红景天有效组分对缺氧缺糖心肌细胞损伤保护作用的研究

【作者】 龙怡；

【导师】 孟宪丽；

【作者基本信息】 成都中医药大学 ， 药理学， 2007， 硕士

【摘要】 【目的】研究藏药红景天中对缺氧缺糖心肌细胞损伤起保护作用的药效物质基础,并初步探讨其作用机制。【方法】制备红景天水提部位、乙酸乙酯部位及正丁醇部位,采用小鼠常压耐缺氧实验与血清药效学方法,筛选出红景天抗缺氧的有效部位;进一步提取乙酸乙酯及正丁醇部位单体成分红景天苷、酪醇、没食子酸、德钦红景天苷、草质素-7-0-(3’-β-D-葡萄糖)-α-L-鼠李糖苷、×××(未定名)共6个成分,用依次稀释法,直接作用于缺氧缺糖心肌细胞,测定其细胞活力MTT值及细胞培养上清液中LDH值与细胞中MDA值的变化,筛选出其抗缺氧的有效单体成分;运用(45)~Ca跨膜内流测量技术,检测红景天抗缺氧有效成分对钙内流的影响;采用反转录聚合酶链式反应(R7-PCR)法检测在不同药物作用下心肌细胞HIF-1αmRNA表达的变化;采用原位末端脱氧核苷酸转移酶标记TUNEL法,测定不同药物对细胞凋亡的影响。【结果】红景天乙酸乙酯部位和正丁醇部位有促进常压小鼠耐缺氧能力及提高缺氧缺糖心肌细胞活力作用;红景天苷、酪醇、没食子酸、德钦红景天苷、草质素-7-0-(3’’-β-D-葡萄糖)-α-L-鼠李糖苷均有不同程度的促细胞活力及减少LDA、MDA水平;而此5个单体成分也有较明显的钙拮抗作用;红景天苷、酪醇都有较明显的增加HIF-1αmRNA表达及抑制细胞凋亡作用。【结论】红景天中对缺氧缺糖心肌细胞起保护作用的有效部位为乙酸乙酯部位及正丁醇部位:有效成分为乙酸乙酯部位的酪醇、没食子酸、德钦红景天苷,正丁醇部位红景天苷、草质素-7-0-(3’’-β-D-葡萄糖)-α-L-鼠李糖苷;红景天抗缺氧的作用机制可能与钙拮抗、增强HIF-1αmRNA表达及抑制细胞凋亡有关。更多还原

【Abstract】 Objective:Research the effective fractions and the effective components of Radix et Rhizoma Rhoddoae,which plays protective roles on hypoxia. Identify the foundation of the substance and approach the mechanism of action initially.Method andMaterial:Extract different fractions using water,acetoacetate and n-butyl alcohol one by one and combine the resistance of hypoxia test of mouse in vivo,use 20%of serum of the rats after second administration of the medicine acting on the hypoxic myocardial cells which have been purified,mensurate the value of MTT of the cell’s energy,sieve the effective fraction of antihypoxia.Further more we extract 6 monosomic components including rhodioside,butyl alcohol,galic acid, rhodiolatuntoside,rhamnoside、×××（unnamed） and then dilute these components in order,use them acting on the hypoxic myocardial cells straightly,mensurate MTT of the cell’s energy and the change of LDH of supematant liquid of the cell culture,find the transmutation of MDA of cell,sieve the effective monosomic components of antihypoxia.We use the method of ⁴⁵Ca transmembrane influx technique to detect the affect of antihypoxic effective components of Radix et Rhizoma Rhoddoae acting on the influx of Ca.Apply the method of RT-PCR and TUNEL-POD to detect the change of HIF-1αmRNA expression of myocardial cells and the affect to cell apoptosis which have been acted on different medicines respectively,approach the mechanism of action of Radix et Rhizoma Rhoddoae initially.Results:Combining the test of mouse in vivo,20%of serum which has the extracted fractions using acetoacetate and n-butyl alcohol can both promote the energy of the hypoxic myocardial cell;Rhodioside,butyl alcohol,galic acid,rhodiolatuntoside and rhamnoside can promote the energy of cell and decreace the level of LDA and MDA in some degree;And 5 monosomic components have the comparative obvious antagonistic action of Ca²⁺.While rhodioside and butyl alcohol both can increse the expression of HIF-1αmRNA and inhibit the cell apoptosis comparatively obviously.Conclution:The effective components of Radix et Rhizoma Rhodoloae which can protect hypoxic myocardial cells are those which has the extracted fractions using acetoacetate and n-butyl alcohol.And the effective components is butyl alcohol,galic acid,rhodiolatuntoside of acetoacetate and rhodioside,rhamnoside of n-butyl alcohol,all these components have obvious antagonistic action of Ca²⁺.While butyl alcohol of acetoacetate and rhodioside of n-butyl alcohol both can increse the expression of HIF-1αmRNA and inhibit the cell apoptosis comparatively obviously.This may be the antihypoxia mechanism of effection components of Radix et Rhizoma Rhoddoae.更多还原