基于白芍养血柔肝功效的芍药苷、芍药内酯苷药理作用研究

发布时间：2018-05-28 10:10

本文选题：芍药苷 + 芍药内酯苷　；参考：《北京中医药大学》2017年硕士论文

【摘要】：背景血虚肝郁证是肝血亏虚所引起的肝失疏泄调达,既有肝经血分的亏虚,又有肝经气机的疏泄失常,是肝血亏虚和肝失疏泄并存的复合中医证候。中医学肝藏血功能与现代医学肝脏在物质和能量代谢的作用有着密切的关系,均可参与神经-内分泌-免疫(NEI)网络中肽类激素、免疫细胞因子等小分子物质的合成与存储。而肝主疏泄生理功能与神经系统对下丘脑-垂体-肾上腺(HPA)轴及其他机体反应轴和神经递质的调节有关,也参与了 NEI网络的调控机制。据此可得,血虚肝郁证证候本质应与NEI的整体调控机制具有高度相似性。因此,以血虚肝郁证动物模型中NEI网络生理和病理变化为研究对象,可初步阐明血虚肝郁证的证候的实质。白芍,味苦、酸,性微寒,归肝经、脾经,即可"养血敛阴"为中医传统补血药,又可"柔肝缓急"主治肝失调达、气郁不舒。肝气多郁,养血柔肝解郁是白芍临床应用的一个重要特色。白芍既能滋肝阴以养肝血,又能敛肝阴来柔肝体,最顺应肝之生理特性,为养血柔肝的代表性中药。白芍,主入肝、脾经。肝藏血主疏泄,主要体现在对气机运行和血液循行的调控上。其作用机制与现代医学中内分泌、免疫、循环、神经、消化及生殖系统有关。脾为后天之本,主运化水谷和水液,其生理功能与现代医学消化、泌尿系统相关;主生血、统血,提示其与内分泌、循环系统相关。据此,可认为白芍的功效与NEI网络密切相关。芍药苷、芍药内酯苷为白芍主要有效成分,芍药内酯苷为白芍特征性成分。现代药理研究表明,芍药苷作为白芍的单萜类成分中含量最高的主要有效成分,大量实验证明芍药苷主要作用于血液系统、神经系统、内分泌系统,有抑制凝血酶原的生成、抗血小板聚集、扩张静脉及外周小血管、改善血液微循环、抗氧化、抗惊厥等多种生物学效应,提示其有补血、抗抑郁、镇痛解痉和改善学习记忆以及对心脑血管的保护等作用。芍药内酯苷与芍药苷化学结构相似,是芍药苷的同分异构体;因其含量较芍药苷低且难以分离纯化,故药理作用研究较少。本课题组基于自有专利技术,对芍药内酯苷现有药理作用研究结果表明,芍药内酯苷可作用于免疫系统中脾脏、胸腺和血液系统中造血细胞因子具有明确的补血作用,同时还可作用于神经系统中HPA轴和脑内单胺类神经递,具有明显的抗抑郁作用。目的"证-效"相关理论提示中医证候与中药功效密切相关,本研究以白芍"养血柔肝"功效为依据,以血虚肝郁证动物模型为切入点,在系统梳理血虚肝郁证证候特点及芍药苷、芍药内酯苷相关药理作用研究的基础上,观察芍药苷、芍药内酯苷对血虚肝郁证动物模型大鼠的干预作用,通过检测相关生理生化指标以期从整体、组织、细胞和蛋白水平,探讨白芍"养血柔肝"功效物质基础及作用机制和血虚肝郁证的证候实质。方法本研究通过复制血虚肝郁证动物模型和课题组自有白芍有效成分芍药苷、芍药内酯苷分离纯化专利技术的支撑下,以经典补血方剂四物颗粒(含白芍且中国药典以芍药苷的含量作为质量控制标准)和传统解郁方剂逍遥丸(含白芍且中国药典以芍药苷的含量作为质量控制标准)为阳性对照药,并通过检测各组大鼠体质量,行为学实验(糖水消耗实验、敞箱实验、高架十字迷宫)以及外周血中WBC、RBC、HGB、脾脏指数、胸腺指数、肾上腺指数和血清中IL-3、IL-6、GM-CSF、TNF-α的含量和血浆中ACTH、CORT和下丘脑中CRH的水平、大脑皮质中PKA、cAMP的活性、海马组织中NE、DA、5-HT、E、5-HIAA的含量及cAMP/PKA信号通路相关神经蛋白PKA、p-CREB、BDNF的蛋白表达水平的变化。结果1文献研究1.1血虚肝郁证的辩证论治及其与神经-内分泌-免疫网络关系的探讨通过阅读大量文献,较系统的梳理了中医学对血虚肝郁证的辩证论治以及神经-内分泌-免疫网络学说与中医学相关理论的关系。不仅掌握了中医学和现代医学对血虚肝郁证的认识,而且明确了 NEI网络与血虚肝郁证的相互关系,为血虚肝郁证的证候实质研究提供了前期工作准备。1.2芍药苷、芍药内酯苷对神经内分泌免疫网络药理作用研究进展通过阅读大量文献,分别概括了白芍主要有效成分芍药苷、芍药内酯苷对神经系统、内分泌系统、免疫系统药理作用,揭示了白芍多环节、多靶点的药理作用机制与神经-内分泌-免疫网络密切相关,即可为整体上系统研究白芍的传统功效提供研究思路,也可为血虚肝郁证和白芍养血柔肝功效的"证-效"相关性研究提供借鉴意义。2实验研究2.1芍药苷、芍药内酯苷对血虚肝郁证大鼠外周血细胞、脏器指数及造血细胞因子的影响结果:与模型组比较,PF30mg·kg-1组和AF30mg·kg-1组体质量、脾脏指数、白细胞数量增加(P0.05、P0.01)。RIA结果显示,PF30mg·kg-1和AF30mg·kg-1组均可增加该组 IL-3 含量(P0.05、P0.05)和减少该组 TNF-α 含量(P0.05、P0.05)。2.2芍药苷、芍药内酯苷对血虚肝郁证大鼠行为学、HPA轴和单胺类神经递质的影响结果:与模型组比较,PF30mg·kg-1组和AF30mg·kg-1组体质量、糖水消量、旷场实验穿行次数增加(P0.05、P0.01)而滞留时间减少(P0.05、P0.05),高架十字迷宫实验开放臂进入次数和停留时间明显增加(P0.05、P0.05),而封闭臂停留时间减少(P0.05);RIA 结果显示,PF30mg·kg-1 组和 AF30mg·kg-1 组均可降低该组 CORT、ACTH含量(P0.05、P0.05);HPLC-ECD 结果显示,AF30mg·kg-1 组、PF30mg·kg-1 组均可不同程度的升高NE、DA、5-HA的含量(P0.05、P0.01)。2.3芍药苷、芍药内酯苷对血虚肝郁证大鼠大脑皮层和海马中cAMP、PKA含量及PKA、p-CREB、BDNF的蛋白水平的影响结果:与模型组比较,RIA结果显示,PF30mg·kg-1组和AF30mg·kg-1组可使PKA含量升高,且AF30mg·kg-1组还可升高cAMP含量(P0.05、P0.05)。Western Blot结果显示,与模型组比较,PF30mg·kg-1组和AF30mg·kg-1组均可增加该组PKA蛋白水平含量(P0.01、P0.05)。结论结论1:实验一结果表明,芍药苷和芍药内酯苷作为白芍的主要有效成分,可通过对造血细胞因子的调控作用,调节免疫系统的紊乱,维持骨髓造血内环境的稳态。结论2:实验二结果表明,芍药苷、芍药内酯苷作为白芍主要有效成分均可在一定程度上改善血虚肝郁大鼠行为学改变,其改善机制可能与二者调节神经递质、减少应激激素的分泌、抑制HPA轴的过度激活,调控神经-内分泌系统的功能有关。结论3:实验三结果表明,芍药苷、芍药内酯苷可调控血虚肝郁证大鼠细胞内cAMP/PKA信号通路中相关信号蛋白,发挥神经保护作用。提示二者均为白芍养血柔肝功效的物质基础。综上所述:本研究揭示白芍主要有效成分芍药苷、芍药内酯苷可通过调节神经-内分泌-免疫(NEI)网络对放射线辐照结合慢性束缚致血虚肝郁证动物模型发挥养血柔肝功效,且提示中药白芍与中医血虚肝郁证的"证-效"相关性及血虚肝郁证的证候本质的探讨,可以神经、内分泌、免疫系统中相关细胞因子、激素、递质为突破口进行NEI网络的研究。并在此基础上,本研究通过观察芍药苷、芍药内酯苷对血虚肝郁证动物模型胞内cAMP/PKA信号通路的影响,进一步揭示白芍养血柔肝功效的作用机制还与对该信号通路中相关受体及靶点的调节作用有关。本研究不仅揭示了白芍"养血柔肝"功效的科学内涵,探讨了白芍主要有效成分芍药苷、芍药内酯苷的药理作用及机制,为白芍临床应用提供了科学依据,而且通过血虚肝郁证和白芍养血柔肝功效"证-效"相关性的研究,既丰富了中医肝脏证候的基础研究,又可为中医药相关理论的研究提供借鉴意义。
[Abstract]:The syndrome of stagnation of the liver in the background of blood deficiency is the transfer of liver loss and relief caused by deficiency of liver blood deficiency, which not only has the deficiency of the liver and blood, but also the disorder of the liver Qi machine. It is a compound TCM syndrome of the coexistence of liver blood deficiency and liver loss and drainage. The function of liver and liver is closely related to the role of the liver in the metabolism of substance and energy in modern medicine, and can be involved in the liver. The synthesis and storage of peptide hormones and immune cell factors in the neural endocrine immune (NEI) network are synthesized and stored. The physiological function of liver catharsis is related to the regulation of the hypothalamus pituitary adrenal (HPA) axis and other body reaction axis and neurotransmitters, and also participates in the regulation mechanism of the NEI network. The essence of the syndrome of Qi syndrome should be highly similar to the overall regulation mechanism of NEI. Therefore, the physiological and pathological changes of NEI network in the animal model of blood deficiency liver depression are studied, and the essence of the syndrome can be clarified preliminarily. The main characteristic of the clinical application of Paeonia lactiflora is Paeonia lactiflora, which can not only nourish liver yin to nourish liver blood, but also converge liver yin to soft liver. It is the most conforming to the physiological characteristics of the liver. It is the representative traditional Chinese medicine for raising blood and liver. The mechanism is related to the endocrine, immune, circulation, nerve, digestion and reproductive systems in modern medicine. The spleen is the day after the spleen, the main transport of water and water, its physiological function with the modern medicine digestion, urinary system related; the main blood, the blood, suggesting that it is related to endocrine, circulation system. It is considered that the efficacy of Paeonia lactiflora is closely related to the NEI network. Paeoniflorin, paeoniflorin is the main effective component of Paeonia lactiflora, and paeoniflorin is a characteristic component of Paeonia lactiflora. Modern pharmacological studies have shown that paeoniflorin is the main effective component in the monoterpenoids of Paeonia lactiflora. A large number of experiments have proved that paeoniflorin mainly acts on blood. System, nervous system, endocrine system, inhibiting the generation of prothrombin, anti platelet aggregation, dilated veins and peripheral blood vessels, improving blood microcirculation, antioxidation, anticonvulsant and other biological effects, suggesting that it has the effect of blood supplement, antidepressant, analgesic and antispasmodic and improvement of learning and memory as well as the protection of heart and brain blood vessels. The chemical structure of glucoside and paeoniflorin is similar to the isomer of paeoniflorin. Because its content is lower than paeoniflorin and it is difficult to be separated and purified, the pharmacological action of paeoniflorin is less. Based on the patent technology, the research group has studied the pharmacological effects of paeoniflorin on the spleen, thymus and blood in the immune system. The hematopoietic cell factor in the system has a clear blood supplement and can also act on the HPA axis of the nervous system and the monoamine neurotransmitters in the brain and has an obvious antidepressant effect. On the basis of a systematic review of the characteristics of the syndrome of stagnation of the liver stagnation and the related pharmacological effects of paeoniflorin and paeoniflorin, the intervention effect of paeoniflorin and paeoniflorin on the rat model of liver depression with blood deficiency syndrome was observed, and the level of the whole, tissue, cell and protein level was discussed by detecting the related physiological and biochemical markers. Radix Paeoniae Paeoniae "nourishing blood and suppressing liver" effect material basis and function mechanism and syndrome essence of blood deficiency liver depression syndrome. Methods the study was carried out by replicating blood deficiency liver depression animal model and subjects group own Paeoniae paeoniflorin and paeoniflorin isolation and purification patent technology supported by the Chinese Pharmacopoeia (including Paeonia lactiflora and Chinese Pharmacopoeia). Taking the content of paeoniflorin as the quality control standard) and the traditional Jieyu prescription, Xiaoyao Pill (including Paeonia lactiflora and the content of paeoniflorin in Chinese Pharmacopoeia as the quality control standard) as the positive control drug, and by testing the body mass of rats in each group, behavior test (sugar water consumption experiment, open box experiment, elevated cross maze) and WBC, RBC, HGB in peripheral blood The contents of spleen index, thymus index, adrenal index, adrenal index and serum IL-3, IL-6, GM-CSF, TNF- alpha, the level of ACTH, CORT and CRH in the hypothalamus, the activity of PKA, cAMP in the cerebral cortex, the content of NE, DA, 5-HT, and related neuroproteins in the hippocampus, and the changes in the protein expression level. Results 1 literature study on the dialectical treatment of 1.1 blood deficiency syndrome of liver depression and its relationship with the neural endocrine immune network, through reading a large number of documents, systematically combed the dialectical treatment of blood deficiency syndrome in TCM and the relationship between the theory of neural endocrine immune network and the related theories of traditional Chinese Medicine. The mutual relationship between the NEI network and the syndrome of blood deficiency liver depression was clarified, and the relationship between the syndrome of blood deficiency and stagnation of liver depression was clarified, and the preliminary work for the study of syndrome essence of the syndrome of blood deficiency liver depression was prepared by.1.2 paeoniflorin, and the research progress on the pharmacological effects of paeoniflorin on the neuroendocrine immune network had passed through a large number of literature, and the main contents of Paeonia lactiflora were summarized. The pharmacological effects of paeoniflorin and paeoniflorin on the nervous system, the endocrine system and the immune system reveal the multiple links of Paeonia lactiflora. The pharmacological mechanism of the multiple targets is closely related to the neural endocrine immune network. It can provide a research idea for the study of the traditional work effect of Paeonia lactiflora on the whole and the syndrome of blood deficiency and the stagnation of the liver and the Paeonia lactiflora. The study of "syndrome effect" related research on the efficacy of nourishing blood and suppressing liver to provide reference significance.2 experimental study 2.1 paeoniflorin, paeoniflorin, the effects of paeoniflorin on peripheral blood cells, organ index and hematopoietic cytokines in rats with blood deficiency liver depression: compared with the model group, the body mass of PF30mg kg-1 group and AF30mg kg-1 group, the spleen index, and the number of white blood cells increased (P0.05, P0). .01).RIA results showed that the group of PF30mg / kg-1 and AF30mg / kg-1 could increase the IL-3 content of the group (P0.05, P0.05) and reduce the TNF- alpha content of the group (P0.05, P0.05) paeoniflorin, the effect of paeoniflorin on the behavior, the axis and the monoamine neurotransmitters of the blood deficiency liver depression rats: compared with the model group, the group and the group of minorities were compared with the model group. Body mass, sugar water dissipation, open field experiment transit times increased (P0.05, P0.01) and the retention time decreased (P0.05, P0.05). The entry times and residence time of the open arm labyrinth experiment increased significantly (P0.05, P0.05), while the retention time of the closed arm decreased (P0.05). RIA results showed that the PF30mg kg-1 group and AF30mg kg-1 group could reduce the group. RT, ACTH content (P0.05, P0.05); HPLC-ECD results showed that AF30mg kg-1 group, PF30mg. Kg-1 group can increase NE, DA, 5-HA content, paeoniflorin, the effect of paeoniflorin on the cerebral cortex and hippocampus of rats with blood deficiency liver depression The results of RIA showed that the content of PKA in the group of PF30mg / kg-1 and AF30mg. Kg-1 could increase the content of PKA, and the AF30mg / kg-1 group could also increase the cAMP content (P0.05, P0.05).Western. As the main effective component of paeoniflorin, glucoside and paeoniflorin can regulate the disturbance of the immune system and maintain the homeostasis of the hematopoietic environment in the bone marrow by regulating the hematopoietic cell factors. Conclusion the results of 2: experiment two show that paeoniflorin and paeoniflorin can improve blood deficiency liver depression to a certain extent as the main effective component of Paeonia lactiflora. Behavioral changes, its improvement mechanism may regulate neurotransmitters, reduce the secretion of stress hormones, inhibit the overactivation of HPA axis and regulate the function of the nervous endocrine system. Conclusion 3: experiment three showed that paeoniflorin and paeoniflorin could regulate the related signal eggs in the intracellular cAMP/PKA signaling pathway of blood deficiency liver depression rats. White Paeonia lactiflora is the material basis for the efficacy of Paeonia lactiflora to nourish blood and soften the liver. To sum up, this study reveals the main effective component of paeoniflorin, paeoniflorin, paeoniflorin, and paeoniflorin by regulating the neural endocrine immunization (NEI) network to nourish blood and soften the liver with radiation irradiation combined with chronic binding of blood deficiency syndrome. On the basis of the study of paeoniflorin and paeoniflorin on blood deficiency, the study on the correlation of "syndrome effect" and the essence of syndrome of stagnation of liver stagnation syndrome of Chinese traditional Chinese medicine and the syndrome essence of stagnation of liver Qi syndrome of blood deficiency can be suggested, and the related cytokines, hormones and transmitters in the immune system can be studied by NEI network. On this basis, the study was conducted by observing paeoniflorin and paeoniflorin in blood deficiency. The effect of the intracellular cAMP/PKA signaling pathway in the animal model of liver qi stagnation, further reveals that the mechanism of the effect of Paeonia lactiflora nourishing the blood and the liver is related to the regulation of the related receptors and targets in the signal pathway. This study not only revealed the scientific connotation of the efficacy of "nourishing the blood and liver" of Paeonia lactiflora, and discussed the main effective components of Paeonia Paeoniae paeoniflorin and Paeonia lactiflora. The pharmacological action and mechanism of ester glycosides provide scientific basis for the clinical application of Paeonia lactiflora, and the research on the correlation of "syndrome effect" through the syndrome of blood deficiency and stagnation of liver and the efficacy of Paeonia lactiflora, not only enriches the basic research of liver syndrome in Chinese medicine, but also provides reference for the research of TCM related theories.
【学位授予单位】：北京中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285.5

【参考文献】