归术益坤汤治疗多囊卵巢综合征的临床与实验研究

【作者】 佟庆；

【导师】 金哲；

【作者基本信息】 北京中医药大学 ， 中西医结合临床， 2011， 博士

【摘要】 多囊卵巢综合征(polycystic ovary syndrome,PCOS)又称为Stein-Leventhal综合征,是以发病多因性、临床症状呈多态性为主要特征的一种内分泌综合征。其病因复杂,涉及范围广泛,至今病因尚不明确,一直以来是国内外众多学者努力完善的课题之一。其典型的临床表现为卵巢多囊性改变、高雄激素血症和黄体生成素(LH)/促卵泡激素(FSH)比值增高,不同程度的月经异常(月经稀发、量少、闭经、功能失调性子宫出血)、不孕、多毛、痤疮、肥胖等,并常伴有胰岛素抵抗或高胰岛素血症和高脂血症。根据其症状,中医学可归于“闭经”、“崩漏”“癥瘕”的病症范畴,古代医家和现代中医学者辨证论治,收到了很好的疗效,显示了中医药在治疗PCOS领域中的巨大潜力。因此,探寻PCOS的病因病机和有效治疗方法,是当前临床研究的重大课题,具有重要社会意义和临床价值。本研究从临床和实验两方面探索PCOS的治疗规律和作用机制,为中医药治疗PCOS贡献力量。1临床研究1.1 PCOS患者舌象的研究1.1.1目的：从中医四诊中最有特色及客观化的舌诊角度探讨PCOS的中医证候规律性,探讨PCOS舌象特征与数字化舌图的运用。1.1.2方法：利用数码相机拍摄101例PCOS患者的舌象照片,通过图像统一化处理,运用统计学方法,对舌象的颜色、形态、舌苔等多方面进行分析,总结规律。1.1.3结果：舌色可分为3类：淡紫舌占34.65%,淡舌占45.54%,紫红舌占19.80%；胖大舌、齿痕舌、嫩舌出现的比例高,且常合并出现,舌苔以腻苔和滑苔多见。1.1.4结论：临床所见PCOS患者以脾肾不足多见,可同时出现血瘀、痰湿的病理产物。1.2归术益坤汤治疗脾肾不足、湿浊结聚型PCOS的疗效观察1.2.1目的：探索临床常见的脾肾不足、湿浊结聚型PCOS的有效治疗方法。1.2.2方法：通过对比用药前后妊娠率、临床症状评分、BMI指数、激素水平、B超等方面,评价有补肾健脾、养血通利作用的归术益坤汤治疗脾肾不足、湿浊结聚型PCOS的疗效。1.2.3结果：归术益坤汤在对改善PCOS患者临床症状、高雄激素水平、卵巢增大等方面,疗效极为显著。就PCOS最典型的月经后错或闭经、月经量少、肥胖、痤疮、多毛等症状而言,治疗6个月后症状均明显改善。治疗6个月内妊娠率为10.2%,治疗12个月内妊娠率为37.5%。1.2.4结论：归术益坤汤能够有效改善脾肾不足、湿浊结聚型PCOS患者的临床症状和实验室指标,促进排卵,提高妊娠率。2实验研究2.1归术益坤汤对PCOS大鼠血清激素水平和组织学的影响2.1.1目的：通过动物实验,从血清激素和卵巢组织学方面检验归术益坤汤治疗PCOS的疗效。2.1.2方法：利用来曲唑诱导建立PCOS大鼠动物模型,灌服归术益坤汤给予治疗,以氯米芬为阳性对照组,检测大鼠用药后正常对照组、模型对照组、阳性对照组、中药组之间激素水平、卵巢结构的变化。2.1.3结果：模型组大鼠血清睾酮(T)的含量明显升高,与正常组、中药组、阳性药组之间均有显著差异(P<0.05),正常组、中药组、阳性药组之间均无组间差异(P>0.05)。模型组的促黄体生成素(LH)水平与阳性药组相比有显著差异(P<0.05),其他各组间差异不明显。而各组间雌二醇(E2)与促卵泡生成素(FSH)之间的差异不明显。模型组大鼠卵泡多数成囊性扩张,可见囊性窦卵泡,颗粒细胞层数减少,黄体组织数量减少。中药组卵巢可见少许囊性窦卵泡的出现,可见成熟卵泡,颗粒细胞数较模型组明显增加,为多层,可见黄体。氯米芬阳性药组亦可见部分囊性窦卵泡,颗粒细胞为多层,排列比较整齐,黄体组织较少。2.1.4结论：归术益坤汤能够有效改善大鼠内分泌,可恢复卵巢卵泡结构,使颗粒细胞增殖,促进卵泡发育。2.2归术益坤汤对PCOS大鼠颗粒细胞自噬的影响2.2.1目的：通过观察归术益坤汤对PCOS大鼠卵巢颗粒细胞自噬发生的影响,发现PCOS颗粒细胞减少的新机制,探索中药治疗PCOS的相关作用机制。2.2.2方法：实验利用来曲唑诱导建立PCOS大鼠动物模型,灌服归术益坤汤给予治疗,以氯米芬为阳性对照组,用药后提取大鼠卵巢颗粒细胞,应用MDC荧光染色及流式细胞术测定鼠颗粒细胞自噬率。采用western blot方法测定各组动物颗粒细胞中自噬特异性标志LC3和自噬相关蛋白Bclin-1的表达,验证各组间自噬发生的差异。2.2.3结果：MDC荧光染色后荧光强度测定：模型组>阳性药组>中药组>正常组。组间两两比较,模型组与正常组、中药组、阳性药组之间均存在显著性差异(P<0.05),而正常组、中药组、阳性药组无组间差异。正常对照组LC3Ⅰ和Ⅱ、Bclin-1蛋白的表达量均较低,PCOS模型组的LC3、Bclin-1呈现高表达(与正常组比较P<0.01)。经中药作用后可见LC3、Bclin-1平呈明显降低趋势(与模型组比较P<0.05),但仍高于阳性药组。2.2.4结论：PCOS模型大鼠的卵巢颗粒细胞确实存在有自噬高发的现象,与PCOS颗粒细胞减少相关。归术益坤汤可以显著降低细胞自噬的发生,有效保护了颗粒细胞,从而对卵巢功能起到了维护作用。2.3归术益坤汤对PCOS大鼠颗粒细胞自噬PI3k/Akt通路的调节作用2.3.1目的：通过检测归术益坤汤对PCOS大鼠卵巢颗粒细胞PI3K、AKT和mTOR的磷酸化蛋白表达水平的影响,对中药干预PCOS卵巢颗粒细胞自噬的信号转导通路加以研究。2.3.2方法：实验利用来曲唑诱导建立PCOS大鼠动物模型,将动物灌服归术益坤汤给予疗,以氯米芬为阳性对照组,用药后取各组实验大鼠卵巢颗粒细胞,提取蛋白后进行蛋白定量,采用western blot方法测定PI3K、Akt、mTOR的蛋白表达。2.3.3结果：模型组PI3K、Akt、mTOR的表达均明显降低,中药与阳性药干预后PI3K、AktmTOR表达明显提高。2.3.4结论：归术益坤汤可有效调节PI3K的活性,提高其蛋白表达量,从而影响mTOR的表达,对自噬发挥了负向调控的作用。更多还原

【Abstract】 Polycystic ovary syndrome, PCOS, also known as Stein-Leventhal syndrome, Is a kind of endocrine syndrome with the main characteristics is caused by Polygenetic, clinical symptoms is polymorphism. The etiology is complex and wide, so far still unclear, and is one of task for world researchers try to improve. The typical clinical manifestations is polycystic ovarian changes in hyperandrogenism and luteinizing hormone (LH)/follicle-stimulating hormone (FSH) ratio increased. Varying degrees of abnormal menstruation (menstrual thin hair, less, amenorrhea, dysfunctional uterine bleeding), infertility, hirsutism, acne, obesity, etc. And of ten accompanied by the emergence with ages insulin resistance or hyperinsulinemia, and hyperlipidemia. According to their symptoms, the Traditional Chinese Medicine can be attributed to "amenorrhea", "bleeding", "Zheng Jia" of the disease areas. The ancient and modern Chinese medicine scholars’diagnosis and treatment has received good results, showing great potential of Chinese medicine in the treatment of PCOS. Therefore, to explore the etiology and pathogenesis of PCOS and effective treatment methods is a major topic of current clinical research with great social significance and clinical value. This thesis from both clinical and experimental to explore the treatment of PCOS and mechanisms, and contribute to Chinese medicine of PCOS.Clinical Research 11.1 Research of PCOS patients’tongue1.1.1 Objective:From Chinese medicine four diagnostic’s features and with the objective point of view of the tongue to study the PCOS regularity, Study the features of PCOS tongue and tongue map digital usage.1.1.2 Methods:By study 101 PCOS patients’tongue digital camera photos, through a unified image processing method then using statistical methods to analyze tongue color, shape, tongue, and many other features to sum up the rule.1.1.3 Results:The tongue can be divided into three categories:purple tongue,34.65%, pale tongue 45.54%, purple tongue 19.80%; fat, large tongue, tongue indentation, tender tongue ratio appears high, and of ten appear together. The tongue coating mostly is greasy and slippery.1.1.4 Conclusion: The clinical findings of PCOS patients more common with spleen deficiency, which can occur and blood stasis, phlegm in the pathological product. 1.2 Use Gui Zhu Jian Kun Tang to cure the PCOS patients with yang deficiency of spleen and kidney and phlegm-dampness in body. To compare the differents between the symptoms before and after the treatment.1.2.1 Objective: To explore the effective treatment for PCOS with yang deficiency of spleen and kidney and phlegm-dampness in body. 1.2.2 Methods:By comparing the pregnancy rate, clinical score, BMI index, hormone levels, type-B ultrasonic before and after treatment.1.2.3 Results:Gui Zhu Jian Kun Tang can improving clinical of PCOS patients symptoms, androgen levels, ovarian increases, etc., the effect is extremely significant. For the most typical PCOS symptoms e.g. late menstruation or amenorrhea, less menstruation, obesity, acne, hirsutism, after treatment 6 months the symptoms were significantly improved. Pregnancy rate within 6 months of treatment was 10.2%, pregnancy rate within 12 months of treatment was 37.5%.1.2.4 Conclusion: Gui Zhu Jian Kun Tang can improve the spleen and kidney deficiency, yang deficiency of spleen and kidney and phlegm-dampness type PCOS patients with clinical symptoms and laboratory indicators improved, to promote ovulation and improve the pregnancy rate.2 Experimental Study2.1 Gui Zhu Jian Kun Tang impact to PCOS rats on serum hormone levels and histological2.1.1 Objective: Through animal experiments, from the serum hormone and ovarian histology verify the efficacy of Gui Zhu Jian Kun Tang treatment on PCOS.2.1.2 Methods:Setup PCOS rat model by letrozole induced, fed animals with Gui Zhu Jian Kun Tang, compare with clomiphene as a positive control group, analyze hormone levels and ovarian changes within normal treatment control group, model control group, positive control group, Chinese medicines group.2.1.3 Results:The model control group rats serum testosterone (T) were significantly increased, compare with the normal group, Chinese medicines group and the positive drug group the difference was significantly between groups (P<0.05), normal group, Chinese medicines group, positive medicine group no differences between each groups (P> 0.05). On model group the luteinizing hormone (LH) levels was significantly different with positive drug group (P<0.05), no significant difference between the other groups. The estradiol (E2) and follicle-stimulating EPO (FSH) no significant difference betweer each groups. Most of model rats the follicles were cystic expansion, showing cystic antral follicles, granulosa cell layer decreased, luteal tissue decreased. Chinese medicines group showing a few Graafianfollicle, shows mature follicles, granulosa cells were significantly increased compared with the model group, was multi-layer and luteal visible. Clomiphene group has also seen some Graafian follicle, granulosa cells is multi-layer, arranged orderly, less luteal tissue visible.2.1.4 Conclusion:Gui Zhu Jian Kun Tang can improve the Rat Endocrinology, restore ovarian follicle structure, make the granulosa cell proliferation and follicular development.2.2 Gui Zhu Jian Kun Tang impact to PCOS rats granulosa cells autophagy rate. ovarian morphology and granulosa cell autophagy to explore the role of traditional Chinese medicine treatment of PCOS related mechanisms.2.2.2 Methods:The experiment setup PCOS rat model by using letrozole induce, fed animals with Gui Zhu Jian Kun Tang, use clomiphene as a positive control group, extract rats ovarian granule cells after treatment, apply with MDC fluorescence staining and flow cytometry methord check rat granulosa cells autophagy difference. Use western blot to test animal’sdeliverance of LC3 and Bclin-1, verify the autophagy difference of each groups.2.2.3 Results:MDC measured fluorescence intensity after staining: the model group> positive drug group> Chinese medicine group> normal group. Between any two groups, model group and normal group, Chinese medicine group and the positive drug groups all have significantly difference (P<0.05). While the normal group, Chinese medicine group, positive medicine group had no difference. Normal control group LC3ⅠandⅡ, Bclin-1 protein expression levels low, On PCOS model group, LC3, Bclin-1 shows high expression (compared with normal group P<0.01). After treatment by Chinese medicine the LC3, Bclin-1 level had significantly lower trend (compared with model group P<0.05), but still higher than the positive drug group.2.2.4 Conclusion: PCOS rat model the ovarian granulosa cells exist a high incidence of the autophagy, associated with PCOS granulosa cells reduced. Gui Zhu Jian Kun Tang can significantly reduce the occurrence of autophagy, have effective protection of the granule cells, and thus piay a maintenance role for ovarian function.2.3 Gui Zhu Jian Kun Tang regulate PCOS rat granulosa cells autophagy pathway PI3k/Akt2.3.1 Objective: To analysis the Gui Zhu Jian Kun Tang impact to PCOS rat ovarian tissue PI3K I, AKT and mTOR phosphorylation level of protein expression and study the Chinese medicine intervention in PCOS ovaries autophagy signaling pathway.2.3.2 Methods:The experiment setup PCOS rat model by using letrozole induce, fed animals with Gui Zhu Jian Kun Tang, use clomiphene as a positive control group, extract rats ovarian granule cells after treatment, carry on protein quantification after extracted proteins, determined by western blot PI3K, Akt, mTOR protein.2.3.3 Result:The model group PI3K, Akt, mTOR expression was significantly decreased, after the intervention of traditional Chinese medicine and positive drug the PI3K, Akt, mTOR expression was significantly increased.2.3.4 Conclusion: Gui Zhu Jian Kun Tang can regulate the activity of PI3K to increase their protein expression, thus affecting the expression of mTOR and plays a negative regulatory role to autophagy.更多还原