6和IL-10的mRNA表达。在预防性和治疗性治疗中，TCDCA显著抑制了AA大鼠的足肿胀和多发性关节炎指数，增加了体重损失和胸腺和脾脏指数，并改善了放射学变化。所有TCDCA治疗的大鼠血清和滑膜组织中TNF-α、IL-1β和IL-6的过量产生和mRNA表达均受到显著抑制，但预防性治疗中IL-10显著增加。在300μg/mL至500μg/mL的特定浓度范围内，TCDCA以浓度依赖的方式显著抑制滑膜细胞中TNF-α、IL-1β和IL-6的过度产生和mRNA表达，但对IL-10的作用相反。综上所述，TCDCA治疗大鼠具有良好的抗佐剂性关节炎活性，其修复作用可能是通过降低大鼠TNF-α、IL-1β和IL-6的蛋白质和mRNA表达，以及增加IL-10来介导的[4]。

流式细胞术分析[1]
FITC膜联蛋白V和碘化普罗迪（PI）结合用于鉴定凋亡的存在。细胞用不同浓度的TCDCA（100μM、10μM、1μM）处理48小时，而对照NR8383细胞仅在DMEM中孵育。然后，根据制造商的协议执行所有步骤。简而言之，用预冷PBS洗涤NR8383细胞2-3次，离心并用1×结合缓冲液以1×106个细胞/ml的浓度重新悬浮。然后将100μl溶液即1×105个细胞转移到1.5ml离心管中，加入FITC-膜联蛋白V（终浓度5μl/100μl）和PI（终浓度为5μg/ml）。在25°C的黑暗中孵育20分钟后，使用BD FACSAria™流式细胞仪立即检测到细胞凋亡。每个样本收集约1×104个细胞，并使用Cell Lab Quanta™SC Analysis软件进行分析。

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 RNA分离和qPCR检测[1]
定量实时PCR（qPCR）用于分析各种细胞因子的mRNA水平。将NR8383细胞加入24孔板中并培养过夜以进行附着。此后，细胞用不同的抑制剂预处理1小时，然后用TCDCA共处理另一个小时。使用Tripre™RNA试剂从24孔板中提取总细胞mRNA。通过琼脂糖凝胶电泳和OD260/280比值测定mRNA的质量。根据制造商的方案，使用PrimeScript™RT Master Mix试剂盒进行cDNA合成。使用SYBR®Premix Ex Tag™试剂盒在ViiA™7系统上扩增cDNA。简而言之，总共25μl反应混合物，包括2μl cDNA、12.5μl 2×SYBR®Premix Ex Tag™、1μl正向和反向特异性靶引物（10μM）和8.5μl ddH2O。qPCR热循环设置为95°C下30秒，然后是95°C上5秒的39个循环，Tm上30秒，95°C时15秒。使用特异性引物进行qPCR（表1）。所有数据均基于比较Ct公式计算（Liu等人，2011a，Liu等人，2011b），每个样本均用β-actin进行归一化。根据Ct值，基于以下方程式分析相对mRNA表达：2−ΔCt[ΔCt=Ct（PKC、JNK、caspase-3、caspase-8）-Ct（β-actin）]。熔解曲线保证了每个反应的纯度。

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 Caspase-3和Caspase-8活性测定[1]
根据制造商的方案，使用caspase-Glo®3/7和caspase-Glo®8检测试剂盒检测胱天蛋白酶-3和胱天蛋白酶-8的酶活性。简而言之，将NR8383细胞以6000个细胞/孔的密度加入白色96孔板中，一式三孔，培养过夜以附着。然后用JNK抑制剂预处理细胞1小时，然后用TCDCA共处理另一个小时。此后，将相同体积的Caspase-Glo®试剂加入每个孔中。样品在25°C下孵育，1小时后使用平板读数光度计检测发光。

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我们以前的研究表明，牛磺脱氧胆酸（TCDCA）作为一种信号分子，具有明显的抗炎和免疫调节特性。在本研究中，我们初步探讨了TCDCA诱导NR8383细胞凋亡的潜在作用和可能的机制。采用流式细胞术分析细胞凋亡率。通过qPCR测定基因表达水平。用蛋白质印迹法检测蛋白激酶C（PKC）、Jun N-末端激酶（JNK）的表达及其磷酸化。我们用caspase-Glo®试剂观察了胱天蛋白酶-3和胱天蛋白酶-8的活性。结果表明，TCDCA以浓度依赖的方式显著提高NR8383细胞的凋亡率。同时，TCDCA处理显著增强了PKC mRNA水平和活性。此外，TCDCA可提高JNK、胱天蛋白酶-3和胱天蛋白酶-8的mRNA表达水平和活性，而特异性抑制剂可显著降低它们的表达水平和活力。我们的结论是，TCDCA通过激活NR8383细胞中的caspase级联反应来促进细胞凋亡，PKC/JNK信号通路可能参与了这一过程。这些结果表明，TCDCA可能是治疗细胞凋亡相关疾病的潜在有效药物[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

135.14

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

注意： 请将本产品存放在密封且受保护的环境中，避免吸湿/受潮。

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)

配方 1 中的溶解度: ≥ 2.08 mg/mL (3.99 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 20.8 mg/mL澄清DMSO储备液加入400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.08 mg/mL (3.99 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 20.8 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.08 mg/mL (3.99 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 20.8 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。