周小娟,孔祥敏,王迎超,蒋程,金兆祥,艾乐,张玲,王毅
ZHOU Xiao-juan,KONG Xiang-min,WANG Ying-chao,JIANG Cheng,JIN Zhao-xiang,AI Le,ZHANG Ling,WANG Yi

• 1:浙江省立同德医院药学部
• 2:浙江省心血管病诊治重点实验室
• 3:天津中新药业集团股份有限公司乐仁堂制药厂
• 4:浙江大学药学院

摘要(Abstract)：

为研究通脉养心丸提取物(TMYX)对大鼠离体肠系膜动脉的舒张作用并探讨其作用机制,采用在大鼠离体肠系膜动脉血管上观察累积浓度TMYX对基础状态血管、苯肾上腺素(PE,1×10~(-5) mol·L~(-1))和氯化钾(KCl,60 mmol·L~(-1))预收缩的血管环的作用,并观察去除血管内皮细胞对TMYX舒张作用的影响。进一步采用一氧化氮合酶抑制剂L-硝基-精氨酸甲酯(L-NAME)和环氧合酶抑制剂吲哚美辛预孵育观察TMYX的舒血管效果。同时在人脐静脉内皮细胞上观察TMYX对细胞NO释放和一氧化氮合酶eNOS磷酸化的影响。结果发现,TMYX在1～750 mg·L~(-1)对基础状态的内皮完整血管张力无明显影响,对KCl预收缩的内皮完整血管也无明显舒缩作用。但TMYX能有效舒张苯肾上腺素收缩的大鼠肠系膜动脉,最大舒张率为(64.71±10.03)%。用一氧化氮合酶抑制剂(L-NAME,1×10~(-4) mol·L~(-1))预孵育或去除肠系膜动脉血管内皮后,TMYX的最大舒张率分别降为(35.77±8.93)%,(25.85±10.84)%,与未加L-NAME组和内皮完整组比较有显著差异(P<0.01)。但吲哚美辛对TMYX的舒张作用没有影响。此外TMYX 50 mg·L~(-1)可以显著增加人脐静脉内皮细胞P-eNOS的蛋白表达,促进NO释放,而L-NAME处理后则下调细胞P-eNOS蛋白表达水平,抑制NO的释放。结果说明TMYX具有内皮依赖性的血管舒张作用,其对苯肾上腺素预收缩的大鼠离体肠系膜动脉的舒张作用与一氧化氮-环磷酸鸟苷(NO-cGMP)通路有关。
The aim of the present study is to evaluate the vasodilation effects of Tongmai Yangxin Pills(TMYX) on rat mesenteric artery as well as its mechanism of action. The relaxation effects of TMYX extracts with different concentrations were determined on isolated rat mesenteric artery in normal condition as well as pretreating by phenylephrine and KCl. Vascular relaxation effects of TMTX were also determined in mesenteric artery preincubated with L-ANME and indomethacin or in endothelium denuded mesenteric artery. Moreover, effects of TMYX by 50 mg·L~(-1) on NO secretion and the phosphorylation of eNOS in a cellular model of human umbilical vein endothelial cell(HUVEC) pretreated with or without L-NAME were also observed. The experimental results showed that TMYX has no obvious effect on vasodilation of arteries in normal or KCl pretreated condition, while it can dose-dependently relax the rat mesenteric artery with intact endothelium stimulated with phenylephrine at a maximal diastolic rate of(64.71±10.03)%. After preincubating with L-NAME for 15 min or removal of mesenteric artery endothelium, the maximal diastolic rate was decreased to(35.77±8.93)% and(25.85±10.84)% respectively. However, preincubating with indomethacin had no inhibitory effect on TMYX induced vascular relaxation. Meanwhile, TMYX at 50 mg·L~(-1) could increase the expression of P-eNOS and the secretion of NO in HUVEC. L-NAME significantly inhibited NO release and phosphorylation of eNOS induced by TMYX. The results suggested TMYX exerted endothelium-dependent relaxation effects against PE-induced contractions of isolated rat mesenteric artery through NO-cGMP signaling pathway.

关键词(KeyWords)： 通脉养心丸;肠系膜;血管舒张;人脐静脉内皮细胞;一氧化氮
Tongmai Yangxin Pills(TMYX);mesenteric artery;vasodilation;HUVEC;NO

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金面上项目(81573641);; 浙江省科技计划项目(2017C37075);; 浙江省自然科学基金项目(LY16H280003);; 浙江省公益性技术应用研究计划项目(2016C33127);; 浙江省医学会临床科研基金项目(2017ZYC-A06)

作者(Author): 周小娟,孔祥敏,王迎超,蒋程,金兆祥,艾乐,张玲,王毅
ZHOU Xiao-juan,KONG Xiang-min,WANG Ying-chao,JIANG Cheng,JIN Zhao-xiang,AI Le,ZHANG Ling,WANG Yi

参考文献(References)：

• [1] 阎维维,张咏梅. 通脉养心丸质量控制研究[J].中草药, 2011,42(9):1751.
• [2] 王怡, 张玲, 肖扬, 等. 通脉养心丸对缺氧诱导心肌细胞损伤炎症因子及氧化应激的影响[J].中医杂志, 2011,52(4):326.
• [3] 陶珊,梁欣莹,王怡,等. 通脉养心丸中心肌保护活性物质的筛选[J].浙江大学学报:医学版, 2015, 44(2):145.
• [4] Tao S, Huang Y, Chen Z, et al. Rapid identification of anti-inflammatory compounds from Tongmai Yangxin Pills by liquid chromatography with high-resolution mass spectrometry and chemometric analysis[J].J Sep Sci, 2015,38(11):1881.
• [5] 吴丽虹,陈桥,张波.通脉养心丸和复方丹参片治疗气虚血瘀证慢性稳定性心绞痛54例临床观察[J].中国实验方剂学杂志,2015,21(15):172.
• [6] 李浩,张苏丽,杨艳,等. 利用微血管环技术检测肠系膜动脉三级分支张力的方法[J].中国应用生理学杂志, 2014(3):214.
• [7] 肖扬,张家福,张玲,等. 从钙超载角度探讨通脉养心丸心肌保护作用的机制[J].中西医结合心脑血管病杂志, 2011, 9(5):562.
• [8] 许文平,李来来,王艳艳,等. 通脉养心丸对异丙肾上腺素所致大鼠心肌肥厚的影响[J].天津中医药, 2014,31(9):552.
• [9] 任建勋,李磊,尹云泽,等. 通脉养心丸对小型猪心肌缺血再灌注损伤后期气阴两虚证的干预作用[J].中草药, 2018,49(7):1612.
• [10] 陈锥. 一种基于磁珠富集的中药药效物质快速发现方法研究[D].杭州:浙江大学,2014.
• [11] 陈然,张玉青,王颖,等. 黄芪甲苷对大鼠离体肠系膜动脉血管舒缩功能的影响[J].温州医学院学报, 2008,38(3):217.
• [12] 张寒,刘欢欢,贾敏,等. 原儿茶醛对大鼠离体肠系膜上动脉血管环的舒张作用及机制[J].西安交通大学学报:医学版, 2018,39(1):31.
• [13] 刘晓丹,潘振伟,庄须国,等. 锦灯笼水提物对大鼠胸主动脉的舒张作用及机制[J].中草药, 2008,39(11):1709.
• [14] 韩军,宣佳利,胡浩然,等. 金丝桃苷对大鼠离体大脑中动脉血管内皮功能的影响及其内在机制的研究[J].中国中药杂志, 2014,39(24):4849.
• [15] Durand M J, Gutterman D D. Diversity in mechanisms of endothelium-dependent vasodilation in health and disease[J].Microcirculation, 2013, 20(3):239.
• [16] 杨耀,陈波,梁凯伦,等. 蒙花苷与木犀草素联用对离体血管的舒张作用及其机制研究[J].中国中药杂志, 2017,42(7):1370.
• [17] Tushar V Gadkari, Natalie Cortes, Kumpal Madrasi, et al. Agmatine induced NO dependent rat mesenteric artery relaxation and its impairment in salt-sensitive hypertension[J].Nitric Oxide, 2013,35:65.
• [18] Feletou M, Huang Y, Vanhoutte P M. Endothelium-mediated control of vascular tone: COX-1 and COX-2 products[J].Br J Pharmacol, 2011, 164(3):894.
• [19] Bystrom J, Wray J A, Sugden M C, et al. Endogenous epoxygenases are modulators of monocyte/macrophage activity[J].PLoS ONE, 2011,6(10):e26591.
• [20] 徐道情,耿彤. 通脉养心方提取工艺优选[J].药物评价研究, 2012,35(4):265.