Endogenous Metabolite; ERK; Caspase-3/12

牛磺熊去氧胆酸 (TUDCA) 通过 PKCα 激活丝裂原激活的磷酸磷酸酶 1 (MKP-1) 并抑制 ERK 磷酸化，最终降低血管平滑肌细胞 (VSMC) 的存活和迁移。牛磺熊去氧胆酸通过 Ca2+ 诱导 PKCα 易位抑制 ERK 牛磺熊去氧胆酸 (200 μM) 可以恢复牛磺熊去氧胆酸 (200 μM) 降低的 VSMC，从而限制 VSMC 的增殖和迁移 活性，这意味着牛磺熊去氧胆酸的抗应激作用取决于 MKP-1 的表达 [1]。

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TUDCA转运机制及作用通路研究 [1]
通过RT-PCR和蛋白质印迹法分析了人血管平滑肌细胞(hVSMCs)摄取TUDCA的转运蛋白。特异性siRNA敲降实验证实，TUDCA通过有机阴离子转运蛋白2(OATP2)进入hVSMCs。TUDCA通过蛋白激酶Cα(PKCα)诱导丝裂原活化蛋白激酶磷酸酶-1(MKP-1)，进而抑制细胞外信号调节激酶(ERK)的活性，最终降低hVSMCs的存活率。PKC抑制剂7-羟基星形孢菌素或MKP-1敲降均可逆转TUDCA的抗增殖作用。此外，TUDCA通过ERK抑制下调基质金属蛋白酶9(MMP-9)的表达，从而抑制hVSMCs迁移能力。
结论 [1]
hVSMCs对TUDCA的摄取由OATP2介导。TUDCA通过PKCα/MKP-1通路抑制ERK磷酸化，从而同时抑制VSMCs的存活和迁移能力。研究还证实TUDCA可显著抑制PDGF诱导的VSMCs增殖和迁移。[1]

使用转移酶 dUTP 缺口切割标记 (TUNEL) 和免疫组织化学进行肿胀细胞核因子 (PCNA) 测定，研究牛磺熊去氧胆酸 (TUDCA) 对体内 VSMC 肿胀和炎症的影响。牛磺熊去氧胆酸盐（10、50 和 100 mg/kg）实验性地增强了受损组织中的 Caspase 3 活性，并且牛磺熊去氧胆酸盐引起了新内膜中 VSMC 的突变。损伤后一周，使用损伤组织测量 ERK 和 MMP-9 表达的磷酸化水平，并与正常对照进行比较。球囊损伤会增加组织中 MMP-9 的产生和 ERK 的磷酸化。在模拟调节方法中，牛磺熊去氧胆酸（10、50 和 100 mg/kg）抑制 ERK 和 MMP-9 的磷酸化 [1]。胆汁酸牛脱氧胆酸盐 (TUDCA) 是亲水性的。牛脱氧胆酸盐可减少内质网中间细胞和胰腺的数量，从而作为细胞保护剂增强肝功能，并可能预防肝细胞癌。当tauodeoxycholate给予Ang II诱导的ApoE-/-小鼠时，它显着降低Andrew分子的表达，包括eIF2α、caspase-3、caspase-12、C/EBP同源蛋白、c-Jun N-终止子蛋白(JNK) ）、激活转录因子 4 (ATF4)、X-box 结合蛋白 (XBP) 和 JNK。这种影响是显着的（p<0.05）。在 ApoE-/- 小鼠中，牛磺熊去氧胆酸可减少血管紧张素 II 引起的腹主动脉收缩。将 0.5 g/kg/天的牛磺熊去氧胆酸盐给予 Ang II 诱导的 ApoE-/- 小鼠（ER 对）。 ..总胆固醇水平（663.6±88.7 mg/dL 与 655.7±65.4 mg/dL；p>0.05）或收缩压（141.3±5.6 mmHg 与 145.9±8.9 mmHg；p>0.05）之间没有差异。牛磺熊去氧胆酸盐组和AAA模型组。此外，与AAA模型组相比，牛磺熊去氧胆酸盐组的AAA损伤面积小于AAA模型组的损伤面积(0.37±0.03mm 2 比1.51±0.06mm；p<0.05)。 ）[2]。

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TUDCA在体内减少新生内膜增生的作用[1]
体外实验结果表明，TUDCA可能通过PKCα介导的ERK抑制作用抑制血管损伤后的新生内膜形成。采用大鼠颈动脉损伤模型分析TUDCA对新生内膜形成的影响。球囊损伤后，口服TUDCA治疗2周能显著抑制新生内膜形成，且呈剂量依赖性（内膜/中膜比值；50 mg/kg组1.48±0.11；100 mg/kg组1.17±0.16 vs 对照组1.86±0.17，n=8，P<0.05）（图4A和B）。此外，TUDCA给药显著减少了新生内膜面积（对照组100±12.9%；TUDCA 10 mg/kg组80.0±19.7%；50 mg/kg组75.5±6.8%；100 mg/kg组56.9±20%，每组n=8）和新生内膜厚度（对照组133.5±26.7 μm；10 mg/kg组92.1±23.3 μm；50 mg/kg组78.5±15.7 μm；100 mg/kg组61.8±16.3 μm，每组n=8）（图4C和D）。同时，TUDCA以剂量依赖性方式增加了管腔面积（正常组100±5.6%；对照组63.3±7.1%；10 mg/kg组73.1±11.1%；50 mg/kg组78.7±5.8%；100 mg/kg组91.2±8.2%，每组n=8），该增加与新生内膜面积的减少呈正相关（图4E）。值得注意的是，中膜面积和中膜细胞数量未受影响（在线补充材料，图S5A和B）。

FITC-TUDCA摄取实验 [1]
人血管平滑肌细胞(hVSMCs)使用平滑肌细胞生长培养基2(Smooth Muscle Cell Growth Medium 2)培养。本研究采用4名供体来源、传代4-7次的hVSMCs。将1×10⁴个hVSMCs接种于96孔板培养后，分别加入指定浓度的FITC-TUDCA，并用预冷磷酸盐缓冲液洗涤。通过检测488 nm处吸光度评估FITC信号。竞争实验中将100 μM FITC-TUDCA与不同浓度的未标记TUDCA混合，处理hVSMCs 15分钟。具体方法详见在线补充材料。

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明胶酶谱分析 [1]
细胞经TUDCA处理24小时后收集上清，上样至含1 mg/mL明胶的SDS-PAGE凝胶。电泳结束后，凝胶在反应缓冲液中孵育24小时，随后用0.15%考马斯亮蓝R250染色。具体方法详见在线补充材料。

细胞活力与增殖实验 [1]
采用Ez-Cytox试剂检测细胞活力与增殖能力。将血管平滑肌细胞(VSMCs，5×10³个/孔)接种于96孔板，使用平滑肌细胞生长培养基2(Smooth Muscle Cell Growth Medium 2)培养。血清饥饿处理后，分别向hVSMCs中加入不同浓度TUDCA(0、50、100及200 μM)，并设置添加/不添加1,2-双(邻氨基苯氧基)乙烷-N,N,N′,N′-四乙酸四(乙酰氧甲酯)(BAPTA，10 μM)和7-羟基星形孢菌素(H7，10 μM)的对照组，继续培养24小时。为评估TUDCA对PDGF刺激的hVSMCs增殖影响，在血清饥饿处理后同时加入TUDCA(浓度梯度同上)与PDGF-BB(50 ng/mL)。每孔加入10 μL Ez-Cytox后，通过检测450 nm波长光密度评估细胞活力。

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划痕愈合实验 [1]
采用划痕法评估hVSMCs迁移能力。将1×10⁶个hVSMCs接种于60 mm培养皿，培养至细胞完全融合。分别用不同浓度TUDCA预处理1小时后，使用改良枪头在细胞单层中央制造划痕。划痕后用无血清SMCGM2培养基洗涤，继续含TUDCA培养36小时。由三位独立研究者对划痕边缘迁移细胞进行计数分析。

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流式细胞术检测 [1]
通过流式细胞术评估TUDCA诱导的hVSMCs凋亡。血清饥饿处理24小时后，分别用浓度梯度(0、50、100及200 μM)TUDCA处理细胞。收集固定细胞后，通过DNA含量分析检测凋亡率。具体方法详见在线补充材料。

Rat carotid artery balloon injury [1]
The rats were anaesthetized with a combined anaesthetic (ketamine, 70 mg/kg; xylazine, 7 mg/kg ip). Noxious stimuli were applied to a limb occasionally throughout the experiments, while monitoring changes in the end-tidal carbon dioxide, heart rate, blood pressure, and cardiac rhythm, in order to ascertain the level of anaesthesia. After the left external carotid artery was exposed, a 2 F Fogarty embolectomy catheter was introduced through an external carotid arteriotomy incision, advanced to the common carotid artery, inflated with 0.2 mL of saline, and withdrawn 10 times, with rotation.TUDCA was then administered orally once a day, in different concentrations (i.e. vehicle, 10, 50, and 100 mg/kg) for 2 weeks. The carotid arteries were fixed by perfusion with 4% formaldehyde, then the tissues were embedded in paraffin, and sections (8 μm) were stained with H&E. The detailed methods were described in Supplementary material online.

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Ang II induced AAA model in mice [2]
Thirty ApoE−/− C57BL/6 male mice aged 8 weeks were randomly divided into three groups (n = 10 in each group): (i) sham operated and injected with physiologic (0.9%) saline as vehicle (“normal: group); (ii) mini-osmotic pumps were implanted subcutaneously into the right flank of ApoE−/− mice to release Ang II (1000 ng/kg/min) over the course of 28 days (“AAA model” group); (iii) AAA model mice treated with ER stress inhibitor (TUDCA) daily for 4 weeks at a dosage of 0.5 g/kg/day in drinking water (“ER stress inhibitor” group). Mice were sacrificed after 28 days of Ang II infusion. As described previously,22 systolic blood pressure was obtained 1 week before the implantation of the mini-osmotic pumps in mice and after 28 days of infusion, by use of a non-invasive tail cuff system. Blood was collected from the retro-orbital sinus under isoflurane anaesthesia. Serum total cholesterol levels were measured using an enzymatic method.

Absorption, Distribution and Excretion
Evidence suggests that tauroursodeoxycholic acid (TAO) can cross the blood-brain barrier in humans. Metabolism/Metabolites TAO undergoes minimal biotransformation. It is partially deconjugated by gut microbiota, forming unconjugated bile acids.

9848818 Oral LD50 in rats >5 gm/kg (Japan Patent Association Tokyo Gazette, Patent No.: 92-235918)
9848818 Intravenous LD50 in rats 300 mg/kg (Japan Patent Association Tokyo Gazette, Patent No.: 92-235918)
9848818 Oral LD50 in mice >6 gm/kg (Japan Patent Association Tokyo Gazette, Patent No.: 92-235918)
9848818 Intravenous LD50 in mice 350 mg/kg (Japan Patent Association Tokyo Gazette, Patent No.: 92-235918)

[1]. Tauroursodeoxycholate (TUDCA) inhibits neointimal hyperplasia by suppression of ERK viaPKCα-mediated MKP-1 induction. Cardiovasc Res. 2011 Nov 1;92(2):307-16.

[2]. Tauroursodeoxycholic Acid Attenuates Angiotensin II Induced Abdominal Aortic Aneurysm Formation in Apolipoprotein E-deficient Mice by Inhibiting Endoplasmic Reticulum Stress. Eur J Vasc Endovasc Surg. 2017 Mar;53(3):337-345.

Tauroursodeoxycholic acid (TAO) is a taurine-bile acid conjugate derived from ursodeoxycholic acid. It is a human metabolite with anti-inflammatory, neuroprotective, apoptosis-inhibiting, cardioprotective, and bone density-maintaining effects. Its function is related to ursodeoxycholic acid, being a conjugated acid of tauroursodeoxycholic acid salt. Tauroursodeoxycholic acid, also known as ursodeoxycholic acid base, is a highly hydrophilic tertiary bile acid, present in low amounts in the human body. It is a taurine conjugate of ursodeoxycholic acid, possessing comparable therapeutic efficacy and safety, but with higher hydrophilicity. Normally, hydrophilic bile acids modulate the cytotoxic effects of hydrophobic bile acids. Tauroursodeoxycholic acid can reduce the absorption of cholesterol in the small intestine, thereby lowering dietary cholesterol intake and cholesterol levels in the body. Currently, tauroursodeoxycholic acid is used in Europe as a bile acid derivative for the treatment and prevention of gallstones. Due to its molecular properties—particularly its anti-apoptotic effects—tauroursodeoxycholic acid (TAO) has been used in research on inflammatory metabolic diseases and neurodegenerative diseases. Studies on TAO in humans have been reported, and relevant data are available. Pharmaceutical Indications: Tauroursodeoxycholic acid is used for the prevention and treatment of gallstone formation. It is also used in combination with phenylbutyric acid (PBA) to treat adult-onset amyotrophic lateral sclerosis (ALS). Mechanism of Action: Approximately 90% of gallstones are formed from cholesterol, and cholesterol formation may be associated with gut microbiota dysbiosis caused by a high-fat diet and other factors. Gut microbiota regulates bile acid metabolism; therefore, changes in gut microbiota composition may significantly alter the bile acid pool and affect cholesterol secretion. While the exact mechanism by which TAO reduces and prevents gallstone formation is not fully understood, it likely works through multiple pathways. A recent mouse study showed that tauroursodeoxycholic acid (TAO) inhibits intestinal cholesterol absorption and reduces hepatic cholesterol levels by upregulating bile acid excretion from the liver to the gallbladder. TAO reduces cholesterol saturation in gallbladder bile, thereby increasing cholesterol solubility in bile. It also maintains specific gut microbiota composition, promotes bile acid synthesis, and alleviates liver inflammation induced by lipopolysaccharide in the blood. Ultimately, TAO enhances bile acid synthesis in the liver and reduces serum and hepatic cholesterol levels. TAO inhibits apoptosis by interfering with mitochondrial cell death pathways. Its mechanisms of action include inhibiting oxygen free radical production, improving endoplasmic reticulum stress, and stabilizing unfolded protein responses. Other anti-apoptotic processes mediated by TAO include cytochrome C release, caspase activation, DNA and nuclear fragmentation, and inhibition of p53 transcriptional activation. TAO is believed to act on multiple cellular targets, inhibiting apoptosis and upregulating cell survival pathways. Tauroursodeoxycholic acid (TUDCA) is a small molecule drug with its highest clinical trial stage being Phase IV (covering all indications). It was first approved in 2022 and has two investigational indications. Objective: Vascular smooth muscle cell (VSMC) proliferation after vascular injury is one of the main pathophysiological mechanisms associated with neointimal hyperplasia. Tauroursodeoxycholic acid (TUDCA) is a cytoprotective agent effective against various cell types, including hepatocytes, and is also an inducer of apoptosis in cancer cells. This study aimed to investigate whether TUDCA could prevent neointimal hyperplasia by inhibiting the growth and migration of VSMCs. Methods and Results: RT-PCR and Western blot analysis were used to analyze the TUDCA transporter proteins in human VSMCs. Gene knockdown experiments using specific siRNA showed that TUDCA enters human vascular smooth muscle cells (hVSMCs) via organic anion transporter 2 (OATP2). TUDCA induces the expression of mitogen-activated protein kinase phosphatase-1 (MKP-1) through protein kinase Cα (PKCα), thereby inhibiting extracellular signal-regulated kinase (ERK) and reducing hVSMC activity. Treatment with the PKC inhibitor 7-hydroxyastrosporin or knockdown of MKP-1 reverses the antiproliferative effect of TUDCA. Furthermore, TUDCA inhibits hVSMC migration and reduces hVSMC activity by inhibiting ERK and reducing matrix metalloproteinase-9 (MMP-9) expression. Oral administration of TUDCA to rats with carotid balloon injury reduced the elevated levels of ERK and MMP-9 induced by balloon injury. TUDCA significantly reduced the intima-to-media ratio by decreasing vascular smooth muscle cell (VSMC) proliferation and inducing apoptosis. Conclusion: TUDCA inhibits ERK through PKCα-mediated MKP-1 induction, thereby reducing smooth muscle cell proliferation and inducing apoptosis, thus inhibiting neointimal hyperplasia. [1]

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Objective/Background: Abdominal aortic aneurysm (AAA) is characterized by smooth muscle cell (SMC) infiltration, apoptosis, inflammatory cell infiltration, angiogenesis, and extracellular matrix degradation. Previous studies have shown that endoplasmic reticulum (ER) stress and SMC apoptosis are increased in mouse models and human thoracic aortic aneurysms. However, it remains unclear whether ER stress is activated during AAA formation and whether inhibiting ER stress can alleviate AAA. Methods: Human AAA and control aortic samples were collected. Immunohistochemical staining was used to detect the expression of endoplasmic reticulum stress molecular chaperones glucose-regulated protein (GRP)-78 and GRP-94. The effect of the endoplasmic reticulum stress inhibitor tauroursodeoxycholic acid (TUDCA) on angiotensin II (Ang II)-induced apolipoprotein E-/- (ApoE-/-) mouse abdominal aortic aneurysm (AAA) formation was investigated. Elastin staining was used to observe elastin breakage. Immunohistochemical and Western blot analysis were used to detect the protein expression of endoplasmic reticulum stress molecular chaperones and apoptosis-related molecules. Results: GRP-78 and GRP-94 expression was significantly upregulated in the aneurysm region of human abdominal aortic aneurysms and Ang II-induced ApoE-/- mice (p < 0.05). TUDCA significantly reduced the maximum diameter of the abdominal aorta in Ang II-induced ApoE-/- mice (p < 0.05). TUDCA significantly reduced the expression of endoplasmic reticulum stress chaperones and the number of apoptotic cells (p < 0.05). Furthermore, TUDCA significantly reduced the expression of apoptotic molecules, such as caspase-3, caspase-12, C/EBP homolog, c-Jun N-terminal kinase-activated transcription factor 4, X-box binding protein, and eukaryotic initiation factor 2α (eIF2α) in angiotensin II (Ang II)-induced ApoE-/- mice (p < 0.05). Conclusion: These results indicate that endoplasmic reticulum stress is involved in the formation of abdominal aortic aneurysms (AAA) in humans and Ang II-induced ApoE-/- mice. TUDCA alleviates Ang II-induced AAA formation in ApoE-/- mice by inhibiting endoplasmic reticulum stress-mediated apoptosis. [2]

C26H49NO8S

535.734167814255

535.317

117609-50-4

Tauroursodeoxycholate;14605-22-2;Tauroursodeoxycholate sodium;35807-85-3;Tauroursodeoxycholate-d4 sodium;2410279-95-5

71306862

White to off-white solid powder

134

6

8

7

36

858

10

S(CCNC(CC[C@@H](C)[C@H]1CC[C@H]2[C@@H]3[C@H](C[C@@H]4C[C@@H](CC[C@]4(C)[C@H]3CC[C@@]21C)O)O)=O)(=O)(=O)O.O.O

BNXLUNVCHFIPFY-GUBAPICVSA-N

InChI=1S/C26H45NO6S.2H2O/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);2*1H2/t16-,17+,18-,19-,20+,21+,22+,24+,25+,26-;;/m1../s1

2-[[(4R)-4-[(3R,5S,7S,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;dihydrate

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 : ~83.33 mg/mL (~155.54 mM)

配方 1 中的溶解度: ≥ 2.08 mg/mL (3.88 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 (3.88 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 (3.88 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));
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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；
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