绵羊下丘脑Ghrlein通过神经元投射参与繁殖调控的研究

Ghrelin from gastrointestinal peptide hormone induces food intake and is associated with increased NPY/AgRP expression by ERα in the hypothalamus. It is well known fact that reproductive function is highly dependent on food intake, body status or metabolic disorders. However, it remains unclear the relationship of neurons of ghrelin involve the reproduction by metabolism balance within sheep hypothalamus. we therefore investigate connection with hypothalamus and ovary using pseudorabies virus (PRV) for the retrograde transsynaptic. Then confirm co-expression neurons of Ghrelin/AgRP/ERα and NPY/GHSR/ERα by triple fluorescence immunohistochemistry at different estrus cycle, also plasma concentrations of Ghrelin and E2 were compared. Based on above evidences we next will evaluate the level of Ca2+ and cAMP in different treatment after culturing the neurons from hypothalamus, and ERα/UCP1/pERK-MAPK/pAMPK/pAKT/CaMKII were analyzed in vitro and vivo to understand the how the ghrelin neurons within hypothalamus involve the regulation of never pathway. Then make sure the distribution net and potential roles of ghrelin in ovine reproduction and provided some theories for the hypothesis that the relationship between energy metabolism and neuroendocrinology mediate the reproduction.

胃肠道产生的肽类激素Ghrelin可通过雌二醇受体(ERα)激活下丘脑内NPY/AgRP神经元促进采食，已知能量代谢平衡是生殖系统的重要物质基础，但绵羊下丘脑Ghrelin神经元通过能量代谢平衡参与繁殖调控的作用方式尚有诸多问题有待澄清。为此本研究首先通过PRV病毒逆行追踪确定下丘脑与卵巢间是否存在与Ghrelin相关的神经作用通路，免疫荧光三标检测发情前、中、后Ghrelin/AgRP/ERα和NPY/GHSR/ERα在下丘脑内的神经投射差异，分析血液内Ghrelin和E2间的作用关系，并将下丘脑神经元体外培养通过正调和负调检测第二信使Ca2+和cAMP的变化，以及ERα/UCP1/pERK-MAPK/pAMPK/pAKT/CaMKII分子和蛋白水平的变化，以期从神经通路、神经元间投射、电生理和信号通路的角度，解释Ghrlein在“能量代谢-神经内分泌”联动中的作用及与繁殖间的关系。

下丘脑Ghrelin神经元参与繁殖调控方式尚有诸多不解之处，为此本研究探讨了ghrelin与相关神经肽投射调节繁殖周期，结果如下：. 1 羊脑Ghrelin神经肽呈广泛性表达，尤其前脑、中脑、后脑以及脊髓膨大部均有密集分布，与卵巢上亦呈广泛性表达一致。. 2 羊脑内表达已知能量代谢调节因子与小鼠脑内有相似性，即可卡因-苯丙胺调节转录肽(CART)、催产素(OXT )、细胞色素P450(CYP17A1)、3β-羟类固醇脱氢酶(3β-HSD)、黑皮质素4型受体(MC4R)、细胞外信号调节激酶(pERK)，且存在一定的共表达。. 3 类固醇合成相关基因类固醇生成急性调节蛋白(StAR)、CYP17A1、HSD3B1参与绵羊发情周期变化的进程，且与AMPK和ERK1/2通路积极相关，提示类固醇激素合成相关基因与卵泡发育和黄体功能的变化密切相关。. 4 Ghrelin上调ERα/CamK-II/pERK蛋白表达，同时具有随时间递增效果持。. 5 外周注射Ghrelin (50μg/kg)后，在发情周期内激活雌激素受体α(ERα)阳性神经信号的表达，并刺激刺鼠相关肽(AgRP)mRNA的表达。此外，Ghrelin在发情期抑制OXT阳性神经元表达，同时降低了雌二醇的血清浓度。. 6 侧脑室内注射Ghrelin (1 nmol)，可降低雌二醇和孕酮的血清浓度，并抑制促卵泡激素和黄体生成激素的分泌。尽管Ghrelin抑制了下丘脑中3β-HSD mRNA和蛋白表达水平，但并未影响下丘脑StAR和CYP17A1的表达。Ghrelin的中枢调节作用间接抑制卵巢中StAR，CYP17A1和3β-HSD mRNA和蛋白表达水平。. 综上，Ghrelin可通过弓状核AgRP回路抑制催产素阳性神经元的表达，从而参与繁殖调控。位于下丘脑Ghrelin神经元可通过调节卵巢中StAR，CYP17A1和3β-HSD表达，抑制血清雌二醇和孕酮分泌。