The effects of the phytoestrogen genistein on the postnatal development of the rat.

The present studies report the effects on neonatal rats of oral exposure to genistein during the period from birth to postnatal day (PND) 21 to generate data for use in assessing human risk following oral ingestion of genistein. Failure to demonstrate significant exposure of the newborn pups via the mothers milk led us to subcutaneously inject genistein into the pups over the period PND 1-7, followed by daily gavage dosing to PND 21. The targeted doses throughout were 4 mg/kg/day genistein (equivalent to the average exposure of infants to total isoflavones in soy milk) and a dose 10 times higher than this (40 mg/kg genistein). The dose used during the injection phase of the experiment was based on plasma determinations of genistein and its major metabolites. Diethylstilbestrol (DES) at 10 micro g/kg was used as a positive control agent for assessment of changes in the sexually dimorphic nucleus of the preoptic area (SDN-POA). Administration of 40 mg/kg genistein increased uterus weights at day 22, advanced the mean day of vaginal opening, and induced permanent estrus in the developing female pups. Progesterone concentrations were also decreased in the mature females. There were no effects in females dosed with 4 mg/kg genistein, the predicted exposure level for infants drinking soy-based infant formulas. There were no consistent effects on male offspring at either dose level of genistein. Although genistein is estrogenic at 40 mg/kg/day, as illustrated by the effects described above, this dose does not have the same repercussions as DES in terms of the organizational effects on the SDN-POA.     

Kinetics of genistein and its conjugated metabolites in pregnant Sprague-Dawley rats following single and repeated genistein administration.

Diets high in soy-based products are well known for their estrogenic activity. Genistein, the predominant phytoestrogen present in soy, is known to interact with estrogen receptors (ER) alpha and beta and elicits reproductive effects in developing rodents. In the rat, genistein is metabolized predominantly to glucuronide and sulfate conjugates, neither of which is capable of activating ER. Therefore, it is critical to understand the delivery of free and conjugated genistein across the placenta to the fetus following maternal genistein exposure such that the potential fetal exposure to free genistein can be assessed. Genistein (4 or 40 mg/kg) was administered to pregnant Sprague-Dawley rats by oral gavage daily from gestation day (GD) 5 through 19 or on GD 19 alone. Maternal and GD 19 fetal tissues were collected 0.5, 1, 2, 4, 6, 8, 12, and 24 h following administration of the final dose on GD 19. Concentrations of genistein, genistein glucuronide, and genistein sulfate were quantitated by LC-MS/MS. In maternal plasma, genistein glucuronide was the predominant metabolite. In the fetal plasma, genistein glucuronide and genistein sulfate were the primary metabolites. Genistein levels in maternal and fetal plasma were much lower than its conjugates. The concentration of genistein in placental tissue was higher than either conjugate. Fetal concentrations of unconjugated genistein following administration of 40 mg/kg were above the EC50 for ERbeta activation. Repeated administration of 40 mg/kg genistein resulted in minor changes in genistein kinetics in the pregnant rat compared to single administration of the same dose. These data suggest that conjugated forms of genistein are not transported across the placenta. High placental concentrations of genistein indicate the placenta is a potential target organ for genistein action during gestation.     

染料木素对异丙肾上腺素诱导的心肌肥厚大鼠血管活性物质的影响

目的观察染料木素(Gen)对异丙肾上腺素(Iso)所致大鼠心肌肥厚的保护作用及其作用机制。方法大鼠背部sc给予Iso 1 mg.kg-1.d-1造模。第2天在给Iso 30 min后,再背部sc给予Gen 0.03,0.1和0.3μmol.kg-1,连续14 d。末次药后12 h检测左心质量参数;放免法测定血浆血管紧张素Ⅱ(AngⅡ),心钠素(ANP)和内皮素(ET)的含量。结果与正常对照组相比,模型组左心室质量参数由(1.87±0.09)mg.kg-1明显提高至(2.48±0.11)mg.kg-1(P<0.01),血浆AngⅡ含量由(245±74)ng.L-1提高至(354±51)ng.L-1(P<0.05),ET含量由(37±17)ng.L-1提高至(66±20)ng.L-1(P<0.05),ANP含量由(506±115)ng.L-1降低至(265±125)ng.L-1(P<0.05)。与模型组相比,Gen 0.03,0.1和0.3μmol.kg-1组左心室质量指数下降,分别为:(2.32±0.15)mg.kg-1(P>0.05),(2.28±0.10)mg.kg-1(P<0.05),(2.25±0.13)mg.kg-1(P<0.05);血浆AngⅡ含量下降,分别为:(242±77)ng.L-1(P<0.05),(198±40)ng.L-1(P<0.01),(196±53)ng.L-1(P<0.05);血浆ET含量下降,分别为:(36±17)ng.L-1(P<0.05),(38±9)ng.L-1(P<0.05),(45±17)ng.L-1;血浆ANP含量升高,分别为:(695±177)ng.L-1(P<0.01),(574±219)ng.L-1(P<0.05),(544±231)ng.L-1(P<0.05)。结论 Gen可能通过影响大鼠体内血管活性物质的平衡,从而抑制Iso诱导的心肌肥厚。     

Modulation of mammary gland development in prepubertal male rats exposed to genistein and methoxychlor.

The estrogenic isoflavone genistein is a common dietary component that has been shown to affect reproductive development in experimental animals at high doses. The objective of the present study was to examine interactions of genistein and the hormonally active pesticide methoxychlor on mammary gland development in juvenile rats. Timed-pregnant Sprague-Dawley rats were fed a soy- and alfalfa-free diet containing different combinations of genistein (300 and 800 ppm) and methoxychlor (800 ppm). Rats were fed these diets starting on gestation day (GD)1 and continuing through pregnancy and lactation until postnatal day (PND) 22, when the pups were killed. Inguinal mammary glands from both female and male pups were processed as whole-mount preparations for morphometric analysis. The total glandular area and the numbers of branch points, lateral buds, and terminal end buds in the male rats were found to be significantly greater in the groups exposed to methoxychlor than those exposed to genistein only. These effects were not observed in the female rats. In the male rats, methoxychlor had the most prominent effect on elongating the glandular ducts, while genistein enhanced the ductile branching. The 2 compounds in combination promoted the development of alveolar-lobular structure, an effect not observed with either compound alone. Immunostaining for proliferating cell nuclear antigen revealed a high percentage of immunopositive cells in the mammary epithelia of the males exposed to methoxychlor and genistein (800 ppm) compared to the controls. While no significant changes in serum levels of mammotrophic hormones were detected, increased immunostaining for insulin-like growth factor-1 receptor, estrogen receptor alpha, and progesterone receptor in the genistein + methoxychlor group suggested that local factors involved in regulating mammary growth may have played a role in propagating the endocrine effects of these two compounds. These results indicated that the mammary glands of juvenile male rather than juvenile female rats may be more sensitive to certain endocrine-active compounds and that high levels of phytoestrogens have the potential to alter the toxicological behaviors of other hormone mimics.     

液相色谱-串联质谱法测定人血浆中染料木素及其葡萄糖醛酸代谢物

目的建立液相色谱-串联质谱联用法测定人血浆中染料木素(雌激素类药物)及其葡萄糖醛酸代谢物(GGS)并研究其在健康人体中的药代动力学。方法10例健康受试者空腹口服染料木素50 mg,血浆加入内标物地塞米松后,经液-液萃取,色谱柱为Capcell Pak C8(2 mm×150 mm,5μm),流动相为:甲醇-5 mmol.L-1乙酸铵-乙腈(70∶20∶10),以ESI源正离子MRM模式测定染料木素的血药浓度,GGS先酶解为原形后间接测定,用DAS 2.0程序计算药代动力学参数。结果线性范围为0.3~500.0μg.L-1(γ=0.9983),回收率在92.9%~104.9%,绝对回收率在74.2%~89.2%,日内、日间变异(RSD)均<15%。染料木素和GGS的主要药代动力学参数:tmax分别为(5.0±1.3),(6.0±2.4)h,Cmax分别为(10.1±6.3),(218.7±68.6)μg.L-1,AUC0-t分别为(31.2±10.3),(3344.0±1635.0)μg.h.L-1,AUC0-∞分别为(33.9±11.4),(3703.0±2031.0)μg.h.L-1。结论测定方法灵敏、准确...     

高效液相色谱法测定大鼠血清中染料木黄酮浓度

目的:建立高效液相色谱法测定大鼠血清中染料木黄酮浓度.方法:血清样品用叔丁基甲醚萃取,萃取物用氮气吹干后,用乙腈-0.05 mol·L-1甲酸胺缓冲液(27:73,pH 4.0)溶解用于色谱分析.色谱务件:采用Thermo C18柱(4.6 mm×250 mm,5μm);以乙腈-0.05 mol·L-1甲酸胺缓冲液(27:73,pH 4.0)为流动相;流速为1.0 mL·min-1;检测波长为260 nm;柱温为25℃;进样量为10μL.结果:染料木黄酮最低检测浓度为0.01 mg·L-1;血清样品浓度在0.01～10.00 mg·L-1范围内呈良好的线性关系,回归方程为:y=0.079 8 6.976 4X,r=0.999 2(n=5);相对回收率为(100.1±1.8)%;日内RSD与日间RSD均小于10.00%(n=5).结论:该方法灵敏,简便、快速、重复性好,适用于大鼠血药浓度测定及药代动力学研究.     

金雀异黄素对低密度脂蛋白诱导的内皮细胞c-myc mRNA表达的影响(英文)

目的 :研究金雀异黄素对氧化型低密度脂蛋白诱导的人血管内皮细胞c mycmRNA表达的影响。方法 :低密度脂蛋白采用密度梯度超速离心法从健康人血浆中提取 ,经铜离子氧化制备成氧化型低密度脂蛋白。培养的人血管内皮细胞给予氧化型低密度脂蛋白 2 0 0mg·L- 1或同时给予金雀异黄素 10 0 μmol·L- 1诱导不同时间 (1,2 ,4h) ,提取细胞总RNA进行Northernblot杂交。结果 :2 0 0mg·L- 1氧化型低密度脂蛋白刺激内皮细胞c mycmR NA在 1h增加为对照水平的 3倍 ,2h增加为对照水平的 3.3倍 ,4h降到对照水平以下 ;给予氧化型低密度脂蛋白诱导的同时给予金雀异黄素 10 0 μmol·L- 1其c mycmRNA表达量在 1h为单独给予氧化型低密度脂蛋白诱导的 80 % ,2h为 6 0 %。结论 :金雀异黄素能有效抑制氧化型低密度脂蛋白诱导的人血管内皮细胞c mycmRNA表达增高     

Genistein对他莫昔芬预处理后多巴胺能神经元的保护作用

目的：观察他莫昔芬阻断雌激素受体后,genistein（GST）对多巴胺能神经元是否具有保护作用。方法：取孕14～16天SD大鼠胚胎中脑腹侧部,常规体外培养后分为4组：正常对照组、GST＋MPP^＋组、TAM＋GST-4-MPP^＋组和MPP^＋组。用TH免疫细胞化学染色观察各组TH阳性神经元的生长状况。用四甲基偶氮唑盐（MTT）法测定细胞活力和代谢状态。用Western Blot观察各组bax蛋白的表达情况。结果：TAM＋GST＋MPP^＋组与GST＋MPP^＋组相比,TH阳性神经元数量有所减少,突起长度变短,细胞活力差,bax蛋白的表达量增加,差异有统计学意义。同时MPP^＋组与TAM＋GST＋MPP^＋组相比,TH阳性神经元数量减少,突起长度短,细胞活力最差,bax蛋白的表达量最多,差异均有统计学意义。结论：TAM能部分下调GST对多巴胺能神经元的保护作用;GST除了通过ER途径发挥保护作用外,还可能经其他途径发挥保护作用。     

染料木素对大鼠食饵性高脂血症PON1及CRP的影响

目的探讨染料木素(genistein,Gen)对大鼠食饵性高脂血症对氧磷酶(paraoxonase,PON1)活性及C-反应蛋白(C-reactive protein,CRP)的影响。方法健康SD大鼠40只,♀♂各半,随机分为以下5组:①普食组;②高脂血症模型组;③低剂量Gen(0.5 mg.kg-1.d-1)组;④高剂量Gen(5 mg.kg-1.d-1)组;⑤辛伐他汀(5 mg.kg-1.d-1)阳性药物对照组;除普食组给以普通饲料喂养外,其他组均饲以高脂饲料喂养加敌百虫(10 mg.kg-1.d-1)腹腔注射,建立食饵性高脂血症模型。各组大鼠连续喂养及同时用药8周,每2周测1次体重,根据体重调整用药量;定期取血检测胆碱酯酶(acetylcholine esterase,AChE)、血脂、丙二醛(ma-lonaldehyde,MDA)、PON1和CRP的含量,用苏木精/伊红(hematoxylin/eosin,HE)染色检测大鼠肝脏及主动脉弓病理形态变化。结果与模型组相比,高剂量Gen组与辛伐他汀组大鼠总胆固醇(total cholesterol,TC)、低密度脂蛋白胆固醇(low density lipoprotein cholesterol,LDL-C)及动脉粥样硬化指数(atherosclerotic index,AI)明显降低,高密度脂蛋白胆固醇(high density lipoprotein cholesterol/total cholesterol,HDL-C)与TC比值(HDL-C/TC)明显升高,血清MDA含量也明显降低,同时还能明显降低高脂饲料喂养加低剂量敌百虫腹腔注射致高脂血症大鼠血清CRP含量,提高PON1活性,改善大鼠肝与主动脉弓形态变化。结论Gen可以减轻高脂饲料喂养加敌百虫诱发的大鼠高脂血症;其作用机制可能与逆转血脂障碍,抑制过氧化脂质作用抗氧化,降低炎症因子CRP抗炎及通过PON1酶的调节有关。     

三羟异黄酮对大鼠缺血性脑损伤神经组织的保护作用

目的探讨植物性雌激素三羟异黄酮（GST）对局灶性永久性脑缺血成年雄性大鼠大脑皮层神经组织的保护作用。方法实验分为假手术组、缺血组、GST组和溶剂对照组,采用HE染色、甲苯胺蓝染色和电子显微镜观察的方法,研究颈内动脉注射GST应急治疗对雄性大鼠局灶性永久性脑缺血模型中大脑皮层神经组织神经元,突触和血-脑脊液屏障的保护作用。结果GST组与缺血组比较,神经元、突触和血-脑脊液屏障的损伤均明显减轻。结论GST应急治疗对缺血性脑损伤的神经组织具有明显的保护作用。     

植物雌激素大豆苷原(daidzein)对结肠癌LoVo细胞生长的影响

目的 了解植物雌激素大豆苷原(daidzein)对结肠癌细胞生长的影响。方法 取对数生长期的人结肠癌细胞株LoVo细胞,以不同浓度的大豆苷原处理细胞2,3,4和6d,然后应用MTT法测定细胞的生长率。结果 大豆苷原在高浓度100μmol/L时在体外对LoVo有明显的抑制作用(P＜0．01),而在低浓度＜5μmol/L时对LoVo细胞又具有促增殖作用。结论 大豆苷原在体外对LoVo细胞的生长具有促进和抑制的双向作用。     

Mammary gland morphology in Sprague-Dawley rats following treatment with an organochlorine mixture in utero and neonatal genistein.

In a related reproductive toxicology study designed to investigate the effects of in utero exposure to environmental toxicants and potential interaction with postnatal genistein, gross enlargement of thoracic mammary glands was observed in female offspring at 200 days of age. Therefore, the objective of this study was to analyze the effect of in utero exposure to a mixture of toxicants on mammary gland morphology. Time-mated Sprague-Dawley rats were treated on days 9-16 of gestation with vehicle or a mixture of environmental toxicants at 1x the acceptable daily intake. Furthermore, it is unclear whether postnatal exposure to phytoestrogens in soy formulas poses breast cancer benefit or risk, and potential interactions with environmental toxicants are unknown. Therefore, half the female pups from each treatment group received either subcutaneous vehicle or genistein (10 microg/g body weight [bw]/day) on postnatal days 2-8. Following necropsy at 200 days of age, a pathologist, blinded to treatment groups, examined mammary gland histopathology. Only mild histological changes were found in mammary glands of rats exposed to the mixture in utero while pronounced ductal hyperplasia, lactational changes, and fibrosis were observed in mammary glands from the genistein group and were more prominent in the mixture + genistein group. Mammary glands of the control group were histologically normal. Collectively, our results reveal that postnatal exposure to pharmacological levels of genistein induces profound morphological changes in the mammary glands of adult female rats, and that high levels of phytoestrogens possess the potential to modulate the toxicological effects of toxicant mixtures.     

体外受精-胚胎移植技术中囊胚培养密度对胚胎发育潜能的影响

目的 分析体外受精-胚胎移植技术（IVF-ET）中囊胚培养密度对胚胎发育潜能的影响。方法 选取2019年1月至12月佛山市妇幼保健院接受体外受精移植的235例体外受精胚胎移植患者为研究对象,将顺利移植的1181个裂卵期胚胎按照囊胚培养密度差异分为四组,单独培养为A组,2～3个集中培养为B组,4～5个集中培养为C组,6个以上集中培养为D组,其中A组培养囊胚共269个,B组培养囊胚共298个,C组培养的囊胚共304个,D组培养囊胚共310个。比较四组的囊胚形成率、可利用囊胚率、优质囊胚率、囊胚等级差异、囊胚着床率及临床妊娠率。结果 C组的囊胚形成率、可利用囊胚率、优质囊胚率均高于A、B、D组,差异有统计学意义（P<0.05）;C组的囊胚4～6期占比高于A、B、D组,差异有统计学意义（P<0.008）。四组胚胎共形成778个胚囊,C组形成囊胚的囊胚着床率、临床妊娠率高于A、B、D组,差异有统计学意义（P<0.008）。结论 囊胚培养密度为4～5个囊胚集中培养时囊胚的发育潜能好,各方面结果均较其余数量培养结果更好,囊胚培养时密度不宜过大也不宜过小。     

植物性雌激素金雀异黄素对人心房肌的电生理效应

目的:研究植物性雌激素金雀异黄素(genistein,GST)对人心房肌的电生理效应及其作用机制.方法:应用经典玻璃微电极方法.结果:GST(10-100μmol/L)抑制人心房肌纤维的舒张期(4相)除极化速率(VDD)和起搏细胞放电频(RPF),此外,GST(100 μmol/L)缩短APD_(90).应用L型钙通道开放剂BayK8644(0.5 μmol/L)可拮抗GST对人心房肌纤维的上述电生理效应,但NO合酶阻断剂L-NAME(1 mmol/L)对GST的效应并无影响.结论:GST对人心房肌具有负性变时作用,并可缩短复极化时程.这些效应可能与其抑制钙离子内流有关.     

植物性雌激素金雀异黄素对家兔窦房结起搏细胞的电生理效应

目的:研究植物性雌激素金雀异黄素(genistein,GST)对家兔窦房结起搏细胞的电生理效应及其作用机制.方法:应用经典玻璃微电极方法.结果:GST(10-150μmol/L)不仅以剂量依赖性方式抑制窦房结起搏细胞的零相最大上升速度(V_(max)),舒张期除极速度(VDD),起搏放电频率(RPF)和动作电位幅度(APA),而且延长复极化90％时间(APD_(90)).提高灌流液中钙离子浓度以及应用L型钙通道开放剂Bay K8644(0.5μmol/L)均可逆转GST对起搏细胞的上述电生理效应,但NO合酶阻断剂L-NAME(1 mmol/L)对GST的效应并无影响.结论:GST对家兔窦房结具有负性变时作用,并可延长复极化时程.这些效应可能与其抑制钙离子内流及钾离子外流有关.     

染料木黄酮对人白血病K562细胞凋亡及增殖的影响

目的:观察染料木黄酮(GEN)对人白血病细胞增殖及凋亡的影响,探讨其抗白血病的可能性。方法:不同浓度(10、20、30、40、50 mg/L),不同时间(6、12、24、48、72 h)染料木黄酮作用后,甲基噻唑基四唑(MTT)法检测K562细胞(慢性髓细胞性白血病细胞株)及人外周血单个核细胞增殖;DNA断裂梯带电泳、Annexin-V/PI双染色流式细胞仪分析检测GEN诱导K562细胞凋亡的作用。结果:GEN可有效地抑制肿瘤细胞增殖,而对正常细胞毒性较小(PBM-CIC50=35.5 mg/L和K562 IC50=13.2 mg/L),抑制作用呈明显的时间、剂量—效应关系(P<0.05);GEN体外能够诱导K562细胞凋亡,诱导作用呈时间、剂量依赖关系(P<0.05);结论:GEN具有抗白血病细胞的作用。     

染料木黄酮在Beagle犬体内的药代动力学染料木黄酮在Beagle犬体内的药代动力学

目的研究染料木黄酮(genistein)在Beagle犬体内的药代动力学。方法染料木黄酮ig后用反相高效液相色谱法测定犬血浆、尿及粪便中原型药物浓度,血浆药物浓度-时间数据用3P97药代动力学软件分析。结果Genistein在Beagle犬体内的代谢符合一室模型,ig后0.29 h达药峰浓度,t1/2 Ke为0.52 h。给药后24 h内有10.79%的原型药物由尿排出,21.55%的原型药物由粪便排出。60 h内有13.00%的原型药物由尿排出,有52.46%的原型药物由粪便排出。结论Beagle犬ig染料木黄酮后吸收迅速,血浆中药物的消除速度快,药物主要以原型经尿和粪便排出体外。     

染料木素对离体豚鼠左室乳头肌收缩功能的影响

目的观察染料木素对离体豚鼠左室乳头肌收缩功能的影响,并探讨其作用机制。方法将离体豚鼠乳头肌置于装有KH液的灌流肌槽中,待平衡后,加入各种药物观察乳头肌收缩活动的变化。结果染料木素和异丙肾上腺素相似,可增强豚鼠左室乳头肌的收缩活动,染料木素(1、100μmol.L-1)的作用还具有明显的剂量依赖性。心得安(1μmol.L-1)和异搏定(0.5μmol.L-1)虽可明显阻断异丙肾上腺素(2μmol.L-1)的正性肌力作用,但对染料木素(100μmol.L-1)的心肌收缩增强效应无明显改变;同时发现染料木素(1、10μmol.L-1)对细胞外液Ca2+浓度升高而诱发的心肌收缩力增强也无明显影响。另外,它莫西芬(1μmol.L-1)可明显减弱染料木素的正性肌力作用,但正矾酸钠(1μmol.L-1)对其作用无明显影响。结论染料木素可增强心肌收缩活动,其作用与心肌细胞膜上的β肾上腺素能受体和钙通道激活以及酪氨酸激酶途径无关,可能与肌质网Ca2+的摄取和利用有关。     

染料木黄酮对大鼠下丘脑脑片室旁核神经元放电的影响(英文)

目的研究染料木黄酮(GST)在心血管中枢神经系统的作用。方法应用细胞外记录单位放电技术,在下丘脑脑片上观察GST对静息状态下的室旁核神经元放电的影响。结果①26个脑片分别灌流GST 10,50,100μmol·L~(-1)2 min,有25个脑片放电频率明显降低,且呈浓度依赖性;②用0.2 mmol·L~(-1) L-谷氨酸灌流脑片,7/7个脑片放电频率明显增加,表现为癫痫样放电,在此基础上加灌GST 50μmol·L~(-1)2 min,其癫痫样放电被抑制;③用G蛋白激活的内向整流型钾通道阻断剂四乙胺1 mmol·L~(-1)灌流脑片,约10 min后加入GST 50μmol·L~(-1),8/8个脑片的放电抑制效应被完全阻断;④用一氧化氮合酶抑制剂左旋硝基精氨酸甲酯50μmol·L~(-1)灌流脑片,7/7个脑片的放电频率增加,在此基础上加灌GST 50μmol·L~(-1)2 min,放电被抑制。结论GST可抑制下丘脑室旁核神经元自发放电,并抑制L-谷氨酸诱发的神经元癫痫样放电。这种抑制作用可能与激活G蛋白激活的内向整流型钾通道,促进K~+外流,从而引起细胞膜超极化有关;而与NO释放无关。GST可能通过降低心血管中枢的活动性而产生一定的心血管系统保护作用。