CN111743898A - 11β-HSD1抑制剂在应激环境下保护神经干细胞的应用 - Google Patents
11β-HSD1抑制剂在应激环境下保护神经干细胞的应用 Download PDFInfo
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Abstract
本发明提供了11β‑HSD1抑制剂用于制备神经保护剂,特别是应激环境下神经干细胞保护剂以及用于制备促进神经干细胞增殖和迁移的药物的用途。本发明首次证实神经干细胞上存在糖皮质激素代谢酶11β‑HSD1,并且发现11β‑HSD1抑制剂,例如BVT‑14225、BVT‑2733、AZD4017、AZD8329、姜黄素及类似物、以及它们的药学上可接受的盐或酯等,通过抑制神经干细胞上11β‑HSD1酶的还原活性,可促进应激环境下神经干细胞的发育,降低糖皮质激素浓度,控制应激水平,为围术期应激或社会应激情况下的神经保护提供了一条新的途径。
Description
技术领域
本发明属于神经保护和应激研究领域,具体涉及11β-羟基类固醇脱氢酶1型(11β-HSD1)抑制剂(例如BVT-14225)用于制备神经干细胞保护剂的用途以及用于制备促进神经干细胞增殖和迁移的药物的用途。
背景技术
神经干细胞(Neural stem cell,NSC)是中枢神经系统的多能干细胞,具有不断自我更新的增殖能力,具有向神经元、星形胶质细胞、少突胶质细胞的分化能力及向特定脑区的迁移能力,对于保持中枢神经系统的稳态和正常脑组织的发育具有重要的作用。另外NSC的发育也发挥着修复受损的神经细胞,保持认知功能的重要作用。胎儿期和新生儿期大脑含有大量的神经干细胞。目前已开展神经干细胞移植用于治疗神经损伤性疾病的临床前研究或临床试验,疗效显著,包括中风、阿兹海默病、帕金森综合征,颅脑创伤后遗症等疾病。
本发明中所指的应激环境为手术等急性应激环境或慢性社会应激环境。与神经干细胞发育相关的急性应激环境具体指孕期母体手术、孕期胎儿手术、新生儿手术或神经干细胞移植手术等。与神经干细胞发育相关的慢性社会应激环境具体指孕期、新生儿期的母婴分离,暴力经历以及不当的家庭护理等。
糖皮质激素是机体内极为重要的一类调节分子,它对机体的发育、生长、代谢以及免疫功能等起着重要调节作用,是机体应激反应最重要的调节激素,也是临床上使用最为广泛而有效的抗炎和免疫抑制剂。作为重要的应激激素,糖皮质激素在孕妇、胎儿及婴幼儿手术的围术期显著增高,在慢性社会应激的情形下也会升高。增高的糖皮质激素或者大剂量或长期应用糖皮质激素会对中枢神经系统,特别是NSC的发育产生一定的不良影响。在NSC的体外培养中,活性糖皮质激素(CORT)会显著抑制神经干细胞的增殖;高浓度的皮质醇会抑制神经干细胞株(HPC03A/07)向神经元的分化,但不影响向星形胶质细胞的分化;孕鼠束缚应激模型中,后代仔鼠神经干细胞的迁移能力也明显降低,因此,应激水平的CORT影响会神经发育。胎鼠早期应激模型的水迷宫实验表明,与对照组相比,应激组的SD大鼠在获得性训练中潜伏期和轨迹总距离均显著延长。因此,应激水平的CORT会损伤认知功能。
11β-羟基类固醇脱氢酶1型(11β-Hydroxysteroid dehydrogenase type 1,11β-HSD1)是糖皮质激素的代谢酶,具有催化非活性的可的松(在啮齿动物为11-去氢皮质酮,即11-Dehydrocorticosterone,DHC)和活性的氢化可的松(即皮质醇,在啮齿类动物为CORT)的相互转化的作用,从而维持局部组织活性糖皮质激素浓度的稳定。11β-HSD1抑制剂包括BVT和AZD系列,BVT系列如BVT-14225和BVT-2733,AZD系列如AZD4017和AZD8329,它们都具有抑制11β-HSD1活性的药理作用;此外,姜黄素及其类似物也具有抑制11β-HSD1活性的作用。
发明内容
本发明的发明人以DHC建立了大鼠原代神经干细胞的应激模型,用神经干细胞标记物巢蛋白(Nestin)和性别决定基因相关转录因子-2(Sex determining region Y-boxtranscription factor 2,Sox2)的免疫荧光染色鉴定原代细胞。然后提取神经干细胞核糖核酸(Ribonucleic acid,RNA)并进行逆转录聚合酶链式反应(Polymerase chainreaction,PCR)、普通扩增PCR和核酸凝胶电泳实验鉴定神经干细胞上的11β-HSD1。
发明人的研究分为两部分,第一部分用EdU实验检测CORT和DHC对神经干细胞增殖能力的影响,确定了应激时DHC的浓度。随后以此浓度进行第二部分实验,研究了BVT通过调整11β-HSD1酶活性而影响神经干细胞发育。
发明人采用用乳酸脱氢酶(Lactate dehydrogenase,LDH)释放实验检测DHC和BVT的细胞毒性;采用超高效液相色谱-质谱法(Ultra performance liquid chromatography-mass spectrometry,UPLC-MS)检测BVT对11β-HSD1活性影响;采用蛋白印迹实验(Westernblot,WB)检测DHC和BVT对神经干细胞的11β-HSD1蛋白表达水平的影响;采用EdU实验检测DHC和BVT对神经干细胞的增殖能力的影响;采用神经元标志物-神经元核抗原抗体(Neurona-l Nuclei,NeuN)和星形胶质细胞标志物-胶质纤维酸性蛋白(Glialfibrillary acidic protein,GFAP)的免疫荧光实验检测DHC和BVT对神经干细胞分化能力的影响;采用划痕实验检测DHC和BVT对神经干细胞迁移能力的影响。
基于上述实验研究及研究结果,发明人完成了本发明。
本发明提供了11β-HSD1抑制剂用于制备神经保护剂的用途。
根据本发明,所述11β-HSD1抑制剂选自BVT-14225(CAS登记号:376638-65-2)及其药学上可接受的盐、BVT-2733(CAS登记号:376641-65-5)及其游离碱以及其游离碱与其他药学上可接受的酸形成的盐、AZD4017(CAS登记号:1024033-43-9)及其药学上可接受的盐或酯、AZD8329(CAS登记号:1048668-70-7)及其药学上可接受的盐或酯、姜黄素(CAS登记号:458-37-7)及其类似物等。其中所述姜黄素类似物可以为发明人等人的文章(Han Lin等人,”Mono-carbonyl curcumin analogues as 11β-hydroxysteroid dehydrogenase1inhibitor,Bioorganic&Medicinal ChemistryLetters,第23卷第15期,2013年8月,第4362-4366页)中记载的化合物A01-A17、B01-B17以及C01-C17,特别是化合物A02、A06、A10、B02、B06、B13、B14、C02、C06和C13;以及发明人等人的文章(Guo-Xin Hu,Han Lin等人,Curcumin as apotent and selective inhibitor of 11β-hydroxysteroiddehydrogenase 1:improving lipid profiles in high-fat-diet-treated rats,PLOSONE,第8卷第3期,2013年3月,e49976)中记载的姜黄素类似物2-16。
所述11β-HSD1抑制剂的结构如下表1所示:
表1
姜黄素类似物A01-A17、B01-B17以及C01-C17的结构式如下表2-4所示:
表2
表3
表4
姜黄素类似物2-16的结构式如下表5所示:
表5
本发明中所提及的药学上可接受的盐可以是酸加成盐,也可以是碱加成盐,这取决于化合物的结构。例如,可以是与以下的无机酸或有机酸形成的酸加成盐:盐酸、硫酸、硝酸、磷酸、甲酸、乙酸、酒石酸、富马酸、柠檬酸、苹果酸、草酸、抗坏血酸、琥珀酸、甲磺酸、乙磺酸、苯磺酸等,或者可以是与钠离子、钾离子、钙离子、铵离子等形成的盐。
本发明中所提及的药学上可接受的酯是指含有羧基或羟基的化合物与醇类化合物、氨基化合物、羧基化合物形成的酯,例如可以是碳酸酯或氨基甲酸酯。
根据本发明,所述神经保护剂是应激环境下神经干细胞保护剂。
根据本发明,所述应激环境包括但不限于以下疾病或手术等急性应激环境以及慢性应激环境:
1)胎儿手术:胎儿脊柱裂,双胎输血综合征,前置血管,先天性膈疝等;
2)产时手术或新生儿手术:脐膨出,先天性双主动脉弓性心脏病,食管闭锁,食管气管瘘,食道裂孔疝等;
3)包括但不限于以下的慢性社会应激环境:孕期、新生儿期的母婴分离,暴力经历以及不当的家庭护理等。
本发明还提供11β-HSD1抑制剂用于制备促进神经干细胞增殖和迁移的药物的用途。
根据本发明,所述促进神经干细胞增殖和迁移的药物能够用于预防和/或治疗以下疾病或病症:中风、阿兹海默症、帕金森综合征、颅脑创伤后遗症;还可用作以下手术前、手术中或手术后的辅助药物:胎儿手术(如因胎儿脊柱裂、双胎输血综合征、前置血管、先天性膈疝而等进行的胎儿手术)、产时手术或新生儿手术(如因脐膨出、先天性双主动脉弓性心脏病、食管闭锁、食管气管瘘、食道裂孔疝等而进行的产时手术或新生儿手术)。
本发明涉及的研究首次证实神经干细胞上存在糖皮质激素代谢酶11β-HSD1,并且发现11β-HSD1抑制剂——如BVT-14225(目前被用作治疗糖尿病的药物)、BVT-2733、AZD4017、AZD8329、姜黄素以及它们的类似物、药物上可接受的盐或酯等——通过抑制神经干细胞上11β-HSD1酶的还原活性,可促进应激环境下神经干细胞的发育,降低糖皮质激素浓度,控制应激水平,为围术期应激或社会应激情况下的神经保护提供了一条新的途径。
附图说明
图1示出了原代神经干细胞增殖形成的多细胞神经球的倒置荧光显微镜照片。
图2示出了神经球(A)和单层神经干细胞(B)的Nestin免疫荧光染色照片,4′,6-二脒基-2-苯基吲哚(4′,6-Diamidino-2-phenylindole,DIPA)复染细胞核,merge为同一位置Nestin和DIPA免疫荧光信号的重叠。
图3示出了神经球(A)和单层神经干细胞(B)的Sox2免疫荧光染色照片,DIPA复染细胞核,merge为同一位置Sox2和DIPA免疫荧光信号的重叠。
图4示出了神经干细胞表达11β-HSD1的电泳鉴定结果。
图5示出了神经干细胞增殖实验的免疫荧光照片。EdU代表具有增殖能力的细胞核,DIPA复染所有细胞核,merge为同一位置EDU和DIPA免疫荧光信号的重叠。
图6示出了神经干细胞11β-HSD1蛋白表达的WB条带。以β-微管蛋白(β-Tubulin)作为内参蛋白。
图7示出了神经干细胞增殖实验的免疫荧光照片。EdU代表具有增殖能力的细胞核,DIPA复染所有细胞核,merge为同一位置EDU和DIPA免疫荧光信号的重叠。
图8示出了神经干细胞分化实验的免疫荧光照片。NeuN代表神经元,GFAP代表星形胶质细胞,DIPA复染所有细胞核,merge为同一位置EDU和DIPA免疫荧光信号的重叠。
图9示出了神经干细胞划痕实验的照片。
具体实施方式
下文将结合具体实施例对本发明的用途做更进一步的详细说明。应当理解,下列实施例仅为示例性地说明和解释本发明,而不应被解释为对本发明保护范围的限制。凡基于本发明上述内容所实现的技术均涵盖在本发明旨在保护的范围内。
除非另有说明,以下实施例中使用的原料和试剂均为市售商品,或者可以通过已知方法制备。以下实施例中所使用的BVT是指BVT-14225。
实例1原代神经干细胞的鉴定
原代神经干细胞经过5-7天的培养,可逐渐增殖,形成具有良好折光性的多细胞的球体(如图1),一般选取400-500um的神经球进行接种贴壁以便进行鉴定和后续研究。
通过免疫荧光染色的方法对提取的原代细胞进行神经干细胞标志物Nestin和Sox2的免疫荧光染色(见附图2和附图3),结果表明,Nestin和Sox2阳性的细胞比例可达96%以上。证明所培养的原代细胞为神经干细胞。
除划痕实验细胞接种前培养板不需要基质胶预处理外,其他实验细胞培养板均用基质胶预处理,并置于细胞培养箱内6~12h,以促进神经干细胞贴壁生长。
实例2神经干细胞上11β-HSD1的鉴定
提取神经干细胞RNA并进行逆转录PCR、普通扩增PCR和核酸凝胶电泳实验鉴定神经干细胞上的11β-HSD1(见附图4),并以管家基因RS16作为内参。结果表明,11β-HSD1表达于神经干细胞上。
实例3以CORT建立应激模型以确定应激时DHC的毒性浓度
将神经干细胞随机分为6组:正常对照组(C组)、溶剂对照组(D组)、CORT组,0.1μMDHC组(DHC O.1组)、1μM DHC组(DHC 1.0组)、10μM DHC组(DHC 10组),将NSC以每孔0.5×105/mL的密度接种于预先放入爬片的24孔板,每孔加入500μL增殖培养基,处理48h后,在实验结束前4h加入终浓度为10μM的5-乙炔基-2’脱氧尿嘧啶核苷(EdU),然后通过Click-iTTMEdUAlexa Fluor488试剂盒说明书(试剂配方见表6)进行免疫荧光实验。用正置莱卡显微镜拍摄照片(见附图5),ImageProPlus 6.0软件计数EdU+细胞数和总的细胞数,并计算增殖率,以此代表NSC的增殖能力。增殖率(%)=EdU+的细胞数/细胞总数×100%。
表6 EdU反应液成分及配比
结果表明,C组、D组、DHC 0.1组和DHC 1.0组的NSC增殖能力无统计学差异;CORT组和DHC 10组的NSC增殖能力与D组相比均明显受到抑制(P<0.05),而CORT组和DHC10组的神经干细胞增殖能力无统计学差异(P>0.05)(见表7)。上述结果说明,1μM CORT和10μM DHC对神经干细胞的增殖具有相同程度抑制作用,此后实验均以10μM DHC作为造模浓度,建立应激环境。
与C组相比,*P<0.05;与D组相比,#P<0.05;和CORT组相比,$P>0.05。
实例4 LDH实验检测BVT(即BVT-14225)和DHC的细胞毒性
将神经干细胞随机分为5组:正常对照组(C组)、溶剂对照组(D组)、10μM DHC组(DHC组)、10μM BVT组(BVT组)和10μM DHC组+10μM BVT组(BD组,BVT先于DHC 1h开始处理)。以每孔1.0×105/mL的密度接种于96孔板,每孔加入100μL培养基,处理48h后按照CytoTox96非放射性细胞毒性试剂盒说明书进行LDH释放实验,用Bio-tek酶标仪检测吸光度(OD490)并计算细胞死亡率,以代表各处理组药物对NSC的细胞毒性作用。细胞毒性(%)=处理孔LDH释放(OD490)/最大LDH释放(OD490)×100%。
CytoTox 96试剂盒包括以下试剂:底物混合物(Substrate Mix),检测缓冲液(Assay Buffer),LDH阳性对照(LDH Positive Control),裂解液(10X)(Lysis Solution(10X)),终止液(Stop Solution)。
结果表明,C组、D组、DHC组、BVT组、BD组的LDH释放量无明显变化(见表8),各组之间均无统计学意义(P>0.05)。即BVT和DHC均不会增加细胞死亡率。
实例5超高效液相色谱-质谱(UPLC-MS)实验检测11β-HSD1酶活性
将神经干细胞细胞按照实例4随机分组,将NSC以每孔0.5×105/mL的密度接种于预先放入爬片的24孔板,每孔加入500uL增殖培养基,处理48h后收集各组的细胞培养基并继续进行如下UPLC-MS处理并计算培养基中DHC和BVT的浓度。
分别配制浓度梯度的CORT(0.01、0.02、0.1、0.5、1、2和5μM)及DHC(0.03、0.06、0.3、1.5、3、6和15μM)(标准品)原液及0.2μM氢化可的松(Hydrocortisone)(内标),取90μL空白培养基+10μL各梯度标准品原液置于1.5mL塑料离心管中,加入10μL内标,涡旋2min混匀,随后加入300μL甲醇沉淀蛋白,涡旋3min,然后4℃,12000rpm离心10min,取100μL上清于进样小瓶中进行UPLC-MS分析,以标准品与内标的峰面积比值与相应的浓度进行加权线性回归,分别得到CORT及DHC的标准曲线方程。然后取100μL细胞培养基和100μL空白培养基进行上述同样的操作,随后将样本与内标的峰面积比值代入标准曲线方程,分别得出各培养基样品中CORT及DHC的浓度,并计算转化率(酶活性)。酶活性=conc(CORT)/conc(DHC)×100%。
由于本实验只有DHC组和BD组细胞培养基中含有外源DHC,同时,质谱实验也证实,C组、D组和BVT组中均不含DHC,因此,本实验只对DHC和BD组进行统计学分析。
结果表明,BD组转化率(酶活性)显著低于DHC组(P<0.01)(见表9)。说明,BVT对11-HSD1还原活性有显著的抑制作用。
和DHC组相比,△P<0.05
实例6WB实验检测11β-HSD1蛋白的表达水平
将神经干细胞细胞按照实例4随机分组,以每孔5×105/mL的密度接种于6孔板,每孔加入2mL培养基,培养24h后分组处理,48h后配置蛋白裂解试剂依次加入至6孔板中,于冰上静置10min,收集裂解液,超声。4℃、12000×g离心30min,收取上清液。用BCA工作试剂盒测定蛋白浓度,每条泳道蛋白上样量25μg,电泳、转膜后用5%脱脂奶粉封闭1h,加入内参蛋白兔β-Tubulin抗体(稀释比例1:2000)和目的蛋白兔11β-HSD1抗体(稀释比例1:1000),4℃摇床孵育18-20h。TBST洗膜,加入稀释比例为1:5000的羊抗兔生物素二抗,室温孵育1h,TBST充分洗膜,通过曝光后显示条带(见附图6)。Image J软件分析目标蛋白条带和内参蛋白条带的积分吸光度(Integrated absorbance,IA),11β-HSD1蛋白表达水平=IA(11β-HSD1)/IA(β-Tubulin)。
BCA试剂盒包括以下试剂:胎牛血清(BSA)原液,反应液A和Cu试剂。
结果表明,C组、D组、DHC组、BVT组和BD组的11β-HSD1蛋白表达水平无明显变化(见表10),各组之间均无统计学意义(P>0.05)。即BVT不改变神经干细胞的11β-HSD1蛋白表达水平。
实例7 BVT调节11β-HSD1酶活性对神经干细胞增殖能力的影响
将神经干细胞细胞按照实例4随机分组,处理48h后,然后按照实例3进行实验(见附图7)。
结果表明,和D组相比,DHC组的神经干细胞增殖能力明显下降(P<0.01),而BVT组和BD组的神经干细胞增殖能力均无统计学意义(P>0.05)。和DHC组相比,BD组神经干细胞的增殖能力明显增强(P<0.05)(见表11),说明在应激条件下,BVT具有促进神经干细胞增殖的作用。
和C组相比,*P<0.05;和D组相比,#P<0.05;和DHC组相比,△P<0.05。
实例7 BVT调节11β-HSD1酶活性对神经干细胞分化能力的影响
将神经干细胞细胞按照实例4随机分组,将NSC按1.0×105/mL的细胞密度接种于预先放入爬片的24孔板,每孔加入500uL增殖培养基,在细胞培养箱内培养24h后,更换为分化培养基,含有1%N-2添加剂(100X)和1%青链霉素混合液的DMEM/F12(3:1)并分组处理,48h后更换分化培养基,结束药物处理。以后每两天更换一次分化培养基,直至细胞总分化时间达到7d。然后对各组细胞进行GFAP、NeuN以及DAPI的免疫荧光染色实验,用正置莱卡显微镜拍摄照片(见图8),ImageJ 1.51软件和ImageProPlus 6.0软件分别计数GFAP+、NeuN+细胞数和总的细胞数,并分别计算GFAP、NeuN的分化率,以此代表NSC的星形胶质细胞和神经元分化能力。GFAP分化率(%)=GFAP+的细胞数/细胞总数×100%,NeuN分化率(%)=NeuN+的细胞数/细胞总数×100%。
结果表明,C组、D组、DHC组、BVT组和BD组的NeuN+、GFAP+细胞所占的比例几乎无明显变化,各组之间的差异均无统计学意义(P>0.05)(见表12)。
实例8 BVT调节11β-HSD1酶活性对神经干细胞迁移能力的影响
将神经干细胞细胞按照实例4随机分组,将神经干细胞以每孔2.5×105/mL的密度接种于24孔板,每孔加入500μL增殖培养基,在细胞培养箱内培养,待培养孔底部完全被细胞覆盖,使用200μL无菌枪头对所有实验孔的细胞进行划痕处理并更换分化培养基,立即用倒置莱卡显微镜拍摄此时细胞划痕(0h)的照片(见附图9)。然后分组处理48h,然后在48h拍照位点处复拍此时的划痕照片(48h)(见附图9)。ImageJ 1.51软件计算处理前后细胞划痕间距的“愈合”率,以此代表NSC的迁移能力。迁移率(%)=(0h的划痕间距面积-48h的划痕间距面积)/0h的划痕间距面积×100%。
结果表明,与D组相比,DHC组神经干细胞的迁移能力稍有下降,但是尚无统计学意义(P>0.05);与DHC组相比,BD组神经干细胞的迁移能力显著增强(P<0.05)(见表13),说明在应激条件下,BVT具有促进神经干细胞迁移作用。
和DHC组相比,△P<0.05
结论:
以上研究结果证明,神经干细胞上存在11β-HSD1。还证明,在本实验条件下,非活性糖皮质激素(DHC)抑制神经干细胞的增殖,对分化和迁移没有影响;BVT(BVT-14225)通过抑制11β-HSD1酶的还原活性,促进应激环境下神经干细胞的增殖和迁移,但对分化没有影响;并且BVT无细胞毒性作用,不影响11β-HSD1蛋白表达的水平。
以上对本发明的实施方式进行了说明。但是,本发明不限定于上述实施方式。凡在本发明的精神和原则之内,所做的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (8)
1.11β-HSD1抑制剂用于制备神经保护剂的用途。
2.根据权利要求1的用途,其中所述11β-HSD1抑制剂选自BVT-14225及其药学上可接受的盐、BVT-2733及其游离碱以及其游离碱与其他药学上可接受的酸形成的盐、AZD4017及其药学上可接受的盐或酯、AZD8329及其药学上可接受的盐或酯、姜黄素及其类似物。
3.根据权利要求1或2的用途,其中所述神经保护剂是应激环境下神经干细胞保护剂。
4.根据权利要求1-3中任一项的用途,其中所述应激环境包括以下疾病或手术的急性应激环境以及慢性应激环境:
1)胎儿手术,例如因胎儿脊柱裂、双胎输血综合征、前置血管、先天性膈疝而进行的胎儿手术;
2)产时手术或新生儿手术,例如因脐膨出、先天性双主动脉弓性心脏病、食管闭锁、食管气管瘘、食道裂孔疝而进行的产时手术或新生儿手术;
3)慢性社会应激环境,例如孕期、新生儿期的母婴分离,暴力经历以及不当的家庭护理造成的慢性应激环境。
5.11β-HSD1抑制剂用于制备促进神经干细胞增殖和迁移的药物的用途。
6.根据权利要求5的用途,其中所述11β-HSD1抑制剂选自BVT-14225及其药学上可接受的盐、BVT-2733及其游离碱以及其游离碱与其他药学上可接受的酸形成的盐、AZD4017及其药学上可接受的盐或酯、AZD8329及其药学上可接受的盐或酯、姜黄素及其类似物。
7.根据权利要求5或6的用途,其中所述促进神经干细胞增殖和迁移的药物能够用于预防和/或治疗以下疾病或病症:中风、阿兹海默症、帕金森综合征、颅脑创伤后遗症;还可用作以下手术前、手术中或手术后的辅助药物:胎儿手术(如因胎儿脊柱裂、双胎输血综合征、前置血管、先天性膈疝而进行的胎儿手术)、产时手术或新生儿手术(如因脐膨出、先天性双主动脉弓性心脏病、食管闭锁、食管气管瘘、食道裂孔疝而进行的产时手术或新生儿手术)。
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