神经肽Y及其受体的生物信息学和医学生理学的研究

神经肽Y（NPY）作为一种具有高度生物活性的神经递质及调节肽能与其G蛋白偶联受体家族（Y1R、Y2R、Y3R、Y4R、Y5R、Y6R）进行特异性结合,且NPY受体作为NPY作用的特定靶点在特定条件下均能被完整的NPY分子激活并通过细胞信号传导广泛参与机体生理功能的调节,使之在临床老年相关性疾病发生和发展过程中发挥重要的调控作用。而NPY及其受体的特异性结合和有效调控之所以能对机体多种生理作用及代谢功能发挥重要影响,与它们各自的生物信息学特性（包括理化性、疏水性、跨膜性、信号肽、二级结构、三级结构等）密切相关。因此,本文从生物信息学及医学生理学两个角度对NPY及其受体进行综述,为NPY及其受体今后在老年医学应用和研究中奠定理论基础。     

人神经肽Y及其受体对脂肪生成影响的研究

人神经肽Y（NPY）作为神经系统影响脂肪生成的调控递质和新近发现的外周脂肪增殖和分化相关细胞、特别是外周脂肪细胞的生物效应分子,通过与其受体家族中不同受体的特异性结合而参与或发挥对机体一系列生理功能的调节和影响,尤其是与肥胖的发生、发展及改变密切相关。而且,NPY对机体肥胖及代谢的影响义呵进一步导致一系列老年相关性疾病、特别是肥胖代谢综合症（如老年人群中常见的高血压、高血脂、冠心病、糖尿病、骨质疏松、老年痴呆及一些恶生肿瘤等）等加快出现、持续发展和不断加重。因此,对NPY及其受体在肥胖发生及介导过程中的作用及其相关机制研究显得尤为重要,也是未来老年生物学和老年医学研究的重点和热点。本文从细胞生理学水平,就NPY及其受体促进脂肪组织生成等研究作一综述,为今后老年相关性疾病的基础研究和临床应用及相关理论的系统阐述奠定基础。     

神经肽Y及其受体与某些老年病及作用机制的研究

高血压、冠心病、肺心病、支气管哮喘、慢性阻塞性肺病、糖尿病、老年性痴呆、帕金森氏病、老年抑郁症与焦虑症、骨质疏松、肿瘤等均是老年人较为常见的疾病,它们直接或间接地威胁甚至危害着老年人的健康和生命。然而,大量基础医学和临床研究发现,神经肽Y及其受体在机体的分布、含量、合成、分泌及表达水平和调控功能等,不仅有可能介导或影响着这些老年性疾病的发生、发展、加重或预后,而且还有可能与治疗这些疾病的生物学效应、作用靶点、用药途径及治疗对策等密切相关。因此,本文就神经肽Y及其受体与这些老年病之间的关系及其作用机制方面的研究进展作一综述,为今后探索以神经肽Y及其受体为靶点的有效治疗方式奠定基础,也为老年医学的科学研究和临床防治提供一些理论和实验依据。     

用于老年基础医学研究与临床辅助诊断的神经肽Y及其受体Y1-6检测方法的初步设想

神经肽Y（NPY）作为机体内一种主要的神经递质和能量调控因子，广泛参与机体各种生理功能的调节，是维持机体内环境稳态的重要物质之一。随着对其研究的不断深入，人们发现它可通过与特定靶细胞膜上不同NPY受体Y1-6的特异性结合而启动并激活相应细胞信号传导通路，从而参与对机体生理功能和靶细胞增殖分化的调控，且NPY及其受体在体内的表达水平又可影响机体的正常生理功能、细胞代谢活动、以及这种改变所诱导或促进或参与的某些老年性疾病的发生、发展及转归，并对相关药物的防治带来明显影响。因此，对NPY及其受体含量的检测项目已成为相关老年性疾病与合并症的发病机制分析、疾病进程观察、治疗效果评价、疾病痊愈判断及预后跟踪随访的重要辅助诊断指标之一。为此，本文通过对可用于老年基础医学研究与临床辅助诊断的NPY及其受体检测的酶联免疫吸附法、放射免疫测定法、荧光免疫检测法、化学发光免疫分析法、免疫组织化学法、压电石英晶片免疫传感器法、免疫印迹法、斑点免疫层析及胶体金法、受体放射配基结合分析法、放射自显影法、液体芯片技术、微粒子酶免分析法等进行NPY及其受体检测方法的综述，并提出前瞻性设想和设计，希望这些用于检测NPY及其受体指标的方法能为未来老年医学的研究和临床应用奠定基础。     

受体靶向干预与肝星状细胞活化功能的调控研究

异常的生物信息分子调控并维持肝星状细胞(HSC)活化及其高功能、永生性等生物学行为。它们的分子信息转导依赖于细胞膜不同的受体-配体间的特异性结合。针对HSC细胞膜表面6-磷酸甘露糖/胰岛素生长因子Ⅱ型受体(M6P/IGF-ⅡR)、精氨酸-甘氨酸-天门冬氨酸(RGD)受体及TGF-βⅡ型受体,人们设计了人工配体或修饰配体、以载体形式连接药物等特异性靶向干预的生物大分子,试图调控或阻断这些分子信息通路的转导,实现抑制HSC的活化与功能表达。     

肥胖蛋白的研究进展

近年来,在肥胖的分子遗传学研究中人们陆续发现了与肥胖有关的基因ｏｂ,ｄｂ,ｆａ,ｆａｔ和ｔｕｂ。肥胖蛋白则为肥胖基因编码的蛋白质,它是激素样代谢因子,具有调节食欲、能量消耗及体内脂肪贮存等生物学效应。人和动物的肥胖蛋白具有高度同源性及组织分布的特异性,其基因表达受营养等环境因子及多种生理因子的调控。肥胖蛋白的生物学效应与其在体内的正常转运及下丘脑中的受体、神经肽Ｙ、黑皮素、刺蛋白等神经调节因子有密     

重组人神经肽Y受体Y2融合蛋白的表达、纯化及其生物信息学分析研究

目的 在大肠杆菌中表达人神经肽Y Y2受体,并对之进行纯化、鉴定及生物信息学分析.方法 取已构建好且经测序确认无误的重组质粒pET28a-Y2转化大肠杆菌BL21(DE3),IPTG诱导表达融合蛋白,并经SDS-PAGE检测和Western 印迹鉴定,表达产物包涵体经Ni~(2+)-NTA亲和层析纯化.然后利用相关在线软件进行生物信息学分析Y2受体蛋白.结果 经IPTG诱导含有pET28a-Y2重组质粒的DE3菌,表达出重组人Y2融合蛋白.重组蛋白经Ni~(2+)-NTA亲和层析进行纯化后,得到了较高纯度的融合蛋白.经相关在线软件分析后获得了Y2受体的相关生物学特性.结论 重组质粒pET28a-Y2在大肠杆菌DE3中成功表达,亲和层析纯化后获得较高纯度融合蛋白,并对Y2受体蛋白的生物学特征进行了预测,为进一步研究其生物学功能及其抗体的研制奠定了基础.     

针刺对谷氨酸钠诱导的肥胖大鼠脂代谢及相关因素的影响

目的探讨针刺减肥效应的某些机理。方法将实验大鼠分为空白组、模型组、针刺组、西布曲明组，观察各组大鼠体重指数(Lee指数)、脂质代谢、胰岛素、瘦素及神经肽Y(NPY)等变化，分析相互之间的作用与联系。结果针刺组大鼠体重指数、脂质代谢的改善度比模型组显著，与西布曲明组比较针刺组效果似有更好的减肥趋势；模型组血胰岛素、瘦素水平呈抵抗状态，而针刺组趋于缓解，西布曲明组无明显改善；模型组瘦素与神经肽Y处于高水平，针刺组瘦素与神经肽Y虽有协调趋向，但分布并不一致；西布曲明组无明显变化。结论针刺具有递减体重指数、调节脂代谢功能，且可能是通过协调机体胰岛素、瘦素及神经肽Y分泌水平而达到降脂减肥目的。     

泽泻汤对代谢综合征大鼠神经肽及其Y1受体的影响

目的观察泽泻汤对代谢综合征(MS)大鼠血浆神经肽(NPY),下丘脑NPY及其Y1R表达的影响,探讨其可能机制。方法用高糖高脂饲料喂饲建立MS大鼠。将23只MS大鼠分为3组,分别给盐水、泽泻汤、盐酸西布曲明。4 w后,测体重、血糖、三酰甘油(TG),放免法检测血浆NPY;免疫组化法检测下丘脑NPY及其Y1R表达。结果与盐水组相比,泽泻汤组大鼠体重、血糖、TG、血浆NPY水平、下丘脑NPY及其Y1R表达的平均光密度均显著降低(P0.05)。结论泽泻汤对MS大鼠有良好的治疗作用,其机制可能与抑制下丘脑NPY及其Y1R表达有关。     

神经肽Y影响人血管平滑肌细胞低密度脂蛋白受体表达

通过观察神经肽Y对人血管平滑肌细胞低密度脂蛋白受体表达的影响 ,以探讨神经体液因素在动脉粥样硬化发生中的重要作用。将体外培养的人血管平滑肌细胞分成对照组和不同浓度神经肽Y组 (浓度分别为 1 0 8mol L、1 0 7mol L、1 0 6 mol L和 1 0 5mol L) ,各组分别培养 6h、1 2h、2 4h和 4 8h ,然后经免疫荧光组织化学染色 ,在激光扫描共聚焦显微镜下定量检测神经肽Y对人血管平滑肌细胞的低密度脂蛋白受体表达的影响。结果发现 ,对照组低密度脂蛋白受体表达的平均荧光值在 2 4 2 8～ 2 5 2 7之间 ,且不同培养时间无显著性差异 (P >0 .0 5 )。而各神经肽Y组低密度脂蛋白受体表达的平均荧光值与对照组之间有显著性差异 (P <0 .0 5或P <0 .0 1 ) ,在不同的培养时间中 ,1 0 8mol L神经肽Y组平均荧光值在 1 798～ 2 4 0 8之间 ;1 0 7mol L神经肽Y组在 1 783～ 2 332之间 ;1 0 6 mol L神经肽Y组在 1 72 2～ 2 2 81之间 ;1 0 5mol L神经肽Y组在 1 5 90～ 2 0 1 0之间。与对照组相比 ,分别平均下降 1 5 .5 9%、1 9.78%、2 1 .91 %和 2 6 .83% ,且不同浓度神经肽Y组之间多有显著性差异 (P <0 .0 5 ) ,神经肽Y对成人血管平滑肌细胞的低密度脂蛋白受体表达的下调作用呈现一定的剂量和时间依赖效应。结果提示 ,     

Neuropeptide Y modulates steroid production of human adrenal H295R cells through Y1 receptors

Neuropeptide Y (NPY) is abundantly expressed in the nervous system and acts on target cells through NPY receptors. The human adrenal cortex and adrenal tumors express NPY receptor subtype Y1, but its function is unknown. We studied Y1-mediated signaling, steroidogenesis and cell proliferation in human adrenal NCI-H295R cells. Radioactive ligand binding studies showed that H295R cells express Y1 receptor specifically. NPY treatment of H295R cells stimulated the MEK/ERK1/2 pathway, confirming that H295R cells express functional Y1 receptors. Studies of the effect of NPY and related peptide PYY on adrenal steroidogenesis revealed a decrease in 11-deoxycortisol production. RIA measurements of cortisol from cell culture medium confirmed this finding. Co-treatment with the Y1 antagonist BIBP2336 reversed the inhibitory effect of NPY on cortisol production proving specificity of this effect. At mRNA level, NPY decreased HSD3B2 and CYP21A2 expression. However NPY revealed no effect on cell proliferation. Our data show that NPY can directly regulate human adrenal cortisol production.     

Evidence for further heterogeneity of the receptors for neuropeptide-Y and peptide-YY in tumor cell lines derived from neural crest.

The expression and structure of the receptors for neuropeptide-Y (NPY) and peptide-YY (PYY) were studied in 16 human and rodent tumor cell lines derived from the neural crest by ligand binding and cross-linking techniques using [125I]Bolton-Hunter-NPY, [125I]PYY, and various forms of monoiodinated NPY and PYY. Although NPY-binding sites were observed in most of the tumor cells, PYY-binding sites were found only on the human neuroblastoma cell lines SMS-MSN, SMS-KAN, SK-N-MC, and MC-IXC and the human Ewing's sarcoma cell line SK-ES. The differential labeling of the NPY/PYY receptors on these cell lines suggests that the NPY/PYY receptors are more heterogeneous than previously described as the Y1, Y2, and Y3 receptor subtypes. Cross-linking studies demonstrate that the Y1 and Y2 receptors for NPY/PYY are structurally different (mol wt, 70 and 50 kilodaltons, respectively) and that the 70- and 50-kilodalton receptor proteins are coexpressed in certain tumor cell lines. This could explain at least in part why cell lines show a relative specificity for Y1/Y2 classification, observed as the inhibition by both C-terminal fragments and Y1-specific analogs on the NPY/PYY binding to membrane receptors. Collectively, the present study suggests further heterogeneity of the NPY/PYY receptors and the existence of multiple receptor proteins in the tumor cell lines derived from the neural crest.     

[Leu31, Pro34]neuropeptide Y: a specific Y1 receptor agonist.

Two types of binding sites have previously been described for 36-amino acid neuropeptide Y (NPY), called Y1 and Y2 receptors. Y2 receptors can bind long C-terminal fragments of NPY-e.g., NPY-(13-36)-peptide. In contrast, Y1 receptors have until now only been characterized as NPY receptors that do not bind such fragments. In the present study an NPY analog is presented, [Leu31, Pro34]NPY, which in a series of human neuroblastoma cell lines and on rat PC-12 cells can displace radiolabeled NPY only from cells that express Y1 receptors and not from those expressing Y2 receptors. The radiolabeled analog, [125I-Tyr36] monoiodo-[Leu31, Pro34]NPY, also binds specifically only to cells with Y1 receptors. The binding of this analog to Y1 receptors on human neuroblastoma cells is associated with a transient increase in cytoplasmic free calcium concentrations similar to the response observed with NPY. [Leu31, Pro34]NPY is also active in vivo as it is even more potent than NPY in increasing blood pressure in anesthetized rats. It is concluded that [Leu31, Pro34]NPY is a specific Y1 receptor agonist and that the analog or variants of it can be useful in delineating the physiological importance of Y1 receptors.     

Estrogen Induces Neuropeptide Y (NPY) Y1 receptor gene expression and responsiveness to NPY in gonadotrope-enriched pituitary cell cultures

We showed previously that neuropeptide Y1 receptor (Y1R) expression is increased in the hypothalamus on proestrus afternoon and that this up-regulation of Y1R mRNA may permit neuropeptide Y (NPY) to facilitate release of the preovulatory GnRH surge. Because NPY also modulates LH release directly, we examined steroid regulation of Y1R expression in the female rat anterior pituitary. Treatment of female rats with estrogen in vivo decreased the levels of Y1R mRNA in the whole pituitary gland. In lactotrope/somatotrope-enriched pituitary cells separated by unit gravity sedimentation, 17beta-estradiol (E(2)) treatment likewise suppressed Y1R expression. In contrast, E(2) elevated Y1R mRNA in gonadotrope-enriched cell populations, indicating that estrogen regulates Y1R mRNA expression differently in gonadotropes vs. other pituitary cell types. After exposure to E(2), NPY augmented GnRH-induced LH release from gonadotrope-enriched cells in a manner requiring Y1R activation. Without steroid exposure, this augmentation disappeared, and with progesterone alone, NPY reduced GnRH-induced LH release. In addition, NPY inhibited prolactin secretion from primary pituitary cells in a steroid-free environment, but not in the presence of estrogen. These findings demonstrate that E(2) can directly up-regulate gonadotrope responsiveness to NPY and suggest that this action is mediated at least in part by E(2)'s ability to stimulate Y1R gene expression in gonadotropes. Our observations are consistent with the idea that this regulatory mechanism represents a component of E(2)'s positive feedback actions in pituitary gonadotropes. The biological importance of E(2)'s opposite effects on Y1R expression in other pituitary cell types remains to be determined.     

A neuropeptide Y Y5 antagonist selectively ameliorates body weight gain and associated parameters in diet-induced obese mice

Neuropeptide Y (NPY) is thought to have a major role in the physiological control of energy homeostasis. Among five NPY receptors described, the NPY Y5 receptor (Y5R) is a prime candidate to mediate some of the effects of NPY on energy homeostasis, although its role in physiologically relevant rodent obesity models remains poorly defined. We examined the effect of a potent and highly selective Y5R antagonist in rodent obesity and dietary models. The Y5R antagonist selectively ameliorated diet-induced obesity (DIO) in rodents by suppressing body weight gain and adiposity while improving the DIO-associated hyperinsulinemia. The compound did not affect the body weight of lean mice fed a regular diet or genetically obese leptin receptor-deficient mice or rats, despite similarly high brain Y5R receptor occupancy. The Y5R antagonist acts in a mechanism-based manner, as the compound did not affect DIO of Y5R-deficient mice. These results indicate that Y5R is involved in the regulation and development of DIO and suggest utility for Y5R antagonists in the treatment of obesity.     

High expression of NPY receptors in the human testis

NPY receptors represent novel molecular therapeutic targets in cancer and obesity. However, the extent of NPY receptor expression in normal human tissues is poorly investigated. Based on the role of NPY in reproductive functions, the NPY receptor expression was studied in 25 normal human testes and, additionally, 24 testicular tumors using NPY receptor autoradiography. In the normal testis, Leydig cells strongly expressed NPY receptor subtype Y2, and small arterial blood vessels Y1. Y2 receptors were found to be functional with agonist-stimulated [(35)S]GTPγS binding autoradiography. Full functional integrity of the NPY system was further suggested by the immunohistochemical detection of NPY peptide in nerve fibers directly adjacent to Leydig cells and arteries. Germ cell tumors expressed Y1 and Y2 on tumor cells in 33% and Y1 on intratumoral blood vessels in 50%. Based on its strong NPY receptor expression in Leydig cells and blood vessels, the normal human testis represents a potentially important physiological and pharmalogical NPY target.     

Role of the Y1 receptor in the regulation of neuropeptide Y-mediated feeding: comparison of wild-type, Y1 receptor-deficient, and Y5 receptor-deficient mice

Neuropeptide Y (NPY) increases food intake through the action of hypothalamic NPY receptors. At least six subtypes of NPY, peptide YY (PYY), and pancreatic polypeptide (PP) receptors have been identified in mice. Although the involvement of Y1 and Y5 receptors in feeding regulation has been suggested, the relative importance of each of these NPY receptors and the participation of a novel feeding receptor are still unclear. To address this issue, we generated a Y1 receptor-deficient (Y1-/-) and a Y5 receptor-deficient (Y5-/-) mouse line in which we directly compared the orexigenic effects of NPY and its analogs after intracerebroventricular (icv) administration. The icv NPY-induced food intake was remarkably reduced in Y1-/- mice, but was not significantly altered by inactivation of the Y5 receptor. The Y1 receptor therefore plays a dominant role in NPY-induced feeding. Stimulation of feeding by moderately selective Y5 agonists [PYY-(3-36), human PP, and bovine PP] was reduced in Y5-/- mice, although food intake did not decrease to vehicle control levels. These results indicate that the Y5 receptor functions as one of the feeding receptors. In addition, the finding that Y5-preferring agonists still induce food intake in Y5-/- mice suggests a role for another NPY receptor(s), including the possibility of novel NPY receptors. Surprisingly, despite the limited efficacy of PYY-(3-36) and PPs at the Y1 receptor, food consumption induced by these agonists was significantly diminished in Y1-/- mice compared with that in wild-type controls. These observations suggest that the feeding stimulation induced by NPY and its analogs may be directly or indirectly modulated by the action of the Y1 receptor. We conclude that multiple NPY receptors, possibly including the novel feeding receptor, are involved in the feeding response evoked by NPY and its analogs. Among them, the Y1 receptor plays a key role in NPY-induced feeding in mice.