畜牧兽医学报  2019, Vol. 50 Issue (11): 2365-2370. DOI: 10.11843/j.issn.0366-6964.2019.11.022    PDF    
引用本文
曹晓娟, 李强, 范奎奎, 潘登, 刘昊东, 王昆, 海日汗, 杜晨光. 胰淀素对小鼠脑内类固醇合成急性调节蛋白表达的影响[J]. 畜牧兽医学报, 2019, 50(11): 2365-2370.  
CAO Xiaojuan, LI Qiang, FAN Kuikui, PAN Deng, LIU Haodong, WANG Kun, HAI Rihan, DU Chenguang. Effect of Amylin on the Expression of Steroidogenic Acute Regulatory Protein in Mouse Brain[J]. Acta Veterinaria et Zootechnica Sinica, 2019, 50(11): 2365-2370.  
胰淀素对小鼠脑内类固醇合成急性调节蛋白表达的影响
曹晓娟1, 李强2, 范奎奎2, 潘登2, 刘昊东1, 王昆3, 海日汗1, 杜晨光1,2     
1. 内蒙古农业大学职业技术学院, 包头 014109;
2. 内蒙古农业大学兽医学院, 呼和浩特 010018;
3. 河北省农林科学院粮油作物研究所, 石家庄 050000
收稿日期：2019-05-29
基金项目：国家自然科学基金（31660701）；内蒙古自然科学基金（2018MS03046）；河北省现代农业产业技术体系肉牛产业创新团队建设（HBCT20181302）
作者简介：曹晓娟(1979-), 女, 山西太谷人, 硕士, 主要从事动物营养与饲料研究, E-mail:zykyk@163.com.
通信作者：杜晨光, 主要从事动物组织胚胎与发育生物学研究, E-mail:dcglxj@163.com.
摘要：本试验旨在定位类固醇激素合成急性调节蛋白（StAR）在小鼠脑内的分布，并对胰淀素（amylin）是否影响小鼠脑内StAR的表达进行初步研究。购买C57BL/6品系小鼠，标准环境下饲养一个月后，将获得的子一代饲养7周，选取9只体重及健康状况相近的雄性小鼠为实验动物，随机分为3组：生理盐水对照组、amylin注射组和amylin+AC187（amylin特异性抑制剂）注射组，按体重（100 μg·kg-1）连续注射3 h后，剖取脑组织，采用免疫荧光染色技术研究StAR在脑内的分布规律，同时采用Western blot技术比较不同组中StAR的表达变化规律。试验结果表明，StAR免疫阳性信号主要表达在不定带（ZI），零星分布于蓝斑核（LC）、下丘脑腹内侧核（VMH）、中缝背核（DR）。与对照组相比，腹腔注射amylin（100 μg·kg-1）极显著降低ZI内StAR表达量（P < 0.01）；联合抑制剂处理组amylin+AC187能极显著抑制amylin对ZI内StAR表达的影响（P < 0.01）。结果表明，腹腔注射amylin可显著抑制ZI内StAR的表达，由于StAR是调节类固醇激素的限速酶，推测amylin除可调节进食及能量代谢外，还可通过调节StAR的表达影响中枢神经系统内类固醇激素的合成速率。
关键词：脑    胰淀素    类固醇合成急性调节蛋白    蛋白表达    
Effect of Amylin on the Expression of Steroidogenic Acute Regulatory Protein in Mouse Brain
CAO Xiaojuan1, LI Qiang2, FAN Kuikui2, PAN Deng2, LIU Haodong1, WANG Kun3, HAI Rihan1, DU Chenguang1,2     
1. Vocational and Technical College, Inner Mongolia Agricultural University, Baotou 014109, China;
2. College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot 010018, China;
3. Institute of Grain and Oil Crops, Hebei Academy of Agricultural and Forestry Sciences, Shijiazhuang 050000, China
Abstract: This study aims to localize the distribution of steroidogenic acute regulatory (StAR) protein in the brain of small mice, and to preliminarily investigate whether amylin affects the expression of StAR in the brain of small mice. C57BL/6 strain mice were purchased and fed under standard environment for one month. The offspring were fed for seven weeks. Nine male mice with similar weight and health status were selected as experimental animals, they were randomly divided into 3 groups:normal saline control group, amylin injection group and amylin + AC187 (amylin specific inhibitor) injection group. After 3 hours' continuous injection according to body weight (100 μg·kg-1), brain tissue was dissected. The distribution of StAR in brain was studied by immunofluorescence staining, and the expression of StAR in different groups was compared by Western blot. The results showed that StAR immunopositive signals were mainly expressed in zone incerta (ZI), sporadically distributed in locus coeruleus (LC), ventromedial hypothalamic nucleus (VMH) and dorsal raphe nucleus (DR). Compared with the control group, intraperitoneal injection of amylin significantly reduced the expression of StAR in ZI (P < 0.01); and amylin +AC187 significantly inhibited the effect of amylin on StAR expression in ZI (P < 0.01). The results showed that intraperitoneal injection of amylin could significantly inhibit the expression of StAR in ZI. StAR is a rate-limiting enzyme that regulating steroid hormones, we speculate that amylin can not only regulate food intake and energy metabolism, but also affect the synthesis rate of steroids in the central nervous system by regulating the expression of StAR.
Key words: brain     amylin     steroidogenic acute regulatory protein     protein expression    

机体稳态的调节过程中类固醇激素(如雌激素、糖皮质激素)对能量代谢和生殖调控机能发挥重要作用[1]。而类固醇激素合成急性调节蛋白(steroidogenic acute regulatory, StAR)是类固醇激素合成过程中的重要调节因子。StAR具有高度的组织特异性，位于相关细胞的线粒体膜上，参与类固醇激素的前体胆固醇由线粒体外膜向内膜的转运，此过程是类固醇激素合成的限速步骤[2]。然而，现有关于StAR的研究主要集中在睾丸和卵巢等性腺组织内类固醇激素的合成，对其在中枢神经系统内的表达分布及作用机制的研究尚未见报道。同时，研究证实肥胖和2型糖尿病模型小鼠体内类固醇激素的含量显著增高[1]，其类固醇激素的合成关键调节因子(如StAR)mRNA表达量亦显著增加。猜测是否能量代谢相关肽可影响StAR的表达。

胰淀素(amylin)与胰岛素(insulin)在胰腺β细胞内共同合成和释放[3]。长期以来普遍认为胰腺衍生的amylin是控制能量代谢的主要来源，然而，研究证实amylin亦表达在中枢神经系统中，尤其是涉及能量代谢的核团内，如下丘脑外侧区(LH)[4]。amylin作为饱食信号在能量代谢领域被广泛研究[5]。整体上，amylin通过减少能量摄入、调节营养物利用率及增加能量消耗进而调控营养物质转换[6]。证实amylin给药4 d对小鼠体重有明显的降低作用，通过减少总能量摄入，最终导致体重减轻[7-8]。

考虑到amylin作为能量调控因子，可引起大鼠血浆乳酸和葡萄糖含量变化[9]，与糖皮质激素对葡萄糖的影响相类似，而StAR是调节糖皮质激素在内的类固醇激素合成的限速酶[10]，因此猜测amylin可能在调节能量代谢过程中影响StAR的表达。

目前有关于amylin对StAR表达的影响还未见相关报道，尤其是中枢神经系统内StAR的分布及其作用机制研究更是空白。因此，本研究首先定位中枢神经系统内StAR的分布区域，然后拟通过腹腔注射amylin，探究amylin对StAR表达的影响，为进一步丰富amylin调节能量代谢的作用方式提供新的思路。

1 材料与方法 1.1 动物饲养及分组

C57BL/6小鼠购自北京维通利华生物科技有限公司，饲养于标准条件鼠房内，一个月后，经自群繁育(避免近交)，获得适应现有饲养条件的6~8周龄健康雄性小鼠。试验用小鼠从试验前3 d开始每天定时(9:00)腹腔注射生理盐水，避免试验应激。

9只小鼠随机分为3组，分别为生理盐水对照组、amylin (100 μg·kg-1)注射组，amylin+AC187(amylin特异性抑制剂)注射组。连续注射3 h(时间点分别为9:00、10:00、11:00)后获取小鼠脑组织进行检测。

1.2 主要试剂及仪器

主要试剂：amylin(PHOENIX, 432888)、SDS-PAGE制胶试剂盒(自配，所有药品购自Sigma)；一抗：Rabbit anti-steroidogenic acute regulatory (StAR, CST, 8449)；二抗：Alexa647 Donkey anti-Rabbit (Abcam, ab150075)；IR800 (1:20 000, Odyssey, IRDyeⓇ 800LT Donkey anti-Rabbit, 926-32210)。

主要仪器：恒温孵育仪(Eppendorf ThermoMixerTM，5382CH800706)，灌注泵(SENZ, 310D)，冰冻切片机(Lecia, CM1950)，激光共聚焦显微镜(Nikon Confocal C2), 酶标仪(Bio-Tek, Synergy HT)，蛋白扫描仪(Li-Cor, Odyssey-CLX)，电泳仪, 转膜仪(Bio-Rad, Mini-PROTEAN Tetra Cell for Mini Precast Gels)，超声破碎仪(QSONICA, Q700)。

1.3 方法 1.3.1 免疫荧光试验

小鼠经4%多聚甲醛(polyformaldehyde, PFA)灌注固定后取全脑，置于固定液中4 ℃过夜，然后转移至30%蔗糖溶液中直至沉底后进行脑组织冠状面连续冰冻切片。切片厚度为30 μm，恒温孵育仪4 ℃通透(1%triton+0.4%SDS，pH=8.70)处理4 h，3%NDS室温封闭1 h。漂片法孵育一抗StAR(CST, 8449)，孵育二抗Alexa647 Donkey anti-Rabbit (Abcam, ab150075)。贴片，封片，使用激光共聚焦显微镜(Nikon Confocal C2)扫描成像。

1.3.2 Western blot

采用本实验室前期建立的方法[11]。具体操作：剖剪小鼠脑组织，留取未定带核团区域组织块，按照既定蛋白提取流程提取脑组织总蛋白。按比例添加5×蛋白上样缓冲液进行蛋白变性，SDS-PAGE凝胶电泳，转膜，5%脱脂奶粉封闭，孵育抗体。一抗为Rabbit anti-steroidogenic acute regulatory (CST, 8449)，二抗为Alexa标记的Donkey anti-Rabbit IR800 (1:20 000, Odyssey, IRDyeⓇ 800LT Donkey anti-Rabbit, 926-32210)；用蛋白扫描仪(Li-Cor, Odyssey-CLX)扫描成像，并分析蛋白条带。

1.4 统计分析

使用GraphPad Prism 6软件(GraphPad Software Inc., San Diego, CA)对目的蛋白的表达量进行统计学分析。其中数据组之间使用非配对t检验进行分析。数据以x±sx的方式进行统计，P＜0.05视为具有显著性差异；* P < 0.05，** P＜0.01。

2 结果 2.1 小鼠中枢神经系统内StAR的免疫荧光定位

从图 1可知，StAR在小鼠脑内呈区域性集中表达，免疫阳性神经元荧光信号主要位于不定带(zone incerta，ZI)内(图 1A~G)，此外在蓝斑核(locus coeruleus，LC)(图 1I~L)、下丘脑弓状核(arcuate nucleus of hypothalamus, ARC)(图 1H)及脑干中缝背核(dorsal raphe nucleus，DR)(图 1M~P)内可见StAR呈零星散状分布。

DR.中缝背核；LC.蓝斑核；ZI.不定带；ARC.下丘脑弓状核；A~D.小鼠脑矢状面不定带内StAR免疫阳性神经元分布；E~G.小鼠脑冠状面不定带内StAR免疫阳性神经元分布；H.小鼠下丘脑弓状核内StAR免疫阳性神经元分布；I~L.小鼠后脑蓝斑核内StAR免疫阳性神经元分布；M~P.小鼠后脑脑干中缝背核内StAR免疫阳性神经元分布 DR.Dorsal raphe nucleus; LC.Locus coeruleus; ZI.Zone incerta; ARC.Arcuate nucleus of hypothalamus; A-D. The distribution of StAR immunoreactive neurons in the sagittal plane of rat brain; E-G. Distribution of StAR immuno-positive neurons in the adventitious zone of the coronal plane of the brain in rats; H.Distribution of StAR immunoreactive neurons in arcuate nucleus of hypothalamus; I-L. Distribution of StAR immuno-positive neurons in the posterior cerebral locus cyanoplakis of mice; M-P. Distribution of StAR immunoreactive neurons in dorsal raphe nucleus of posterior brain stem of mice 图 1 StAR在小鼠中枢神经系统内的分布 Fig. 1 Distribution of StAR in the central nervous system of mouse
2.2 amylin对小鼠脑不定带内StAR表达的影响

Western blot结果显示(图 2)，与对照组相比，腹腔注射amylin可极显著抑制不定带内StAR蛋白表达量(P＜0.01)，腹腔联合注射amylin+AC187可减弱对不定带内StAR蛋白的抑制作用(P＜0.01)。

**. P＜0.01 图 2 Amylin对小鼠脑不定带内StAR表达的影响 Fig. 2 Effect of amylin on the expression of StAR in the zone incerta of mouse brain
3 讨论

类固醇激素对维持能量代谢和生殖能力至关重要，而合成类固醇细胞必须将大量胆固醇从线粒体外膜转移到内膜。在类固醇生成细胞中，胆固醇首先转化为孕烯酮，最终转化为孕酮[12]。线粒体膜中胆固醇的初始转运是类固醇形成的限速步骤，受StAR介导[13]。如在黄体早期和中期，StAR表达量最多，在黄体期末期显著下降。这种上调使黄体产生大量的黄体酮，以保证妊娠的维持[14]。雌激素是经StAR作用合成的类固醇激素的一种，除了调节生殖功能外，雌激素在能量平衡的中枢调节中起着关键作用，包括调节进食行为和能量消耗[15-16]。尽管雌激素缺乏与肥胖之间的相互关系是一直以来备受争议的主题，但激素替代疗法可减少腹部肥胖、胰岛素抵抗和新发糖尿病[17]。

不定带亦称疑带，是脑内一个重要的神经核团，涉及内脏活动、唤醒、注意、运动及帕金森病[18]。随着研究的深入，发现未定带与区内的γ-氨基丁酸神经元和暴食行为有关[19]。证实不定带-丘脑室旁核神经通路(ZI-PVT)参与饮食调控方面。并且激活未定带GABA神经元所诱发的暴食行为比激活下丘脑弓状核刺鼠相关肽(AgRP)神经元所引起的促食效应及激活AgRP-PVT轴所引起的促食效应快百倍有余[20]。本文结果显示，StAR免疫阳性荧光信号密集分布于不定带内，同时下丘脑和不定带内表达有食欲素[21]、血管紧张素Ⅱ[22]、CART、黑色素浓集激素(melanin-concentrating hormone，MCH)、黑素细胞刺激激素(melanocyte stimulating hormone，αMSH)[23]等进食控制激素。据此，进一步证实笔者的猜测，StAR可能在不定带内通过调节相关激素的表达，进而影响能量代谢。

作为饱食和肥胖信号，amylin在机体能量代谢调节过程中主要促进负能量平衡，抑制进食、降低体重[11, 24]。女性绝经后，机体内分泌会发生一些列变化，从而导致内环境稳态改变[25]，尤其是雌激素减少可促进脂肪沉积及葡萄糖代谢减慢。而amylin有助于治疗绝经后肥胖[26]。同时，leptin受体敲除小鼠表现血浆糖皮质激素、11-脱氧皮质酮和孕酮水平升高，醛固酮转化为孕酮及皮质酮转化为孕酮的速率明显提升，证实类固醇激素水平的升高是由于db/db小鼠肾上腺中胆固醇转运体StAR的转录增加所致[1]。结合本试验中amylin可抑制不定带内StAR的结果，及StAR作为类固醇激素合成的限速酶[27]和amylin可影响雌激素分泌[28]等结论，表明amylin除具有调节进食及能量代谢功能外，还可通过调节StAR的表达影响中枢神经系统内类固醇激素的合成速率。

4 结论

通过检测小鼠神经系统内StAR的分布，以及证实amylin可抑制丘脑不定带内StAR的蛋白表达，提示amylin可通过StAR影响类固醇激素的合成速率进而参与调控摄食及能量代谢的过程。

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