(+)-儿茶素缓解大鼠低氧性肺动脉高压的作用和机制
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摘要
研究了(+)-儿茶素对SD大鼠低氧性肺动脉高压(PAH)的影响及其机制。采用27只SD雄性大鼠按随机数字表法分为3组(对照组、缺氧模型组、(+)-儿茶素干预组)。通过检测大鼠平均肺动脉压(Mean pulmonary arterialpressure,mPAP)和肺血管阻力(Pulmonaryvascularresistance,PVR)反应体内肺血流动力学变化;处死大鼠后测算右心室肥厚指数(Rightventriclehypertrophyindex,RVHI)及肺动脉血管壁厚度和外周血管直径的比值(Vascular wall thickness of outer circumference ratio,WT%);WB检测肺动脉内皮细胞一氧化氮合酶(eNOS),用NO试剂盒检测NO表达量;用CCK-8法检测肺动脉平滑肌细胞增殖以确定细胞活力,利用WB检测分析肺动脉平滑肌细胞钙敏感受体(CaSR)、荧光探针法检测细胞内总Ca~(2+)的浓度。研究结果表明,与缺氧模型组相比,(+)-儿茶素能够显著减缓肺缺氧诱导的肺动脉压力升高(n=9,P<0.05;);通过逆转缺氧引起的肺内皮细胞一氧化氮合酶(eNOS)显著减少(n=3,P<0.05)和NO合成显著减少(n=5,P<0.05),达到抑制肺动脉内皮血管收缩作用;通过下调缺氧诱导的平滑肌细胞内CaSR表达(n=3,P<0.05),逆转缺氧的升钙效应(n=27,P<0.05),达到抑制平滑肌细胞增殖的作用(n=30,P<0.05)。综上可见,(+)-儿茶素通过抑制肺血管收缩和血管平滑肌细胞增殖来缓解缺氧诱导的PAH,为PAH的治疗方法提供了新思路。
To study the effects and mechanism of(+)-catechins((+)-C) on hypoxia pulmonary arterial hypertension(PAH) in rats, twenty seven SD male rats were randomly divided into 3 groups with equalnumber(control group, hypoxia group and hypoxia+(+)-C group). Mean pulmonary arterial pressure(mPAP), pulmonary vascular resistance(PVR) were measured to indicate the hemodynamic changes in vivo. After executing the rats, the right ventricle hypertrophy index(RVHI) and the vascular wall thickness of outer circumference ratio(WT%) were calculated. The expression of nitric oxide synthase(eNOS) and NO in pulmonary artery endothelial cells was analyzed by WB and NO kit respectively. Cellular viability of pulmonary arterial smooth muscle cell was determined by CCK-8 assay. The expression of calcium sensing receptor(CaSR) and total intracellular Ca~(2+) were analyzed by WB and fluorescent probe. The results showed that(+)-C could decrease the pulmonary arterial pressure induced by hypoxia(n=9, P<0.05). Increase eNOS in pulmonary endothelial cells(n=3, P<0.05) and increase NO synthesis in pulmonary endothelial cells(n=5, P<0.05). The expression of CaSR in smooth muscle cells induced by hypoxia was inhibited(n=3, P<0.05), while the calcium concentration was induced by(+)-C(n=27, P<0.05). Vascular smooth muscle cell proliferation was inhibited by(+)-C(n=30, P<0.05). In conclusion,(+)-C can alleviate hypoxia induced PAH by inhibiting pulmonary vasoconstriction and vascular smooth muscle cell proliferation, providing a new approach for the treatment of PAH.
To study the effects and mechanism of(+)-catechins((+)-C) on hypoxia pulmonary arterial hypertension(PAH) in rats, twenty seven SD male rats were randomly divided into 3 groups with equalnumber(control group, hypoxia group and hypoxia+(+)-C group). Mean pulmonary arterial pressure(mPAP), pulmonary vascular resistance(PVR) were measured to indicate the hemodynamic changes in vivo. After executing the rats, the right ventricle hypertrophy index(RVHI) and the vascular wall thickness of outer circumference ratio(WT%) were calculated. The expression of nitric oxide synthase(eNOS) and NO in pulmonary artery endothelial cells was analyzed by WB and NO kit respectively. Cellular viability of pulmonary arterial smooth muscle cell was determined by CCK-8 assay. The expression of calcium sensing receptor(CaSR) and total intracellular Ca~(2+) were analyzed by WB and fluorescent probe. The results showed that(+)-C could decrease the pulmonary arterial pressure induced by hypoxia(n=9, P<0.05). Increase eNOS in pulmonary endothelial cells(n=3, P<0.05) and increase NO synthesis in pulmonary endothelial cells(n=5, P<0.05). The expression of CaSR in smooth muscle cells induced by hypoxia was inhibited(n=3, P<0.05), while the calcium concentration was induced by(+)-C(n=27, P<0.05). Vascular smooth muscle cell proliferation was inhibited by(+)-C(n=30, P<0.05). In conclusion,(+)-C can alleviate hypoxia induced PAH by inhibiting pulmonary vasoconstriction and vascular smooth muscle cell proliferation, providing a new approach for the treatment of PAH.
引文
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[16]Peng X,Li HX,Shao HJ,et al.Involvement of calcium-sensing receptors in hypoxia-induced vascular remodeling and pulmonary hypertension by promoting phenotypic modulation of small pulmonary arteries[J].Molecular&Cellular Biochemistry,2014,396(1/2):87-98.
[17]Yamamura A,Yagi S,Ohara N,et al.Calcilytics enhance sildenafil-induced antiproliferation in idiopathic pulmonary arterial hypertension[J].Eur J Pharmacol,2016,784:15-21.DOI:10.1016/j.ejphar.2016.04.059.
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[3]Tan X,Feng L,Huang X,et al.Histone deacetylase inhibitors promote eNOS expression in vascular smooth muscle cells and suppress hypoxia-induced cell growth[J].Journal of Cellular&Molecular Medicine,2017,21(9):2022-2035.
[4]Colella M,Gerbino A,Hofer AM,et al.Recent advances in understanding the extracellular calcium-sensing receptor[J].F1000Res,2016,5(F1000 Faculty Rev):2535.DOI:10.12688/f1000research.8963.1.http://dx.doi.org/10.12688/f1000research.8963.1.
[5]Yang J,Li X,Allamki RS,et al.Smad-dependent and smad-independent induction of id1 by prostacyclin analogues inhibits proliferation of pulmonary artery smooth muscle cells in vitro and in vivo[J].Circ Res,2010,107(2):252-262.
[6]刘超,陈若芸.儿茶素及其类似物的化学和生物活性研究进展[J].中国中药杂志,2004,29(10):1017-1021.
[7]付静,江海,李新生.超高效液相色谱法测定绿茶中5种儿茶素[J].分析试验室,2013,32(7):75-80.
[8]Breen EC,Scadeng M,Lai NC,et al.Functional magnetic resonance imaging for in vivo quantification of pulmonary hypertension in the Sugen 5416/hypoxia mouse[J].Exp Physiol,2017,102(3):347-353.https://doi:10.1113/EP086067.
[9]陈丽英,应可净,黄斐斐,等.低氧性肺动脉高压大鼠模型的建立[J].中华危重症医学杂志(电子版),2015(5):312-314.
[10]章琦,胡立群,尹长森,等.儿茶素对实验性大鼠心力衰竭模型的保护作用[J].安徽医科大学学报,2014(6):764-767.
[11]何小解,易著文,杨华彬,等.微胶囊化儿茶素对阿霉素肾病大鼠VEGF表达的影响[J].中国病理生理杂志,2007,23(8):1570-1573.
[12]Afolayan AJ,Eis A,Alexander M,et al.Decreased endothelial nitric oxide synthase expression and function contribute to impaired mitochondrial biogenesis and oxidative stress in fetal lambs with persistent pulmonary hypertension[J].Am JPhysiol Lung Cell Mol Physiol,2016,310(1):L40-L49.https://doi.org/10.1152/ajplung.00392.2014.
[13]Firth AL,Remillard CV,Platoshyn O,et al.Functional ion channels in human pulmonary artery smooth muscle cells:Voltage-dependent cation channels[J].Pulm Circ,2011,1(1):48-71.
[14]Tang H,Yamamura A,Yamamura H,et al.Pathogenic role of calcium-sensing receptors in the development and progression of pulmonary hypertension[J].Am J Physiol Lung Cell Mol Physiol,2016,310(9):L846-L859.https://doi.org/10.1152/ajplung.00050.2016.
[15]Schreckenberg R,Schlüter KD.Calcium sensing receptor expression and signalling in cardiovascular physiology and disease[J].Vascul Pharmacol,2018,107:35-42.DOI:10.1016/j.vph.2018.02.007.
[16]Peng X,Li HX,Shao HJ,et al.Involvement of calcium-sensing receptors in hypoxia-induced vascular remodeling and pulmonary hypertension by promoting phenotypic modulation of small pulmonary arteries[J].Molecular&Cellular Biochemistry,2014,396(1/2):87-98.
[17]Yamamura A,Yagi S,Ohara N,et al.Calcilytics enhance sildenafil-induced antiproliferation in idiopathic pulmonary arterial hypertension[J].Eur J Pharmacol,2016,784:15-21.DOI:10.1016/j.ejphar.2016.04.059.