【作者】 宋俐；

【导师】 黎烈荣；

【作者基本信息】 湖北中医药大学 ， 中医妇科学， 2011， 博士

【摘要】 目的：多囊卵巢综合征(poly cystic ovarian syndrome, PCOS)是育龄期妇女常见生殖内分泌紊乱性疾病,临床上以持续无排卵、高雄激素血症和胰岛素抵抗(insulin resistance, IR)为特征,是女性无排卵性不孕的主要原因之一。其发病率在生育期妇女中高达5-10%,占无排卵性不孕的70%左右。随着年龄的增长,PCOS也是妇女Ⅱ型糖尿病、心血管疾病等发生的主要诱因。有研究表明由于多毛、肥胖及不孕等临床表现导致高达33%的PCOS患者伴有焦虑和抑郁,对女性的身心健康和生活质量造成极大影响。从1935年Stein和Leventhal首次报道本病以来,各国学者对其研究不断深入,但由于其临床表现呈多态性,发病原因至今尚未阐明。目前国内外较为一致的看法认为下丘脑-垂体-卵巢轴(hypothalamus-pituitary-ovarian axis, HPOA)调节功能异常是其主要发病机制之一。临床研究表明,中医治疗生殖内分泌疾病有很好的优势和经验。中医治疗PCOS,可促进生殖功能恢复,调节内分泌及代谢紊乱,作用持久,毒副作用小。导师黎烈荣教授从医四十载,治疗中医妇科疑难病经验丰富。固本祛瘀方是黎教授治疗PCOS的经验方。临床观察表明,固本祛瘀方能降低PCOS患者雄激素水平,降低黄体生成素(luteinizing hormone, LH)水平及LH/卵泡刺激素(follicle stimulating hormone, FSH)比值,有效调节月经周期,促进排卵,提高妊娠率。本课题拟应用双氢睾酮(dihydrotestosterone, DHT)诱导复制的PCOS大鼠模型,采用放射免疫法、组织病理学方法、免疫组织化学染色法、荧光免疫双标记组织化学法等实验方法,从整体-细胞-分子水平,较为全面地探讨固本祛瘀方对DHT诱导复制的PCOS模型大鼠HPOA功能影响的作用机制,以了解固本祛瘀方治疗PCOS的可能作用机理,为其临床推广应用提供依据。方法：雌性Wistar新生幼鼠,饲养于12h光照/12h黑暗的环境中,至21日龄时随机分为4组：正常对照组、模型对照组、西药对照组和固本祛瘀组,每组12只。模型对照组、西药对照组和固本祛瘀组大鼠颈部埋植双氢睾酮缓释片剂(DHT,7.5mg,90d缓释,相当于1.66m/kg,每天释放83μg)制备PCOS大鼠模型。造模6周后,即大鼠64日龄起,固本祛瘀组和西药对照组开始分别给药治疗。固本祛瘀方由菟丝子等12味中药组成,由本院制剂室按特定流程煎制、浓缩,制成浓缩液。西药对照组治疗用药为非甾体类抗雄激素制剂氟他胺(Flutamide)。通过人与大鼠体表面积比率换算法计算给药量。固本祛瘀组、西药对照组大鼠每日灌胃给药,每次1ml,早晚各1次/天,连续4周。模型对照组、正常对照组大鼠每日灌胃蒸馏水,每次1ml,早晚各1次/天,连续4周。治疗时,每周观察各组大鼠体重、活动、饮食等变化；每天阴道脱落细胞学检查研究各组大鼠的动情周期变化；治疗结束后,组织病理学方法研究各组大鼠卵巢、子宫及垂体的形态及组织病理学改变；放射免疫法(radioimmunoassay, RIA)研究各组大鼠血清性激素雌二醇(estradiol,E2)、睾酮(testosterone, T)、孕酮(progesterone, P)、LH、FSH水平变化,结合垂体组织离体静态孵育方法研究垂体离体活细胞LH释放水平；免疫组织化学染色法、荧光免疫双标记组织化学法研究各组大鼠垂体促性腺激素释放激素受体(gonadotropin releasing hormone receptor, GnRH-R)表达水平变化,下丘脑内侧视前区(medial preoptic area, MPO)及斜角带核水平支(horizontal limb of diagonal band, HDB)促性腺激素释放激素(gonadotropin releasing hormone, GnRH)和GnRH-R、雄激素受体(androgen receptor, AR)表达水平变化,及GnRH与AR在下丘脑的共表达情况。结果：1、体重变化：大鼠21日龄时造模开始,模型对照组、西药对照组和固本祛瘀组大鼠皮下DHT埋药。此时各组间大鼠体重比较无统计学差异(P>0.05)。大鼠63日龄时,即埋药后6周,3组造模大鼠体重与正常对照组比较均有非常显著性差异(P<0.01),3组造模大鼠体重均明显高于正常对照组,与PCOS (?)患者代谢特征一致。此后固本祛瘀组、西药对照组开始给药,治疗4周后,固本祛瘀组大鼠体重与模型对照组比较有非常显著性差异(P<0.01),与正常对照组、西药对照组比较无统计学差异(P>0.05)。提示经过治疗,固本祛瘀组PCOS大鼠体重明显下降,基本恢复正常水平。2、动情周期的影响：治疗开始至实验结束,连续每天记录各组大鼠动情周期变化。正常对照组大鼠表现规律的连续4-5天动情周期。模型对照组大鼠表现动情周期延长,主要表现为间期延长,无动情表现等现象。固本祛瘀组大鼠治疗开始阶段表现同模型对照组,主要表现为间期,无动情表现,治疗2周左右,间期时间明显缩短,逐渐出现动情周期性变化,但欠规则,提示连续规律动情周期逐渐建立,卵巢功能逐步恢复,尤其在治疗第3、4周左右,可见明显好转趋势。3、脏器系数的影响：固本祛瘀组大鼠卵巢系数与模型对照组、西药对照组比较分别有非常显著、显著性差异(P<0.01、P<0.05),固本祛瘀组大鼠卵巢系数明显低于模型对照组、西药对照组；与正常对照组比较无统计学差异(P>0.05)。提示经过治疗,固本祛瘀组PCOS大鼠卵巢系数明显降低,基本恢复正常水平。固本祛瘀组大鼠子宫系数与各对照组比较均无统计学差异(P>0.05)。提示造模及治疗对各组大鼠子宫系数无明显影响。固本祛瘀组大鼠垂体系数与各对照组比较均无统计学差异(P>0.05)。提示固本祛瘀方对PCOS大鼠垂体系数无明显影响。4、卵巢组织结构的影响：固本祛瘀组大鼠光镜下见皮质表层细胞增多,结缔组织致密,包膜下可见处于不同发育期的卵泡,包括窦状卵泡及成熟卵泡,表现出排卵迹象。卵巢髓质可见疏松结缔组织及血管。卵母细胞外围颗粒细胞层逐渐增厚,PCOS增厚的卵巢包膜变薄,增厚的卵泡膜细胞层变薄。表明固本祛瘀方具有促进颗粒细胞增生、分化和使增厚的卵泡膜细胞层、卵巢包膜变薄的作用。5、性激素水平的影响：固本祛瘀组大鼠血清E2水平与模型对照组比较有显著性差异(P<0.05),固本祛瘀组大鼠血清E2水平明显高于模型对照组；与正常对照组、西药对照组比较均无统计学差异(P>0.05)。固本祛瘀组大鼠血清T水平与模型对照组比较有非常显著性差异(P<0.01),固本祛瘀组大鼠血清T水平明显低于模型对照组；与正常对照组、西药对照组比较均无统计学差异(P>0.05)。固本祛瘀组大鼠血清P水平与各对照组比较均无统计学差异(P>0.05)。固本祛瘀组大鼠血清LH水平与模型对照组比较有非常显著性差异(P<0.01),固本祛瘀组大鼠血清LH水平明显低于模型对照组；与正常对照组、西药对照组比较均无统计学差异(P>0.05)。固本祛瘀组大鼠LH/FSH比值与模型对照组比较有非常显著性差异(P<0.01),固本祛瘀组大鼠LH/FSH比值明显低于模型对照组；与正常对照组、西药对照组比较均无统计学差异(P>0.05)。固本祛瘀组大鼠血清FSH水平与各对照组比较均无统计学差异(P>0.05)。提示固本祛瘀方能降低PCOS大鼠血清T、LH水平,降低LH/FSH比值,升高血清E2水平,对血清P及FSH无明显影响。6、垂体释放LH水平的影响：固本祛瘀组、西药对照组、模型对照组大鼠垂体释放LH水平分别与正常对照组比较均有非常显著性差异(P<0.01),3组大鼠垂体释放LH水平明显高于正常水平。固本祛瘀组、西药对照组大鼠垂体释放LH水平与模型对照组比较分别有非常显著性、显著性差异(P<0.01、P<0.05),2组大鼠垂体释放LH水平明显低于模型对照组。提示经过治疗,固本祛瘀组PCOS大鼠垂体释放LH水平有一定程度降低,但尚未恢复正常水平。7、垂体GnRH-R表达的影响：固本祛瘀组大鼠垂体GnRH-R阳性表达细胞数与模型对照组比较有显著性差异(P<0.05),固本祛瘀组大鼠垂体GnRH-R阳性表达细胞数明显高于模型对照组；与正常对照组、西药对照组比较均无统计学差异(P>0.05)。提示经过治疗,固本祛瘀组PCOS大鼠垂体GnRH-R表达明显升高,基本恢复正常水平。8、下丘脑GnRH、GnRH-R、AR表达的影响：(1)GnRH:固本祛瘀组大鼠下丘脑MPO及HDB部位GnRH阳性表达神经元数与模型对照组比较分别有非常显著性、显著性差异(P<0.01、P<0.05),固本祛瘀组大鼠下丘脑MPO及HDB部位GnRH阳性表达神经元数明显低于模型对照组。固本祛瘀组大鼠下丘脑MPO部位GnRH阳性表达神经元数与西药对照组比较有显著性差异(P<0.05),固本祛瘀组大鼠下丘脑MPO部位GnRH阳性表达神经元数明显低于西药对照组。固本祛瘀组大鼠下丘脑MPO及HDB部位GnRH阳性表达神经元数与正常对照组比较均无统计学差异(P>0.05)。提示经过治疗,固本祛瘀组PCOS大鼠下丘脑MPO及HDB部位GnRH神经元表达明显降低,基本恢复正常水平。(2)GnRH-R:固本祛瘀组大鼠下丘脑MPO及HDB部位GnRH-R阳性表达细胞数与正常对照组比较均有显著性差异(P<0.05),固本祛瘀组大鼠下丘脑MPO及HDB部位GnRH-R阳性表达细胞数明显低于正常对照组；而与模型对照组、西药对照组比较均无统计学差异(P>0.05)。提示经过治疗,固本祛瘀组PCOS大鼠下丘脑MPO及HDB部位GnRH-R表达有所升高,但无统计学意义,没有恢复正常水平。(3)AR：固本祛瘀组大鼠下丘脑MPO部位AR阳性表达细胞数与模型对照组比较有显著性差异(P<0.05),固本祛瘀组大鼠下丘脑MPO部位AR阳性表达细胞数明显低于模型对照组；而与正常对照组、西药对照组比较均无统计学差异(P>0.05)。提示经过治疗,固本祛瘀组PCOS大鼠下丘脑MPO部位AR表达明显降低,基本恢复正常水平。结论：1、固本祛瘀方能显著改善PCOS肥胖、卵巢性周期紊乱、高雄激素血症、LH/FSH比值升高等病理改变。2、固本祛瘀方能直接改善卵巢组织结构的病理改变,调节卵巢功能。主要表现在减轻PCOS增重的卵巢重量,促进卵巢颗粒细胞增生、分化,使增厚的卵泡膜细胞层、卵巢包膜变薄,改善卵巢功能,促进卵泡发育成熟并排卵。3、固本祛瘀方不能改善垂体系数,但能通过改善垂体细胞的功能发挥治疗作用；能直接降低PCOS大鼠垂体释放LH的水平,但不能恢复完全正常水平。4、固本祛瘀方能分别通过影响垂体GnRH-R表达水平,下丘脑MPO及HDB部位GnRH、GnRH-R、AR表达水平,调节PCOS异常增加的GnRH脉冲频率,降低垂体对GnRH的敏感性,使垂体分泌LH频率及幅度降低,恢复LH周期性改变及峰值出现,促进排卵。更多还原

【Abstract】 Objective:Polycystic ovarian syndrome (PCOS), the common female reproductive endocrine disease, associated with anovulation, hyperandrogenism and insulin resistance, is one of the main reasons for female anovulatory infertility. PCOS incidence is 5-10% among reproductive women, which leads to 70% anovulatory infertility. With age, PCOS increases the risk for type 2 diabetes and cardiovascular diseases. The etiology of PCOS is incompletely understood. Dysfunction of hypothalamus-pituitary-ovarian axis (HPOA) likely plays key role in it.Clinical researches indicate that Chinese medical therapies have good effects on endocrinopathy. Chinese medical therapies may restore PCOS patients’ reproductive function and rectify their endocrinal and metabolic disorders without side or adverse effects. The tutor, Professor Li Lierong, with 40-year clinical experience in gynecopathy, has rich experiences on complicated diseases treated with Chinese medicine. Gubenquyu Decoction is created by Prof. Li according to her clinical experience on PCOS. The clinical observation shows Gubenquyu Decoction may effectively decrease elevated plasma T, LH levels and LH/FSH, restore PCOS patients’ menstruation, improve their ovulation and increase the pregnancy rate. We are meant to from organic-cellular-molecular levels, systemically research the therapeutic mechanism of Gubenquyu Decoction on the hypothalamus-pituitary-ovarian axis, on PCOS rat model induced by DHT, with molecular biology methods, in order to provide the scientific basis for its future promotion in clinical application.Methods:New born female Wistar rats, fed in 12h light/12h dark environment, till 21-day age, were randomly divided into 4 groups:①Control,②PCOS,③Flutamide,④Gubenquyu, each group 12 rats. Rats in PCOS, Flutamide and Gubenquyu groups implanted subcutaneously with dihydrostestosterone (DHT, 7.5mg,90-d continuous release pellets, equal to 1.66m/kg,83μg per day) to induce PCOS model. From 64-day age, Rats in Gubenquyu and Flutamide groups started to receive treatment. Gubenquyu Decoction was one kind of pure herbal extract made of 12 herbs. Flutamide was an anti-androgen hormone. Gubenquyu group and Flutamide group received treatment twice per day for 4 weeks.During treatment, the changes of weight, activity, diet, etc. of the rats in each group were observed once per week; changes of estrus cycle of the rats in each group were observed once per day. After treatment, tissue structures of ovaries, uterus, and pituitary were observed by histopathologic methods; plasma E2、T、P、LH and FSH levels were measured by RIA; expressions of GnRH-R in pituitary were measured by immunohistochemistry; expressions of GnRH, GnRH-R and AR at MPO and HDB in hypothalamic were measured by immunohistochemistry.Results:1. Changes of body weight:At 21-day age, the body weight of rats had no significant difference among each group (P> 0.05). At 63-day age, implanted with DHT for 6 weeks, the body weight of rats in 3 DHT model groups had very significant difference between in Control group (P<0.01), the body weight of rats in 3 DHT model groups were obviously higher than in Control group, which was similar to PCOS human metabolic changes. After 4 weeks’treatment, the body weight of rats in Gubenquyu group had very significant difference between in PCOS group (P<0.01), while had no significant difference between in both Control and Flutamide groups (P> 0.05), which indicated the weights of PCOS rats were obviously decreased treated by Gubenquyu Decoction for 4 weeks.2. Effects on estrus cycle:From the beginning of the treatment (namely at 64-day age), continuously recorded rats’ estrus cycles of each group for 4 weeks until the experiment ended. We found that Control rats exhibited regular 4-5 days estrus cycle including diestrus, proestrus, estrus and metestrus stages; while PCOS rats exhibited no dynamic change in estrous cycle and were constantly in diestrus phase; meanwhile Gubenquyu rats exhibited recovery of regular estrus cycle after treatment, which indicated the recovery of PCOS rats’ ovary functions gradually.3. Effects on organ coefficient:Ovarian coefficient of Gubenquyu rats had very significant, significant differences between of PCOS rats, Flutamide rats respectively (P<0.01、P<0.05), ovarian coefficient of Gubenquyu rats were lower than of PCOS and Flutamide rats; while had no significant difference between of Control rats(P> 0.05). Uterus and pituitary coefficients of rats had no significant difference among each group (P> 0.05)4. Effects on ovarian morphology:Ovary weight and size were higher in DHT rats than in control and Gubenquyu rats. Slices of Gubenquyu group exhibited different stages follicle, including primordial follicle, preantral follicle, primary follicle, zona pellucida, antral follicle, secondary follicle, provulatory follicle, and ovulation; diminished granulosa cell layer increased, thickened theca interna cell layer reduced, and thickened cystic wall decreased compared with PCOS rats. 5. Effects on plasma sex hormone levels:Plasma E2 level in Gubenquyu rats had very significant difference between in PCOS rats(P<0.01); while no significant difference between in both Control and flutamide rats (P> 0.05) Plasma T level in Gubenquyu rats had very significant difference between in PCOS rats(P<0.01); while no significant difference between in both Control and Flutamide rats (P>0.05). Plasma P level of rats had no significant difference among each group (P> 0.05). Plasma LH level in Gubenquyu rats had very significant difference between in PCOS rats(P<0.01); while had no significant difference between in both Control and Flutamide rats (P> 0.05) LH/FSH in Gubenquyu rats had very significant difference between in PCOS rats(P<0.01); while had no significant difference between in both Control and Flutamide rats (P>0.05). Plasma FSH level of rats had no significant difference among each group (P> 0.05)6. Effects on pituitary releasing LH level:Pituitary releasing LH level of 3 DHT implanted groups had very significant difference between Control group (P<0.01); Pituitary releasing LH level of Gubenquyu rats had very significant difference between of PCOS group (P<0.01), while of Flutamide rats had significant difference between of PCOS rats (P<0.05).7. Effects on GnRH-R expression in pituitary:The number of GnRH-R cells in pituitary of Gubenquyu rats had significant difference between of PCOS rats(P<0.05); while had no significant difference between of Control and Flutamide rats (P> 0.05)8. GnRH、GnRH-R、AR expression in MPO and HDB of hypothalamus:(1)GnRH:The number of GnRH cells of Gubenquyu rats had very significant, significant differences between of PCOS rats in MPO and HDB respectively(P<0.01、P<0.05); while had significant difference between of Flutamide rats in MPO (P<0.05); meanwhile had no significant difference between of Control rats in MPO and HDB (P>0.05) (2)GnRH-R:The number of GnRH-R cells of Gubenquyu rats had significant differences between of Control group in both MPO and HDB (P<0.05); while had no significant difference between of both PCOS and Fflutamide rats (P> 0.05)(3)AR:The number of AR cells of Gubenquyu rats had significant difference between of PCOS rats in MPO (P<0.05); while had no significant differences between of both Control and Flutamide rats (P> 0.05)Conclusions:1. Gubenquyu Decoction can significantly reduce PCOS increased weight; restore estrus cycle; decrease plasma T level and LH/FSH, etc.2. Gubenquyu Decoction can rectify pathologic changes of ovarian structures, decrease the increased ovarian weight and improve the ovulation.3. Gubenquyu Decoction can’t rectify pituitary coefficient, but can modulate the function of pituitary:it can decrease pituitary releasing LH level, but can not restore normal level.4. Gubenquyu Decoction can effect the protein expression of GnRH-R in pituitary and GnRH、GnRH-R、AR expression in MPO and HDB of hypothalamus, to modulate increased GnRH pulse frequency, decrease sensitivity for GnRH in pituitary, reduce the frequency and amplitude of LH releasing, to improve ovulation.更多还原