黄芪甲苷对微血管自律性舒缩活动及大鼠心肌微血管内皮细胞NO、ET-1的影响
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摘要
为探讨黄芪甲苷(Ast)对人合谷穴区皮内微血管自律性舒缩活动振幅及大鼠心肌微血管内皮细胞(RMMECs)NO、ET-1分泌量的影响,利用激光多普勒血流测定仪结合离子导入仪检测微血管自律性舒缩活动的振幅;利用ELISA方法分别检测5、10、25μg·mL~(-1)Ast孵育3、6、9、12 h对RMMECs分泌NO和ET-1的影响;利用Real-Time PCR检测10μg·mL~(-1)Ast作用RMMECs后ET-1和NO基因转录量的变化。结果显示,人合谷穴区皮下导入Ast后,微血管舒缩振幅提高。与对照组比较,低、中、高浓度的Ast均能促进RMMECs分泌NO、ET-1,以及NO/ET-1的比值升高。10μg·mL~(-1) Ast与RMMECs作用3 h时,NO/ET-1值最高,NO、ET-1基因转录水平升高,与分泌水平的结果一致。25μg·mL~(-1) Ast与RMMECs作用3 h时,其NO/ET-1值略低于10μg·mL~(-1) Ast组,但其比率可相对稳定地维持在较高水平。结果表明,中药成分Ast能提高微血管舒缩振幅,其机制可能是通过调节RMMECs释放NO和ET-1在循环系统中的动态平衡。
The study was conducted to investigate the effects of astragaloside(Ast) on microvascular vasomotion at Hegu acupoint, nitric oxide and endothelin-1 released by rat myocardial microvascular endothelial cells(RMMECs). Laser Doppler flowmetry combined with iontophoresis was used to detect the amplitude of microvascular vasomotion. ELISA method was used to detect the levels of NO and ET-1 released by RMMECs incubated with different doses of Ast for 3, 6, 9 and 12 hours. Real-Time PCR was used to detect the gene transcription of NO and ET-1 in RMMECs treated with 10 μg·mL~(-1)Ast. The results were as follows: After introduction of Ast, the amplitude of microvascular vasomotion at Hegu acupoint was improved. Compared with control group(RMMECs), the levels of NO and ET-1 were improved, NO/ET-1 value was increased by Ast in Ast incubated RMMECs. When RMMECs were incubated with 10 μg·mL~(-1) Ast for 3 hours, the NO/ET-1 value was the highest, and the gene transcription levels of NO and ET-1 were improved, which were consistent with the results of secretion level. When 25 μg·mL~(-1) Ast incubated RMMECs for 3 hours, its NO/ET-1 value was slightly lower than that of 10 μg·mL~(-1) Ast group, but its ratio could be maintained at a relatively high level. The results showed that Ast could increase the amplitude of microvascular vasomotion, and its mechanism might be through regulating the dynamic balance of NO and ET-1 released by RMMECs in circulatory system.
The study was conducted to investigate the effects of astragaloside(Ast) on microvascular vasomotion at Hegu acupoint, nitric oxide and endothelin-1 released by rat myocardial microvascular endothelial cells(RMMECs). Laser Doppler flowmetry combined with iontophoresis was used to detect the amplitude of microvascular vasomotion. ELISA method was used to detect the levels of NO and ET-1 released by RMMECs incubated with different doses of Ast for 3, 6, 9 and 12 hours. Real-Time PCR was used to detect the gene transcription of NO and ET-1 in RMMECs treated with 10 μg·mL~(-1)Ast. The results were as follows: After introduction of Ast, the amplitude of microvascular vasomotion at Hegu acupoint was improved. Compared with control group(RMMECs), the levels of NO and ET-1 were improved, NO/ET-1 value was increased by Ast in Ast incubated RMMECs. When RMMECs were incubated with 10 μg·mL~(-1) Ast for 3 hours, the NO/ET-1 value was the highest, and the gene transcription levels of NO and ET-1 were improved, which were consistent with the results of secretion level. When 25 μg·mL~(-1) Ast incubated RMMECs for 3 hours, its NO/ET-1 value was slightly lower than that of 10 μg·mL~(-1) Ast group, but its ratio could be maintained at a relatively high level. The results showed that Ast could increase the amplitude of microvascular vasomotion, and its mechanism might be through regulating the dynamic balance of NO and ET-1 released by RMMECs in circulatory system.
引文
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[2] 周芹.黄芪甲苷抗凋亡作用机制的研究进展[J].中国现代应用药学,2017,34(5):783-788.ZHOU Q.Process of the anti-apoptotic mechanism of astragaloside Ⅳ[J].Chinese Journal of Modern Applied Pharmacy,2017,34(5):783-788.(in Chinese)
[3] 梁瑾,刘小花,任远,等.HPLC-DAD-ELSD法同时测定黄芪中5个成分的含量[J].药物分析杂志,2013,33(2):210-213.LIANG J,LIU X H,REN Y,et al.Simultaneous determination of five compounds in Radix Astragali by high-performance liquid chromatography coupled with diode arry and evaporative light scattering detectors[J].Chinese Journal of Pharmaceutical Analysis,2013,33(2):210-213.(in Chinese)
[4] 杜文沛.肺积方和黄芪甲苷干预IDO及肺癌微环境免疫逃逸作用研究[D].上海:华东理工大学,2016.DU W P.The influence of Feiji recipe and Astragaloside Ⅳ on IDO and immune escape in tumor microenvironment[D].Shanghai:East China University of Science and Technology,2016.(in Chinese)
[5] 司静文.基于血管新生与成熟的Hif-1α信号机制揭示黄芪甲苷IV治疗心肌梗死的新机制[D].西安:第四军医大学,2014.SI J W.Astragaloside IV (As- IV) stimulate angiogenesis to treat myocardial infarction through Hif-1α signaling[D].Xi'an:The Fourth Military Medical University,2014.(in Chinese)
[6] 李敏.黄芪甲苷对大鼠脑缺血再灌注损伤后血脑屏障及脑的保护作用机制研究[D].西安:第四军医大学,2012.LI M.The Neuroprotective role and mechanism of Astragaloside Ⅳ against cerebral ischemia/reperfusion injury in rats[D].Xi'an:The Fourth Military Medical University,2012.(in Chinese)
[7] 王琼英.黄芪及其单体黄芪甲苷对代谢综合征和高血压心脏损害的影响[D].兰州:兰州大学,2015.WANG Q Y.The effect of radix astragali and its monomer astragaloside Ⅳ on cardiac damage in metabolic syndrome and hypertension[D].Lanzhou:Lanzhou University,2015.(in Chinese)
[8] 缪天玲.特发性肺纤维化大鼠不同时期微血管变化特征及黄芪甲苷的干预研究[D].兰州:甘肃中医药大学,2015.MIU T L.Research on the characteristics of microvascular changes in different stages of idiopath dulmonary fibrosis and on the intervention effects of astragaloside on the syndrome[D].Lanzhou:Gansu University of Chinese Medicine,2015.(in Chinese)
[9] 张玲.芪参益气方促血管新生机制及抗心肌缺血作用研究[D].杭州:浙江大学,2011.ZHANG L.Studies on angiogenic mechanisms and protective effect against myocardial ischemia of QI-SHEN-YI-QI Formula[D].Hangzhou:Zhejiang University,2011.(in Chinese)
[10] 孙豪栋.黄芪总皂苷的微生物转化及其条件优化的研究[D].开封:河南大学,2011.SUN H D.Microbial transformation of total saponins of astragalus and optimization of its conditions [D].Kaifeng:Henan University,2011.(in Chinese)
[11] CHU Y F,YAN X M,LI X R,et al.Effect of Chinese herbal medicinal ingredients on IL-2 mRNA levels of T lymphocytes in mice measured using semiquantification RT-PCR[J].J Integr Agric,2006,5(11):873-878.
[12] 杨珍,周惠芬,周鹏,等.川芎、黄芪有效成分配伍对缺氧脑微血管内皮细胞的影响[J].中草药,2015,46(9):1326-1332.YANG Z,ZHOU H F,ZHOU P,et al.Effects of compatibility of main components from Chuanxiong Rhizoma and Astragali Radix on hypoxia damaged brain microvascular endothelial cells[J].Chinese Traditional and Herbal Drugs,2015,46(9):1326-1332.(in Chinese)
[13] DE BOER M P,WIJNSTOK N J,SERNé E H,et al.Body mass index is related to microvascular vasomotion,this is partly explained by adiponectin[J].Eur J Clin Invest,2014,44(7):660-667.
[14] 王冠楠,张涛,董虹,等.肥大细胞脱颗粒物质中提高微血管舒缩活动振幅的成分筛选[C]//第三届京津冀畜牧兽医科技创新研讨会暨“瑞普杯”新思想、新方法、新观点论坛论文集.天津:天津市畜牧兽医学会,2012:209-215.WANG G N,ZHANG T,DONG H,et al.Screening of components increasing the amplitude of microvessel vasomotion activities in mast cell departiculate matter[C]//Papers Collection of Third Beijing-Tianjin-Hebei Animal Husbandry and Veterinary Science and Technology Innovation Seminar and "Rip Cup" Forum on New Ideas,New Methods and New Views.Tianjin:Tianjin Association of Animal Science and Veterinary Medicine,2012:209-215.(in Chinese)
[15] 王冠楠,张涛,张爽,等.麻醉对比格犬胃腧穴区真皮层微血管舒缩活动的影响[J].微循环学杂志,2014,24(3):12-14,17.WANG G N,ZHANG T,ZHANG S,et al.Effects of anesthesia on dermal microvascular vasomotion at Weishu Acupoints of Beagles[J].Chinese Journal of Microcirculation,2014,24(3):12-14,17.(in Chinese)
[16] PRADHAN R K,CHAKRAVARTHY V S.Informational dynamics of vasomotion in microvascular networks:a review[J].Acta Physiol,2011,201(2):193-218.
[17] ROSSI M,PISTELLI F,PESCE M,et al.Impact of long-term exposure to cigarette smoking on skin microvascular function[J].Microvasc Res,2014,93:46-51.
[18] XIA J,WANG H,ZHANG Q M,et al.The therapeutic effect of curcumin in male albino rats and its putative mechanisms on cerebral microvascular flow[J].Brain Res,2016,1642:131-135.
[19] FURCHGOTT R F,ZAWADZKI J V.The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine[J].Nature,1980,288(5789):373-376.
[20] GMC R,TOUSOULIS D,MCFADDEN E,et al.Effects of neuropeptide Y on coronary artery vasomotion in patients with microvascular angina[J].Int J Cardiol,2017,238:123-127.
[21] 刘小瑜,董虹,张涛,等.几种中药有效成分对微血管舒缩活动振幅的影响[C]//中国畜牧兽医学会中兽医学分会2011年学术年会论文集.广州:中国畜牧兽医学会,2011:281-285.LIU X Y,DONG H,ZHANG T,et al.Effects of several active ingredients of traditional Chinese medicine on the amplitude of microvascular vasomotion[C]//Symposium of Annual Meeting of Association of Chinese Animal Science and Veterinary Medicine's Chinese Traditional Veterinary Medicine Chapter,2011.Guangzhou:Association of Chinese Animal Science and Veterinary Medicine's Chinese Traditional Veterinary Medicine Chapter,2011:281-285.(in Chinese)
[22] MU Y P,HUANG Q H,ZHU J L,et al.Magnesium attenuates endothelin-1-induced vasoreactivity and enhances vasodilatation in mouse pulmonary arteries:Modulation by chronic hypoxic pulmonary hypertension[J].Exp Physiol,2018,103(4):604-616.
[23] WANG R,CAO L,SHEN Z X,et al.Up-regulation of contractile endothelin receptors by airborne fine particulate matter in rat mesenteric arteries via activation of MAPK pathway[J].Environ Sci Pollut Res Int,2018,25(15):14713-14725.
[24] XI C,XU J Q,XIANG M,et al.Effects of rhynchophylline and isorhynchophylline on nitric oxide and endothelin-1 secretion from RIMECs induced by Listeriolysin O in vitro[J].Vet Microbiol,2010,143(2-4):262-269.
[25] 武煜,顾振纶.一氧化氮的心血管作用研究进展[J].中国血液流变学杂志,2004,14(1):143-145.WU Y,GU Z L.The research progress of effect of NO on cardialvascular[J].Chinese Journal of Hemorheology,2004,14(1):143-145.(in Chinese)
[26] 张涛,索占伟,胡格,等.提高微血管舒缩活动振幅是中药的作用机理之一[J].中兽医学杂志,2009(S1):494-498.ZHANG T,SUO Z W,HU G,et al.Increasing the amplitude of microvascular vasomotion is one of the mechanisms of traditional Chinese medicine[J].Chinese Journal of Traditional Veterinary Science,2009(S1):494-498.(in Chinese)
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