CN105920015B - AMPK抑制剂Compound C在制备激活坐骨神经髓鞘形成制剂中的应用 - Google Patents

AMPK抑制剂Compound C在制备激活坐骨神经髓鞘形成制剂中的应用 Download PDF

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CN105920015B
CN105920015B CN201610301629.9A CN201610301629A CN105920015B CN 105920015 B CN105920015 B CN 105920015B CN 201610301629 A CN201610301629 A CN 201610301629A CN 105920015 B CN105920015 B CN 105920015B
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孙诚
刘晓宇
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Suzhou Qingyaqirui Biotechnology Co ltd
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Abstract

本发明公开了一种AMPK抑制剂Compound C在制备激活坐骨神经髓鞘形成制剂中的应用。本发明AMPK抑制剂Compound C可有效促进外周神经髓鞘的形成。

Description

AMPK抑制剂Compound C在制备激活坐骨神经髓鞘形成制剂中 的应用
技术领域
本发明涉及一种AMPK抑制剂Compound C在制备激活坐骨神经髓鞘形成制剂中的应用。
背景技术
AMPK(Adenosine Monophosphate-activated Protein Kinase)是细胞能量的关键传感器。该蛋白质激酶能够通过感受细胞能量状态来维持真核细胞的ATP生成和消耗的平衡，即能量稳态，它的激活有助于纠正代谢紊乱，使细胞代谢趋向生理平衡，由此成为治疗代谢紊乱和癌症的可能靶点。很多研究表明，AMPK的激活可刺激机体产生ATP，为生理活动提供能量；同时，AMPK的激活可强烈抑制蛋白及脂肪的合成，以减少ATP的消耗。
髓鞘包裹在神经细胞轴突外面的一层膜，由雪旺细胞或少突胶质细胞组成，其生理作用表现为绝缘作用，防止神经电冲动从神经元轴突传递至另一神经元轴突。地震，车祸，事故等各种自然灾害或意外都有可能造成外周神经损伤，因此，在我们的日常生活中，外周神经损伤是一种常见病症。神经损伤后髓鞘的形成是神经功能得以恢复的重要保证。大量研究表明，髓鞘的主要组分为脂类及蛋白分子。
发明内容
本发明的目的在于提供一种可促进外周神经髓鞘形成的AMPK抑制剂Compound C在制备激活坐骨神经髓鞘形成制剂中的应用。
本发明的技术解决方案是：
一种AMPK抑制剂6‐[4‐[2‐(1‐哌啶基)乙氧基]苯基]‐3‐(4‐吡啶基)吡唑并[1,5‐A]嘧啶(6-[4-[2-(4-Morpholinyl)ethoxy]phenyl]-3-phenyl-pyrazolo[1,5-a]pyrimidine)在制备激活坐骨神经髓鞘形成制剂中的应用。
本发明AMPK抑制剂Compound C(6-[4-[2-(4-Morpholinyl)ethoxy]phenyl]-3-phenyl-pyrazolo[1,5-a]pyrimidine)可有效促进外周神经髓鞘的形成。
附图说明
下面结合附图和实施例对本发明作进一步说明。
图1是羟甲基戊二酰辅酶A还原酶(HMGCR)基因表达分析示意图。其中上方的线为实验组大鼠数据；下方的线为对照组大鼠数据。***p<0.001，Student's t test分析。
图2是固醇调节元件结合蛋白-2(SREBP-2)基因表达分析示意图。其中上方的线为实验组大鼠数据；下方的线为对照组大鼠数据。***p<0.001，Student's t test分析。
图3是大鼠出生后7天(P7)坐骨神经组织是p-ACC、ACC、SERBP2及HMGCR蛋白表达分析示意图。
图4是对图3中的蛋白表达定量分析结果示意图。Vehicle：对照组，ComC：实验组。**p<0.01，Student's t test分析。
图5是大鼠坐骨神经组织胆固醇含量测定示意图。Vehicle：对照组，ComC：实验组。*p<0.05，Student's t test分析。
图6是大鼠坐骨神经组织蛋白表达分析示意图。
图7是大鼠坐骨神经组织蛋白表达(图6)定量分析。*p<0.05，**p<0.01,***p<0.001,Student's t test分析。
图8是大鼠坐骨神经电镜照片示意图。Vehicle：对照组，ComC：实验组。
图9是大鼠坐骨髓鞘厚度分析示意图。Vehicle：对照组，ComC：实验组。*p<0.05,Student's t test分析。
具体实施方式
一种AMPK抑制剂Compound C在制备激活坐骨神经髓鞘形成制剂中的应用。
一、实验步骤
1、动物处理
实验材料为刚出生的SD大鼠，向每只大鼠皮下注射6‐[4‐[2‐(1‐哌啶基)乙氧基]苯基]‐3‐(4‐吡啶基)吡唑并[1,5‐A]嘧啶(6-[4-[2-(4-Morpholinyl)ethoxy]phenyl]-3-phenyl-pyrazolo[1,5-a]pyrimidine)，以下简称为ComC，剂量为50mg/kg体重/天，溶剂为二甲基亚砜(DMSO)。对照大鼠注射等剂量的DMSO。在处理后的不同天数收集大鼠坐骨神经组织用于后续分析。每组实验动物6只，实验重复三次。
2、坐骨神经组织胆固醇含量的测定
胆固醇含量的测定利用Sigma-Aldrich的试剂盒(MAK043)进行。取10mg坐骨神经组织置于200μl匀浆液(氯仿：异丙醇：IGEPAL CA-630＝7：11：0.1)中，用组织匀浆进行匀浆，将所得匀浆液进行离心(13000rpm,10分钟)，去除不溶物质。上清有机相转移到一个新的小管，50℃下干燥去除氯仿，并用真空干燥器将去除剩余溶剂。用胆固醇测定液(试剂盒所附)溶解干燥所得的脂类，进一步测定参照试剂盒操作步骤进行。
3、坐骨神经RNA提取及基因表达检测
使用Trizol试剂(Invitrogen公司产品)提取坐骨神经组织的RNA，再经cDNA合成试剂盒(Bio-Rad公司产品)转录成cDNA。用SYBR Green Supermix(Bio-Rad公司产品)来做基因表达分析，仪器为iQ5Multicolor Real-Time PCR Detection System(Bio-Rad公司产品)。结果2-ΔCt方法计算mRNA水平。以18S持家基因来校准mRNA的水平。所使用的引物序列如下：
18S rRNA正向引物:5’-AGT CCC TGC CCT TTG TAC ACA-3’；
18S rRNA负向引物:5’-CGT TCC GAG GGC CTC ACT-3’；
Hmgcr正向引物:5'-AGC TTG CCC GAA TTG TAT GTG-3'；
Hmgcr负向引物:5；-TCT GTT GTG AAC CAT GTG ACT TC-3'.
Srebp2正向引物:5'-GCA GCA ACG GGA CCA TTC T-3'；
Srebp2负向引物:5'-CCC CAT GAC TAA GTC CTT CAA CT-3'.
4、坐骨神经蛋白提取及检测
动物组织样品裂解液配方：25mM Tris-HCl，pH 7.4；100mM NaF；50mM Na4P2O7；10mM Na3VO4；10mM EGTA；10mM EDTA；1％NP-40；10μg/ml Leupeptin；10μg/ml Aprotinin；2mM PMSF；20nM Okadaic acid。用台式匀浆器(Polytron,PT2100)匀浆，样品于4℃旋转裂解1小时后离心(13000rpm，4℃)20min，离心后小心去除上层脂质，其余上清转至另一离心管并再次离心。这一过程重复2次以彻底去除蛋白样品中的脂质。样品蛋白含量用蛋白测定试剂盒测定，根据所得结果将所有样品的蛋白浓度调至相同水平，加入上样缓冲液，混匀后于100℃煮5min，冷却至室温用于Western blot分析。
将上述步骤所得的样品用聚丙烯酰胺凝胶电泳(SDS-PAGE)分离，并将胶上蛋白转至PVDF膜。转膜结束后的PVDF膜用含5％牛血清白蛋白的TBST(Tris-buffered salinesolution/Tween)缓冲液室温封闭1小时。封闭后的PVDF与一抗共孵过夜(4℃)。与一抗作用结束后，用TBST洗PVDF膜三次，再与二抗室温作用1小时。二抗作用结束后用TBST洗三次。最后PVDF膜用化学发光反应体系(chemiluminescence assay system，Roche)反应，并曝光至X胶片(Kodak)。各蛋白表达水平定量用软件Quantity-One(Bio-Rad)分析。所有抗体均购自于Cell Signaling Technology公司。
5、坐骨神经电镜观察
各组随机抽取3只动物取坐骨神经作为电镜标本。动物在深度麻醉下，经心脏依次灌注0.85％氯化钠溶液和固定液(1％多聚甲醛+1.25％戊二醛)。灌注固定后，取坐骨神经组织并修切成电镜标本大小(1.5mm x 1mm x 1mm)，将标本浸入4％戊二醛溶液进行前固定，并用1％锇酸进行后固定，醋酸铀块染，梯度乙醇脱水，Epon812环氧树脂包埋，半薄切片定位，超薄切片(70nm，横切)，常规枸橼酸铅复染，透射电镜观察。所得照片在图象分析系统上测量有髓神经直径和轴突直径，以轴突直径/有髓神经直径数值(g-ratio)衡量髓鞘厚度。
二、实验结果
为研究抑制AMPK是否可促进外周神经髓鞘的形成，我们用AMPK的抑制剂6‐[4‐[2‐(1‐哌啶基)乙氧基]苯基]‐3‐(4‐吡啶基)吡唑并[1,5‐A]嘧啶(6-[4-[2-(4-Morpholinyl)ethoxy]phenyl]-3-phenyl-pyrazolo[1,5-a]pyrimidine)处理刚出生的大鼠(剂量为50mg/kg/day，皮下注射)，在处理后的不同时间点(出生后2天、5天、7天和11天，图中分别标注为P2、P5、P7、P11)收集坐骨神经组织用于后续分析。羟甲基戊二酰辅酶A还原酶(HMGCR)和固醇调节元件结合蛋白-2(SREBP2)是脂肪合成过程中的两个关键基因，我们结果表明Compound C(ComC)处理可有效激活这两个基因的表达(图1；图2)。同时，我们也检测了这两个基因的蛋白表达水平。结果表明ComC处理可显著促进羟甲基戊二酰辅酶A还原酶(HMGCR)和固醇调节元件结合蛋白-2(SREBP2)这两个基因的蛋白水平(图3)。此外，ACC是AMPK信号通路的下游分子，我们结果表明ComC处理可降低磷酸化ACC(p-ACC)和ACC的表达(图3)。对上述结果进行蛋白定量分析，结果表明p-ACC水平显著下降，SREBP2和HMGCR的表达则显著上升(图4)。胆固醇是髓鞘的一个重要组分，其在坐骨神经中的水平在ComC处理下显著升高(图5)。除脂肪合成被ComC激活外，蛋白的合成也被激活。如图6所示，蛋白合成的负调控因子(p-AMPK,p-TSC2,p-Raptor)在ComC作用下均呈下降趋势；而蛋白合成的正调控因子(p-P70S6K,p-4E-BP1)则呈上升趋势。对上述蛋白表达差异进行定量分析，结果表明p-AMPK,p-TSC2,p-Raptor蛋白表达在ComC处理下均显著下降，而p-P70S6K,p-4E-BP1均显著升高(图7)。在接下来的实验中，我们用电镜观察了坐骨神经的超微结构，结果显示ComC处理的大鼠坐骨神经髓鞘套厚度较对照组变厚(图8)。定量数据也表明ComC处理可促进新生大鼠坐骨神经髓鞘的厚度(图9)。以上结果说明抑制AMPK抑制剂ComC可加速外周神经髓鞘的形成。

Claims (1)

1.一种AMPK抑制剂6-[4-[2-(1-哌啶基)乙氧基]苯基]-3-(4-吡啶基)吡唑并[1,5-A]嘧啶在制备激活坐骨神经髓鞘形成制剂中的应用。
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