酒石酸美托洛尔调节TGF-β/Smad信号通路抑制大鼠胸主动脉瘤进程的机制研究
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摘要
目的研究酒石酸美托洛尔通过调节转化生长因子-β(TGF-β)/Smad信号通路抑制大鼠胸主动脉瘤(TAA)进程的分子机制。方法暴露大鼠胸降主动脉1 cm,将已用0.5 mol/L的氯化钙浸泡好的棉纱覆盖血管外膜15～20 min,建立TAA模型,设置模型组和酒石酸美托洛尔高、中、低剂量(0.60、0.30、0.15 mg/kg)组,另取10只大鼠作为对照组,每天1次,连续ig给药4周,模型组和对照组ig等体积蒸馏水。对大鼠生理活动进行观察和记录,HE染色观察动脉管腔面改变;实时荧光定量PCR(qRT-PCR)和Western blotting对大鼠TAA组织中的TGF-β、Smad2、Smad3的表达水平进行检测。结果与对照组比较,模型组大鼠进食较少,体质量减轻,精神萎靡,毛发杂乱,无光泽,活动减少;酒石酸美托洛尔组与模型组比较,生理状态好转;HE染色显示,对照组主动脉壁弹力板排列规则、紧密,呈波浪形膜状;模型组动脉瘤壁的弹力板平直,并且有断裂现象;与模型组比较,酒石酸美托洛尔组弹力板断裂减少,动脉壁呈波浪形膜状。与对照组比较,模型组瘤组织中的TGF-β、Smad2、Smad3的mRNA、蛋白表达水平均显著上调(P<0.05);与模型组织比较,酒石酸美托洛尔组TGF-β、Smad2、Smad3的mRNA、蛋白表达水平均显著下调(P<0.05),且呈剂量相关性。结论酒石酸美托洛尔通过调节TGF-β/Smad信号通路抑制大鼠TAA的进程。
Objective To investigate metoprolol tartrate regulated TGF-β/Smad signal pathway suppressed thoracic aortic aneurysms progression. Methods After exposing the descending thoracic aorta of rats for 1 cm, the cotton yarn immersed in 0.5 mol/L calcium chloride was covered with adventitia for 15 ～ 20 minutes to establish TAA model. The model group, the high, medium and low dose of metoprolol tartrate(0.60, 0.30, 0.15 mg/kg) group were set up, another 10 rats were taken as control group. Corresponding drugs was ig administered once a day for four weeks, distilled water was ig administered in model group and control group. The physiological activities of rats were observed and recorded, HE staining was used to observe the changes of arterial lumen surface.TGF-β, Smad2, Smad3 mRNA detected by real time PCR, and TGF-β, Smad2, Smad3 protein level analyzed by western blotting.Results Compared with control group, rats in model group ate less, had lighter body weight, mental retardation, disordered hair, no luster and less activity; compared with model group, the physiological state of the rats in the metoprolol tartrate group improved. HE staining showed that the elastic plates in the aortic wall of the control group were arranged regularly and tightly in a wavy membrane shape; the elastic plates in the aneurysm wall of the model group were straight and fractured; compared with the model group, the elastic plates in the metoprolol tartrate group were less fractured and the arterial wall was wavy membrane shape. Compared with the control group, the expression levels of TGF-β, Smad2 and Smad3 in model group were significantly increased(P < 0.05); compared with model group, the expression levels of TGF-β, Smad2 and Smad3 in the metoprolol tartrate group were significantly decreased(P < 0.05), and there was a dose-dependent relationship. Conclusion metoprolol regulated TGF-β/Smad signal pathway suppressed thoracic aortic aneurysms progression.
Objective To investigate metoprolol tartrate regulated TGF-β/Smad signal pathway suppressed thoracic aortic aneurysms progression. Methods After exposing the descending thoracic aorta of rats for 1 cm, the cotton yarn immersed in 0.5 mol/L calcium chloride was covered with adventitia for 15 ～ 20 minutes to establish TAA model. The model group, the high, medium and low dose of metoprolol tartrate(0.60, 0.30, 0.15 mg/kg) group were set up, another 10 rats were taken as control group. Corresponding drugs was ig administered once a day for four weeks, distilled water was ig administered in model group and control group. The physiological activities of rats were observed and recorded, HE staining was used to observe the changes of arterial lumen surface.TGF-β, Smad2, Smad3 mRNA detected by real time PCR, and TGF-β, Smad2, Smad3 protein level analyzed by western blotting.Results Compared with control group, rats in model group ate less, had lighter body weight, mental retardation, disordered hair, no luster and less activity; compared with model group, the physiological state of the rats in the metoprolol tartrate group improved. HE staining showed that the elastic plates in the aortic wall of the control group were arranged regularly and tightly in a wavy membrane shape; the elastic plates in the aneurysm wall of the model group were straight and fractured; compared with the model group, the elastic plates in the metoprolol tartrate group were less fractured and the arterial wall was wavy membrane shape. Compared with the control group, the expression levels of TGF-β, Smad2 and Smad3 in model group were significantly increased(P < 0.05); compared with model group, the expression levels of TGF-β, Smad2 and Smad3 in the metoprolol tartrate group were significantly decreased(P < 0.05), and there was a dose-dependent relationship. Conclusion metoprolol regulated TGF-β/Smad signal pathway suppressed thoracic aortic aneurysms progression.
引文
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[16] Chen X Y, Ye S B, Xiao W, et al. ERK1/2 pathway mediates epithelial-mesenchymal transition by crossinteracting with TGFβ/Smad and Jagged/Notch signaling pathways in lens epithelial cells[J]. International Journal of Molecular Medicine, 2014, 33(6):1664-1670.
[17] Lee I H, Sohn M, Lim H J, et al. Ahnak functions as a tumor suppressor via modulation of TGFβ/Smad signaling pathway[J]. Oncogene, 2014, 33(38):4675-4684.
[18] Park J H, Yoon J, Lee K Y, et al. Effects of geniposide on hepatocytes undergoing epithelial-mesenchymal transition in hepatic fibrosis by targeting TGFβ/Smad and ERK-MAPK signalingpathways[J].Biochimie,2015,113:26-34.
[19] Wang Z J, Song Y H, Tu W, et al.β-2 spectrin is involved in hepatocyte proliferation through the interaction of TGFβ/Smad and PI3K/AKT signalling[J]. Liver Int,2012, 32(7):1103-1111.
[20] Xu F Y, Liu C W, Zhou D D, et al. TGF-β/SMAD pathway and its regulation in hepatic fibrosis[J]. J Histochem Cytochem, 2016, 64(3):157-167.
[21] Tu B, Peng Z X, Fan Q M, et al. Osteosarcoma cells promote the production of pro-tumor cytokines in mesenchymal stem cells by inhibiting their osteogenic differentiation through the TGF-β/Smad2/3 pathway[J].Exp Cell Res, 2014, 320(1):164-173.
[22] Zhang L, Cheng X, Gao Y Y, et al. Curcumin inhibits metastasis in human papillary thyroid carcinoma BCPAP cells via down-regulation of the TGF-β/Smad2/3 signaling pathway[J]. Exp Cell Res, 2016, 341(2):157-165.
[23] Zhao H W, Li Y W, Feng R, et al. TGF-β/Smad2/3 signal pathway involves in U251 cell proliferation and apoptosis[J]. Gene, 2015, 562(1):76-82.
[24] Yuan S M, Wang J, Hu X N, et al. Fator transformador de crescimento-β/Smad como via de sinaliza??o em aortopatias[J].RevBrasCirCardiovasc,2011,26(3):393-403.
[25] CAO, J, WU, Q, GENG, L, et al. Rapamycin inhibits CaCl2-induced thoracic aortic aneurysm formation in rats through mTOR-mediated suppression of proinflammatory mediators[J]. Mol Med Rep, 2017, 16(2):1911-1919.
[26] Franken R, den Hartog A W, Radonic T, et al. Beneficial outcome of losartan therapy depends on type of FBN1mutation in marfan syndrome[J]. Circ Cardiovasc Genet,2015, 8(2):383-388.
[27] Lerner-Ellis J P, Aldubayan S H, Hernandez A L, et al.The spectrum of FBN1, TGFβR1, TGFβR2 and ACTA2variants in 594 individuals with suspected marfan syndrome, loeys-dietz syndrome or thoracic aortic aneurysms and dissections(TAAD)[J]. Mol Genet Metab, 2014, 112(2):171-176.
[28] Meienberg J, Rohrbach M, Neuenschwander S, et al.Hemizygous deletion of COL3A1, COL5A2, and MSTN causes a complex phenotype with aortic dissection:a lesson for and from true haploinsufficiency[J]. Eur J Hum Genet, 2010, 18(12):1315-1321.
[29] Regalado E S, Guo D C, Prakash S, et al. Aortic disease presentation and outcome associated with ACTA2mutations[J]. Circ Cardiovasc Genet, 2015, 8(3):457-464.
[30] Antunes N d e J, Cavalli R C, Marques M P, et al.Influence of gestational diabetes on the stereoselective pharmacokinetics and placental distribution of metoprolol and its metabolites in parturients[J]. Br J Clin Pharmacol, 2015, 79(4):605-616.
[31] Karlson B W, Dellborg M, Gullestad L, et al. A pharmacokinetic and pharmacodynamic comparison of immediate-release metoprolol and extended-release metoprolol CR/XL in patients with suspected acute myocardial infarction:a randomized, open-label study[J]. Cardiology, 2014, 127(2):73-82.
[32] Mateos A, García-Lunar I, García-Ruiz J M, et al.Efficacy and safety of out-of-hospital intravenous metoprolol administration in anterior ST-segment elevation acute myocardial infarction:insights from the METOCARD-CNIC trial[J]. Ann Emerg Med, 2015, 65(3):318-324.
[33] Liu F L, Mo E P, Yang L, et al. Autophagy is involved in TGF-β1-induced protective mechanisms and formation of cancer-associated fibroblasts phenotype in tumor microenvironment[J]. Oncotarget, 2016, 7(4):4122-4141.
[34] Gao J F, Zhu Y H, Nilsson M, et al. TGF-βisoforms induce EMT independent migration of ovarian cancer cells[J]. Cancer Cell Int, 2014, 14(1):72.
[35] Tao M Z, Gao X, Zhou T J, et al. Effects of TGF-β1 on the proliferation and apoptosis of human cervical cancer hela cells in vitro[J]. Cell Biochem Biophys, 2015, 73(3):737-741.
[36] Wang Y C, Liu J S, Chen J Y, et al. MiR-29 mediates TGFβ1-induced extracellular matrix synthesis through activation of Wnt/β-catenin pathway in human pulmonary fibroblasts[J]. THC, 2015, 23(s1):S119-S125.
[37] Shi L, Dong N, Fang X C, et al. Regulatory mechanisms of TGF-β1-induced fibrogenesis of human alveolar epithelial cells[J]. J Cell Mol Med, 2016, 20(11):2183-2193.
[38] Macias M J, Martin-Malpartida P, MassaguéJ. Structural determinants of Smad function in TGF-βsignaling[J].Trends Biochem Sci, 2015, 40(6):296-308.
[39] Yoshida K, Murata M, Yamaguchi T, et al. TGF-β/Smad signaling during hepatic fibro-carcinogenesis(review)[J]. Int J Oncol, 2014, 45(4):1363-1371.
[40] Chen Z W, Qian J Y, Ma J Y, et al. Glucocorticoid ameliorates early cardiac dysfunction after coronary microembolization and suppresses TGF-β1/Smad3 and CTGFexpression[J].IntJCardiol,2013,167(5):2278-2284.
[2] Adamo L, Braverman A C. Surgical threshold for bicuspid aortic valve aneurysm:a case for individual decision-making[J]. Heart, 2015, 101(17):1361-1367.
[3] Liu S, Xie Z W, Daugherty A, et al. Mineralocorticoid receptor agonists induce mouse aortic aneurysm formation and rupture in the presence of high salt[J].Arterioscler Thromb Vasc Biol, 2013, 33(7):1568-1579.
[4] Coulon C. Thoracic aortic aneurysms and pregnancy[J].La Presse Médicale, 2015, 44(11):1126-1135.
[5] De Backer J, Renard M, Campens L, et al. Marfan syndrome and related heritable thoracic aortic aneurysms and dissections[J].CurrPharmDes,2015,21(28):4061-4075.
[6] Humphrey J D, Schwartz M A, Tellides G, et al. Role of mechanotransduction in vascular biology:focus on thoracic aortic aneurysms and dissections[J]. Circ Res,2015, 116(8):1448-1461.
[7] Yokoyama E, Tsuruoka S, Saitou Y, et al. Isolation of Listeria monocytogenes from a patient with sealed ruptured thoracoabdominal aortic aneurysm[J].Kansenshogaku Zasshi, 2004, 78(12):1016-1019.
[8] Iakoubova O A, Tong C H, Rowland C M, et al. Genetic variants in FBN-1 and risk for thoracic aortic aneurysm and dissection[J]. PLoS One, 2014, 9(4):e91437.
[9] Isselbacher E M, Lino Cardenas C L, Lindsay M E.Hereditary influence in thoracic aortic aneurysm and dissection[J]. Circulation, 2016, 133(24):2516-2528.
[10] Boileau C, Guo D C, Hanna N, et al. TGFB2mutations cause familial thoracic aortic aneurysms and dissections associated with mild systemic features of Marfan syndrome[J]. Nat Genet, 2012, 44(8):916-921.
[11] Bertoli-Avella A M, Gillis E, Morisaki H, et al. Mutations in a TGF-βligand, TGFB3, cause syndromic aortic aneurysms and dissections[J]. J Am Coll Cardiol, 2015,65(13):1324-1336.
[12] Dong S B, Zheng J, Ma W G, et al. Identification and surgical repair of familial thoracic aortic aneurysm and dissection caused by TGFBR1 mutation[J]. Ann Vasc Surg, 2014, 28(8):1909-1912.
[13] Panesi P, Foffa I, Sabina S, et al. Novel TGFBR2 and known missense SMAD3 mutations:two case reports of thoracic aortic aneurysms[J]. Ann Thorac Surg, 2015, 99(1):303-305.
[14] Wischmeijer A, Van Laer L, Tortora G, et al. Thoracic aortic aneurysm in infancy in aneurysms-osteoarthritis syndrome due to a novel SMAD3 mutation:further delineation of the phenotype[J]. Am J Med Genet A,2013, 161A(5):1028-1035.
[15] Teekakirikul P, Milewicz D M, Miller D T, et al. Thoracic aortic disease in two patients with juvenile polyposis syndrome and SMAD4 mutations[J]. Am J Med Genet A, 2013, 161A(1):185-191.
[16] Chen X Y, Ye S B, Xiao W, et al. ERK1/2 pathway mediates epithelial-mesenchymal transition by crossinteracting with TGFβ/Smad and Jagged/Notch signaling pathways in lens epithelial cells[J]. International Journal of Molecular Medicine, 2014, 33(6):1664-1670.
[17] Lee I H, Sohn M, Lim H J, et al. Ahnak functions as a tumor suppressor via modulation of TGFβ/Smad signaling pathway[J]. Oncogene, 2014, 33(38):4675-4684.
[18] Park J H, Yoon J, Lee K Y, et al. Effects of geniposide on hepatocytes undergoing epithelial-mesenchymal transition in hepatic fibrosis by targeting TGFβ/Smad and ERK-MAPK signalingpathways[J].Biochimie,2015,113:26-34.
[19] Wang Z J, Song Y H, Tu W, et al.β-2 spectrin is involved in hepatocyte proliferation through the interaction of TGFβ/Smad and PI3K/AKT signalling[J]. Liver Int,2012, 32(7):1103-1111.
[20] Xu F Y, Liu C W, Zhou D D, et al. TGF-β/SMAD pathway and its regulation in hepatic fibrosis[J]. J Histochem Cytochem, 2016, 64(3):157-167.
[21] Tu B, Peng Z X, Fan Q M, et al. Osteosarcoma cells promote the production of pro-tumor cytokines in mesenchymal stem cells by inhibiting their osteogenic differentiation through the TGF-β/Smad2/3 pathway[J].Exp Cell Res, 2014, 320(1):164-173.
[22] Zhang L, Cheng X, Gao Y Y, et al. Curcumin inhibits metastasis in human papillary thyroid carcinoma BCPAP cells via down-regulation of the TGF-β/Smad2/3 signaling pathway[J]. Exp Cell Res, 2016, 341(2):157-165.
[23] Zhao H W, Li Y W, Feng R, et al. TGF-β/Smad2/3 signal pathway involves in U251 cell proliferation and apoptosis[J]. Gene, 2015, 562(1):76-82.
[24] Yuan S M, Wang J, Hu X N, et al. Fator transformador de crescimento-β/Smad como via de sinaliza??o em aortopatias[J].RevBrasCirCardiovasc,2011,26(3):393-403.
[25] CAO, J, WU, Q, GENG, L, et al. Rapamycin inhibits CaCl2-induced thoracic aortic aneurysm formation in rats through mTOR-mediated suppression of proinflammatory mediators[J]. Mol Med Rep, 2017, 16(2):1911-1919.
[26] Franken R, den Hartog A W, Radonic T, et al. Beneficial outcome of losartan therapy depends on type of FBN1mutation in marfan syndrome[J]. Circ Cardiovasc Genet,2015, 8(2):383-388.
[27] Lerner-Ellis J P, Aldubayan S H, Hernandez A L, et al.The spectrum of FBN1, TGFβR1, TGFβR2 and ACTA2variants in 594 individuals with suspected marfan syndrome, loeys-dietz syndrome or thoracic aortic aneurysms and dissections(TAAD)[J]. Mol Genet Metab, 2014, 112(2):171-176.
[28] Meienberg J, Rohrbach M, Neuenschwander S, et al.Hemizygous deletion of COL3A1, COL5A2, and MSTN causes a complex phenotype with aortic dissection:a lesson for and from true haploinsufficiency[J]. Eur J Hum Genet, 2010, 18(12):1315-1321.
[29] Regalado E S, Guo D C, Prakash S, et al. Aortic disease presentation and outcome associated with ACTA2mutations[J]. Circ Cardiovasc Genet, 2015, 8(3):457-464.
[30] Antunes N d e J, Cavalli R C, Marques M P, et al.Influence of gestational diabetes on the stereoselective pharmacokinetics and placental distribution of metoprolol and its metabolites in parturients[J]. Br J Clin Pharmacol, 2015, 79(4):605-616.
[31] Karlson B W, Dellborg M, Gullestad L, et al. A pharmacokinetic and pharmacodynamic comparison of immediate-release metoprolol and extended-release metoprolol CR/XL in patients with suspected acute myocardial infarction:a randomized, open-label study[J]. Cardiology, 2014, 127(2):73-82.
[32] Mateos A, García-Lunar I, García-Ruiz J M, et al.Efficacy and safety of out-of-hospital intravenous metoprolol administration in anterior ST-segment elevation acute myocardial infarction:insights from the METOCARD-CNIC trial[J]. Ann Emerg Med, 2015, 65(3):318-324.
[33] Liu F L, Mo E P, Yang L, et al. Autophagy is involved in TGF-β1-induced protective mechanisms and formation of cancer-associated fibroblasts phenotype in tumor microenvironment[J]. Oncotarget, 2016, 7(4):4122-4141.
[34] Gao J F, Zhu Y H, Nilsson M, et al. TGF-βisoforms induce EMT independent migration of ovarian cancer cells[J]. Cancer Cell Int, 2014, 14(1):72.
[35] Tao M Z, Gao X, Zhou T J, et al. Effects of TGF-β1 on the proliferation and apoptosis of human cervical cancer hela cells in vitro[J]. Cell Biochem Biophys, 2015, 73(3):737-741.
[36] Wang Y C, Liu J S, Chen J Y, et al. MiR-29 mediates TGFβ1-induced extracellular matrix synthesis through activation of Wnt/β-catenin pathway in human pulmonary fibroblasts[J]. THC, 2015, 23(s1):S119-S125.
[37] Shi L, Dong N, Fang X C, et al. Regulatory mechanisms of TGF-β1-induced fibrogenesis of human alveolar epithelial cells[J]. J Cell Mol Med, 2016, 20(11):2183-2193.
[38] Macias M J, Martin-Malpartida P, MassaguéJ. Structural determinants of Smad function in TGF-βsignaling[J].Trends Biochem Sci, 2015, 40(6):296-308.
[39] Yoshida K, Murata M, Yamaguchi T, et al. TGF-β/Smad signaling during hepatic fibro-carcinogenesis(review)[J]. Int J Oncol, 2014, 45(4):1363-1371.
[40] Chen Z W, Qian J Y, Ma J Y, et al. Glucocorticoid ameliorates early cardiac dysfunction after coronary microembolization and suppresses TGF-β1/Smad3 and CTGFexpression[J].IntJCardiol,2013,167(5):2278-2284.