Leptin effects on the hypothalamic-pituitary-gonadal axis by activating proopiomelanocortin neurons Correlation between regulatory effects and duration

BACKGROUND：Leptin preserves reproductive functions by stimulating hypothalamic-pituitary-gonadal axis activities at different levels.Some interneurons play an important role in leptin regulation of the gonadal axis.It remains uncertain whether leptin regulates reproductive functions by activating proopiomelanocortin （POMC） neurons.OBJECTIVE：To investigate leptin effects on secretory function of the neuroendocrine-gonadal axis by activating POMC neurons and to observe and verify the relationship between leptin effects and various time points.DESIGN,TIME AND SETTING：The randomized,controlled,animal study was performed at the Basic Institute of Chengde Medical University and the Research Room of Reproductive Immunology of National Research Institute for Family Planning from June to September 2008.MATERIALS：Leptin （Peprotech,USA）,a-melanocyte stimulating hormone and rabbit anti-POMC polyclonal antibody （SC-20148） （Santa Cruz Biotechnology,USA）,follicle stimulating hormone,luteinizing hormone,and gonadotropin releasing hormone enzyme-labeled immunosorbent assay （ELISA） kit （ADL,USA） were used in the present study.METHODS：A total of 60 healthy,female,adult,Wistar rats received 17 （3-estradiol for 5 consecutive days at 15 days after ovariectomy.The rats were randomly assigned to physiological saline （n= 35）,leptin （n = 35）,and a-melanocyte stimulating hormone （n = 20） groups.MAIN OUTCOME MEASURES：Changes in gonadotropin releasing hormone,luteinizing hormone,and follicle stimulating hormone were compared following intraventricular injection of physiological saline,leptin,and a-melanocyte stimulating hormone at various time points.Changes in POMC mRNA and protein expression in the hypothalamus were measured following physiological saline and leptin injection via the lateral ventricle.RESULTS：Compared to the physiological saline group,leptin and a-melanocyte stimulating hormone affected secretion in the hypothalamus-pituitary axis.Leptin affected secretion of gonadotropin releasing hormone,luteinizing hormone,and follicle stimulating hormone,whereas a-melanocyte stimulating hormone inhibited secretion of these hormones.Compared to the physiological saline group,POMC mRNA expression was significantly increased in the hypothalamus at 2 and 4 hours after leptin injection （P〈 0.05）,but expression recovered to physiological saline group levels at 6 hours after injection （P 〉 0.05）.POMC protein expression was significantly increased in the hypothalamus at 4 and 6 hours after leptin injection （P〈 0.05）.CONCLUSION：Leptin affects secretory function of the neuroendocrine-gonadal axis through combined effects on POMC neurons and other pathways.Results suggested that the regulatory effects of POMC neurons were later compared to other neurons.

TEMPORAL ALTERATIONS OF PROOPIOMELANOCORTIN mRNA LEVEL IN RAT HYPOTHALAMIC ARCUATE NUCLEUS FOLLOWING ELECTROACUPUNCTURE

Alterations of proopiomelanocortin (POMC) rnRNA level in the hypothalamic arcuatenucleus were studied in rats grouped under 6h, 24h l 72h, 96h and 96h (EAX2 ) after elec-troacupuncture(EA) using rnolecular hybridization techniques. It was found that after EA, the levelof POMC mRNA rose significantly (P【0.001 ), a maximun being in the 72h group and declining inthe 96h group. In the 96h (EAX2 ) group, where EA was given at the beginning of the 72nd hour after the first one, instead of declining, the mRNA leve1 rose again. The findings may provide an expla-nation for the long postueffect of EA and its accumulation.

HFD-exacerbated Metabolic Side Effects of Olanzapine Are Suppressed by ER Stress Inhibitor

Objective Numerous schizophrenic patients are suffering from obesity primarily attributed to antipsychotic medication and poor dietary habits.This study investigated the progressive deterioration of olanzapine-induced metabolic disorders in the presence of a high-fat diet(HFD)and explored the involvement of endoplasmic reticulum(ER)stress.Methods Female Sprague-Dawley rats fed on a standard chow diet or HFD were treated with olanzapine(3 mg/kg/day)and the ER stress inhibitor 4-phenylbutyric acid(4-PBA,1 and 0.5 g/kg/day)for 8 days.Changes in body weight,food intake,and plasma lipids were assessed.Hepatic fat accumulation was evaluated using oil red O staining.Western blotting and immunofluorescence assays were employed to examine the expression of ER stress markers,NOD-like receptor pyrin domain-containing protein 3(NLRP3),and proopiomelanocortin(POMC)in the hypothalamus or liver.Results Compared to olanzapine alone,olanzapine+HFD induced greater weight gain,increased hyperlipidemia,and enhanced hepatic fat accumulation(P<0.05).Co-treatment with 4-PBA exhibited a dose-dependent inhibition of these effects(P<0.05).Further mechanistic investigations revealed that olanzapine alone activated ER stress,upregulated NLRP3 expression in the hypothalamus and liver,and downregulated hypothalamic POMC expression.The HFD exacerbated these effects by 50%–100%.Moreover,co-administration of 4-PBA dose-dependently attenuated the olanzapine+HFD-induced alterations in ER stress,NLRP3,and POMC expression in the hypothalamus and liver(P<0.05).Conclusion HFD worsened olanzapine-induced weight gain and lipid metabolic disorders,possibly through ER stress-POMC and ER stress-NLRP3 signaling.ER stress inhibitors could be effective in preventing olanzapine+HFD-induced metabolic disorders.

2Hz和100Hz电针加速脑内三种阿片肽基因表达

我室以往的工作证明2Hz和100Hz电针可引起中枢释放不同种类的阿片肽，本工作试图阐明不同频率的电针是否影响三种阿片肽的基因转录。用地高辛标记的反义cRNA探针进行原位杂交，显示大鼠脑内前脑啡肽原（preproenkephalin，PPE），前强啡肽原（preprodynorphin，PPD）和前阿黑皮素原（proopiomelanocortin，POMC）mRNA。结果：（1）低、高频电针均不影响POMCmRNA的水平。（2）对PPE的影响，两种频率电针诱导脑干网状结构头端腹内侧区PPEmRNA升高的幅度相似，2Hz电针诱导视上核、视交叉上核、下丘脑室分核、弓状核、腹内侧核反外侧丘系核的PPEmRNA表达比100Hz电外高得多。（3）对PPD的表达，2Hz电针不改变脑内PPDmRNA的水平，100Hz电针可使现上核、下丘脑室旁核、腹内侧核及脑干臂旁核的PPDmRNA明显升高。鉴于肽类的加速释放必然引起合成的增加，上述结果为不同频率电针加速不同内源性阿片肽的释放和合成，从而发挥镇痛作同，提供了有力的佐证。

不同间隔时间电针对佐剂性关节炎大鼠炎症局部前阿黑皮素和前脑啡肽原mRNA表达的影响

目的:观察不同间隔时间电针对佐剂性关节炎(AA)大鼠的镇痛效应,以探讨针刺镇痛的最佳间隔时间和作用机制。方法:将96只大鼠随机分为对照组、模型组和电针组,每组再分为3 h、6 h、12 h和24 h小组,以Freund’s完全佐剂造成AA大鼠模型,电针选取悬钟和昆仑穴,分别以3 h、6 h、12 h和24 h作为治疗间隔时间,连续治疗6 d,以痛阈、炎症局部前阿黑皮素(POMC)mRNA和前脑啡肽原(PENK)mRNA表达作为观察指标。结果:间隔24 h电针能显著提高AA大鼠的痛阈;不同间隔时间电针均能促进炎症局部POMC mRNA和PENK mRNA的表达。结论:间隔24 h电针可明显提高对AA大鼠的镇痛效应;不同间隔时间电针镇痛效应与促进炎症局部阿片肽基因表达有关。

急性操作胁迫对刀鲚应激反应相关神经内分泌因子的影响

为深入了解刀鲚应激反应中相关神经内分泌因子作用的分子机理,采用手工捕捉的方式对刀鲚进行了急性操作胁迫。通过放射免疫法和化学发光法测定刀鲚应激反应后头肾和血浆皮质醇含量的变化,结果显示,刀鲚胁迫刺激后血浆皮质醇含量极显著性升高,血浆皮质醇浓度平均升高56.48%,头肾皮质醇含量显著性升高,头肾皮质醇浓度平均升高49.68%,表明急性操作胁迫确实引起刀鲚的应激反应。通过同源克隆的方法获得刀鲚促肾上腺皮质激素释放激素(CRH)、硬骨鱼紧张肽(UI)、阿黑皮素原(POMC)基因的部分序列,并应用实时荧光定量PCR(RT-qPCR)方法检测上述神经内分泌因子mRNA的表达变化,结果显示,CRH基因的表达水平极显著性下降,POMC基因的表达水平显著性下降,UI基因的表达水平有下降趋势但不显著。上述结果显示,皮质醇、CRH、UI和POMC等神经内分泌因子通过鱼类下丘脑—脑垂体—肾间腺轴参与刀鲚应激反应的调节,为进一步了解刀鲚胁迫应答的作用机理,实现对其有效调控打下基础。

白芷总挥发油对疼痛模型大鼠的β-内啡肽、促肾上腺皮质激素、一氧化氮及前阿黑皮素的影响

目的 :探讨白芷总挥发油 (essentialoilofRadixAngelicaeDahuricae ,EOAD)对甲醛所致的伤害性疼痛模型大鼠的β 内啡肽、促肾上腺皮质激素 (ACTH)、一氧化氮 (NO)及前阿黑皮素 (Proopiomelanocortin ,POMC)的影响。方法 :分别采用放射免疫法测定 β 内啡肽、ACTH ,化学法测定NO的含量 ;并用原位杂交方法研究EOAD对前阿黑皮素信使核糖核酸 (POMCmRNA)基因表达的影响。结果 :EOAD能显著升高甲醛所致的伤害性疼痛模型大鼠下丘脑的 β 内啡肽、NO含量 ;使下丘脑、脑干POMCmRNA表达阳性细胞数大幅度增加 ;而对血中的ACTH没有影响。结论 :EOAD可能通过增加中枢POMCmRNA表达阳性细胞数和内源性镇痛物质含量 ,激活内源性镇痛机制而镇痛 ,其作用部位可能在中枢。

冷应激对大鼠垂体前叶离体细胞ACTH分泌和POMC基因表达的影响及锌的保护作用

目的 :研究冷应激对大鼠垂体前叶离体细胞ACTH分泌和POMC基因表达的影响及锌的保护作用 .方法 :采用原代培养的大鼠垂体前叶离体细胞 ,培养液中加入不同剂量的锌 ,并给予低温处理 .放射免疫法测定培养液中的ACTH ,斑点杂交法 (Dotblot)检测POMCmRNA水平 .结果 :一定浓度的锌对垂体前叶离体细胞ACTH的分泌有明显的促进作用 ;冷刺激后 ,垂体前叶细胞ACTH的分泌明显增加 ,加锌组ACTH增加的幅度有所降低 ;冷刺激使POMCmRNA的表达明显增强 ,补锌可使冷暴露大鼠垂体前叶POMCmRNA的表达水平有所下降 .结论

针刺足三里穴对冷应激性胃溃疡大鼠下丘脑与肾上腺SP和POMC表达的影响

目的:研究针刺足三里穴对冷应激性急性胃溃疡的保护作用,以及应激大鼠下丘脑和肾上腺SP和POMCmRNA的表达情况.方法:大鼠22只随机分成3组:正常对照组(6只)、单纯应激组(8只)、针刺预防应激组(8只).针刺预防组先给于针刺足三里穴,再冷应激.胃黏膜溃疡指数和RT-PCR法研究针刺足三里对大鼠的冷应激性溃疡的保护作用和下丘脑、肾上腺的SP、POMC的表达,经图像分析系统照相并进行半定量.结果:与单纯应激组比较,针刺足三里穴可以明显减少冷应激造成的溃疡指数(9.75±1.91vs26.25±4.40,P<0.01),抑制应激造成的皮质醇分泌增加(66.83nmol/L±12.25nmol/Lvs104.38nmol/L±8.31nmol/L,P<0.01),上调下丘脑的SP的表达(1.02±0.42vs0.45±0.12,P<0.05),下调肾上腺SP的表达(1.88±0.82vs2.93±1.08,P<0.05);下丘脑POMC在应激过程中表达增高,针刺能抑制POMC的表达,刺预防应激组与单纯应激组相比差异显著(0.56±0.14vs0.82±0.19,P<0.01);肾上腺中无POMC表达,不参与应激过程.结论:针刺对冷应激溃疡具有保护作用,该种保护作用是通过对生理性上调下丘脑SP的表达,下调POMC的病理性表达及抑制肾上腺SP而实现的.

调节食欲生殖的信号肽在雄激素致不孕大鼠中的表达及滋肾阴药的作用

目的 探讨雄激素不孕大鼠 (ASR)肥胖及无排卵的中枢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ASR模型。用原位杂交、双标原位杂交的方法检测与肥胖发生有关的几种信号肽 /激素如神经肽Y(NPY)、瘦素 (leptin)受体长体 (OB Rb)、前阿黑皮素原 (POMC)mRNA在ASR下丘脑弓状核 (ARC)的表达含量和其关系 ,以及灌服滋肾阴药后上述指标的变化。结果 ASR呈肥胖、无排卵状态。ASR的ARC中NPYmRNA神经元上有OB RbmRNA表达 ,NPYmRNA神经元与POMCmRNA神经元有突触联系。NPY和POMC基因表达水平明显增加 (P <0 .0 1) ,OB Rb 基因表达水平明显降低 (P <0 .0 1)。中药治疗ASR后 ,大鼠出现减肥和排卵现象 ,ARC中NPY、POMC基因表达下降 ,OB Rb 基因表达上升至正常大鼠水平 (P >0 .0 5 )。结论 ASR肥胖及无排卵可能与NPY、POMC基因过表达、OB Rb mRNA数目减少有关 。

下丘脑黑皮质素及中脑多巴胺系统对奖赏行为的调控

动机行为受生理需求相关神经环路的调控,包括管理摄食、能量代谢等内在动机行为的下丘脑黑皮质素(melanocortin,MC)系统和负责奖赏环路的中脑多巴胺(dopamine,DA)系统.MC系统中前阿黑皮素原(proopiomelanocortin,POMC)神经元与刺豚鼠相关蛋白(agouti-related protein,AgRP)神经元合成与分泌的递质及神经肽协同完成了对摄食等动机行为的调控,且DA系统通过调节奖赏环路参与摄食等动机行为的发生过程.此外,高强度的激活DA系统是成瘾性药物的共同特征,当DA系统激活与用药行为反复关联后,部分使用者进入药物成瘾状态,他们表现出强迫性的觅药动机.已有研究提示,药物成瘾的过程可能是药物导致动机行为调控中枢发生适应性改变的过程,这个变化反之促发了强迫性觅药行为的形成.本文将从下丘脑黑皮质素系统两种主要的神经元——POMC神经元和AgRP神经元——在对于摄食和用药相关的奖赏行为调控作用入手,分析它们与DA系统的相互作用模式,论述下丘脑黑皮质素系统的功能失调与药物成瘾的关系.

体复康对运动性疲劳大鼠脑组织中阿黑皮素原基因表达动态变化的影响

目的 :从基因转录水平阐明运动性疲劳产生及药物调节的机理。方法 :采用生物素标记阿黑皮素原 (POMC)cRNA探针 (Bio cRNA)原位杂交法结合图像分析 ,观察中药复方体复康对运动性疲劳大鼠不同脑区POMCmRNA表达。结果 :对于慢性运动应激 ,体复康能增强POMC在海马CA1 区、下丘脑部位的基因表达 ,而能降低POMC在额叶皮层的基因表达。长时期 (共 7周 )大强度的跑台运动 (速度由 1 5m/min递增至 3 5m/min ,运动时间为每天 2 0～2 5min)的大鼠 ,末次运动后POMC基因表达在额叶皮层、海马CA1 下降 (前者至运动结束后 3h仍未恢复 ,后者至运动结束后 3 0min即恢复 ) ,在下丘脑则增强 ;体复康能增强末次运动后POMC在额叶皮层、海马CA1 区、下丘脑等脑区的基因表达 ,尤其是在运动结束后 3h最为显著。结论 :POMCmRNA表达在运动性疲劳大鼠不同脑区呈现不同的动态变化趋势 ,体复康对其POMCmRNA表达有重要动态调节作用。

瘦素经POMC神经元对神经内分泌-生殖轴的影响

目的:探讨瘦素(leptin)对神经内分泌-生殖轴的可能调节机制。方法:建立卵巢摘除补充雌激素(OEP)大鼠模型,动物随机分为leptin组、α-黑素细胞刺激素(α-MSH)组和生理盐水对照组,于侧脑室注射后1h、2h、4h和6h分别取血和脑组织。ELISA法检测血中促性腺释放激素(Gn-RH)、黄体生成素(LH)和卵泡刺激素(FSH)浓度。Western印迹检测下丘脑组织中阿片促黑激素皮质素原(POMC)蛋白量的变化。结果:侧脑室注射leptin可促进GnRH、FSH、LH的分泌,而注射α-MSH则抑制GnRH、FSH和LH的分泌。注射leptin显著增加下丘脑中的POMC含量。结论:Leptin可能通过POMC神经元或与其他途径共同影响下丘脑-垂体生殖轴的分泌功能;POMC神经元发挥的调节作用可能晚于或弱于其它神经元。

侧脑室注射瘦素对大鼠迷走复合体阿黑皮素原的影响

目的研究侧脑室注射瘦素对大鼠迷走复合体阿黑皮素原的影响,探讨瘦素是否同下丘脑弓状核一样也在迷走复合体对阿黑皮素原进行调节,从而发现瘦素在迷走复合体上发挥作用的可能路径。方法 SD大鼠36只,随机分为试验组和对照组,每组18只,试验组根据注射后处死的时间(1、3、5 h)分成3个亚组,每组6只。大鼠侧脑室留置不锈钢导管1周,自由恢复及驯养,实验动物在清醒状态下于侧脑室注射瘦素3.5μg/μl,对照组注射3.5μg/μl0.9%氯化钠溶液。苏木精-伊红染色定位迷走复合体,测定阿黑皮素原在该处的表达。结果中枢注射瘦素组大鼠侧脑室注射瘦素后1、3、5 h采用免疫组化检测迷走复合体上阿黑皮素原的表达阳性细胞计数、表达阳性细胞积分光密度与生理盐水和对照组比较,差异均无统计学意义(P>0.05),中枢注射瘦素组1、3、5 h亚组组间迷走复合体上阿黑皮素原的表达阳性细胞计数、表达阳性细胞积分光密度比较,差异无统计学意义(P>0.05)。结论侧脑室注射瘦素后不能引起迷走复合体上阿黑皮素原的增多。

鲇POMC cDNA的克隆与表达分析

采用RT-PCR和cDNA末端快速扩增法(RACE)分离、克隆出鲇(Parasilurus asotus)垂体阿黑皮素原(proopiomelanocortin,POMC)基因全长cDNA序列,共1 099 bp,包括129 bp 5′非翻译区、648 bp阅读框以及322 bp 3′非翻译区,阅读框共编码215个氨基酸,POMC前体蛋白的结构主要由信号肽(Met1-Ala28)、N端多肽(Gln29-Asp102)、促肾上腺皮质激素(ACTH,Ser 105-Met144)、α-黑色素细胞刺激激素(α-MSH,Ser105-Val 117)、促肾上腺皮质激素样中叶肽(CLIP,Arg123-Met144)、γ-促脂解激素(γ-LPH,Glu145-Ser158)、β-黑色素细胞刺激激素(β-MSH,Asp161-Ser177)和β-内啡肽(β-EP,Tyr178-Leu215)构成。分子量计算值为24.66 kD,理论等电点为7.25。同源性分析表明鲇与斑点叉尾的相似性高达88%,其构成多肽中α-MSH、β-MSH、β-EP、CLIP保守性逐渐降低,而γ-MSH、连接肽(JP)、γ-LPH是变异比较大的区域,同时鲇POMC中不含有γ-MSH,连接肽和部分γ-LPH也不存在。生物信息学分析显示鲇POMC基因所编码的蛋白质以亲水性区域为主,具有丰富的B细胞抗原位点。定量分析结果表明POMC mRNA主要在垂体中高表达,在非垂体组织的脑中表达量最高,鳃、头肾、肝、脾、肌肉、皮肤等组织也有少量表达。

斜带石斑鱼阿片黑素促皮质素原全长cDNA克隆及序列分析

通过构建斜带石斑鱼垂体cDNA文库和随机测序 ,克隆了其阿片黑素促皮质素原 (Proopiomelanocortin ,POMC)全长cDNA。斜带石斑鱼POMC全长cDNA为 86 3bp(含Poly(A) ) ,编码的POMC多肽前体为 2 19aa。氨基酸序列比较分析表明 :斜带石斑鱼POMC前体包含有促肾上腺皮质激素 (Adrenocorticotropin ,ACTH) ,α 促黑素 (α melanocytestim ulatinghormone ,α MSH) ,β 促黑素 ( β MSH) ,γ 促脂素 (γ lipotrophichormone,γ LPH) ,β 内啡肽 ( β endorphin)等 ,但缺失了γ 促黑素 (γ MSH)和大部分连接区。斜带石斑鱼POMC与哺乳动物POMC的同源性为 39%— 4 2 %左右 ,与鸟类的同源性为 4 2 %左右 ,与两栖类的同源性为 36 %— 4 1% ,与其他鱼类POMC的同源性为 38%— 77%。斜带石斑鱼和罗非鱼的POMC的ACTH功能区都为 4 0个氨基酸残基 ,而其他脊椎动物为

调神益肾针法对更年期大鼠下丘脑阿片促黑素皮质素原mRNA表达的影响

目的 :研究调神益肾针法对更年期雌性大鼠下丘脑阿片促黑素皮质素原mRNA表达的影响。方法 :采用逆转录 聚合酶链反应技术。结果 :更年期大鼠下丘脑阿片促黑素皮质素原mRNA表达明显降低 ;经针刺干预后 ,调神益肾组该基因的表达明显上调 ,补肾健脾组则无明显变化。结论 :增强下丘脑阿片促黑素皮质素原mRNA的表达可能是调神益肾针法治疗更年期综合征的机制之一。

The effect of traditional Chinese medicine──Tian Gui Recipeon obesity and anovulation in androgen-sterilized rats

Objective: A traditional Chinese medicine Tian Gui Recipe (TGR) has been used to effectively treat clomiphene resistant anovulatory disease and obesity, especially in polycystic ovary syndrome (PCOS) cases with hyperinsulinemia. The effect of TGR on obesity and anovulation was investigated in androgen-sterilized rats (ASR). Methods: Female SD rats at the age of 9 days were divided into 3 groups. Group ASR (n=15): anovulation was demonstrated at the age of 70 days by vaginal smear in rats while 1. 25 mg of testosterone propionate was injected subcutaneously on its 9th neonatal day. Group A+H (n = 25): TGR were administered to ASR at the age of 80 days for 3 weeks. Rats with regular estrous cycle and ovulation after herbal treatment were included in this group. Group C (n = 15): normal ovulated rats were recruited. Around the age of 112 days or on proestrous day, all rats were sacrificed under anesthesia. Serum leptin, testosterone (T), estrogen (E2), follicular stimulating hormone (FSH), luteinizing hormone (LH) levels were measured with radioimmunoassay (RIA). Double immunofluorescent staining combined with confocal laser scanning microscope and dual in situ hybridization were carried out on sections through areas of arcuate nucleus (ARC) to determine whether estrogen receptor (ER) immunoreactivity (IR) or long form of leptin receptor (OB-Rb) mRNA were expressed in neuropeptide Y (NPY) immuno-and mRNA-containing neurons, and its relationship to proopiomelanocortin (POMC) mRNA-containing neurons. Immunohistochemistry and in situ hybridization were used to observe the levels of ER, NPY, gonadotrophin releasing hormone (GnRH) and gene expression levels on NPY, OB-Rb, POMC. Meanwhile, the criteria of energy state, including daily food intake, retroperitoneal fat depot pad and body weight, were measured and evaluated. Values of immunohistochemistry and in situ hybridization were expressed in mean optic density (MOD). Results: Seventeen out of the 25 rats (68%) in Group A+H were ovulated after herbal treatment. ASR characterized with significantly high metabolic rate, energy imbalance and obesity (P<0. 01, P<0. 01, P<0. 01). The mean serum leptin, T, E2 levels of group ASR were significantly higher than those of Group C and Group A+H (P< 0. 01,P<0. 01 ), while FSH, LH levels were lower (P<0. 01, P<0. 01 ). ERs were proved to be distributing in NPY-containing neurons, OB-Rb coexpressed in NPY-syn- thesizing neurons, and both NPY and POMC mRNA expressing neurons overlapped in ARC. Compared to Group C, levels of NPY mRNA and NPY, POMC mRNA expressions were increased (P<0. 01, P<0. 01, P<0. 01 ), OB-Rb mRNA, ER and GnRH immunoreactive expression were decreased significantly in the hypothalamus of ASR (P<0. 01, P<0. 01, P<0. 01 ). These dramatic neuroendocrine changes became normal in ovulated ASR after the herbal treatment (all P>0. 05). Conclusion: The elevated peripheral E2 caused by high T and leptin levels in ASR may down-regulate their corresponding receptors oriented on hypothalamic NPY- containing neurons respectively, therefore challenge the NPY mRNA and NPY, POMC mRNA overexpressions. The overexpression, of NPY, POMC gene transcription and translation stimulate food intake, promote the deposit of adipocyte tissue, and inhibit GnRH expression and GnRH/FSH, LH release, which contribute to the occurrence of obesity and anovulation in ASR. TGR may reverse these abnormal neuroendocrine cascades by lowering the levels of T, E2 and leptin.

The effect of traditional Chinese medicine“Tian Gui Recipe (TGR)”on obesity and anovulation in androgen-sterilized rats(ASR)

Objectives: A traditional Chinese medicine “Tian Gui Recipe (TGR)” has been used to effectively treat clomiphene resistant anovulatory disease and obesity, especially in polycystic ovary syndrome (PCOS) cases with hyperinsulinemia. The effect of TGR on obesity and anovulation was investigated in androgen-sterilized rats (ASR).Methods: Female SD rats at the age of 9 days were divided into 3 groups. Group ASR (n=15): anovulation was demonstrated at the age of 70 days by vaginal smear in rats while 1.25 mg of testosterone propionate was injected subcutaneously on its 9th neonatal day. Group A+H (n=25): TGR were administered to ASR at the age of 80 days for 3 weeks. Rats with regular estrous cycle and ovulation after herbal treatment were included in this group. Group C (n=15): normal ovulated rats were recruited. Around the age of 112 days or on proestrous day, all rats were sacrificed under anesthesia. Serum leptin, testosterone (T), estrogen (E2), follicular stimulating hormone (FSH), luteinizing hormone (LH) levels were measured with radioimmunoassay (RIA). Double immunofluorescent staining combined with confocal laser scanning microscope and dual in situ hybridization were carried out on sections through areas of arcuate nucleus (ARC) to determine whether estrogen receptor (ER) immunoreactivity (IR) or long form of leptin receptor (OB-Rb) mRNA were expressed in neuropeptide Y (NPY) immuno- and mRNA-containing neurons, and their relationship to proopiomelanocortin (POMC) mRNA-containing neurons. Immunohistochemistry and in situ hybridization were used to observe the levels of ER-, NPY-,gonadotrophin releasing hormone (GnRH)-IR and gene expression levels of NPY, OB-Rb, and POMC in ARC. Meanwhile, the criteria of energy state,including daily food intake, retroperitoneal fat depot pad and body weight,were measured and evaluated. Values of immunohistochemistry and in situ hybridization were expressed in mean optic density (MOD).Results:Seventeen out of the 25 rats (68%) in Group A+H were ovulated after herbal treatment. ASR characterized with significantly high metabolic rate, energy imbalance and obesity (P<0.01,P<0.01,P<0.01).The mean serum leptin, T, E2 levels of ASR were significantly higher than that of Group C and Group A+H (P<0.01,P<0.01), while being contraryto that of FSH, LH levels (P<0.01,P<0.01). ERs were proved distributing in NPY-containing neurons, OB-Rb coexpressed in NPY-synthesizing neurons, and both NPY- and POMC- mRNA expressing neurons overlapped in ARC. Compared to Group C, levels of NPY mRNA and NPY, POMC mRNA expressions increased (P<0.01,P<0.01,P<0.01),OB-Rb mRNA, ER and GnRH immunoreactive expression decreased significantly in the hypothalamus of ASR (P<0.01,P<0.01,P<0.01).These dramatic neuroendocrine changes became normal in ovulated ASR after the herbal treatment (all P>0.05).Conclusion: The elevated peripheral estrogen caused by high testosterone and leptin levels in ASR may down-regulate their corresponding receptors oriented on hypothalamic NPY-containing neurons respectively, therefore challenge the NPY mRNA and NPY,POMC mRNA overexpression. The overexpression of NPY, POMC gene transcription and translation stimulate food intake, promote the deposit of adipocyte tissue, and inhibit GnRH expression and GnRH/FSH, LH release, which contribute to the occurrence of obesity and anovulation in ASR. “TGR” may reverse these abnormal neuroendocrine cascades by lowering levels of testosterone, estrogen and leptin.

半滑舌鳎(Cynoglossus semilaevis)体表色素细胞观察及POMC表达特性分析

为认识养殖半滑舌鳎无眼侧黑化的细胞学特性,利用显微观察方法研究了其皮肤黑色素细胞、黄色素细胞和虹彩细胞的形态特征,比较了3种色素细胞在有眼侧皮肤、无眼侧正常和黑化皮肤中的数量分布特征。为进一步揭示无眼侧黑化的分子机制,克隆了半滑舌鳎POMC基因的c DNA序列。结果显示,黑色素细胞较大,含黑色和棕色的色素颗粒,有树突状分枝不明显和延伸成放射状两种形态;黄色素细胞较小,含黄色素颗粒;虹彩细胞最小,含鸟粪素颗粒。半滑舌鳎POMC基因的c DNA序列长910 bp,包括一个114 bp的5'非翻译区和一个154 bp的3'非翻译区,开放阅读框长度为642 bp,共编码213个氨基酸,包含ACTH、α-MSH、β-MSH、γ-LPH、β-内啡肽5个多肽序列,但缺失γ-MSH和大部分连接区。半滑舌鳎POMC基因的氨基酸序列与其他鱼类的同源性为30%-64%。定量PCR分析显示,POMC m RNA主要在垂体中表达,其次是脑、性腺和无眼侧黑化皮肤。正常与黑化皮肤中的差异表达结果显示,无眼侧黑化皮肤中POMC m RNA表达量最高,并与有眼侧皮肤和无眼侧正常皮肤中的POMC m RNA表达量差异显著,揭示了POMC的表达与无眼侧黑化性状密切相关。