关键词： 雄激素；大鼠；雌激素受体；神经肽Y；瘦素；中药

【摘要】目的探讨9日龄雄激素致不孕大鼠(9d-ASR)肥胖-无排卵机制及滋肾阴药的作用。方法给出生9日龄SD雌性大鼠一次性皮下注射丙酸睾丸酮，建立9d-ASR模型。检测雌激素受体(ER)和神经肽Y(NPY)神经元共存情况；检测9d-ASR下丘脑ER、NPY及促性腺激素释放激素(GnRH)含量变化；测定血雌二醇(E₂)、睾酮(T)、瘦素、促卵泡激素(FSH)、促黄体激素(LH)的变化。结果ASR呈能量失衡致肥胖状态，血E₂、T、瘦素水平明显增加(P＜0.01)，FSH、LH明显低下(P＜0.01)，下丘脑ER存在于NPY神经元上，其数目减少，NPY表达增强，GnRH分泌下降。NPY与ER或GnRH均呈负相关(r=-0.7104,P＜0.01；r=-0.8802,P＜0.01)。瘦素与体重呈正相关(r=0.8977,P＜0.01)，与FSH、LH呈负相关(r=-0.7517,P＜0.01；r=-0.8444，P＜0.01)。用滋肾阴药治疗后，各神经内分泌-代谢指标均恢复正常。结论9d-ASR模型有神经内分泌-代谢失调而致肥胖并引起低促性腺激素性性腺功能低下性无排卵，滋肾阴中药有纠正这些失调而产生减肥及促排卵的作用。

Mechanism of obesity-anovulation in androgen-sterilized rat and the weight reducing and ovulation inducing effects of tonifying Kidney yin herbs

SUN Fei,YU Jin,DA Cuidi,et al

.Obstetric and Gynecologic Hospital,Shanghai Medical University,Shanghai,200011

【Abstract】ObjectiveTo explore the mechanism of obesity and chronic anovulation in androgen-sterilized rat at age of 9 days (9d-ASR) and the effect of herbs of tonifying Kidney yin.Methods9d-ASR model was established by injecting testosterone propionate subcutaneously to SD female rat at age of 9 days. Double immunofluorescent staining was carried out on sections through arcuate nucleus (ARC) to determine whether neuropeptide Y (NPY)-containing neurons expressed estrogen receptors (ER). The immunoreactive amounts of ER, NPY neurons and gonadotropin-releasing hormone (GnRH) fibers of hypothalamic ARC-median eminence (ME) in 9d-ASR were studied with immunohistochemistry. Serum estrogen (E₂), testosterone (T), leptin, follicular stimulating hormone (FSH) and luteinizing hormone (LH) were measured with radioimmunoassay. All parameters were reevaluated after administration of herbs to 9d-ASR.Results Estrogen receptors were predominantly distributed in the cytoplasm of NPY-containing neurons as well as in the nucleus and axons of NPY-synthesizing neurons. Compared with normal group, ASRs were characterized by high metabolism rate, obesity, increase in hypothalamic NPY expression (P＜0.01), decrease in numbers of ER and GnRH secretion (P＜0.01). Levels of serum E₂, T and leptin were significantly higher and FSH and LH levels significantly lower in 9d-ASR than in normal rats (P＜0.01). Significantly negative correlation between NPY and ER or GnRH (r=0.7104, P＜0.01; r=-0.8802, P＜0.01, respectively) were observed, and so were the correlations between leptin and FSH or LH (r=-0.7517, P＜0.01; r=-0.8444, P＜0.01, respectively). Serum leptin showed positive correlation with body weight in 9d-ASR (r=0.8977, P＜0.01). These dramatic neuroendocrine-metabolic changes became normal after feeding 9d-ASR with herbs.ConclusionsObesity and chronic anovulation coexist in 9d-ASR. Elevated E₂ levels may down-regulate the hypothalamic ER oriented NPY neurons, followed by the release of NPY and suppression of GnRH. Effect of the herbs may be involved in reducing body weight and inducing ovulation.

【Key words】Androgen Rat Estrogen receptor Neuropeptide Y Leptin Traditional Chinese herbs

(Chin J Endocrinol Metab, 1999,15:259-262)

机体的生殖能力与其营养及代谢状况有关^〔1〕，能量摄入与消耗处于不平衡状态时能影响生殖功能，如肥胖不仅在一定情况下影响排卵功能，而且也是发生糖尿病和心血管疾病的重要危险因素。遗传性肥胖小鼠模型(ob/ob鼠)表现为肥胖、中枢性卵巢功能低下、不育、高胰岛素血症和胰岛素抵抗、下丘脑神经肽Y(neuropeptide Y, NPY)分泌明显增加等现象^〔2〕，最近已发现这些现象和肥胖基因编码的产物瘦素(leptin)原发性缺乏有关^〔3〕，NPY刺激进食。NPY神经元与下丘脑促性腺激素释放激素(GnRH)神经元有突触联系，NPY能刺激GnRH-促黄体激素(LH)的分泌，但具有性激素尤其是雌激素依赖性^〔4〕。俞瑾等^〔5〕采用9日龄SD雌性大鼠成功地诱发出高雄激素高胰岛素无排卵动物模型——雄激素致不孕大鼠(androgen-sterilized rat, ASR)，发现ASR具有许多类似ob/ob小鼠的症状，如高胰岛素高雄激素血症、胰岛素抵抗、卵巢呈多囊性变、无排卵、不育、性腺功能低下^〔6-8〕，并发现补肾中药可逆转这些现象。张月萍等^〔6〕于1993年注意到9日龄ASR(9d-ASR)尚有肥胖的特点。本实验研究9d-ASR模型中雌激素受体(ER)与NPY及GnRH的关系及瘦素的作用，以及灌服滋肾阴药后这些指标的变化，以探讨9d-ASR肥胖及与不育的关系及中药的减肥-促排卵效果和中枢作用的环节。

材料和方法

一、9d-ASR模型建立及给药方法^〔5〕

9日龄SD雌性大鼠分3组：正常对照组(C组，15只)；9d-ASR组(A组，15只)；9d-ASR给滋肾阴药组(A+H组，15只)。A组及A+H组颈背部皮下一次性注射丙酸睾丸酮1.25mg，C组注射同等量中性茶油。21日龄断乳，25℃恒温(50%湿度)清洁级饲养，不加饲维生素类制品。12小时光照(6:00～18:00)和12小时黑暗周期交替进行。饲料和水充足供给。70日龄起做连续阴道涂片共11天，阴道上皮细胞持续角化，无周期性，无排卵，作为9d-ASR模型。81日龄起，A+H组灌服滋肾阴药“天癸方”(全国名老中医俞瑾验方：生地12g、仙灵脾12g、补骨脂12g、女贞子12g、菟丝子12g、萸肉12g、淮山药12g、茯苓9g、知母12g。上海医科大学药学院生药教研室提取水溶性部分，制成浸膏342mg/ml，每ml含生药3g，按临床剂量20倍给药，每100g体重给药约1ml)，连续21天。A组及C组同法灌服等量蒸馏水。

二、取材

70～100日龄每天记录进食量。101日龄称体重，大鼠不禁食，于动情期用戊巴比妥钠(50mg/kg,i.p.)麻醉，心室灌注，取脑后固定。根据Koning等^〔9〕大鼠脑定位图谱，从A5150～A3180〔弓状核(ARC)-正中隆起(ME)所在的平面〕用冰冻切片机将组织切成30μm厚的切片，进行免疫组化处理。取腔静脉血，离心后取血清置-20℃待放射免疫测定(RIA)。分离腹膜后脂肪垫(retroperitoneal fat pad, RPFP)并精确称重。

三、实验室检测

1.RIA测定：雌二醇(E₂)、睾酮(T)、大鼠瘦素RIA药盒分别购自DPC和美国LINCO Research INC，批间及批内变异均＜8%。特异性大鼠促卵泡激素(rFSH)及大鼠促黄体激素(rLH)RIA药盒由美国National Institute of Diabetes、Digestive & Kidney (NIDDK)和美国National Hormone & Pituitary Program的Parlow博士惠赠，批间及批内变异分别＜10%及＜7%。

2.免疫组织化学检测：分别取各组ARC-ME头、中、尾三部分切片进行免疫组化检测。兔抗鼠ER_α(1:400)及兔抗NPY(1:8000)分别购自Santa Cruz公司及Sigma公司；兔抗GnRH(1:1000)由中科院动物所生殖生物学国家重点实验室庄临之教授惠赠。采用ABC法(药盒购自Santa Cruz公司)。用正常兔血清代替一抗作为阴性对照。免疫阳性物质的相对含量在Alpha Imager^TM2000 Digital Imaging & Analysis Systems (Alpha Innotech公司)上用OptiGreen软件测定其密度(spot densitometry)，以校正背景后区域像素点(pixel)密度平均值表示。

3.ER和NPY双标荧光免疫组化检测：鼠ER单抗IgG₁购自Santa Cruz公司(1:250)，罗达明(TRITC)标记羊抗鼠IgG(1:20)购自Boehringer Mannheim公司；兔抗NPY(1:4000)购自Sigma公司，荧光素(FITC)标记猪抗兔IgG(1:40)为DAKO公司产品。一抗混合物(同时含ER抗体、NPY抗体)孵育37℃1小时，而后4℃48小时，用正常羊、猪血清混合物代替一抗混合物作为阴性对照。荧光二抗混合物孵育37℃1小时，磷酸甘油封片。结果用Leica TCS-NT激光共聚焦显微镜(CLSM)分别于x、y、z轴作0.1μm厚度的FITC和TRITC双通道荧光断层扫描并作三维重建，观察ER和NPY神经元共存情况。

四、统计方法

用SAS 6.12统计软件包对数据进行ANOVA分析，分别采用Dunnett T-test和Student-Newman-Keuls进行检验。相关检验采用Pearson相关分析。以体重作为协变量，瘦素和NPY关系采用协方差分析和Partial相关分析。