决明子提取物扩血管作用机理的实验研究
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摘要
目的:研究决明子提取物的扩血管作用并探讨其作用机理。
方法:制备SD大鼠去内皮以及有内皮胸主动脉环,分别做氯化钾和去氧肾上腺素(Phenylephrine hydrochloride,PE)量效曲线,依次加入决明子提取物不同剂量预作用后再做量效曲线观察其变化;应用无钙和复钙的实验方法,以PE为血管收缩剂,观察不同剂量决明子提取物对无钙液和1.1 mmol/L、2.2mmol/L复钙液中PE引起血管收缩的影响;培养人脐静脉内皮细胞株,分别用血管紧张素Ⅱ(AngiotensinⅡ,AngⅡ)单独干预和不同剂量决明子提取物与AngⅡ共同干预,细胞计数6天绘制生长曲线,药物干预24小时后MTT比色法检测细胞增殖活性,重氮法检测细胞匀浆中诱生型一氧化氮合酶(inducible nitric oxide synthase,iNOS)活性,化学法检测细胞上清液中一氧化氮(nitric oxide,NO)含量。
结果:与对照组比较,决明子提取物0.1mg/L组和1mg/L组对去内皮血管环KCl致血管收缩率没有差异,10mg/L组血管收缩率减低(P＜0.05);决明子0.1mg/L组对有内皮血管环的血管收缩率无差异,1mg/L、10mg/L组能使血管收缩率降低。与对照组相比,决明子提取物0.1mg/L、1mg/L和10mg/L均能降低PE血管收缩率,有内皮、去内皮的血管环与对照组比较均有差异(P＜0.05)。无钙复钙实验中,与对照组相比,决明子提取对PE引起的无钙液中血管收缩无影响(P＞0.05);
1.1mmol/L复钙后决明子提取物1mg/L组血管收缩率降低(P＜0.05),0.1mg/L组和10mg/L组无变化;2.2mmol/L复钙后,决明子三个剂量组均能降低血管收缩率。内皮细胞实验中,对照组、AngⅡ组与决明子提取物干预组人内皮细胞生长曲线无差异;细胞增殖活力无差异;与对照组相比,AngⅡ组内皮细胞培养液NO含量下降,细胞匀浆iNOS活性增加,决明子提取物(10mg/L、100mg/L)与AngⅡ共同干预组比AngⅡ组NO增加,iNOS降低。
结论:决明子提取物可影响血管上受体操纵的钙通道和电位依赖性钙通道的开放舒张血管,不影响细胞内贮钙释放。决明子提取物扩血管效应有一定内皮依赖性,能减少血管内皮细胞iNOS产生,增加NO释放。
Objective:To study the vasorelaxation action of Cassia Seed and its mechanisms.
Methods:Rat aortic rings with or without endothelium were isolated and suspended in organ chambers for the measurement of contractile force to observe the effects of Cassia Seed on the concentration-response curve for potassium chloride(KC1) or Phenylephrine hydrochloride(PE).The procedure of Ca~(2+) -free and Ca~(2+) addition was designed to observe indirectly the effect of Cassia Seed on intracellular calcium ion.Human umbilical vein endothelial cells(HUECs) were cultured and divided into four groups,control, AngiotensinⅡ(AngⅡ)(stimulated with AngⅡ10~(-6) mol /L for 24 hours),AngⅡand Cassia seed groups(AngⅡ10~(-6)mol/L,Cassia seed 10mg/L and 100mg/L were added to cell culture medium at the same time).The cell viability was observed by cell counting for six days and methyl thiazolyl tetrazolium(MTT).The nitric oxide(NO) and nitric oxide synthase(NOS) in culture medium were assayed.
Result:Cassia Seed at 10mg/L was shown to inhibit the contractile force of rat de-endothelium aortic rings induced by KCL.But it was prove to be effective that the Cassia Seed at 1mg/L and 10mg/L can inhibit the contractive force induced by KCl in endothelium aortic rings.Cassia Seed at 0.1mg/L,1mg/L and 10mg/L significantly inhibited the contractive force induced by PE with endothelium or without endothelium.It was equivalent contractile force between Cassia Seed and control in calcium free Krebs, whereas it was adverse after 1.1mmol/L,2.2mmol/L calcium addition.The growth curve of HUECs and MTT were not affected by AngⅡor cassia seed.But NO concentration of Culture medium was diminished in AngⅡgroup,while the cassia seed increased it.The iNOS in AngⅡgroups was significantly increased compared with the control,and markedly decreased in cassia seed groups.
Conclusion:Cassia Seed has obviously vasorelaxation.It is partially dependent on endothelium and seems to be related to voltage-operated calcium channel and receptor-controled calcium channel in membrane of vascular smooth muscle cells.The NO of endothelial cells may be stimulated by the cassia seed.The iNOS could be up-regulated by AngⅡand be down-regulated by Cassia Seed.
方法:制备SD大鼠去内皮以及有内皮胸主动脉环,分别做氯化钾和去氧肾上腺素(Phenylephrine hydrochloride,PE)量效曲线,依次加入决明子提取物不同剂量预作用后再做量效曲线观察其变化;应用无钙和复钙的实验方法,以PE为血管收缩剂,观察不同剂量决明子提取物对无钙液和1.1 mmol/L、2.2mmol/L复钙液中PE引起血管收缩的影响;培养人脐静脉内皮细胞株,分别用血管紧张素Ⅱ(AngiotensinⅡ,AngⅡ)单独干预和不同剂量决明子提取物与AngⅡ共同干预,细胞计数6天绘制生长曲线,药物干预24小时后MTT比色法检测细胞增殖活性,重氮法检测细胞匀浆中诱生型一氧化氮合酶(inducible nitric oxide synthase,iNOS)活性,化学法检测细胞上清液中一氧化氮(nitric oxide,NO)含量。
结果:与对照组比较,决明子提取物0.1mg/L组和1mg/L组对去内皮血管环KCl致血管收缩率没有差异,10mg/L组血管收缩率减低(P＜0.05);决明子0.1mg/L组对有内皮血管环的血管收缩率无差异,1mg/L、10mg/L组能使血管收缩率降低。与对照组相比,决明子提取物0.1mg/L、1mg/L和10mg/L均能降低PE血管收缩率,有内皮、去内皮的血管环与对照组比较均有差异(P＜0.05)。无钙复钙实验中,与对照组相比,决明子提取对PE引起的无钙液中血管收缩无影响(P＞0.05);
1.1mmol/L复钙后决明子提取物1mg/L组血管收缩率降低(P＜0.05),0.1mg/L组和10mg/L组无变化;2.2mmol/L复钙后,决明子三个剂量组均能降低血管收缩率。内皮细胞实验中,对照组、AngⅡ组与决明子提取物干预组人内皮细胞生长曲线无差异;细胞增殖活力无差异;与对照组相比,AngⅡ组内皮细胞培养液NO含量下降,细胞匀浆iNOS活性增加,决明子提取物(10mg/L、100mg/L)与AngⅡ共同干预组比AngⅡ组NO增加,iNOS降低。
结论:决明子提取物可影响血管上受体操纵的钙通道和电位依赖性钙通道的开放舒张血管,不影响细胞内贮钙释放。决明子提取物扩血管效应有一定内皮依赖性,能减少血管内皮细胞iNOS产生,增加NO释放。
Objective:To study the vasorelaxation action of Cassia Seed and its mechanisms.
Methods:Rat aortic rings with or without endothelium were isolated and suspended in organ chambers for the measurement of contractile force to observe the effects of Cassia Seed on the concentration-response curve for potassium chloride(KC1) or Phenylephrine hydrochloride(PE).The procedure of Ca~(2+) -free and Ca~(2+) addition was designed to observe indirectly the effect of Cassia Seed on intracellular calcium ion.Human umbilical vein endothelial cells(HUECs) were cultured and divided into four groups,control, AngiotensinⅡ(AngⅡ)(stimulated with AngⅡ10~(-6) mol /L for 24 hours),AngⅡand Cassia seed groups(AngⅡ10~(-6)mol/L,Cassia seed 10mg/L and 100mg/L were added to cell culture medium at the same time).The cell viability was observed by cell counting for six days and methyl thiazolyl tetrazolium(MTT).The nitric oxide(NO) and nitric oxide synthase(NOS) in culture medium were assayed.
Result:Cassia Seed at 10mg/L was shown to inhibit the contractile force of rat de-endothelium aortic rings induced by KCL.But it was prove to be effective that the Cassia Seed at 1mg/L and 10mg/L can inhibit the contractive force induced by KCl in endothelium aortic rings.Cassia Seed at 0.1mg/L,1mg/L and 10mg/L significantly inhibited the contractive force induced by PE with endothelium or without endothelium.It was equivalent contractile force between Cassia Seed and control in calcium free Krebs, whereas it was adverse after 1.1mmol/L,2.2mmol/L calcium addition.The growth curve of HUECs and MTT were not affected by AngⅡor cassia seed.But NO concentration of Culture medium was diminished in AngⅡgroup,while the cassia seed increased it.The iNOS in AngⅡgroups was significantly increased compared with the control,and markedly decreased in cassia seed groups.
Conclusion:Cassia Seed has obviously vasorelaxation.It is partially dependent on endothelium and seems to be related to voltage-operated calcium channel and receptor-controled calcium channel in membrane of vascular smooth muscle cells.The NO of endothelial cells may be stimulated by the cassia seed.The iNOS could be up-regulated by AngⅡand be down-regulated by Cassia Seed.
引文
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2.中华人民共和国卫生部.中国居民营养与健康状况调查报告(2002)[M].北京:人民卫生出版社,2005:15-16.
3.顾东风,Jiang He,吴锡桂,等.中国成年人主要死亡原因及其危险因素[J].中国慢性疾病预防和控制,2006,14(3):149-155.
4.周晓明,陈琼华,等.中药大黄的生化学研究Ⅹ Ⅹ Ⅱ葸醌衍生物对兔肾髓质Na~+-K~+-ATP酶活性的抑制和利尿作用[J].药学学报,1988,23(1):17-19.
5.李续娥,马伟,郭宝江.新西兰兔灌胃决明子后吸收入血的氨基酸分析[J].华南师范大学学报:自然科学版,2003(3):112-115
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