【作者】 王文祥；

【导师】 张文昌；

【作者基本信息】 福建医科大学 ， 药理学， 2013， 博士

【摘要】 大豆异黄酮是大豆等豆科植物生长过程中形成的植物化学物，是一种植物雌激素，流行病学和动物实验显示大豆异黄酮对人类有很多益处，如预防癌症，防治心脑血管疾病，改善更年期综合征等。但鉴于未成年体内雌激素水平较低和下丘脑-垂体-性腺轴发育的不完善，使得他们对外源性雌激素高度敏感。因此大豆异黄酮的雌激素样作用对其内分泌产生干扰作用的机会较大。目前对大豆异黄酮的生殖发育毒性国内外做了一些研究，但大部分的研究主要集中在大豆异黄酮的外源性雌激素样效应上，如对子宫、阴道、乳腺等雌激素效应器官的影响。而对于内源性雌激素的产生器官卵巢的影响研究极少，且缺乏深入的分子机制研究。基于以上研究现状，本研究建立Wistar大鼠断乳致性成熟期持续暴露大豆异黄酮的动物模型，研究大豆异黄酮对卵巢发育的一般影响，对卵泡液微环境成分影响的代谢组学变化，对卵巢细胞凋亡及Fas及Bcl-2/Bax信号途径在大豆异黄酮诱导卵泡细胞凋亡中的调控作用。研究结果对于明确大豆异黄酮未成年卵泡发育毒性及其机制以及未成年女性含大豆异黄酮的豆类及其制品的安全推荐量的制定具有重要的指导意义。目的：研究断乳至性成熟期持续大豆异黄酮对卵巢发育的一般影响。进一步采用代谢组学（HPLC-MS）技术研究断乳致性成熟期持续暴露大豆异黄酮大鼠卵巢卵泡液内源性代谢物的变化，筛选出差异代谢标记物。进而探讨大豆异黄酮对卵巢颗粒细胞凋亡的影响及Fas和Bcl-2/Bax信号通路在其中的调控作用。为大豆异黄酮未成年女（雌）性性腺生殖发育干扰作用的预防与控制以及未成年女性含大豆异黄酮的配方制剂和豆类食品安全摄入量的制定提供科学依据。方法：1.清洁级雌性Wistar大鼠80只，21日龄，45±5g,按体重随机分为4组，每组20只，以0、50、100、200mg/kg.BW的大豆异黄酮灌胃，每天1次,直至第80日龄，共灌胃59天。每天记录体重，观察阴门开放时间和动情周期，干预结束后于动情期剖杀大鼠，取血测定血清雌二醇和孕酮。取卵巢、子宫称重。取卵巢固定、包埋、切片、HE染色计数各级卵泡，计算构成比。2.采用显微注射技术提取上述动物模型中右侧卵巢有腔卵泡的卵泡液，每3只卵巢卵泡液混在一起收集为1份，约15μL，每组6份。经离心、甲醇抽提，HPLC-MS检测，色谱和质谱数据经适当降维，去噪。采用主成分分析法（PCA）和偏最小二乘判别法（PLS-DA）进行模式识别，根据变量权重重要性排序值（VIP）和数据库筛选差异代谢标记物，并用标准品进行验证。3.每组随机抽取2个卵巢组织用于透射电镜观察细胞凋亡。取上述研究内容“1”中的切片用TUNEL法检测细胞凋亡。每组随机抽取6个卵巢组织采用real time-PCR检测Caspase-3、8、9、Fas及Bcl-2、Bax mRNA表达，剩下的每组6个卵巢组织采用Western-blot法测定Caspase-3、Fas、Bcl-2及Bax蛋白表达水平。结果：1.大豆异黄酮对卵巢发育的一般影响：（1）与对照组相比，大豆异黄酮剂量组体重无明显差别，卵巢和子宫脏器系数无明显差异（P＞0.05）。（2）与对照组相比，高剂量组阴道开放时间明显提前（P＜0.05），各剂量组动情周期无明显变化（P＞0.05）。（3）与对照组相比，高剂量组血清雌二醇明显降低（P＜0.05），各组间孕酮水平差别无显著意义（P＞0.05）。（4）与对照组相比，中高剂量组闭锁卵泡和黄体比例明显升高（P＜0.05），而原始卵泡和初级卵泡明显减少（P＜0.05）。2.大豆异黄酮对卵泡液影响的代谢组学研究：（1）总离子色谱图：肉眼上看大豆异黄酮剂量组与对照组有些差别。（2）PCA分析：经数据降维和去除噪音，1300多个峰被识别，在PCA得分散点图和3D图上各剂量组与对照组差别明显。（3）PLS-DA分析：在PLS-DA得分散点图和3D图上可看到各剂量组之间以及与对照组之间差异很明显。（4）经VIP和数据库检索以及标准品验证，共筛选出24个差异代谢标记物，涉及性激素代谢、氨基酸代谢、脂肪酸代谢以及能量代谢途径。3.大豆异黄酮对卵巢颗粒细胞凋亡的影响及Fas和Bcl-2/Bax信号通路的调控作用研究：（1）透射电镜发现大豆异黄酮剂量组颗粒细胞凋亡增多，且存在明显的脂肪变性。（2）TUNEL法检测发现高剂量组有腔卵泡的凋亡率显著高于对照组（P＜0.05），同时闭锁卵泡颗粒细胞凋亡率也高于对照组（P＜0.05），中高剂量组黄体颗粒细胞凋亡率显著高于对照组（P＜0.05）（3）中高剂量组Caspase-3、8、9、Fas及Bax mRNA表达均上调，与对照组比较差异具有统计学意义（P＜0.05）。而Bcl-2mRNA各组间差别无统计学意义（P＞0.05）。（4）与real time-PCR结果一致，Caspase-3、Fas、Bax基因蛋白表达均上调（P＜0.05），Bcl-2蛋白表达低中剂量组与对照组差别无显著意义（P＞0.05），而在高剂量组反而下降（P＜0.05），与real time-PCR结果不一致。结论：1．断乳致性成熟期持续暴露大豆异黄酮会影响卵巢发育，导致闭锁卵泡和黄体比例增多，卵巢是大豆异黄酮的敏感器官之一。2.断乳致性成熟期持续暴露大豆异黄酮会使卵巢卵泡液的代谢物发生明显变化，涉及性激素代谢、氨基酸代谢、脂肪酸代谢以及能量代谢途径，提示大豆异黄酮可通过诱导卵泡液代谢成分改变影响卵泡发育。3．大豆异黄酮可诱导卵巢颗粒细胞凋亡和脂肪变性，其可能机制主要是通过激活Bcl-2/Bax和Fas系统，进而激活Caspase家族途径。更多还原

【Abstract】 Soy isoflavones (SIF) are a kind of phytochemicals and phytoestrogens whichare found in soy and other legumes. Epidemiological evidence and experimentalstudies have repeatedly linked the consumption of soy isoflavones or soy products toa variety of beneficial health effects, such as the prevention of cancer andcardiovascular disease, as well as osteoporosis in menopausal and postmenopausalwomen. Children have low estrogen levels and the development of hypothalamic-pituitary-ovarian axis is immature, which makes them more sensitive to exogenousestrogenic compounds. Therefore, it is reasonable to assume that soy isoflavones maypose a greater hazard to the developing reproductive system in children, and publicconcern about this issue has recently increased. Previous studies had shown that soyisoflavones may adversely affect estrogen-sensitive target tissues, including the uterus,oviduct, vagina and mammary gland. However, whether the ovary, especially theovarian follicle, which as an endogenous estrogen-producing tissue, is affected by soyisoflavones remains unclear and is seldom been intensively studied. Based on theseobservations, the purpose of this study was to determine the influence of theadministration of soy isoflavones from weaning to sexual maturity on ovarian follicledevelopment by using the animal model of Wistar rats. The studies of our researchinclude the effects of soy isoflavones on ovarian development in general; themetabolomic variations in follicular fluid; apoptosis of ovarian cells and theregulation function of Fas and Bcl-2/Bax signal pathway. The results would haveguiding instuction for the prevention and control of soy isoflavones on female gonadalreproductive development and for the formulation of soy foods and soy isoflavonesformula in safe intake of immature female. Objective:To investigate the general effect of soy isoflavones on ovarian development byestablishing animal models of soybean isoflavones exposure from weaning to sexualmaturity, then to investigate the metabolomic variations in follicular fluid usingHPLC-MS and uncover useful toxic biomarkers. Finally to explore the effects of soyisoflavones on apoptosis of ovarian granulosa cells and the regulation function of Fasand Bcl-2/Bax signaling pathway. the results would have guiding instruction for theprevention and control of soy isoflavones on female gonadal reproductivedevelopment and for the formulation of soy foods and soy isoflavones formula in safeintake of immature female.Methods:1. Eighty female21day old Wistar rats weighing45±5g were randomly dividedinto four groups of twenty rats each. Rats of each group were treated daily byintragastric administration of SIF (0,50,100or200mg/kg body weight, respectively)until they reached sexual maturity. Weight gain was measured every day. Age ofvaginal opening and estrus cycle were observed before killing. After the treatmentperiod, the animals were killed by decapitation at the oestrus of estrous cycle, andblood, ovarian samples were harvested to determine serum estradiol (E2) andprogesterone (P4) levels and enumerate ovarian follicles in each stage.2. Follicular fluid was collected from the antral follicles of the ovary using amicroinjector under an inverted microscope. Follicular fluid collected from the leftovaries of three rats from the same group was pooled, up to a total volume of15μl.Each group consists of6samples.The follicular fluids were then prepared successivepassing through a series of centrifugation, methanol extraction, HPLC-MS detection,chromatography and mass spectrometry data dimension reduction and denoising.Principal component analysis (PCA) and partial least-squares-discriminant analysis(PLS-DA) was used to recognize pattern. Variable importance in projection (VIP) anddatabases were employed to identify differences in metabolites and reveal useful toxic biomarkers. Finally, Standards of metabolic interest were used to confirm thestructures.3. Two ovarian tissues were randomly selected to observe cell apoptosis by usingtransmission electron microscopy. Ovarian sections in part one were also used todetect cell apoptosis by TUNEL method. Six ovarian tissues were selected to detectmRNA level of Caspase-3,8,9, Fas, Bax and Bcl-2by real time PCR. The rest6ovarian tissues were selected to determine protein expression level of Caspase-3, Fas,Bax and Bcl-2by Western-blot method.Results:1. The general effect of soy isoflavones on ovarian development (1)There was nosignificant difference in body, ovary and uterus weight among any of the groups (P>0.05).(2)Compared with the control group, age of vaginal opening was accelerated inhigh-dose groups (P<0.05). There was no significant change in estrus cycle of SIFgroups (P>0.05).(3)SIF treatment led to significant reductions in serum E2levels inhigh-dose SIF group as compared to the control group (P <0.05). However, there wereno significant differences (P>0.05) in the levels of serum P4among any of thegroups.(4)In the control and low-dose SIF group, a large number of primordial andprimary follicles were found. However, in the middle and high dose SIF groups, thenumbers of atretic follicles and corpora lutea were higher than those of the controlgroup (P <0.05)2. Metabolomic changes in follicular fluid induced by soy isoflavonesadministered to rats from weaning until sexual maturity:(1) Total ion intensitychromatogram: Visual comparison of these chromatograms indicated that themetabolic profiles of the groups treated with soy isoflavones differ somewhat fromthat of control group.(2)PCA analysis: Through data dimension reduction anddenoising, approximately1300peaks (defined by a pair of m/z values and RT) wereidentified. the control and low, middle and high dose SIF rats are appreciablyseparated in the pseudo3D-PCA score plot.(3)PLS-DA analysis: The score plots of the PLS-DA between the control group and the groups treated with soy isoflavonesare clearly separated.(4)Based on the VIP value, database retrieval and standardverification, The24most significantly altered metabolites were identified, includingprimary sex hormones, amino acids, fatty acids and metabolites involved in energymetabolism.3. The effects of soy isoflavones on apoptosis of ovarian cells and the regulationfunction of Fas and Bcl-2/Bax signaling pathway:(1) Granulosa cell apoptosisincreased in SIF groups from Transmission electron microscope. Lipid accumulationwas also found in SIF groups.(2) By using TUNEL assay, the results showed thatgranulosa cell apoptosis rate of the antral follicle in the high-dose group wasobviously higher than that of the control group (P<0.05), while granulosa cellapoptosis rate of the atretic follicle was also higher than that of the control group(P<0.05). Granulosa cell apoptosis rate of corpora lutea in middle and high SIFgroups was significantly higher than that of control group (P<0.05).(3)mRNA levelsof caspase-3,8,9, Fas and Bax in middle and high-dose groups were up-regulated(P<0.05). mRNA level of Bcl-2in low-dose SIF group was higher than that of controlgroup (P<0.05).(4)Consistent with the results of real time PCR, protein expression ofCaspase-3, Fas, Bax gene was significantly up-regulated (P<0.05), but Bcl-2proteinexpression in high-dose group was significance lower than that of control group,which was inconsistent with the results of real time PCR.Conclusions:1. Continuous exposure of soy isoflavones from weaning to sexual maturity canaffect ovarian development, resulting in atretic follicle and corpus luteum increasing,which indicate that the ovary is one of the main target tissues of soy isoflavones.2. Soy isoflavones can induce metabolic alterations in follicular fluid, whichinclude primary sex hormones, amino acids, fatty acids and metabolites involved inenergy metabolism. These findings indicate that soy isoflavones affect ovarian follicledevelopment by inducing metabolomic variations in follicular fluid. 3. Soy isoflavones can induce apoptosis of granulosa cells and lipids accumulationin ovary. the possible mechanism may relate to the activation of Bcl-2/Bax and Fassystem, and then activating the Caspase family signaling pathway.更多还原