补肾调肝法治疗多囊卵巢综合征作用及对雌雄激素合成的分子调控

【作者】 刘新敏；

【导师】 李光荣；

【作者基本信息】 中国中医科学院 ， 中医妇科学， 2007， 博士

【摘要】 多囊卵巢综合征（polycystic ovary syndrome，PCOS）是育龄期女性常见的生殖内分泌紊乱性疾病，其病理改变不仅涉及生殖功能障碍，还有高血脂、高血压、糖尿病和心血管疾病等多种并发症，给患者的身体健康带来极大的危害。因此寻找一种有效的治疗方法阻断PCOS的病程、防止并发症的发生将具有重要的科学意义和社会意义，但目前的治疗方法（包括西医疗法、中医疗法、中西医结合疗法）仅局限在排卵率、受孕率等近期疗效方面，在远期疗效和预防并发症方面仍然没有取得突破。祖国医学没有PCOS这一病名，根据其临床表现，见于“月经后期”、“闭经”、“不孕症”等篇章，多数学者采用“补肾活血”、“补肾化痰”等方法治疗。导师李光荣教授是我国著名中医妇科专家，从事中医妇科临床40余年，在中医药治疗PCOS方面积累了丰富的经验，形成了独树一帜的学术思想。导师强调情志因素的重要性，首创补肾调肝法治疗PCOS并验案屡现，且有一定的远期疗效，展现了中医药能在PCOS远期疗效方面取得突破的迹象。为了验证补肾调肝法治疗PCOS的作用，本论文从理论、临床、实验三个方面进行研究，阐述PCOS的病因病机、补肾调肝法在PCOS治疗中的应用及根据补肾调肝法拟定经验方—多囊饮治疗PCOS的疗效和作用机制，以期为PCOS提供新的更有效的治疗方法。1理论研究1．1祖国医学对PCOS病因病机的认识根据PCOS的临床表现，在中医古籍中，类似的记载散见于闭经、月经后期、不孕症等篇章中。本论文总结了古代医家对上述三种疾病病因病机的认识。总体说来，病因包括：①情志失调；②外感寒邪；③痰湿；④瘀血。其中痰湿、瘀血既是病理产物，又是致病因素。病机包括虚、实两类，虚证有：①脾胃虚弱，气血虚少；②肾阴亏虚；③阴虚血燥；④肾阳虚衰，虚寒内生。实证有：①气滞血瘀；②寒凝血瘀；③痰湿壅塞。这一结论为研究PCOS的病因病机建立了理论框架，起到指导作用。1．2从情志致病探讨PCOS的病因病机根据其临床表现，PCOS属于月经病的范畴，而月经的产生与肾、肝的关系最密切。流行病学研究显示，情志不调在PCOS的发病和病情发展中均起了重要作用，因此，情志不调是PCOS的中医病因之一。祖国医学理论认为，肾主藏精，精能生髓，脑为髓之海，“肾不生，则髓不能满”，“脑为元神之府”，说明脑的精神活动与肾精脑髓有关；“邪之所凑，其气必虚”，可见髓海不充是情志致病所需具备的主要条件，肾虚是PCOS情志致病的重要基础；情志不调能通过引起髓海失司加重肾虚。肝主情志，主疏泄，情志不调能导致肝失疏泄、肝气郁结，气郁血行不畅或肝郁克脾，脾失运化，进而产生痰湿、瘀血等病理产物，故肝郁是PCOS情志致病的结果。综上所述，情志不调是PCOS的中医病因之一，肾虚肝郁为PCOS的核心病机。这一结论与相关疾病的病因病机的理论框架相符。基于肝郁多变证的理论，应用补肾调肝法治疗PCOS方能切中病机。该结论为补肾调肝法的临床应用奠定了理论基础。1．3 PCOS情志致病的现代医学依据PCOS患者的情志不调主要表现为抑郁、紧张、烦躁、焦虑等情绪变化。现代医学认为，下丘脑能通过自主神经系统实现对情绪的调节。下丘脑还是生殖内分泌器官，能通过下丘脑-垂体-卵巢轴完成对生殖功能的调节，可见，下丘脑是联系女性情绪与生殖内分泌的重要解剖结构，女性情绪的变化能通过引起下丘脑内分泌功能的改变而影响下丘脑—垂体—卵巢轴的功能。下丘脑—垂体—卵巢轴功能失调是PCOS的病理改变之一。下丘脑的结构和功能为PCOS情志致病提供了解剖学依据。情绪变化还能引起部分神经递质如多巴胺、β—内非肽、肾素—血管紧张素—醛固酮的改变。紧张能诱导多巴胺的代谢失活，以致不能有效抑制促性腺激素释放激素的脉冲频率，引起黄体生成激素分泌增多，诱发高雄激素血症。焦虑能引起血中β—内非肽的含量升高，引发高胰岛素血症。肾素—血管紧张素—醛固酮的异常分泌是导致卵巢雄激素过多及卵泡发育障碍的原因之一。神经递质的变化为PCOS情志致病提供了内分泌学依据。下丘脑的结构和功能及神经递质的变化所引起的内分泌改变从现代医学角度为PCOS情志致病中医病因提供了有力的佐证。2临床研究2．1李光荣教授治疗PCOS的治法研究本研究以名老中医结构化临床诊疗信息采集系统为技术平台，对导师治疗PCOS的原始病例、原始处方从多角度进行分析对比、综合归纳，探讨补肾调肝法在导师治疗PCOS中的应用，结果显示：①导师治疗PCOS应用频次最多的治法是补肾法、调肝法。②用药类别分析显示，导师治疗PCOS最常用的药物类别是补肾药、调肝药、活血药。③常用药物频次分析显示，导师治疗PCOS最常用的药物有19味，也可分为四类，即补肾药、调肝药、活血药、健脾药，其中应用频次最多的药物是当归、白芍、仙灵脾。治法和用药分析均显示，补肾法、调肝法是导师治疗PCOS使用频次最多的方法。导师认为，PCOS发病机理复杂，治疗应当两法或数法并用方切中病机，因此将补肾法、调肝法合而为一，即用补肾调肝法治疗PCOS。本研究说明，补肾调肝法是导师治疗PCOS最常用的治疗方法。2．2多囊饮治疗PCOS50例疗效总结导师经过四十多年的临床实践，通过对临床经验的不断总结和对药物的反复筛选，凝炼出补肾调肝的经验方—多囊饮治疗PCOS，为了更系统、更全面评估多囊饮治疗PCOS的疗效，本研究对50例导师以多囊饮为基本方加减治疗的PCOS患者的临床疗效进行了总结，结果显示：①50例患者，共治疗414个月，共行经263次，周期行经率63.5％。39例患者基础体温出现双相，26例患者出现排卵，患者恢复排卵的比率为52％。26例不孕症患者中，在治疗期间或停药半年内妊娠者19例，其中3例自然流产，妊娠率73.1％，妊娠成功率61.5％。②经多囊饮治疗后，患者的月经周期、经期、经量较治疗前明显好转。③对13例经多囊饮治疗后恢复排卵并停止治疗的PCOS患者进行随访，在停服中药1年时（或产后月经复潮半年时）其月经状况仍明显好于治疗前。④多囊饮能降低PCOS患者血清促黄体素（LH）、睾酮（T）及LH／促卵泡素（FSH）水平。⑤多囊饮对PCOS患者子宫、卵巢的体积无明显影响。该结果说明根据补肾调肝法拟定的经验方—多囊饮治疗PCOS具有较好的近期疗效和一定的远期疗效。3实验研究本论文实验研究以Poretsky的方法制造的多囊卵巢（PCO）大鼠模型为载体，采用放免法、ELISA、组织病理学、免疫组化、原位杂交、Western-blotting等技术，从整体—细胞—分子水平探讨多囊饮治疗PCOS的作用机制。实验结果显示：①多囊饮能升高PCO模型大鼠血清雌二醇（E₂）水平、降低T水平，还能调节垂体FSH、LH的分泌，对其生殖功能的恢复有促进作用。②Poretsky PCO大鼠模型血清胰岛素样生长因子—1（IGF-1）水平明显降低，与PCOS患者的病理改变不同。多囊饮能升高PCO模型大鼠血清IGF-1、转化生长因子-β₁（TGF-β₁）水平，降低肿瘤坏死因子-α（TNF-α）水平，但PCO模型大鼠血清IGF-1、TGF-β₁、TNF-α水平的改变对血清雌、雄激素水平未产生影响。③多囊饮能改善PCO模型大鼠卵巢的组织结构，表现在促进PCO模型大鼠卵泡的发育，促进卵泡颗粒细胞增生、分化，使增厚的卵泡膜细胞层、卵巢包膜变薄。④多囊饮能上调PCO模型大鼠卵巢与E₂合成有关的颗粒细胞FSH受体的蛋白表达、下调与T合成有关的卵泡膜—间质细胞LH受体的蛋白表达。⑤多囊饮能上调PCO模型大鼠卵巢与E₂合成有关的芳香化酶mRNA和蛋白表达，下调与T合成有关的细胞色素P450c17酶mRNA和蛋白表达。4结论①理论研究显示，情志不调是PCOS的中医病因之一，肾虚肝郁为PCOS的核心病机，补肾调肝法为PCOS的基本治法。②临床研究显示，补肾调肝法是导师治疗PCOS最常用的治疗方法，根据补肾调肝法拟定的处方—多囊饮治疗PCOS具有较好的近期疗效和一定的远期疗效。③实验研究显示，多囊饮能通过上调卵巢颗粒细胞FSH受体蛋白表达、芳香化酶mRNA和蛋白表达促进E₂合成，通过下调卵泡膜细胞LH受体蛋白表达、细胞色素P450c17酶mRNA和蛋白表达抑制T合成。多囊饮通过对卵巢雌、雄激素合成的影响而调节雌、雄激素比例，改善多囊卵巢的组织结构，发挥治疗PCOS的作用。④通过该研究，证实了根据补肾调肝法拟定的经验方—多囊饮治疗PCOS的客观性，从一个侧面说明合成、分泌雌、雄激素的生理功能是“肾主生殖”、“肝肾同源”理论的物质基础之一。5本研究特色和创新之处①本论文根据导师的学术思想，首次提出情志不调是PCOS的中医病因，肾虚肝郁是PCOS的核心病机，补肾调肝法是PCOS的基本治法，并从理论、临床、动物实验等方面证实了其客观性，丰富了PCOS治法的内容，国内外尚未见同类报道。②本论文把中医理论思维与前沿技术有机结合，首次从雌、雄激素合成的角度研究中药治疗PCOS的作用机制，提出合成、分泌雌、雄激素的生理功能是“肾主生殖”、“肝肾同源”理论的物质基础之一，使中医药治疗PCOS从理论到实践达到新的水平。国内尚未见同类报道。更多还原

【Abstract】 Polycystic ovary syndrome is a common disorder syndrome of reproductive endocrine inwomen of childbearing age. Its pathological changes involve not only in reproductivedysfunction, but also in hyperlipemia, hypertension, diabetes and cardiovascular disease andother complications. It brings great harm to the health of the patients. So finding an effectivetreatment of PCOS blocking the course, preventing the occurrence of complications will beimportant scientific significance and social significance. However, the present treatmentmethods （including modern medicine therapy/traditional Chinese medicine therapy/traditionaland western medicine therapy）limit to the short-term efficacy such as ovulation rate andpregnancy rate, the long-term efficacy and prevention of complications are still nobreakthrough.There isn’t the name of PCOS in TCM. According to the clinical manifestation, it is foundin the chapter of "late menstruation", "amenorrhea" and "infertility". Many scholars treatPCOS by BuShenHuoXue therapy or BuShenHuaTan therapy. My supervisor, professor LiGuang-rong is one of the most famous TCM gynecologists of our country. She has been aTCM gynecologist for more than 40 years. She has accumulated a wealth of experience in thetreatment of PCOS by TCM, and has established an unique academic ideas. She stresses theimportance of emotion, and creats BuShenTiaoGan therapy to treat PCOS. In recent years, therepeated testing of professor Li treating PCOS by BuShen TiaoGan therapy is the case. Thetherapy also has the long-term effects. It demonstrates a sign of a breakthrough of TCM inPCOS long-term efficacy. To Certificate the treatment role of BuShen TiaoGan therapy onPCOS and its mechanism, we study from three aspects in this paper: theory, clinical andexperiment. We describe the cause and pathogenesis of PCOS, the utility, the efficacy and themechanism of BuShen TiaoGan therapy. We provide new and more effective therapy.1 Theoretical studies1．1 The recognition of TCM on the cause and pathogenesis of PCOSAccording to the clinical manifestations of PCOS, the similar records scatter among thechapters of late menstruation, amenorrher and infertility in the ancient TCM literature. Wesummarized the recognizations of the ancient doctors to the cause and the pathogenesis about the 3 kinds of diseases in this research. In general, the causes include:emotional disorder,exogenous cold, phlegm and stagnant blood. Phlegm and stagnant blood are eitherpathological products or pathogenic factors. The pathogenesis includes two types ofdeficiency and sthenia syndrome. The deficiency syndromes include:①insufficiency of spleenand stomach, deficiency of Qi and blood;②asthenia of kidney Yin;③Yin deficiency blooddryness;④declination of kidney Yang, cold of insufficiency type internal produce. The stheniasyndromes include:①stagnancy of Qi and blood stasis;②cold stagnation and bloodstasis;③stagnation of phlegm-dampness. The conclusion establishes a theoretical frameworkfor us to study the cause and the pathogenesis of PCOS.1．2 To approach the cause and the pathogenesis of pcos from emotion disorderAccording to its clinical manifestations, PCOS belongs to the area of menstruationdiseases. Menstruation is closely related to liver and kidney. Epidemiological studies haveshown that emotions play an important role in the invasion and the progression of PCOS.Therefore, emotion disorder is one of the causes of PCOS. According to the TCM theory,kidney storing essence; kidney producing marrow; brain as the sea of marrow; kidney doesn’tproduce, and marrow can’t be full of; brain is the house of mental activity, it is demonstratedthat the cerebration of brain is related to kidney essence and brain marrow. Somebody who isexposed to pathogens and deficiency of Qi which must be present. Therefore, we think thatmarrow sea vacuity is needed for emotion to cause diseases. Kidney deficiency is the basis foremotion to cause PCOS.Emotion disorder can bring more serious kidney deficiency bycausing the marrow sea disorder. Liver controls emotion. Liver smoothes and regulates theflow of Qi and blood. Emotion disorder can cause stagnation of liver Qi by causing itdisorder for liver to smooth and regulates the flow of Qi and blood, so as to havingpathological products such as phlegm and blood stasis. Stagnation of liver Qi is the result ofemotion disorder. Above all, emotion disorder is the cause of PCOS. Asthenia of kidney andstagnation of liver Qi are the core pathogenesis. This conclusion is the same as the theoryframework that we have built. According to the theory that stagnation of liver Qi can causemore syndrome, it will be exactly suitable to treat PCOS by strengthening kidney andregulating liver. 1．3 The modern medicine basis of emotion disorder causing PCOSAccording to the modern medicine theory, hypothalamus regulates emotions through theautonomic nervous system. It is also a reproductive endocrine organ, regulating thereproductive function by hypothalamic-pituitary-ovarian axis. Therefore, hypothalamus is animportant anatomical structure which contract the emotion and reproductive endocrine offemale. The changes of female’s emotion can affect the function ofhypothalamic-pituitary-ovarian axis by changing the functions of endocrine of hypothalamus.The dysfunction of hypothalamic-pituitary-ovarian axis in PCOS is one of pathologicalchanges. Hypothalamic structure and function provide anatomical basis for PCOS emotiondisorder cause. Emotional changes can arouse some neurotransmitters such asdopamine,β-peptide, rennin-angiotensin-aldosterone change. Tension can induce themetabolism of dopamine inactivation, which can inhibit GnRH pulse frequency, leadingluteinizing hormone secretion increased, inducing hyperandrogenism. Anxiety can cause thebloodβ-peptide levels increased, triggering hyperinsulinemia. Renin-angiotensin-aldosteroneabnormal secretion is one of the causes of ovarian androgen excess and folliculardevelopment disturbance. Neurotransmitter changes provide an endocrine basis for PCOSemotion disorder etiology. This conclusion provided strong evidence for PCOS emotiondisorder TCM etiology from the perspective of modern medicine.2 Clinical studies2．1 The PCOS therapy study on professor Li Guang-rongOn the basis of the theory study, we proposed that BuShen TiaoGan therapy is the basictreatment of PCOS. In this study, we will regard the old Chinese structured clinicalinformation collection system as a technology platform. The original cases and prescriptionsof my supervisor are analyzed from multiple perspectives. The results showed that:①BuShenTiaoGan therapy is the most common treatment of PCOS of my supervisor.②According tothe analysis of medicine, the most commonly used drugs of my supervisor to treat PCOS arestrengthening kidney drugs, regulating liver drugs and promoting blood drugs.③Drugscommonly used frequency analysis showed that 19 pieces of drugs most commonly used bymy supervisor can also be divided into four categories, that is strengthening kidney drugs,regulating liver drugs, promoting blood drugs and invigorating spleen drugs. The most commonly used drugs are Radix angelicae sinensis, Radix paeomiae alba and Herba epimedii.The treatment and medication analysis show that BuShen and TiaoGan therapy are the mostcommonly used methods for my supervisor to treat PCOS. Because of the complexpathogenesis, my supervisor combined the two methods to treat PCOS. The study shows thatBuShen TiaoGan therapy of PCOS belongs to the famous old Chinese experience in a clinicalbackground.2．2 DUONANG decoction treat PCOS 50 cases clinical summaryWith more than 40 years of clinical practice, continuing review of clinical experienceand repeating drug screen, my supervisor summarized BuShen TiaoGan experienceprescription-DUONANG decoction to treat PCOS. To a more systematic and comprehensiveassessment of BuShen TiaoGan therapy efficacy, we will summarize the clinical efficacy of50 cases which my supervisor treated by DUONANG decoction in this study. The resultsshowed:①50 patients, treated 414 months of a total, menstruation appears 263 times, via ratecycle is 63．5%.39 patients appear biphasic basal body temperature, 26 patients appearovulation, the rate of restore ovulation is 52%.There are 26 infertility patients, 19 patientspregnant during treatment or withdrawal within 6 months. Among the 19 patients,3 patientsabortion, pregnancy success rate is 61．5%.②The patients menstruation is better aftertreatment than before.③13 patients ,who have restored ovulation after treatment and stoppedtreatment, maintained better about menstruation than treatment before.④DUONANGdecoction can lower serum luteinizing hormone（LH）,testosterone（T） and LH/follicularstimulating hormone（FSH）.⑤DUONANG decoction has no effects on the uterus and ovaryvolumn. The results show that DUONANG decoction has good short-term efficacy and somelong-term efficacy.3 Experimental studyIn experimental study of this paper, we create polycystic ovary （PCO） rat models byPoretsky’ way, by RIA, ELISA, histopathology, immunohistochemistry, situ hybridization,Western-blotting, to explore the mechanism of DUONANG decoction from thewhole-cell-molecular levels. The results showed:①DUONANG decoction can increase PCO rat models’ serum E₂, lower T, regulate thesecretion of FSH,LH,and restore reproductive function. ②The serum IGF-1 level of PCO rat models are significantly decreased, differing fromthe PCOS patients pathological changes. DUONANG decoction can increase PCO rat models’serum IGF-1, TGF-β₁level,lower TNF-αlevel. But the changes of PCO rat models’ IGF-1,TGF-βhave no impact on the serum T and E₂．③DUONANG decoction improve the PCO rat models’ ovarian organizational structure,by the way of promoting PCO rat models’ follicles development and granulose cellproliferation, differentiation, thinning the theca cell layer and ovarian capsule.④DUONANG decoction can increase PCO rat model FSH receptor protein expressionof granulose cells that is related to E₂ synthesis, reduce LH receptor protein expression oftheca cells which is related to T synthesis.⑤DUONANG decoction can increase the PCO rat model ovaries’ aromatase mRNA andprotein expression which is related to E₂ synthesis, reduce Cytochrome P450c17 mRNA andprotein expression which is related to T synthesis.4 Conclusion①Theoretical studies indicate that emotion disorder is one of the TCM causes of PCOS,Asthenia of kidney and stagnation of liver Qi are the core pathogenesis. strengthening kidneyand regulating liver is the basic treatment of PCOS.②Clinical studies show that BuShen TiaoGan therapy is the most commonly method thatmy supervisor treats PCOS and has its clinical background. DUONANG decoction has goodshort-term and certain long-term efficacy on PCOS.③Experimental studies have shown that DUONANG decoction can promote E₂synthesis by increasing FSH receptor protein expression, aromatase mRNA and proteinexpression of granulose cells, inhibiting T synthesis by decreasing LH receptor proteinexpression, Cytochrome P450c17 mRNA and protein expression. DUONANG decoctionregulates the ratio of E₂ and T by affecting the synthesis of them so as to improve theorganization structure and play a role in the treatment of PCOS.④The objectivity has been confirmed for BuShen TiaoGan therapy to treat PCOS in thisstudy. It has been shown that the physiological function that synthesizes and secrets E₂ and Tis one of the foundation of kidney dominating reproduction, liver and kidney having acommon source theory. 5 Innovations①We first proposed that emotion disorder is the TCM cause of PCOS, asthenia ofkidney and stagnation of liver Qi are the core pathogenesis, strengthening kidney andregulating liver is the basic treatment of PCOS. We prove the objectivity in theory, clinicaland animal experiments. The similar report was not found.②Combining TCM theoretical with advanced technique, we study the mechanism howTCM treating PCOS from the aspects of E₂ and T synthesis. the physiological function thatsynthesizes and secrets E₂ and T is one of the foundation of kidney dominatingreproduction ,liver and kidney having a common source theory. This study elevated theinvestigation in TCM treating PCOS to advanced level from theory to practice.更多还原