节点文献
沙棘总黄酮抗栓作用机制及有效成分初探
【作者】 程嘉艺；
【导师】 康廷国；
【作者基本信息】 辽宁中医药大学 ， 生药学， 2008， 博士
【摘要】 目的:观察沙棘总黄酮口服给药对光化学反应血栓模型的影响;在在体及细胞水平探讨沙棘总黄酮抗栓作用机制;对沙棘总黄酮抗栓活性成分进行初步分析。方法:采用光化学血栓模型观察沙棘总黄酮口服给药对血栓形成的影响,观察指标为目标血管血流完全停止时间和490nm波长绿色光照射后不同时间血流速度;采用全血血细胞计数法测定小鼠血小板聚集性、采用ELISA法小鼠血浆von Willebrand因子含量;采用人脐静脉内皮细胞系ECV304,以H2O2制备血管内皮细胞损伤模型;采用MTT法测定内皮细胞相对活力;采用流式细胞仪测定内皮细胞坏死率和平均荧光强度的的变化;采用ELISA法检测细胞培养液中t-PA、PAI-1、vWF、TM蛋白的含量;采用荧光分光光度法测定内皮细胞培养液中LDH的含量;采用共聚焦激光扫描显微镜测定细胞内游离钙离子浓度;采用RT-PCR技术测定PAI-1 mRNA的相对表达;采用Western Blot法测定内皮细胞caspase-3活性。结果:1.0、3.0、10.0g/kg剂量的沙棘总黄酮剂量依存性地将血流完全停止时间分别提高至41.0±2.1、50.5±1.6、56.0±2.1min,ρ<0.01;沙棘总黄酮明显提高模型小鼠血流速度,作用时间随剂量增加而延长。各剂量沙棘总黄酮明显增加模型小鼠全血血小板数,中、高剂量明显降低小鼠血浆vWF浓度(ρ<0.05或ρ<0.01),低剂量对小鼠血浆vWF浓度无明显影响。H2O2浓度依赖性抑制ECV304细胞的活性,200μM的H2O2可使内皮细胞活性被抑制66%。400.0、200.0μg/ml的沙棘总黄酮提高H2O2损伤ECV304细胞的活力,ρ值分别小于0.01、0.05; 11.4μg/ml、5.7μg/ml的异鼠李素可提高H2O2损伤后内皮细胞相对活力,但22.8μg/ml异鼠李素则无此作用;400.0μg/ml的沙棘总黄酮组内皮细胞活性被抑制程度明显低于相对应的异鼠李素组。不同浓度的沙棘总黄酮、异鼠李素、槲皮素均显著降低H2O2损伤后内皮细胞死亡率(P<0.05),其中中、低浓度异鼠李素的作用显著强于高浓度组,ρ<0.05或ρ<0.01;高浓度槲皮素降低细胞死亡率的作用明显强于相应浓度的沙棘总黄酮,而高浓度的异鼠李素则明显弱于相应浓度的沙棘总黄酮。沙棘总黄酮和异鼠李素均可剂量依赖性抑制ECV-304细胞PAI-1的分泌,与空白对照组相比,两者高、中剂量组均有显著性差异(ρ<0.01),低剂量组无显著性差异,PAI-1的mRNA表达结果与此相同;沙棘总黄酮和异鼠李素对ECV-304细胞分泌t-PA和vWF均无明显影响。除22.8μg/ml异鼠李素外,各浓度的沙棘总黄酮、槲皮素、异鼠李素均明显抑制H2O2所致内皮细胞培养液中TM蛋白含量增加;沙棘总黄酮与相应浓度的异鼠李素和槲皮素的作用没有差异。沙棘总黄酮、异鼠李素、槲皮素均明显抑制H2O2所致内皮细胞LDH泄露(ρ<0.01),其中槲皮素的作用随浓度增加而有增强的趋势,而高浓度异鼠李素的作用则明显弱于中的浓度组,ρ<0.05。沙棘总黄酮和槲皮素均可对抗H2O2所致ECV304细胞胞浆内钙离子浓度升高,但异鼠李素没有该作用,特别是高浓度的异鼠李素有加重钙超载的作用。沙棘总黄酮、异鼠李素、槲皮素均明显抑制H2O2损伤内皮细胞caspase-3活性(ρ<0.01);高浓度异鼠李素的作用明显弱于相应浓度的沙棘总黄酮(ρ<0.01),高浓度槲皮素的作用则明显强于相应浓度的沙棘总黄酮(ρ<0.05)。结论:(1)沙棘总黄酮口服给药有抗血栓作用。(2)沙棘总黄酮抑制血小板活性,是其抗栓作用的机制之一。(3)沙棘总黄酮对过氧化损伤内皮细胞的保护作用,是其抗栓作用的机制之一。(4)沙棘总黄酮抑制钙超载、降低Caspase-3表达,是其内皮细胞保护作用机制之一。(5)异鼠李素、槲皮素是沙棘总黄酮抗栓作用活性成分。(6)高浓度的异鼠李素可能损伤内皮细胞。(7)沙棘总黄酮抗栓作用是多靶点的。
【Abstract】 Aim:To observe the impact of oral administration of total flavones of Hippophae Rhamnoides (TFH) on photochemical reaction model of thrombosis, investigate the mechanism of action of the anti-thrombosis effect of TFH and initially analyze the active anti-thrombosis ingredients of TFH.Methods:The impact of oral administration of total flavones of Hippophae hamnoides (TFH) on thrombosis was studied in a photochemical reaction model. The fully off-time of blood flow and the different blood flow velocities under the irradiation of 490nm green light of target blood vessels were calculated. The platelet aggregation activity of mice was studied by whole blood hemacytometry and the serum content of von Wilebrand Factor v (vWF) was measured by ELISA. Hydrogen dioxide (H2O2) was used to prepare the vascular endothelial cell injury model in ECV304 human umbilical vein endothelial cells (HUVEC). The relative vigor of endothelial cell was measured by MTT. The necrosis rate of endothelial cell and changes of the mean fluorecence intensity was measured by flow cytometry (FCM). The contents of t-PA, PAI-1, vWF, and TM of cell culture solution were measure by ELISA and the content of LDH was measured by pectrofluorometry. Intracellular free calcium concentration was measured by confocal laser-scan microscopy. The expression level of PAI-1 mRNA was detected by RT-PCR and the activity of caspase-3 in endothelial cell was detected by Western Blot.Results: The fully off-time of blood flow was prolonged to 41.0±2.1, 50.5±1.6 and 56.0±2.1min under the effect of 1.0, 3.0 and 10.0g/kg TFH respectively which showing a dose-dependent tendency (P<0.01); TFH significantly accelerated the velocity of blood flow in mice and the action time prolonged with the increase of dosage and significantly increased the PLT count in mice in all dosage group; Middle and high dose of TFH significantly decreased serum concentration of vWF in mice (P<0.05 or P<0.01) while low dose of TFH showed no obvious influence on it; H2O2 inhibited the vigor of ECV304 cell with a concentration-dependent tendency. 66% ECV304 cell could be inhibited by 200μM H2O2; 400μg/ml and 200μg/ml TFH could enhance the effect of H2O2 on ECV304 cell (P<0.05 and P<0.01 respectively); 11.4μg/ml and 5.7μg/ml isorhamnetin(ISO) could enhance the vigor of endothelial after H2O2 injury, while 22.8μg/ml ISO showed no this effect; The degree of endothelial cell inhibition of 400μg/ml TFH group was obviously lower than the corresponding ISO group; Different concentration of TFH, ISO and quercetin (Que) all cut down the mortality of endothelial cell after H2O2 injury(P<0.05) in which the effects of middle and low concentration of ISO were obviously stronger than that of the high concentration group(P<0.05 or P<0.01); The protective effect of high concentration of Que was obviously stronger than that of the high concentration of TFH while the protective effect of high concentration of ISO was poorer than that of high concentration of TFH. TFH and ISO could both inhibit the secretion of PAI-1 by ECV304 cell which showing a dose-dependent tendency. Compared with the control group, the significant difference was shown in both high concentration and middle concentration group(P<0.01) while not found in low concentration group. The same result was found in PAI-1 mRNA expression; No influence was found about TFH and ISO on the secretion of t-PA and vWF by ECV304 cell; The increase of protein TM in endothelial cell culture solution caused by H2O2 was inhibited by all sorts of concentration of TFH, Que and ISO except 22.8μg/ml ISO. No difference was found in TFH and corresponding concentration of ISO and Que; The leakage of LDH caused by H2O2 was inhibited by TFH, Que and ISO obviously(P<0.01) in which the effect of Que was augmented with the increase of concentration while the effect of high concentration of ISO was obviously poorer than that of the middle and low concentration group(P<0.05); The elevation of intracellular plasma calcium concentration caused by H2O2 was antagonized by TFH and Que , but no this effect was detected in ISO. To the contrary, high concentration of ISO could aggravate calcium overload; The damage of endothelial cell caspase-3 activity caused by H2O2 was inhibited by TFH, Que and ISO(P<0.01) in which the effect of high concentration of ISO was obviously poorer than that of the TFH (P<0.01) and the effect of high concentration of Que was obviously stronger than that of the TFH (P<0.05).Conclusions:(1) Oral administration of TFH has anti-thrombosis effect. (2) The inhibition of PLT activity is one of the mechanisms of its anti-thrombosis effect. (3) The protective effect of TFH on the injury of vascular endothelia caused by H2O2 is one of the mechanisms of its anti-thrombosis effect. (4) TFH can inhibit calcium overload and cut down the expression of Caspase-3, which is one of the mechanisms of its anti-thrombosis effect. (5)ISO and Que are active anti-thrombosis ingredients of TFH. (6) High concentration of ISO may injury endothelial cells. (7) The anti-thrombosis effect of TFH are multi-targetted .