基于特异性敲除技术的葛根素与葛根雌激素样和舒血管作用的相关性研究
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摘要
中药的化学成分与其功效之间相关性的研究是中药研究的重点内容之一。对于绝大多数中药,选择其中典型的单体成分(即代表性成分)作为切入点深入研究,是当前中医药领域采用的主要研究策略之一,但这种方法忽视了中药发挥独特疗效的整体性：即所单体成分的“代表性”如何,是否与中药功效主治相关?因此需要探索完善、科学的方法和技术平台进行药效组分分析,阐明代表性成分与中药功效的相关性。
中药小分子特异性敲除法是研究中药代表性成分与中药功效间相关性的新的技术方法。通过对比研究,分析代表性成分在整体药味中敲除前后药效的变化,揭示该成分与整体药效的相关性。葛根是一种古老而传统的常用中药,葛根素是葛根中具有代表性的异黄酮类化合物,也是葛根质量控制的主要成分。研究发现,异黄酮类化合物作为植物雌激素,能通过调节NO分泌保护心血管。葛根中葛根素等异黄酮类化合物具有不同程度的弱雌激素的作用和扩血管效应,是治疗心血管系统疾病的有效成分。本论文以葛根为研究对象,利用葛根素单抗建立特异性敲除葛根素的技术方法,获得特异性敲除葛根素的葛根水提物,借助与葛根传统主治功效和现代药理作用相对应的离体组织和细胞模型,以药效与生物学指标相结合的评价标准,探索葛根的代表成分葛根素在葛根雌激素样和血管舒张功效的相关性及机理。这不仅有利于深入地开展药理研究,还为葛根素作为葛根药材的质量控制等研究指标成分提供明确依据,奠定了利用特异性敲除法研究中药材指标成分与其功效的相关性研究的实验基础,从而使这一新的技术手段得以更广泛的利用。
1敲除法探讨葛根素在葛根雌激素样活性中的作用
[目的]
比较研究葛根提取液(radix puerariae extract, RPE)、葛根素(puerarin, PRR)以及敲除葛根素的葛根提取液(PRR knock-out RPE, RRR-KO-RPE)对雌激素受体阳性人乳腺癌T47D细胞增殖、细胞周期及ERα, ERβ蛋白表达的影响,探讨葛根素在葛根雌激素样活性药效中的相关性。
[方法]
以T47D细胞为模板,采用MTT方法检测药物及ICI182,780干预下细胞增殖变化,应用流式细胞仪测定细胞周期变化,采用western blot法T47D细胞ERα、ERβ蛋白表达特点。
[结果]
1RPE、PRR和PRR-KO-RPE均具有与雌激素类似的促细胞增殖的作用,且呈现出一定的剂量-时间-效应关系。PRR敲除后细胞增殖显著降低。药物作用48h,ICI182,780对RPE增殖抑制率最强,敲除PRR后细胞抑制率降低。
2三组药物均能降低细胞Go/G1期比例,增加S期细胞比例。RPE和PRR能不同程度提高细胞G2/M期的比例。PRR敲除后,G2/M期的比例及细胞增殖指数显著降低。
3各组均能提高ERα、ERβ的相对表达水平。PRR敲除后,ER α表达无显著变化,ER β表达减低。ICI182,780对各药物ERα、ERβ的表达均有拮抗作用,PRR敲除后,细胞表达ER α的拮抗程度无明显改变。
[结论]
口葛根素在葛根雌激素样活性的药效中具有一定的相关性,其机理与细胞增殖及ERβ表达有关。
2敲除法探讨葛根素在葛根舒血管药效中的作用
[目的]
比较研究RPE、PRR以及PRR-KO-RPE对SD大鼠胸主动脉血管环张力的影响,探讨葛根素在葛根舒血管药效中的作用。
[方法]
采用大鼠胸主动脉环体外实验,观察比较RPE、PRR和PRR-KO-RPE对内皮完整及内皮去除的血管环在基础状态(2g张力)以及去甲肾上腺素(NE)预收缩状态下的作用。
[结果]
1累积浓度的RPE、PRR和PRR-KO-RPE在对内皮完整和去内皮的胸主动脉血管环均有一定的舒张作用,并呈剂量依赖性舒张效应。葛根素敲除后,葛根对血管环的舒张程度显著降低。
2末次给药后PRR较RPE可快速达到最大舒张效应。RPE舒张血管的整体时间较PRR和PRR-KO-RPE长。
[结论]
口葛根素在葛根内皮依赖性舒血管药效中具有一定的相关性。
3敲除法探讨葛根素在葛根的雌激素样活性与内皮依赖性舒血管药效相关性及机理
[目的]
初步研究葛根素在人脐静脉内皮细胞(HUVECs)的分布和代谢特点,比较研究RPE. PRR以及PRR-KO-RPE对HUVECs增殖、NO分泌及eNOS、ERα、ERβ蛋白表达的影响,探讨葛根素在葛根舒血管雌激素样活性与内皮依赖性舒血管药效的关系及机理。
[方法]
以HUVECs为模板,采用细胞免疫组化法和葛根素免疫竞争ELISA法,比较研究葛根素单体及葛根提取液中的葛根素在HUVECs中的代谢分布特点,以及PRR的代谢与NO表达分泌的关系,MTT法测定三种药物及拮抗剂作用下对HUVECs的增殖及NO分泌的作用,western blot法检测HUVECs中ERα、ERβ和eNOS的表达影响。
[结果]
1葛根素能特异性结合在HUVEC细胞上,胞浆及细胞核周围可能是葛根素与HUVEVs特异性结合的位置。
21.0μg/mL的RPE和PRR作用30min、10.0μg/mL的RPE和PRR作用20min时,HUVECs对葛根素单体的吸收程度明显高于对葛根中相同浓度葛根素的吸收。1.0μg/mL葛根素单体对HUVECs作用10min,分泌NO的含量高于相同葛根素含量的葛根提取液,60min后葛根提取液对细胞分泌NO的含量高于葛根素。l0μg/mL的葛根素单体对HUVECs作用10min,分泌NO的含量高于相同葛根素含量的葛根提取液,40min后葛根提取液对细胞分泌NO的含量高于葛根素。
3RPE对细胞的分泌NO作用显著高于PRR和PRR-KO-RPE,敲除PRR后NO分泌减少。L-NAME和ICI182,780均可抑制RPE, PRR和PRR-KO-RPE对HUVECs分泌NO的作用。敲除PRR后L-NAME和ICI182,780对HUVECs分泌NO抑制程度均减弱。
4RPE、PRR和PRR-KO-RPE可以通过eNOS途径和雌激素受体途径促进NO的分泌。RPE组、PRR组和PRR-KO-RPE组均能提高ERα的相对表达水平,敲除前后拮抗剂对ERα拮抗作用无显著改变。RPE组、PRR组和PRR-KO-RPE组均能提高ERβ的相对表达水平。敲除前后ICI182,780对ERβ的拮抗作用显著降低。
[结论]
口葛根素在内皮细胞的分布的不均一性和代谢的差异性可能导致其不同生物效应的基础。eNOS-NO途径和ERβ-NO途径可能是葛根素在葛根促进内皮细胞NO分泌的药效相关性的机制。
The correlation between indicators and its efficacy in the Chinese herbs is the key scientific issues of basic and applied research of Chinese medicine. For most of Chinese medicineherbs, choosingtypical monomer component (representative component) as a starting point in-depth study is one of the main strategies used in the field of Chinese medicine currently,but this approach ignores the unique effect of holistic medicine plays,that is:How representative are the monomer component,if they are associated with traditional Chinese medicineindications?We the need to explore sound, scientific methods and techniques platforms efficacy component analysis to clarify the correlation representative composition and efficacy of traditional Chinese medicine.
The small molecule of Chinese herbsspecifitic knockout technology is new technology and methods in studing the correlationefficacy of representative composition and of Chinese herbs. By comparison study, we can reveal the correlation of the representative component and the herbs by the analysis of representative components in addition to efficacy before and after knockout of representative components.Pueraria is an ancient and traditional Chinese herb which is commonly used,Puerarin is a representative isoflavones in Pueraria.The study found that isoflavones as phytoestrogens can protect cardiovascular by regulating NO secretion.Puerarin and other isoflavones in Pueraria have a weak estrogen and vasodilator effect, whichare effective ingredients for the treatment of cardiovascular diseases.In this study, we choose Radix Puerariae which has exact efficacy and basic research results as a template, Puerarin monoclonal antibody as a tool to establish the "specific knock out the Puerarin" technology, and then get the Pueraria water extracts which has specific knock out Puerarin. In order to clarify the correlation between Puerarin and Radix Puerariae in drug efficacy and contribution, we take difference compared method as research methods, use In vitro models and cell models corresponding to the traditional attending effect and the pharmacological effects of the herb, take a combination of efficacy and biological indicators as evaluations criteria through the establishment of the immunoassay method of high sensitivity, specificity, suitable for micro-biological samples. After complete the above-mentioned study and achieve the desired objectives, we can not only clarify the material basis of Radix Puerariae and Puerarine in pharmacodynamics, but also provide the exact basis for index components in the quality control of Pueraria herbs compatibility mechanism, metabolic studies, preparation process and other aspects. We can lay a solid foundation for the relevance of the research methods in Chinese herbal medicines and their efficacy using specific knock out index components method, so that this new technical means can be more widely available.
1Investigation of puerarin and Pueraria on estrogen-like activity by knockout methods
[OBJECTIVE]
Comparative Study of RPE, PRR and PRR-KO-RPE on estrogen receptor-positive human breast cancer cell T47D proliferation, cell cycle effects of and ERa, ERβ protein expression.Explore estrogen-like activity on puerarin andpueraria vasodilatory estrogen-like activity in the efficacy of effect.
[METHODS]
Take T47D cells as a template, using the MTT method for detecting drugs and IC1182,780intervention changes in cell proliferation, cell cycle was measured by flow cytometry change, using western blot method to detect the protein level of ERa and ERβ protein expression of T47D cells.
[RESULTS]
1Three drugs have the effect of promoting cell proliferation and showed a dose response relationship. After PRR knockout significantly decreased cell proliferation. Drugs48h, ICI182,780on RPE proliferation inhibition rate strongest knockout PRR inhibition rate.
2Three groups of drugs could reduce the proportion of cells in G0/G1phase, increasing the proportion of cells in S phase. RPE and PRR can increase the proportion of cells in different degrees of G2/M phase. After the PRR knockout percentage and proliferation index G2/M phase decreased significantly.
3Each group can raise the relative expression levels of ERa, ERβ's. After the PRR knockout, ERa expression did not change significantly, ERβ expression reduced. IC1182,780for each medication ERa, ERP expression of both antagonism, the PRR knockout, ERa cells expressing the degree of antagonism did not change significantly.
[CONCLOTION]
Puerarin has some relevance in Pueraria efficacy of estrogen-like activity, and the mechanism of cell proliferation and ERβ pathway.
2Investigation of puerarin in Pueraria on vasodilatory efficacy by knockout methods
[OBJECTIVE]
Comparative Studyof radix puerariae extract(RPE), puerarin (PRR) and knockout Puerarin Pueraria Extract (PRR-KO-RPE) impacted on the SD rat thoracic vascular ring tension, explore the role of puerarin in Pueraria vasodilatory efficacy.
[METHODS]
Isometric tension recordings were applied to evaluate the effects of RPE, PRR and PRR-KO-RPE on resting, NE-Preconsrticting baortic ringswith or without endothelium.
[RESULTS]
1Cumulative concentration of RPE,PRR and PRR-KO-RPE in endothelial intact and denuded thoracic aortic rings have some relaxing effect and dose-dependent relaxation effects. Puerarin knockout, Kudzu degree of relaxation of vascular rings significantly reduced.
2PRR after the last administration over the RP E can quickly reach maximum relaxation effects. RP E vasodilation and overall time than PRR PRR-KO-RPE long.
[CONCLOTION]
Puerarin has some relevance in endothelium-dependent vasodilatation Pueraria efficacy.
3Correlation study on Pueraria and Puerarin on the efficiency of estrogen-like activity and efficacy of endothelium-dependent vasodilatation by knockout methods
[OBJECTIVE]
Preliminary study of puerarin in human umbilical vein endothelial cells (HUVECs) distribution and metabolic characteristics.Comparative study of RPE, PRR and PRR-KO-RPE on HUVECs proliferation, NO secretion and eNOS, ERa, ERβ protein expression.Investigate the relationship and mechanism of puerarin andpueraria vasodilatory estrogen-like activity and endo the lium-dependent vasodilator efficacy.
[METHODS]
Take HUVEC cells as a template. Using cell immunohistochemistry and puerarin immune competitive ELISA method, comparative study of puerarin monomer and Pueraria extract puerarin HUVECs metabolic characteristics of the distribution, and metabolic expression of the relationship between PRR and NO secretion; MTT method HUVECs proliferation and secretion of NO, western blot analysis to detect the expression of drugs on HUVECs in ERa, ERβ and eNOS determined under the action of the three drugs and antagonists.
[RESULTS]
1Immunohistochemical staining cells observed in cytoplasm and nucleus PRR role HUVECs after around a large brown granules.
2Take puerarin concentration as standard amount, RPE and PRR role of1.0μg/mL of30min, RPE and PRR role of10.0μg/mL for20min, HUVECs extent of absorption of puerarin monomer is significantly higher than the same concentration of Pueraria Pueraria Su absorption.1.0μg/mL puerarin monomer effect on HUVECs10min, NO secretion levels higher than the same puerarin in Pueraria Extract,60min after Pueraria extract content is higher than that of the cells to secrete NO puerarin.10μg/mL puerarin monomer effect on HUVECs10min, NO secretion was higher than the same puerarin in Pueraria Extract,40min after Pueraria extract content is higher than that of the cells to secrete NO puerarin.
3The secretion of NO on RPE was stronger than PRR and PRR-KO-RPE.Knockout PRR NO secretion. L-NAME and ICI182,780inhibited RPE, PRR and PRR-KO-RPE secretion of NO effect on HUVECs. Knockout PRR L-NAME and ICI182,780on the degree of inhibition of NO secretion HUVECs were weakened.
4Three drugs could increase the eNOS, ERa and ERβrelative expression levels. PRR group has the highest expression of eNOS and lower expression of ERa than RPE and PRR-KO-RPE. RPE, PRR and PRR-KO-RPE group have no different ERβ expression. L-NAME has the strongest degree of antagonism on PRR relative expression of eNOS, ICI182,780has the strongest degree of antagonism on PRR relative expression of ERa and ER(3
[CONCLOTION]
Puerarin in heterogeneity and metabolic differences in the distribution of endothelial cells may lead to the foundation of their different biological effects. eNOS-NO pathway and ERβ-NO pathway may be puerarin in Pueraria pharmacodynamic correlation mechanisms to promote endothelial NO secretion.
中药小分子特异性敲除法是研究中药代表性成分与中药功效间相关性的新的技术方法。通过对比研究,分析代表性成分在整体药味中敲除前后药效的变化,揭示该成分与整体药效的相关性。葛根是一种古老而传统的常用中药,葛根素是葛根中具有代表性的异黄酮类化合物,也是葛根质量控制的主要成分。研究发现,异黄酮类化合物作为植物雌激素,能通过调节NO分泌保护心血管。葛根中葛根素等异黄酮类化合物具有不同程度的弱雌激素的作用和扩血管效应,是治疗心血管系统疾病的有效成分。本论文以葛根为研究对象,利用葛根素单抗建立特异性敲除葛根素的技术方法,获得特异性敲除葛根素的葛根水提物,借助与葛根传统主治功效和现代药理作用相对应的离体组织和细胞模型,以药效与生物学指标相结合的评价标准,探索葛根的代表成分葛根素在葛根雌激素样和血管舒张功效的相关性及机理。这不仅有利于深入地开展药理研究,还为葛根素作为葛根药材的质量控制等研究指标成分提供明确依据,奠定了利用特异性敲除法研究中药材指标成分与其功效的相关性研究的实验基础,从而使这一新的技术手段得以更广泛的利用。
1敲除法探讨葛根素在葛根雌激素样活性中的作用
[目的]
比较研究葛根提取液(radix puerariae extract, RPE)、葛根素(puerarin, PRR)以及敲除葛根素的葛根提取液(PRR knock-out RPE, RRR-KO-RPE)对雌激素受体阳性人乳腺癌T47D细胞增殖、细胞周期及ERα, ERβ蛋白表达的影响,探讨葛根素在葛根雌激素样活性药效中的相关性。
[方法]
以T47D细胞为模板,采用MTT方法检测药物及ICI182,780干预下细胞增殖变化,应用流式细胞仪测定细胞周期变化,采用western blot法T47D细胞ERα、ERβ蛋白表达特点。
[结果]
1RPE、PRR和PRR-KO-RPE均具有与雌激素类似的促细胞增殖的作用,且呈现出一定的剂量-时间-效应关系。PRR敲除后细胞增殖显著降低。药物作用48h,ICI182,780对RPE增殖抑制率最强,敲除PRR后细胞抑制率降低。
2三组药物均能降低细胞Go/G1期比例,增加S期细胞比例。RPE和PRR能不同程度提高细胞G2/M期的比例。PRR敲除后,G2/M期的比例及细胞增殖指数显著降低。
3各组均能提高ERα、ERβ的相对表达水平。PRR敲除后,ER α表达无显著变化,ER β表达减低。ICI182,780对各药物ERα、ERβ的表达均有拮抗作用,PRR敲除后,细胞表达ER α的拮抗程度无明显改变。
[结论]
口葛根素在葛根雌激素样活性的药效中具有一定的相关性,其机理与细胞增殖及ERβ表达有关。
2敲除法探讨葛根素在葛根舒血管药效中的作用
[目的]
比较研究RPE、PRR以及PRR-KO-RPE对SD大鼠胸主动脉血管环张力的影响,探讨葛根素在葛根舒血管药效中的作用。
[方法]
采用大鼠胸主动脉环体外实验,观察比较RPE、PRR和PRR-KO-RPE对内皮完整及内皮去除的血管环在基础状态(2g张力)以及去甲肾上腺素(NE)预收缩状态下的作用。
[结果]
1累积浓度的RPE、PRR和PRR-KO-RPE在对内皮完整和去内皮的胸主动脉血管环均有一定的舒张作用,并呈剂量依赖性舒张效应。葛根素敲除后,葛根对血管环的舒张程度显著降低。
2末次给药后PRR较RPE可快速达到最大舒张效应。RPE舒张血管的整体时间较PRR和PRR-KO-RPE长。
[结论]
口葛根素在葛根内皮依赖性舒血管药效中具有一定的相关性。
3敲除法探讨葛根素在葛根的雌激素样活性与内皮依赖性舒血管药效相关性及机理
[目的]
初步研究葛根素在人脐静脉内皮细胞(HUVECs)的分布和代谢特点,比较研究RPE. PRR以及PRR-KO-RPE对HUVECs增殖、NO分泌及eNOS、ERα、ERβ蛋白表达的影响,探讨葛根素在葛根舒血管雌激素样活性与内皮依赖性舒血管药效的关系及机理。
[方法]
以HUVECs为模板,采用细胞免疫组化法和葛根素免疫竞争ELISA法,比较研究葛根素单体及葛根提取液中的葛根素在HUVECs中的代谢分布特点,以及PRR的代谢与NO表达分泌的关系,MTT法测定三种药物及拮抗剂作用下对HUVECs的增殖及NO分泌的作用,western blot法检测HUVECs中ERα、ERβ和eNOS的表达影响。
[结果]
1葛根素能特异性结合在HUVEC细胞上,胞浆及细胞核周围可能是葛根素与HUVEVs特异性结合的位置。
21.0μg/mL的RPE和PRR作用30min、10.0μg/mL的RPE和PRR作用20min时,HUVECs对葛根素单体的吸收程度明显高于对葛根中相同浓度葛根素的吸收。1.0μg/mL葛根素单体对HUVECs作用10min,分泌NO的含量高于相同葛根素含量的葛根提取液,60min后葛根提取液对细胞分泌NO的含量高于葛根素。l0μg/mL的葛根素单体对HUVECs作用10min,分泌NO的含量高于相同葛根素含量的葛根提取液,40min后葛根提取液对细胞分泌NO的含量高于葛根素。
3RPE对细胞的分泌NO作用显著高于PRR和PRR-KO-RPE,敲除PRR后NO分泌减少。L-NAME和ICI182,780均可抑制RPE, PRR和PRR-KO-RPE对HUVECs分泌NO的作用。敲除PRR后L-NAME和ICI182,780对HUVECs分泌NO抑制程度均减弱。
4RPE、PRR和PRR-KO-RPE可以通过eNOS途径和雌激素受体途径促进NO的分泌。RPE组、PRR组和PRR-KO-RPE组均能提高ERα的相对表达水平,敲除前后拮抗剂对ERα拮抗作用无显著改变。RPE组、PRR组和PRR-KO-RPE组均能提高ERβ的相对表达水平。敲除前后ICI182,780对ERβ的拮抗作用显著降低。
[结论]
口葛根素在内皮细胞的分布的不均一性和代谢的差异性可能导致其不同生物效应的基础。eNOS-NO途径和ERβ-NO途径可能是葛根素在葛根促进内皮细胞NO分泌的药效相关性的机制。
The correlation between indicators and its efficacy in the Chinese herbs is the key scientific issues of basic and applied research of Chinese medicine. For most of Chinese medicineherbs, choosingtypical monomer component (representative component) as a starting point in-depth study is one of the main strategies used in the field of Chinese medicine currently,but this approach ignores the unique effect of holistic medicine plays,that is:How representative are the monomer component,if they are associated with traditional Chinese medicineindications?We the need to explore sound, scientific methods and techniques platforms efficacy component analysis to clarify the correlation representative composition and efficacy of traditional Chinese medicine.
The small molecule of Chinese herbsspecifitic knockout technology is new technology and methods in studing the correlationefficacy of representative composition and of Chinese herbs. By comparison study, we can reveal the correlation of the representative component and the herbs by the analysis of representative components in addition to efficacy before and after knockout of representative components.Pueraria is an ancient and traditional Chinese herb which is commonly used,Puerarin is a representative isoflavones in Pueraria.The study found that isoflavones as phytoestrogens can protect cardiovascular by regulating NO secretion.Puerarin and other isoflavones in Pueraria have a weak estrogen and vasodilator effect, whichare effective ingredients for the treatment of cardiovascular diseases.In this study, we choose Radix Puerariae which has exact efficacy and basic research results as a template, Puerarin monoclonal antibody as a tool to establish the "specific knock out the Puerarin" technology, and then get the Pueraria water extracts which has specific knock out Puerarin. In order to clarify the correlation between Puerarin and Radix Puerariae in drug efficacy and contribution, we take difference compared method as research methods, use In vitro models and cell models corresponding to the traditional attending effect and the pharmacological effects of the herb, take a combination of efficacy and biological indicators as evaluations criteria through the establishment of the immunoassay method of high sensitivity, specificity, suitable for micro-biological samples. After complete the above-mentioned study and achieve the desired objectives, we can not only clarify the material basis of Radix Puerariae and Puerarine in pharmacodynamics, but also provide the exact basis for index components in the quality control of Pueraria herbs compatibility mechanism, metabolic studies, preparation process and other aspects. We can lay a solid foundation for the relevance of the research methods in Chinese herbal medicines and their efficacy using specific knock out index components method, so that this new technical means can be more widely available.
1Investigation of puerarin and Pueraria on estrogen-like activity by knockout methods
[OBJECTIVE]
Comparative Study of RPE, PRR and PRR-KO-RPE on estrogen receptor-positive human breast cancer cell T47D proliferation, cell cycle effects of and ERa, ERβ protein expression.Explore estrogen-like activity on puerarin andpueraria vasodilatory estrogen-like activity in the efficacy of effect.
[METHODS]
Take T47D cells as a template, using the MTT method for detecting drugs and IC1182,780intervention changes in cell proliferation, cell cycle was measured by flow cytometry change, using western blot method to detect the protein level of ERa and ERβ protein expression of T47D cells.
[RESULTS]
1Three drugs have the effect of promoting cell proliferation and showed a dose response relationship. After PRR knockout significantly decreased cell proliferation. Drugs48h, ICI182,780on RPE proliferation inhibition rate strongest knockout PRR inhibition rate.
2Three groups of drugs could reduce the proportion of cells in G0/G1phase, increasing the proportion of cells in S phase. RPE and PRR can increase the proportion of cells in different degrees of G2/M phase. After the PRR knockout percentage and proliferation index G2/M phase decreased significantly.
3Each group can raise the relative expression levels of ERa, ERβ's. After the PRR knockout, ERa expression did not change significantly, ERβ expression reduced. IC1182,780for each medication ERa, ERP expression of both antagonism, the PRR knockout, ERa cells expressing the degree of antagonism did not change significantly.
[CONCLOTION]
Puerarin has some relevance in Pueraria efficacy of estrogen-like activity, and the mechanism of cell proliferation and ERβ pathway.
2Investigation of puerarin in Pueraria on vasodilatory efficacy by knockout methods
[OBJECTIVE]
Comparative Studyof radix puerariae extract(RPE), puerarin (PRR) and knockout Puerarin Pueraria Extract (PRR-KO-RPE) impacted on the SD rat thoracic vascular ring tension, explore the role of puerarin in Pueraria vasodilatory efficacy.
[METHODS]
Isometric tension recordings were applied to evaluate the effects of RPE, PRR and PRR-KO-RPE on resting, NE-Preconsrticting baortic ringswith or without endothelium.
[RESULTS]
1Cumulative concentration of RPE,PRR and PRR-KO-RPE in endothelial intact and denuded thoracic aortic rings have some relaxing effect and dose-dependent relaxation effects. Puerarin knockout, Kudzu degree of relaxation of vascular rings significantly reduced.
2PRR after the last administration over the RP E can quickly reach maximum relaxation effects. RP E vasodilation and overall time than PRR PRR-KO-RPE long.
[CONCLOTION]
Puerarin has some relevance in endothelium-dependent vasodilatation Pueraria efficacy.
3Correlation study on Pueraria and Puerarin on the efficiency of estrogen-like activity and efficacy of endothelium-dependent vasodilatation by knockout methods
[OBJECTIVE]
Preliminary study of puerarin in human umbilical vein endothelial cells (HUVECs) distribution and metabolic characteristics.Comparative study of RPE, PRR and PRR-KO-RPE on HUVECs proliferation, NO secretion and eNOS, ERa, ERβ protein expression.Investigate the relationship and mechanism of puerarin andpueraria vasodilatory estrogen-like activity and endo the lium-dependent vasodilator efficacy.
[METHODS]
Take HUVEC cells as a template. Using cell immunohistochemistry and puerarin immune competitive ELISA method, comparative study of puerarin monomer and Pueraria extract puerarin HUVECs metabolic characteristics of the distribution, and metabolic expression of the relationship between PRR and NO secretion; MTT method HUVECs proliferation and secretion of NO, western blot analysis to detect the expression of drugs on HUVECs in ERa, ERβ and eNOS determined under the action of the three drugs and antagonists.
[RESULTS]
1Immunohistochemical staining cells observed in cytoplasm and nucleus PRR role HUVECs after around a large brown granules.
2Take puerarin concentration as standard amount, RPE and PRR role of1.0μg/mL of30min, RPE and PRR role of10.0μg/mL for20min, HUVECs extent of absorption of puerarin monomer is significantly higher than the same concentration of Pueraria Pueraria Su absorption.1.0μg/mL puerarin monomer effect on HUVECs10min, NO secretion levels higher than the same puerarin in Pueraria Extract,60min after Pueraria extract content is higher than that of the cells to secrete NO puerarin.10μg/mL puerarin monomer effect on HUVECs10min, NO secretion was higher than the same puerarin in Pueraria Extract,40min after Pueraria extract content is higher than that of the cells to secrete NO puerarin.
3The secretion of NO on RPE was stronger than PRR and PRR-KO-RPE.Knockout PRR NO secretion. L-NAME and ICI182,780inhibited RPE, PRR and PRR-KO-RPE secretion of NO effect on HUVECs. Knockout PRR L-NAME and ICI182,780on the degree of inhibition of NO secretion HUVECs were weakened.
4Three drugs could increase the eNOS, ERa and ERβrelative expression levels. PRR group has the highest expression of eNOS and lower expression of ERa than RPE and PRR-KO-RPE. RPE, PRR and PRR-KO-RPE group have no different ERβ expression. L-NAME has the strongest degree of antagonism on PRR relative expression of eNOS, ICI182,780has the strongest degree of antagonism on PRR relative expression of ERa and ER(3
[CONCLOTION]
Puerarin in heterogeneity and metabolic differences in the distribution of endothelial cells may lead to the foundation of their different biological effects. eNOS-NO pathway and ERβ-NO pathway may be puerarin in Pueraria pharmacodynamic correlation mechanisms to promote endothelial NO secretion.
引文
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