妊娠、分娩以及泌乳期大鼠下丘脑中食欲素及其受体表达的变化

目的 观察大鼠下丘脑中食欲素(Orexin)及其受体(OX1R、OX2R)在妊娠、分娩以及泌乳期表达的变化,以探讨Orexin与生殖功能之间的关系.方法 通过竞争性逆转录多聚酶链式反应(Competitive RT-PCR)和免疫组织化学方法测定大鼠下丘脑中Orexin前体(Prepro-OX)、Orexin-A、OX1R和OX2R在妊娠、分娩以及泌乳期的表达.结果 Orexin-A和OX1R阳性神经元主要分别存在于大鼠下丘脑外侧区(LHA)以及妊娠和泌乳期大鼠的下丘脑室旁核 (PVN)和视上核(SON).泌乳第1d的Prepro-Orexin mRNA和OX1R的表达水平明显高于妊娠后期和泌乳期.OX2R的表达在生殖各时期之间没有明显改变.结论 Orexin可能参与大鼠泌乳早期的生殖功能调节,下丘脑的PVN及SON可能是其重要的作用部位.     

Orexin-A对肥胖抵抗大鼠和高脂饮食诱导肥胖大鼠摄食和自由活动的影响

 目的: 探讨肥胖抵抗(OR)大鼠、高脂饮食诱导肥胖(DIO)大鼠及正常SD大鼠的摄食量和自由活动量的差异及食欲肽(orexin-A)在其中的作用。方法: 分别于OR、DIO和SD大鼠下丘脑头端外侧区(rLHa)埋置套管,经套管注射不同剂量(0、31.25、62.5、125和250 pmol)的orexin-A,观察大鼠2 h内摄食量和自由活动情况;采用real-time PCR检测下丘脑和rLHa prepro-orexin、orexin-A受体(OX1R)和 orexin-B受体(OX2R)的 mRNA表达;采用放射免疫分析法检测下丘脑和rLHa OX1R和OX2R的蛋白含量。结果: rLHa微量注射orexin-A可显著增加OR、DIO和SD大鼠的摄食量(P<0.05);rLHa微量注射orexin-A后,OR和SD大鼠的自由活动量比DIO大鼠显著增高(P<0.05)。OR大鼠rLHA OX1R和OX2R的mRNA及蛋白表达均明显高于DIO和SD大鼠(P<0.05)。结论: 下丘脑orexin-A参与肥胖和正常大鼠能量代谢调控,其中对OR大鼠的调控效应最佳。     

食欲素-B抑制大鼠脑缺血再灌注损伤

目的研究食欲素-B(orexin-B,OXB)对大鼠脑缺血再灌注的神经保护作用及其分子机制。方法制备大鼠大脑中动脉栓塞模型(MCAO),将大鼠随机分为:假手术组(control)、缺血再灌注组(I/R)、缺血再灌注+PBS组(I/R+PBS)和缺血再灌注+orexin-B组(I/R+OXB);通过神经功能评分确定模型是否成功;TTC染色法测定大鼠脑梗死体积;Western blot检测海马区食欲素受体2(OX2R)、p-AKT和p-GSK-3β蛋白表达;跳台实验检测大鼠学习与记忆。结果 I/R组海马中OX2R及p-AKT蛋白表达较对照组减少(P<0.05),p-GSK-3β蛋白表达增加(P<0.05),而I/R+OXB组上述变化明显减轻(P<0.05);I/R+OXB组脑梗死体积明显减少,I/R组潜伏期时间减少,错误次数增多(P<0.05),而I/R+OXB组上述变化显著减小(P<0.05)。结论食欲素-B抑制脑缺血再灌注损伤,可能与增强p-AKT活性、抑制p-GSK-3β活性有关。     

肥胖大鼠下丘脑前增食欲素原表达降低

肥胖对人体的健康已构成严重威胁。增食欲素(orex-ins)A和B是2种主要由下丘脑分泌的神经肽,均来源于同一前体———前增食欲素原(prepro-orexin)的蛋白水解产物,通过功能性受体增食欲素受体-1和2(orexin receptor-1OX1R;orexin receptor-2 OX2R)参与摄食和能量代谢调节。本研究     

下丘脑室旁核外源性orexin-A对肥胖大鼠摄食的影响及NPY受体信号的调控

目的:探讨饮食诱导肥胖(DIO)大鼠神经肽Y(NPY)受体信号通路是否参与下丘脑室旁核(PVN)外源性注射食欲素A(orexin-A)对摄食和葡萄糖敏感(GS)神经元兴奋性的调控。方法:采用荧光免疫组织化学实验观察PVN中orexin-A受体(即食欲素1型受体,OX1R)和NPY受体Y5(NPY-5R)的表达;采用单细胞外放电记录观察orexin-A对PVN内GS神经元兴奋性的影响;分别于SD大鼠和DIO大鼠PVN埋置套管,经套管注射orexinA、OX1R拮抗剂SB-334867和NPY-5R拮抗剂CGP-71683,观察大鼠0~2 h和0~4 h摄食量。结果:DIO大鼠PVN中OX1R和NPY-5R的表达显著高于SD大鼠。Orexin-A抑制PVN内葡萄糖抑制性(GI)神经元,兴奋葡萄糖兴奋性(GE)神经元,但是orexin-A对GS神经元的兴奋或抑制效应可被NPY-5R拮抗剂CGP-71683部分阻断,且与SD大鼠相比,orexin-A对DIO大鼠PVN内GS神经元兴奋或抑制效应更加明显。PVN内注射orexin-A可增加SD大鼠和DIO大鼠摄食量。但是orexin-A诱导的促摄食效应被NPY-5R拮抗剂CGP-71683部分阻断。与SD大鼠相比,orexin-A诱导的促摄食效应在DIO大鼠中更加明显。结论:PVN内外源性注射orexin-A可能主要通过OX1R信号通路参与大鼠摄食和GS神经元兴奋性调控,NPY-5R信号也参与了该过程调控,在DIO大鼠中更敏感。     

高原急性缺氧对大鼠食欲及其下丘脑GLP-1 mRNA表达的影响

 目的： 探讨高原急性缺氧对大鼠食欲及其下丘脑胰升血糖素样肽-1 mRNA表达的影响。方法: 雄性Wistar大鼠随机分为平原对照组、3 000 m 缺氧组、4 000 m 缺氧组、5 000 m 缺氧组和 6 000 m 缺氧组，各缺氧组设置缺氧后1、2、3 d 3个时点。观察缺氧环境下大鼠食欲的变化；采用RT-PCR方法检测大鼠下丘脑中GLP-1 mRNA的表达。 结果: ① 高原急性缺氧后，大鼠食欲减退。②对照组和缺氧组大鼠下丘脑中均存在GLP-1 mRNA的表达。高原急性缺氧后，下丘脑GLP-1 mRNA表达增加，并随着海拔高度的上升和缺氧时间的延长逐渐增加 (P<0.05, P<0.01)。结论: 高原急性缺氧后下丘脑GLP-1 mRNA表达增加，可能是大鼠食欲减退的重要原因之一。     

orexin A和orexin 1型受体在新生大鼠与成年大鼠延髓分布的比较

本文研究orexinA(OXA)和orexin 1型受体(OX1R)在新生大鼠与成年大鼠延髓的分布及比较。新生(1～5d)和成年(6～8周)SD大鼠,取其延髓,采用免疫组织化学方法和图像分析技术,观察OXA和OX1R在新生与成年SD大鼠延髓的分布。结果显示,OXA免疫反应阳性纤维和OX1R免疫反应阳性细胞在新生和成年SD大鼠延髓内有广泛分布,主要分布在延髓腹外侧区(ventrolateralmedulla,VLM)和舌下神经核(hypoglossalnucleus,XII)。在这两个区域,新生大鼠组的OX1R免疫反应阳性细胞的相对光密度值均低于成年大鼠组(P<0.001)。结果表明随着大鼠发育成熟,OX1R表达水平增加,可能与其生理功能完善有关。OXA免疫反应纤维和OX1R免疫反应阳性细胞在延髓的VLM和XII的表达提示可能与心血管和呼吸等活动的调节有关。     

食欲素调节心血管活动的研究进展

食欲素(OX)是主要由下丘脑释放的一种神经肽。中枢神经系统中，食欲素神经元与其他神经核团之间构成广泛联系，参与了能量代谢、睡眠周期与心血管活动调节等。食欲素对于正常血压维持具有重要作用，食欲素神经元及其受体高表达参与了自发性高血压、应激性高血压等多种类型高血压的发生发展过程。     

慢性脑缺血大鼠脑OX1R表达的变化

目的：研究慢性脑缺血时脑食欲素受体-1（OX1R）的表达及其随缺血时间的变化。方法：通过结扎双侧颈总动脉建立慢性脑缺血模型，通过水迷宫对慢性脑缺血大鼠的行为学进行评价，免疫组化法观察OX1R的表达，双标免疫荧光进一步确定OX1R表达的定位。结果：缺血15d时大鼠的学习记忆能力明显减退，1月、2月较缺血15 d模型组的学习记忆能力有所好转。同时，从缺血急性期一直持续到15 d OX1R的表达明显增高，1月时OX1R的表达明显低于15d，2月时OX1R的表达再次增高。从组织学看：15d时部分细胞萎缩，1月时大部分细胞变形萎缩，2月时部分细胞形态恢复正常。双标免疫荧光证实OX1R确实在神经元有表达。结论：慢性缺血性脑损伤时OX1R的表达呈双相性变化,食欲素系统可能在缺血性脑损伤与修复过程中发挥一定的调节作用。     

营养性肥胖大鼠的脂代谢紊乱与下丘脑中增食欲素A的表达分析

目的: 研究营养性肥胖大鼠下丘脑中增食欲素(orexin A)表达与脂代谢紊乱的规律。 方法: 高脂饮食诱导并评估营养性肥胖大鼠动物模型;采用化学发光免疫分析法和生化酶法测定大鼠血清胰岛素(Ins)、甘油三酯(TG)和总胆固醇(TC);应用实时定量PCR检测下丘脑组织中orexin A mRNA的表达规律。 结果: 大鼠高脂膳食饲养8周后,营养性肥胖大鼠的体重、体脂含量和Lee's指数均高于对照组,血清Ins增加约50％,TG和TC分别增加约94％和43％(P<0.05);营养性肥胖大鼠下丘脑中orexin A的mRNA表达减少约57 % (P<0.05), 且orexin A的表达量与Lee's指数、Ins、TG、TC呈显著负相关,相关系数分别为r=-0.798 (P<0.05)、r=-0.868(P<0.05)、r=-0.981(P<0.05)和r=-0.815(P<0.05)。结论: 在大鼠高脂膳食诱导的营养性肥胖过程中,下丘脑orexin A的低表达和脂代谢紊乱均与营养性肥胖的发生密切相关。     

增食欲素系统在胰岛素抵抗大鼠胰腺组织中的表达及意义

目的：研究高脂肪膳食诱导的胰岛素抵抗（IR）大鼠胰腺组织中增食欲素（orexin）及其受体的变化规律。方法:IR大鼠模型采用高脂肪膳食诱导并经钳夹技术证实。采用微型血糖仪和化学发光免疫分析法测定全血葡萄糖和血清胰岛素的变化。应用实时定量PCR和Western 印迹杂交分析检测胰腺组织中orexin及orexin受体（OXR）mRNA和蛋白质在IR状态下的表达改变。结果:高脂肪膳食喂养4周后，实验组大鼠葡萄糖输注率由对照组(12.5±0.6) mg·kg-1·min-1下降至(7.6±0.4) mg·kg-1·min-1。实验组大鼠的体重、全血葡萄糖、血清胰岛素水平高于对照组，分别为26％、25％和30％（P<0.05）；高脂肪膳食诱导的IR大鼠胰腺组织中orexin mRNA和蛋白质的表达比对照组减少约71%和63％，而OX1R mRNA和蛋白质的表达比对照组降低约49％和67％（P<0.05），OX2R mRNA和蛋白质的表达改变不明显。结论:高脂肪膳食可诱导大鼠体内产生IR，并抑制胰腺中orexin及其OX1R基因的转录和翻译。Orexin系统可能是参与调节脂肪－胰岛轴的主要因素。     

Localization of orexin B and orexin-2 receptor in the rat epididymis

The peptides orexin A (OXA) and orexin B (OXB) derived from the proteolytic cleavage of a common precursor molecule, prepro-orexin, were originally described in the rat hypothalamus. Successively, they have been found in many other brain regions as well as in peripheral organs of mammals and other less evolved animals. The widespread localization of orexins accounts for the multiple activities that they exert in the body, including the regulation of energy homeostasis, feeding, metabolism, sleep and arousal, stress, addiction, and cardiovascular and endocrine functions. Both OXA and OXB peptides bind to two G-coupled receptors, orexin-1 (OX1R) and orexin-2 (OX2R) receptor, though with different binding affinity. Altered expression/activity of orexins and their receptors has been associated with a large number of human diseases. Though at present evidence highlighted a role for orexins and cognate receptors in mammalian reproduction, their central and/or local effects on gonadal functions remain poorly known. Here, we investigated the localization of OXB and OX2R in the rat epididymis. Immunohistochemical staining of sections from caput, corpus and cauda segments of the organ showed intense signals for both OXB and OX2R in the principal cells of the lining epithelium, while no staining was detected in the other cell types. Negative results were obtained from immunohistochemical analysis of hypothalamic and testicular tissues from OX2R knock-out mice (OX2R^?/?) and OX1R/OX2R double knock-out (OX1R^?/?; OX2R^?/?) mice, thus demonstrating the specificity of the rabbit polyclonal anti-OX2R antibody used in our study. On contrary, the same antibody clearly showed the presence of OX2R in sections from hypothalamus and testis of normal mice and rats which are well known to express the receptor. Thus, our results provide the first definite evidence for the immunohistochemical localization of OXB and OX2R in the principal cells of rat epididymis.     

Reinvestigation of the effect of orexin A on catecholamine release from adrenal chromaffin cells

Orexins have been shown to be implicated in the regulation of adrenal medulla functions. However, there are still inconsistent investigations on the effects of orexins on catecholamine release from chromaffin cells in varying species. In the present study, using the carbon-fiber amperometry, we investigated whether orexin A would stimulate catecholamine release from rat and mouse adrenal chromffin cells. Puff application of orexin A dose-dependently induced amperometric currents in the cultured rat chromaffin cells, which was completely blocked by the selective OX1R antagonist SB-334867 or by the removal of extracellular calcium. Likewise, in the mouse adrenal medulla slices, orexin A also induced catecholamine release mainly through the activation of OX1R. These results gain insight into our understanding of the pharmacological relevance of orexin system in modulating neuroendocrine functions.     

出生前尼古丁暴露增加新生大鼠下丘脑orexin A及延髓内orexin 1型受体表达

流行病学调查显示,出生前暴露于烟雾环境是新生儿猝死综合征发生的首位原因,尼古丁是香烟烟雾中最主要的影响胎儿神经系统发育的成分。为了观察出生前尼古丁暴露对新生大鼠下丘脑orexin A(OXA)及延髓内orexin 1型受体(OX1R)表达的影响,本实验将20只雌性成年大鼠随机均分为二组,怀孕后第5 d开始每天分别皮下注射尼古丁6 mg/kg(模型组)或等量的生理盐水(对照组),直至分娩。随机选取模型组和对照组所产的新生大鼠(1～3 d),采用免疫组织化学方法和图像分析技术,观察新生鼠下丘脑内OXA及延髓内OX1R阳性神经元的分布情况。结果显示:两组新生大鼠下丘脑内OXA免疫阳性细胞均有表达,且都主要存在于下丘脑背内侧区与穹窿周围,模型组的新生大鼠OXA免疫阳性细胞的相对光密度(ROD)值高于对照组(P<0.05)。延髓内OX1R免疫阳性细胞在两组内均有广泛分布,主要分布在腹外侧区和舌下神经核。在这两个区域,模型组新生鼠的OX1R免疫阳性细胞的ROD值均高于对照组(P<0.001)。以上结果表明,出生前尼古丁暴露的新生大鼠,下丘脑OXA及延髓内OX1R的表达均上调,提示出生前尼古丁暴露改变了新生大鼠脑内OXA系统递质的释放和突触传递,这意味着脑内orexin系统参与出生前尼古丁暴露导致的各种疾患。     

Expression of orexin A and its receptor 1 in the human prostate

The peptides orexin A (OXA) and orexin B, deriving from the cleavage of the precursor molecule prepro‐orexin, bind two G‐coupled transmembrane receptors, named as receptor 1 (OX1R) and receptor 2 for orexin, showing different affinity‐binding properties. First discovered in the rat hypothalamus, orexins and their receptors have been also found in many peripheral tissues where they exert neuroendocrine, autocrine and paracrine functions. Because inconclusive data on their localization in the mammalian prostate are reported, the aim of this study was to investigate the presence of prepro‐orexin, OXA and OX1R in the human normal and hyperplastic gland. Immunohistochemistry revealed the localization of both OXA and OX1R in the cytoplasm of the follicular exocrine epithelium of all tested normal and hyperplastic prostates. Positive immunostaining was mainly observed in the basal cells of the stratified epithelium, and only rarely in the apical cells. The expression of mRNAs coding for prepro‐orexin and OX1R and of proteins in the tissues was also ascertained by polymerase chain reaction and Western blotting analysis, respectively. In order to gain insights into the functional activity of OXA in the prostate, we administered different concentrations of OXA to cultured prostatic epithelial cells PNT1A. We first demonstrated that PNT1A cells express OX1R. The addition of OXA did not affect PNT1A cell proliferation, while it enhanced cAMP synthesis and Ca²⁺ release from intracellular storage. Overall, our results definitely demonstrate the expression of OXA and OX1R in the human prostate, and suggest an active role for them in the metabolism of the gland.     

Localization of the orexin system in the gastrointestinal tract of fallow deer

The aim of the present study was to investigate by immunohistochemistry the presence and distribution of the orexin system in the stomach and gut of fallow deer. Abundant orexin A-positive cells were localized in the middle and basal portions of the mucosal glands of the cardial and fundic regions of the stomach. In the same gastric areas, orexin B-positive cells were also found, mainly localized in the basal portion of glands. In the intestinal tract, orexin-containing cells were occasionally found in the duodenal epithelium and in the rectal intestinal glands. Immunoreactivity for orexin receptors, type 1 and 2 (OX1R and OX2R), was not detected in the same stomach regions. OX1R-immunopositivity was observed in the enteric neuron ganglia localized in the submucosal and muscular intestinal layers, while OX2R-immunopositivity was found close in contact with the cytoplasmic membrane of epithelial cells in the small intestine.