Background
Tauroursodeoxycholate?is an endoplasmic reticulum (ER) stress inhibitor. Tauroursodeoxycholate significantly reduces expression of apoptosis molecules, such as caspase-3 and caspase-12. Tauroursodeoxycholate also inhibits ERK.
Tauroursodeoxycholate (TUDCA) suppresses both viability and migration of vascular smooth muscle cells (VSMCs) through inhibition of ERK phosphorylation, by induction of mitogen-activated protein kinase phosphatase-1 (MKP-1) via PKCα. Tauroursodeoxycholate inhibits both the proliferation and migration of VSMCs via inhibition of ERK, through Ca2+-dependent PKCα translocation. Tauroursodeoxycholate prevents platelet-derived growth factor (PDGF) and vascular injury-induced MMP-9 expression. The knock-down of MKP-1 using specific si-RNA restores the reduced VSMC viability by Tauroursodeoxycholate (200 μM), which suggests that anti-proliferative effect of Tauroursodeoxycholate depended on the MKP-1 expression[1].
The effects of Tauroursodeoxycholate (TUDCA) on proliferation and apoptosis of VSMCs in vivo are examined using immunohistochemistry for proliferating cell nuclear antigen (PCNA) and the transferase dUTP nick-end labelling (TUNEL) assay. Tauroursodeoxycholate (10, 50, and 100 mg/kg) increases the caspase 3 activity of injured tissues in a dose-dependent manner, indicating that Tauroursodeoxycholate induces apoptosis of VSMCs in the neointima. Using the injured tissues, further examination and comparison of the phosphorylation level of ERK and MMP-9 expression is performed at 1 week after injury, compared with normal controls. Balloon injury increased both the phosphorylation of ERK and expression of MMP-9 in the tissues. Tauroursodeoxycholate (10, 50, and 100 mg/kg) inhibits phosphorylation of ERK and MMP-9 expression in a dose-dependent manner[1]. Tauroursodeoxycholate (TUDCA) is a hydrophilic bile acid. Tauroursodeoxycholate as a cytoprotective agent improves liver function and can prevent hepatocellular carcinoma by reducing ER stress and apoptosis. Tauroursodeoxycholate significantly reduces expression of apoptosis molecules, such as caspase-3, caspase-12, C/EBP homologous protein, c-Jun N-terminal kinase (JNK), activating transcription factor 4 (ATF4), X-box binding protein (XBP), and eukaryotic initiation factor 2α (eIF2α) in Ang II induced ApoE-/- mice (p0.05) and total cholesterol levels (663.6±88.7 mg/dL vs 655.7±65.4 mg/dL; p>0 .05) do not differ between the AAA model group and Tauroursodeoxycholate group. In addition, maximum aortic diameter is significantly smaller in those in Tauroursodeoxycholate group compared with those in the AAA model group (0.95±0.03 mm vs 1.79±0.04 mm; p<0.05). AAA lesion areas are also smaller in those in Tauroursodeoxycholate group than in those in the AAA model group (0.37±0.03 mm2 vs 1.51±0.06 mm2; p<0.05)[2].
牛磺酸是一种内质网应激抑制剂。牛磺酸可显著降低凋亡分子如胱天蛋白酶-3和胱天蛋白酶-12的表达。牛磺酸脱氧胆酸盐也抑制ERK。
牛磺酸脱氧胆酸盐（TUDCA）通过抑制ERK磷酸化，通过PKCα诱导丝裂原活化蛋白激酶磷酸酶-1（MKP-1），抑制血管平滑肌细胞（VSMCs）的生存能力和迁移。牛磺酸通过抑制ERK，通过Ca2+依赖性PKCα易位，抑制VSMCs的增殖和迁移。牛磺酸可防止血小板衍生生长因子（PDGF）和血管损伤诱导的MMP-9表达。使用特异性siRNA敲除MKP-1可恢复牛磺酸（200μM）降低的VSMC活力，这表明牛磺酸的抗增殖作用取决于MKP-1的表达[1]。
用增殖细胞核抗原（PCNA）免疫组织化学和转移酶dUTP缺口末端标记（TUNEL）法检测了牛磺酸脱氧胆酸（TUDCA）对体内VSMCs增殖和凋亡的影响。牛磺酸（10、50和100 mg/kg）以剂量依赖性方式增加损伤组织的胱天蛋白酶3活性，表明牛磺酸诱导新生内膜中VSMCs的凋亡。使用损伤的组织，与正常对照相比，在损伤后1周进行ERK和MMP-9表达的磷酸化水平的进一步检查和比较。球囊损伤增加了组织中ERK的磷酸化和MMP-9的表达。牛磺酸脱氧胆酸盐（10、50和100 mg/kg）以剂量依赖性方式抑制ERK和MMP-9表达的磷酸化[1]。
牛磺酸脱氧胆酸（TUDCA）是一种亲水性胆汁酸。牛磺酸脱氧胆酸作为一种细胞保护剂可以改善肝功能，并通过减少内质网应激和细胞凋亡来预防肝细胞癌。牛磺酸显著降低凋亡分子的表达，如胱天蛋白酶-3、胱天蛋白酶-12、C/EBP同源蛋白、C-Jun N-末端激酶（JNK）、激活转录因子4（ATF4）、X-box结合蛋白（XBP），和真核起始因子2α（eIF2α）在Ang II诱导的ApoE-/-小鼠中的表达（p0.05）和总胆固醇水平（663.6±88.7mg/dLvs 655.7±65.4mg/dL；p>0.05）在AAA模型组和牛磺酸脱氧胆酸盐组之间没有差异。此外，与AAA模型组相比，牛磺酸组的最大主动脉直径显著较小（0.95±0.03 mm vs 1.79±0.04 mm；p<0.05）。牛磺酸组的AAA病变面积也小于AAA模型组（0.37±0.03 mm2 vs 1.51±0.06 mm2；p<0.05）[2]。
[1]. Kim SY, et al. Tauroursodeoxycholate (TUDCA) inhibits neointimal hyperplasia by suppression of ERK viaPKCα-mediated MKP-1 induction. Cardiovasc Res. 2011 Nov 1;92(2):307-16. [2]. Qin Y, et al. 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.
Protocol
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Cell experiment:
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Cell viability and proliferation are measured using Ez-Cytox. VSMCs (5×103 cells) are seeded onto 96-well plates in Smooth Muscle Cell Growth Medium 2 (SMCGM2) and cultured. After serum starvation, Tauroursodeoxycholate (0, 50, 100, and 200 μM) is added to the hVSMCs, with or without 1,2-bis(o-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA, 10 μM) and 7-hydroxystaurosporine (H7, 10 μM) and cultured for 24 h. To assess the effect of Tauroursodeoxycholate on the PDGF-stimulated hVSMC proliferation, hVSMCs are seeded onto 96-well plates and cultured. After serum starvation, Tauroursodeoxycholate (0, 50, 100, and 200 μM) is added to the hVSMCs, with or without PDGF-BB (50 ng/mL) and cultured. After addition of 10 μL of Ez-Cytox into each well, cell viability is evaluated by measuring the optical density at 450 nm[1].
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Animal experiment:
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Rats[1]Sprague-Dawley rats are anaesthetized with a combined anaesthetic (Ketamine, 70 mg/kg; Xylazine, 7 mg/kg ip). Tauroursodeoxycholate is administered orally once a day, in different concentrations (i.e. vehicle, 10, 50, and 100 mg/kg) for 2 weeks. The carotid arteries are fixed by perfusion with 4% formaldehyde, then the tissues are embedded in paraffin, and sections (8 μm) are stained with H&E[1]. Mice[2]Thirty ApoE-/- C57BL/6 male mice aged 8 weeks are 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 are 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 Tauroursodeoxycholate daily for 4 weeks at a dosage of 0.5 g/kg/day in drinking water (Tauroursodeoxycholate group). Mice are sacrificed after 28 days of Ang II infusion[2].
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参考文献:
[1]. Kim SY, et al. Tauroursodeoxycholate (TUDCA) inhibits neointimal hyperplasia by suppression of ERK viaPKCα-mediated MKP-1 induction. Cardiovasc Res. 2011 Nov 1;92(2):307-16.
[2]. Qin Y, et al. 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.
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