本事琥珀散及其拆方对子宫内膜异位症大鼠模型免疫相关细胞因子的影响
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摘要
子宫内膜异位症(Endometriosis, EMs)是指具有生长能力的子宫内膜组织(腺体和间质)出现在子宫腔被覆黏膜以外的身体其他部位的一种疾病,临床上以腹痛、痛经、性交不适和不孕等为典型症状。EMs虽然是一种良性疾病,但却具有类似恶性肿瘤远处转移和种植生长的能力。大约有3%～10%的生育年龄妇女患有EMs,在不孕症患者中,25%-35%有EMs存在,20%-90%的慢性盆腔痛患者和40%-60%的痛经患者与EMs相关。EMs是妇科常见病、多发病,严重影响妇女的生活质量,其发病率近年来呈逐渐增高的趋势,经过治疗仍有较高的复发率。中药治疗EMs在改善症状、提高受孕率、降低复发率以及调节全身免疫功能方面都有独到之处。
在EMs发病机制中,免疫学说是目前的研究热点。自身免疫平衡的打破使异位内膜得以逃脱免疫监视,顺利在病灶部位种植生长。免疫相关细胞因子在这一过程中起到了重要作用。目前对免疫相关细胞因子的研究表明,它们一方面间接反映了机体的免疫状态,据其可以推测机体免疫环境对EMs发生发展的影响；另一方面,它们还参与到EMs发生发展的各个环节。
中医学认为,EMs的基本病机是血瘀。除此以外,尚有气滞、寒凝、肾虚、痰凝等病机参与其中。在初期EMs患者中,血瘀、气滞、寒凝病机所占比例较大。目前关于中医证候与现代医学相关性的研究认为,血瘀、气滞、寒凝均可造成机体免疫机制的失调,气滞证又多与中枢单胺类神经递质的调控有关。
本事琥珀散出自宋·许叔微的《普及本事方》,由三棱、莪术、赤芍、刘寄奴、丹皮、肉桂、熟地、当归、乌药、元胡组成,具有行气活血,温经止痛的功效。最初主要用来治疗女子痛经,现代临床亦常治疗以痛经、不孕为主要症状的妇科疾病,获得良好效果。目前,对于运用本事琥珀散治疗EMs的研究报道数量不少,但研究重点多集中在临床疗效的观察和运用规律的总结,缺乏深入的基础性研究。
基于上述基础,我们分析认为：本事琥珀散作为治疗EMs的基本方,其对在EMs发生发展过程中具有重要作用的免疫相关细胞因子可能存在调控作用。行气、活血、温经三种不同功效可能在调控作用中分别具有不同的意义。同时,作为气滞证的特征性标志,中枢神经单胺类递质可能也会受到本事琥珀散的调控。于是,围绕上述问题,我们设计了本课题。
研究内容分为3部分：一、本事琥珀散及其拆方对EMs大鼠Th1/Th2细胞漂移的影响。通过研究本事琥珀散对Th1、Th2细胞分泌的特征性细胞因子IFN-γ, IL-2和IL-4、IL-10外周血含量的影响,探讨本事琥珀散及其拆方对Th1/Th2田胞漂移影响的作用机制。二、本事琥珀散及其拆方对免疫相关细胞因子的影响。围绕异位内膜“种植-侵袭-血管生成”的“3A模式”,针对与免疫及异位内膜侵袭基底膜、新生血管生成相关的细胞因子,探讨本事琥珀散及其拆方防治EMs的作用机制。三、本事琥珀散及其拆方对中枢单胺类神经递质的调控作用。以下丘脑单胺类神经递质及其代谢产物为研究对象,探讨本事琥珀散及其拆方对气滞血瘀证治疗作用的发生机制。实验结果和结论如下：
1本事琥珀散及其拆方具有抑制EMs大鼠外周血Th1/Th2细胞漂移的作用
Th1/Th2漂移现象能够反映机体的免疫状态,作为Thl和Th2细胞分泌的特征性因子,IFN-γ和IL-4的比值是衡量Th1/Th2平衡的重要指标。本事琥珀散正是通过调节IFN-γ/IL-4的比值,抑制了Th1/Th2的漂移,调整了机体免疫状态,从而对EMs起到了预防和治疗作用。其中,行气活血、温经补血而以活血为主的药物组合在调节IFN-γ/IL-4比值方面作用最为突出,在本事琥珀散全方中起着重要作用；行气活血以行气为主的药物组合能够降低IFN-γ的含量；温经补血的药物组合在升高IL-2方面具有明显作用；行气活血以活血为主的药物对IL-10含量有明显的升高作用,但若在其基础上加大行气的力量,则反而会明显降低IL-10的含量。
2本事琥珀散及其拆方对EMs免疫相关细胞因子在外周血和异位内膜变化的影响
本事琥珀散有降低EMs大鼠外周血中升高的TNF-α、IL-6和MIF的趋势,能明显降低异位内膜MMP-2、TIMP-2和VEGF水平,说明改善免疫和抑制异位内膜的种植侵袭、血管新生可能是本事琥珀散防治EMs的作用机制之一。TNF-α和IL-6经本事琥珀散拆方及协同作用各组治疗后含量变化的一致性,说明TNF-α同IL-6具有相关性,本事琥珀散及其拆方可能是通过调节TNF-α的含量进而影响了IL-6含量的变化。拆方研究发现,行气、活血、温经补血三种功效均单独对EMs异位内膜的侵袭和血管新生有明显作用；行气活血功效会促进异位内膜的种植侵袭,需同温经补血药物联合使用才能发挥对EMs的防治作用。
3本事琥珀散及其拆方对下丘脑单胺类神经递质影响的作用机制
下丘脑单胺类神经递质的变化是肝郁气滞证的微观表现之一,具体表现为NE含量的降低和5-HT、DA活性的增强。本事琥珀散能够升高下丘脑NE、5-HT和DA的含量,但对5-HT和DA的活性有抑制作用,这可能是其治疗气滞血瘀型EMs的作用机制之一。同时,拆方研究还发现行气为主的药物组合或和活血为主的药物组合对下丘脑NE、5-HT含量均有明显的升高作用,活血为主的药物组合能够降低5-HIAA和HVA的水平,说明行气活血功效能够升高下丘脑单胺类神经递质的含量并抑制其活性,本事琥珀散正是凭借其行气活血的功效实现了治疗肝郁气滞证的作用。
除此以外,本课题发现EMs大鼠外周血Th1/Th2漂移现象主要表现为Th2细胞向Thl细胞的漂移。Thl细胞向Th2细胞的漂移体现了机体细胞免疫的抑制状态,Th2细胞向Th1细胞的漂移则代表了细胞免疫的激活状态。EMs患者常因机体免疫低下,导致异位内膜逃脱免疫监视,种植于子宫腔外。而人为将自体子宫内膜种植于腹腔的EMs大鼠模型没有细胞免疫低下的先决条件,却因异位内膜的种植激活了免疫系统,从而表现出Th1细胞的增多而Th2细胞的减少,或其比值的增高。
同时,在自体内膜移植EMs大鼠模型身上还出现了肝郁气滞的倾向,即5-HT和DA活性的升高。血瘀是EMs的基本病机,EMs大鼠模型存在血瘀状态也在大量实验中得到证实。中医理论认为“血为气之母”,血是气的载体,气依附于血存在于体内。当血行瘀滞时,气亦会因之而运行迟缓,形成气滞之候。因此虽然未对大鼠模型进行证候干预,但由于疾病的发展,其证候已经由血瘀变为了气滞血瘀。这可以为进一步研究EMs证候的演变发展提供线索。
概括上述结论,本课题采用按功效拆分的拆方方法,从Th1/Th2漂移、免疫相关细胞因子和中枢单胺类神经等方面对本事琥珀散及其拆方防治EMs的作用机制进行了研究。结果表明了本事琥珀散及其拆方具有抑制EMs大鼠外周血Th1/Th2细胞漂移的作用；反映了本事琥珀散及其拆方对EMs免疫相关细胞因子在外周血和异位内膜变化的影响,揭示了本事琥珀散及其拆方对下丘脑单胺类神经递质影响的作用机制。
上述结论表明,本事琥珀散通过抑制Th1/Th2漂移,降低免疫相关细胞因子含量,阻止异位内膜的种植侵袭和新生血管生成等作用机制治疗EMs,同时能够调节下丘脑单胺类神经递质的分泌和活性,以改善机体的肝郁气滞状态。行气、活血、温经补血为主的三个有效组份及其协同作用分别在以上各个环节中对治疗EMs有作用。但这三个有效组份的共同作用才能取得在全部环节上的最佳效果,从而体现了本事琥珀散组方的合理性。
总之,本文对本事琥珀散的研究是采用按功效拆分的拆方方法,结合现代医学理论,阐述了本事琥珀散在免疫、“3A"模式和神经-内分泌-细胞因子网络角度治疗EMs的作用机制,在蛋白层面上揭示了本事琥珀散的部分作用靶点,初步探讨了行气、活血、温经补血功效针对各机制、靶点的作用效果,为全面认识本事琥珀散治疗EMs的作用机制,了解各功效的作用靶点,精简优化药方奠定了基础。
It is a kind of disease which we call it endometriosis shortened as EMs that the endometrial tissue consisting of glands and stroma with the ability of growth appears outside of the womb cavity mucous membrane. EMs presents with abdominal pain, dysmenorrhea, sexual intercourse discomfort and infecund etc as typical symptoms. Although EMs is a benign disease, it has the ability of distant metastasis and planting growth as malignant tumor. About 3%～10% women in reproductive age suffer from EMs. About 25%～35% patients with infertility,20%～90% patients with chronic pelvic pain and 40%～60% patients with dysmenorrheal suffer from EMs. EMs is a kind of common and frequently-occurring gynaecologic disease. It has a great effect upon women life quality. In recent years, the incidence of EMs is gradually increasing and the relapse rate is still very high after treatment. Traditional Chinese Medicine have good effects on the EMs, which can improve symptoms, raising pregnancy rate, lowering relapse rate and regulating immunity.
In the seizures of EMs, immunology theory is a popular research. Because organic immune balance is broken, ectopic endometrium can escape from the monitoring of immunization, then plant and grow into the lesion site. Immune related cytokines played an important role in this process. At present, the autoimmunity-associated cytokine studies show that immune related cytokines not only indirectly reflect the immune status of organism, hypothesizing the immune environment on the occurrence and development of EMs, but also are involved in the development of EMs links.
According to Traditional Chinese Medicine, the basic pathogenesis of EMs is blood stasis. In addition, many other pathogenesis, such as qi stagnation, cold coagulation, kidney deficiency, phlegm and so on, are related with EMs as well. Among early EMs patients, blood stasis, qi stagnation and cold coagulation are in large proportion. At present, the thought about Traditional Chinese Medicine and Modern Medicine believe that the disorders of the immune system can be caused by blood stasis, qi stagnation and cold coagulation. Besides that stagnation of qi is involved in the regulation of central monoamine neurotransmitters.
Ben Shi Hu Po San (BSHPS) comes from "Pu Ji Ben Shi Fang", which was written by Xu Shuwei in Song Dynasty. BSHPS is formed by Triangular, Turmeric, Red peony root, Liu Ji Merino, Paeonol, Cinnamon, Radix, Angelica, Radix Linderae and Corydalis tuber. BSHPS has the effect on operating qi, activating blood circulation, warming meridian and relieving pain. It is mainly used in the treatment of women with dysmenorrhea in ancient time. Now it's often used to treat gynecological disease, main symptoms of which are dysmenorrhea or infertility and so on. At present, there are a number of research reports about the use of BSHPS in the treatment of EMs, but the focus is more concentrated on the clinical efficacy observation and the use of rules, lack of basic research.
Based on the above basis, we think that, as a basic method of treating EMs, BSHPS may have regulation effect on the immune cytokines which play an important role in the development of EMs. The three different functions, operating qi, activating blood circulation and warming meridian may have different meanings in regulation. At the same time, as the characteristic sign of qi stagnation, central monoamine neurotransmitters may also be regulated by BSHPS. Therefore we design this research around the above problems.
The research content is divided into 3 parts:Part one is about the effects of BSHPS and its decompositions on the Thl/Th2 cells drift in EMs model rats. We discuss about the mechanisms of BSHPS and its decompositions for regulating Thl/Th2 drift in endometriosis, by comparing the levels of IFN-γ, IL2, IL-4 and IL-10, which are characteristic cytokines secreted by Thl and Th2 cells, in endometriosis model rats before and after using BSHPS and its decompositions. Part two is about the effects of BSHPS and its decompositions on immune cell factors. If ectopic endometriums want to survive at abdominal wall, they must experience "Attachment-Aggression-Angiogenesis" which is shorten for "3A model". Arrounding the "3A model", targeting to cytokines which are related to immunity, ectopic endometrium invasion of basement membrane and angiogenesis, we explore the mechanisms of BSHPS and its decompositions for preventing and treating EMs. Part three is about the regulation function of BSHPS and its decompositions on central monoamine neurotransmitter. Observating the effection of BSHPS and its decompositions on hypothalamus monoamine neurotransmitter and its metabolites, we explore the treatment mechanism of BSHPS and its decompositions for qi stagnancy and blood stasis. The experimental results and conclusions are as follows:
1. BSHPS and its decompositions have effects on inhibiting the Th1/Th2 cell drift in EMs rats peripheral blood.
The phenomenon of Th1/Th2 drift can reflect the immune status of body. As the characteristics cytokines that Thl cell and Th2 cell secreted, the ratio of IFN-y/IL-4 is an important index to measure the balance of Th1/Th2. BSHPS inhibits the Th1/Th2 drift, adjusts the immune function of body and plays a role in preventing and treating EMs, just by modulating IFN-γ/IL-4 ratio. Among above, the drug combination which has the function of activating blood circulation, has the best effect in regulating the ratio of IFN-γ/IL-4.The drug combination which has the function of operating qi can reduce the content of IFN-γ. And the drug combination which has the function of warming meridian and enriching the blood has an obvious effect on increasing the content of IL-2. The drug combination which has greater effect on activating blood circulation than operating qi, can significantly increase the content of IL-10. But if the function of operating qi increases, the effect of the drug will go to the other side.
2. BSHPS and its decompositions have effects on immune cytokines in the peripheral blood and endometrial changes of EMs rats.
BSHPS can reduce the elevated trend of TNF-α, IL-6 and MIF in the peripheral blood of EMs rat, and also can significantly reduce MMP-2, TIMP-2 and VEGF levels in the ectopic endometrium. It illustrates that improvement of immunity, inhibition of ectopic endometrial planting and invasion, and angiogenesis may be the mechanisms of BSHPS in treating EMs. Content of TNF-αand IL-6 in the peripheral blood have consistent changes by using BSHPS in spliting and synergy groups. That means TNF-αand IL-6 have correlation. BSHPS and its decompositions may influence the content of IL-6 by regulating the content of TNF-α. The study of decompositions finds that operating qi, activating blood circulation and warming meridian and enriching the blood can separately inhibit the ectopic endometrial invasion and angiogenesis in EMs. The combination of operating qi and activating blood will promote the planting and invasion of ectopic endometrium, and must be combined with warming meridian and enriching the blood medicine in order to play an obvious role of treating EMs.
3. BSHPS and its decompositions have effects on hypothalamic monoamines neurotransmitter of EMs rats.
The changes of monoamine neurotransmitters in hypothalamus is microcosmic performance of liver-qi stagnation syndrome, which manifests the decrease of NE content and enhanced activity of 5-HT, DA. BSHPS can increase the ability of hypothalamic NE,5-HT and DA content, but inhibite 5-HT and DA activity, which may be one of the mechanism on treating qi stagnation and blood stasis type EMs. At the same time, the research of decompositions also finds that components of operating qi and activating blood significantly increase the NE,5-HT content on the hypothalamus. The components of activating blood can reduce 5-HIAA and HVA levels. These indicates that the functions operating qi and activating blood can increase the content of monoamine neurotransmitters in hypothalamus and inhibit its activity. It is by the function of operating qi and activating blood that BSHPS is successfully applied to treat the syndrome of liver-qi stagnation.
Besides, the study finds that the Th1/Th2 mainly manifest Th2 cells drift toward THl cells on EMs rats. That Thl cells drift toward Th2 cells shows the immune inhibitatory status, while Th2 cells drift toward Th1 cells shows the immune activated status. On patients with EMs, the ectopic endometrium escape from the monitoring of immunization, then plant and grow outside cavity of uterus due to hypoimmunity. There is not the condition of hypoimmunity on the autologous endometrium transplantation model of rats, but the ectopic endometrium planting activates the immunity system, then it manifests that Th1 cells increase and Th2 cells reduce or the ratio of Thl/Th2 increases.
At the same time, there is also the tendency of stagnation in the autologous endometrium transplantation EMs model of rats, namely 5-HT and DA activity increases. Blood stasis is the basic pathogenesis of EMs, and it is proven in a large number of experiments on EMs rat model in the presence of blood stasis. In the theory of Traditional Chinese Medicine "qi is mother of blood ", which means blood is the carrier of qi and qi exists in body attaching to blood. If the blood circulation tends to blood stasis, qi will move slowly, which becomes the syndrome of qi stagnation. Therefore, although there is not syndrome intervention on rat model, but the syndrome has been changed from blood stasis to qi stagnation and blood stasis due to the disease progressing. This can be some clues for further study of EMs syndrome development.
Summing up the above conclusion, this paper uses the method of decomposition according to different effectiveness, and research the prevention and treatment on EMs mechanism by BSHPS and its decompositions in terms of Th1/Th2 drift, immune related cytokines and monoaminergic nerve and so on. The results show that BSHPS and its decompositions has effect of inhibition of Th1/Th2 cell drift of peripheral blood on EMs rats; BSHPS and its decompositions effect on EMs immune cytokines in the peripheral blood and endometrial changes, which reveals the mechanism of action of BSHPS and its decompositions on hypothalamic monoamines neurotransmitter.
Above all, BSHPS is applied to treat EMs by inhibiting Thl/Th2 drift, decreasing immune related cytokine content, preventing ectopic endometrium planting invasion and angiogenesis mechanism. At the same time, it is used to regulate hypothalamic monoamine neurotransmitter secretion and activity so as to improve the state of liver-qi state. The three mainly effective components, operating qi, promoting blood circulation, warming meridian and blood tonic consisting, and its synergistic effect play an important role in treating EMs in every aspect. But the three effective components should work together to achieve the best result in all the links.
In a word, the research about BSHPS uses the method of split demolition according to the different effectiveness, combined with modern medical theory. The research elaborates the effect and mechanism of BSHPS upon immune, "3A" mode and neuroendocrine cytokines network perspective in treating EMs. The research reveals part of target points of BSHPS in protein level. The research discusses the effect of operating qi, activating blood circulation, warming meridian and blood tonic according to the mechanism and target points of effections. This research lays the foundation for comprehensive understanding the mechanisms of BSHPS in treating EMs, knowing the target of all effections, streamlining and optimizating prescription.
在EMs发病机制中,免疫学说是目前的研究热点。自身免疫平衡的打破使异位内膜得以逃脱免疫监视,顺利在病灶部位种植生长。免疫相关细胞因子在这一过程中起到了重要作用。目前对免疫相关细胞因子的研究表明,它们一方面间接反映了机体的免疫状态,据其可以推测机体免疫环境对EMs发生发展的影响；另一方面,它们还参与到EMs发生发展的各个环节。
中医学认为,EMs的基本病机是血瘀。除此以外,尚有气滞、寒凝、肾虚、痰凝等病机参与其中。在初期EMs患者中,血瘀、气滞、寒凝病机所占比例较大。目前关于中医证候与现代医学相关性的研究认为,血瘀、气滞、寒凝均可造成机体免疫机制的失调,气滞证又多与中枢单胺类神经递质的调控有关。
本事琥珀散出自宋·许叔微的《普及本事方》,由三棱、莪术、赤芍、刘寄奴、丹皮、肉桂、熟地、当归、乌药、元胡组成,具有行气活血,温经止痛的功效。最初主要用来治疗女子痛经,现代临床亦常治疗以痛经、不孕为主要症状的妇科疾病,获得良好效果。目前,对于运用本事琥珀散治疗EMs的研究报道数量不少,但研究重点多集中在临床疗效的观察和运用规律的总结,缺乏深入的基础性研究。
基于上述基础,我们分析认为：本事琥珀散作为治疗EMs的基本方,其对在EMs发生发展过程中具有重要作用的免疫相关细胞因子可能存在调控作用。行气、活血、温经三种不同功效可能在调控作用中分别具有不同的意义。同时,作为气滞证的特征性标志,中枢神经单胺类递质可能也会受到本事琥珀散的调控。于是,围绕上述问题,我们设计了本课题。
研究内容分为3部分：一、本事琥珀散及其拆方对EMs大鼠Th1/Th2细胞漂移的影响。通过研究本事琥珀散对Th1、Th2细胞分泌的特征性细胞因子IFN-γ, IL-2和IL-4、IL-10外周血含量的影响,探讨本事琥珀散及其拆方对Th1/Th2田胞漂移影响的作用机制。二、本事琥珀散及其拆方对免疫相关细胞因子的影响。围绕异位内膜“种植-侵袭-血管生成”的“3A模式”,针对与免疫及异位内膜侵袭基底膜、新生血管生成相关的细胞因子,探讨本事琥珀散及其拆方防治EMs的作用机制。三、本事琥珀散及其拆方对中枢单胺类神经递质的调控作用。以下丘脑单胺类神经递质及其代谢产物为研究对象,探讨本事琥珀散及其拆方对气滞血瘀证治疗作用的发生机制。实验结果和结论如下：
1本事琥珀散及其拆方具有抑制EMs大鼠外周血Th1/Th2细胞漂移的作用
Th1/Th2漂移现象能够反映机体的免疫状态,作为Thl和Th2细胞分泌的特征性因子,IFN-γ和IL-4的比值是衡量Th1/Th2平衡的重要指标。本事琥珀散正是通过调节IFN-γ/IL-4的比值,抑制了Th1/Th2的漂移,调整了机体免疫状态,从而对EMs起到了预防和治疗作用。其中,行气活血、温经补血而以活血为主的药物组合在调节IFN-γ/IL-4比值方面作用最为突出,在本事琥珀散全方中起着重要作用；行气活血以行气为主的药物组合能够降低IFN-γ的含量；温经补血的药物组合在升高IL-2方面具有明显作用；行气活血以活血为主的药物对IL-10含量有明显的升高作用,但若在其基础上加大行气的力量,则反而会明显降低IL-10的含量。
2本事琥珀散及其拆方对EMs免疫相关细胞因子在外周血和异位内膜变化的影响
本事琥珀散有降低EMs大鼠外周血中升高的TNF-α、IL-6和MIF的趋势,能明显降低异位内膜MMP-2、TIMP-2和VEGF水平,说明改善免疫和抑制异位内膜的种植侵袭、血管新生可能是本事琥珀散防治EMs的作用机制之一。TNF-α和IL-6经本事琥珀散拆方及协同作用各组治疗后含量变化的一致性,说明TNF-α同IL-6具有相关性,本事琥珀散及其拆方可能是通过调节TNF-α的含量进而影响了IL-6含量的变化。拆方研究发现,行气、活血、温经补血三种功效均单独对EMs异位内膜的侵袭和血管新生有明显作用；行气活血功效会促进异位内膜的种植侵袭,需同温经补血药物联合使用才能发挥对EMs的防治作用。
3本事琥珀散及其拆方对下丘脑单胺类神经递质影响的作用机制
下丘脑单胺类神经递质的变化是肝郁气滞证的微观表现之一,具体表现为NE含量的降低和5-HT、DA活性的增强。本事琥珀散能够升高下丘脑NE、5-HT和DA的含量,但对5-HT和DA的活性有抑制作用,这可能是其治疗气滞血瘀型EMs的作用机制之一。同时,拆方研究还发现行气为主的药物组合或和活血为主的药物组合对下丘脑NE、5-HT含量均有明显的升高作用,活血为主的药物组合能够降低5-HIAA和HVA的水平,说明行气活血功效能够升高下丘脑单胺类神经递质的含量并抑制其活性,本事琥珀散正是凭借其行气活血的功效实现了治疗肝郁气滞证的作用。
除此以外,本课题发现EMs大鼠外周血Th1/Th2漂移现象主要表现为Th2细胞向Thl细胞的漂移。Thl细胞向Th2细胞的漂移体现了机体细胞免疫的抑制状态,Th2细胞向Th1细胞的漂移则代表了细胞免疫的激活状态。EMs患者常因机体免疫低下,导致异位内膜逃脱免疫监视,种植于子宫腔外。而人为将自体子宫内膜种植于腹腔的EMs大鼠模型没有细胞免疫低下的先决条件,却因异位内膜的种植激活了免疫系统,从而表现出Th1细胞的增多而Th2细胞的减少,或其比值的增高。
同时,在自体内膜移植EMs大鼠模型身上还出现了肝郁气滞的倾向,即5-HT和DA活性的升高。血瘀是EMs的基本病机,EMs大鼠模型存在血瘀状态也在大量实验中得到证实。中医理论认为“血为气之母”,血是气的载体,气依附于血存在于体内。当血行瘀滞时,气亦会因之而运行迟缓,形成气滞之候。因此虽然未对大鼠模型进行证候干预,但由于疾病的发展,其证候已经由血瘀变为了气滞血瘀。这可以为进一步研究EMs证候的演变发展提供线索。
概括上述结论,本课题采用按功效拆分的拆方方法,从Th1/Th2漂移、免疫相关细胞因子和中枢单胺类神经等方面对本事琥珀散及其拆方防治EMs的作用机制进行了研究。结果表明了本事琥珀散及其拆方具有抑制EMs大鼠外周血Th1/Th2细胞漂移的作用；反映了本事琥珀散及其拆方对EMs免疫相关细胞因子在外周血和异位内膜变化的影响,揭示了本事琥珀散及其拆方对下丘脑单胺类神经递质影响的作用机制。
上述结论表明,本事琥珀散通过抑制Th1/Th2漂移,降低免疫相关细胞因子含量,阻止异位内膜的种植侵袭和新生血管生成等作用机制治疗EMs,同时能够调节下丘脑单胺类神经递质的分泌和活性,以改善机体的肝郁气滞状态。行气、活血、温经补血为主的三个有效组份及其协同作用分别在以上各个环节中对治疗EMs有作用。但这三个有效组份的共同作用才能取得在全部环节上的最佳效果,从而体现了本事琥珀散组方的合理性。
总之,本文对本事琥珀散的研究是采用按功效拆分的拆方方法,结合现代医学理论,阐述了本事琥珀散在免疫、“3A"模式和神经-内分泌-细胞因子网络角度治疗EMs的作用机制,在蛋白层面上揭示了本事琥珀散的部分作用靶点,初步探讨了行气、活血、温经补血功效针对各机制、靶点的作用效果,为全面认识本事琥珀散治疗EMs的作用机制,了解各功效的作用靶点,精简优化药方奠定了基础。
It is a kind of disease which we call it endometriosis shortened as EMs that the endometrial tissue consisting of glands and stroma with the ability of growth appears outside of the womb cavity mucous membrane. EMs presents with abdominal pain, dysmenorrhea, sexual intercourse discomfort and infecund etc as typical symptoms. Although EMs is a benign disease, it has the ability of distant metastasis and planting growth as malignant tumor. About 3%～10% women in reproductive age suffer from EMs. About 25%～35% patients with infertility,20%～90% patients with chronic pelvic pain and 40%～60% patients with dysmenorrheal suffer from EMs. EMs is a kind of common and frequently-occurring gynaecologic disease. It has a great effect upon women life quality. In recent years, the incidence of EMs is gradually increasing and the relapse rate is still very high after treatment. Traditional Chinese Medicine have good effects on the EMs, which can improve symptoms, raising pregnancy rate, lowering relapse rate and regulating immunity.
In the seizures of EMs, immunology theory is a popular research. Because organic immune balance is broken, ectopic endometrium can escape from the monitoring of immunization, then plant and grow into the lesion site. Immune related cytokines played an important role in this process. At present, the autoimmunity-associated cytokine studies show that immune related cytokines not only indirectly reflect the immune status of organism, hypothesizing the immune environment on the occurrence and development of EMs, but also are involved in the development of EMs links.
According to Traditional Chinese Medicine, the basic pathogenesis of EMs is blood stasis. In addition, many other pathogenesis, such as qi stagnation, cold coagulation, kidney deficiency, phlegm and so on, are related with EMs as well. Among early EMs patients, blood stasis, qi stagnation and cold coagulation are in large proportion. At present, the thought about Traditional Chinese Medicine and Modern Medicine believe that the disorders of the immune system can be caused by blood stasis, qi stagnation and cold coagulation. Besides that stagnation of qi is involved in the regulation of central monoamine neurotransmitters.
Ben Shi Hu Po San (BSHPS) comes from "Pu Ji Ben Shi Fang", which was written by Xu Shuwei in Song Dynasty. BSHPS is formed by Triangular, Turmeric, Red peony root, Liu Ji Merino, Paeonol, Cinnamon, Radix, Angelica, Radix Linderae and Corydalis tuber. BSHPS has the effect on operating qi, activating blood circulation, warming meridian and relieving pain. It is mainly used in the treatment of women with dysmenorrhea in ancient time. Now it's often used to treat gynecological disease, main symptoms of which are dysmenorrhea or infertility and so on. At present, there are a number of research reports about the use of BSHPS in the treatment of EMs, but the focus is more concentrated on the clinical efficacy observation and the use of rules, lack of basic research.
Based on the above basis, we think that, as a basic method of treating EMs, BSHPS may have regulation effect on the immune cytokines which play an important role in the development of EMs. The three different functions, operating qi, activating blood circulation and warming meridian may have different meanings in regulation. At the same time, as the characteristic sign of qi stagnation, central monoamine neurotransmitters may also be regulated by BSHPS. Therefore we design this research around the above problems.
The research content is divided into 3 parts:Part one is about the effects of BSHPS and its decompositions on the Thl/Th2 cells drift in EMs model rats. We discuss about the mechanisms of BSHPS and its decompositions for regulating Thl/Th2 drift in endometriosis, by comparing the levels of IFN-γ, IL2, IL-4 and IL-10, which are characteristic cytokines secreted by Thl and Th2 cells, in endometriosis model rats before and after using BSHPS and its decompositions. Part two is about the effects of BSHPS and its decompositions on immune cell factors. If ectopic endometriums want to survive at abdominal wall, they must experience "Attachment-Aggression-Angiogenesis" which is shorten for "3A model". Arrounding the "3A model", targeting to cytokines which are related to immunity, ectopic endometrium invasion of basement membrane and angiogenesis, we explore the mechanisms of BSHPS and its decompositions for preventing and treating EMs. Part three is about the regulation function of BSHPS and its decompositions on central monoamine neurotransmitter. Observating the effection of BSHPS and its decompositions on hypothalamus monoamine neurotransmitter and its metabolites, we explore the treatment mechanism of BSHPS and its decompositions for qi stagnancy and blood stasis. The experimental results and conclusions are as follows:
1. BSHPS and its decompositions have effects on inhibiting the Th1/Th2 cell drift in EMs rats peripheral blood.
The phenomenon of Th1/Th2 drift can reflect the immune status of body. As the characteristics cytokines that Thl cell and Th2 cell secreted, the ratio of IFN-y/IL-4 is an important index to measure the balance of Th1/Th2. BSHPS inhibits the Th1/Th2 drift, adjusts the immune function of body and plays a role in preventing and treating EMs, just by modulating IFN-γ/IL-4 ratio. Among above, the drug combination which has the function of activating blood circulation, has the best effect in regulating the ratio of IFN-γ/IL-4.The drug combination which has the function of operating qi can reduce the content of IFN-γ. And the drug combination which has the function of warming meridian and enriching the blood has an obvious effect on increasing the content of IL-2. The drug combination which has greater effect on activating blood circulation than operating qi, can significantly increase the content of IL-10. But if the function of operating qi increases, the effect of the drug will go to the other side.
2. BSHPS and its decompositions have effects on immune cytokines in the peripheral blood and endometrial changes of EMs rats.
BSHPS can reduce the elevated trend of TNF-α, IL-6 and MIF in the peripheral blood of EMs rat, and also can significantly reduce MMP-2, TIMP-2 and VEGF levels in the ectopic endometrium. It illustrates that improvement of immunity, inhibition of ectopic endometrial planting and invasion, and angiogenesis may be the mechanisms of BSHPS in treating EMs. Content of TNF-αand IL-6 in the peripheral blood have consistent changes by using BSHPS in spliting and synergy groups. That means TNF-αand IL-6 have correlation. BSHPS and its decompositions may influence the content of IL-6 by regulating the content of TNF-α. The study of decompositions finds that operating qi, activating blood circulation and warming meridian and enriching the blood can separately inhibit the ectopic endometrial invasion and angiogenesis in EMs. The combination of operating qi and activating blood will promote the planting and invasion of ectopic endometrium, and must be combined with warming meridian and enriching the blood medicine in order to play an obvious role of treating EMs.
3. BSHPS and its decompositions have effects on hypothalamic monoamines neurotransmitter of EMs rats.
The changes of monoamine neurotransmitters in hypothalamus is microcosmic performance of liver-qi stagnation syndrome, which manifests the decrease of NE content and enhanced activity of 5-HT, DA. BSHPS can increase the ability of hypothalamic NE,5-HT and DA content, but inhibite 5-HT and DA activity, which may be one of the mechanism on treating qi stagnation and blood stasis type EMs. At the same time, the research of decompositions also finds that components of operating qi and activating blood significantly increase the NE,5-HT content on the hypothalamus. The components of activating blood can reduce 5-HIAA and HVA levels. These indicates that the functions operating qi and activating blood can increase the content of monoamine neurotransmitters in hypothalamus and inhibit its activity. It is by the function of operating qi and activating blood that BSHPS is successfully applied to treat the syndrome of liver-qi stagnation.
Besides, the study finds that the Th1/Th2 mainly manifest Th2 cells drift toward THl cells on EMs rats. That Thl cells drift toward Th2 cells shows the immune inhibitatory status, while Th2 cells drift toward Th1 cells shows the immune activated status. On patients with EMs, the ectopic endometrium escape from the monitoring of immunization, then plant and grow outside cavity of uterus due to hypoimmunity. There is not the condition of hypoimmunity on the autologous endometrium transplantation model of rats, but the ectopic endometrium planting activates the immunity system, then it manifests that Th1 cells increase and Th2 cells reduce or the ratio of Thl/Th2 increases.
At the same time, there is also the tendency of stagnation in the autologous endometrium transplantation EMs model of rats, namely 5-HT and DA activity increases. Blood stasis is the basic pathogenesis of EMs, and it is proven in a large number of experiments on EMs rat model in the presence of blood stasis. In the theory of Traditional Chinese Medicine "qi is mother of blood ", which means blood is the carrier of qi and qi exists in body attaching to blood. If the blood circulation tends to blood stasis, qi will move slowly, which becomes the syndrome of qi stagnation. Therefore, although there is not syndrome intervention on rat model, but the syndrome has been changed from blood stasis to qi stagnation and blood stasis due to the disease progressing. This can be some clues for further study of EMs syndrome development.
Summing up the above conclusion, this paper uses the method of decomposition according to different effectiveness, and research the prevention and treatment on EMs mechanism by BSHPS and its decompositions in terms of Th1/Th2 drift, immune related cytokines and monoaminergic nerve and so on. The results show that BSHPS and its decompositions has effect of inhibition of Th1/Th2 cell drift of peripheral blood on EMs rats; BSHPS and its decompositions effect on EMs immune cytokines in the peripheral blood and endometrial changes, which reveals the mechanism of action of BSHPS and its decompositions on hypothalamic monoamines neurotransmitter.
Above all, BSHPS is applied to treat EMs by inhibiting Thl/Th2 drift, decreasing immune related cytokine content, preventing ectopic endometrium planting invasion and angiogenesis mechanism. At the same time, it is used to regulate hypothalamic monoamine neurotransmitter secretion and activity so as to improve the state of liver-qi state. The three mainly effective components, operating qi, promoting blood circulation, warming meridian and blood tonic consisting, and its synergistic effect play an important role in treating EMs in every aspect. But the three effective components should work together to achieve the best result in all the links.
In a word, the research about BSHPS uses the method of split demolition according to the different effectiveness, combined with modern medical theory. The research elaborates the effect and mechanism of BSHPS upon immune, "3A" mode and neuroendocrine cytokines network perspective in treating EMs. The research reveals part of target points of BSHPS in protein level. The research discusses the effect of operating qi, activating blood circulation, warming meridian and blood tonic according to the mechanism and target points of effections. This research lays the foundation for comprehensive understanding the mechanisms of BSHPS in treating EMs, knowing the target of all effections, streamlining and optimizating prescription.
引文
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