CN107320718B - 促生长激素释放激素激动剂在制备抗血管钙化药物中的应用 - Google Patents
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Abstract
本发明公开了促生长激素释放激素激动剂在医学上的一种新用途,即促生长激素释放激素激动剂在制备抗血管钙化药物中的应用。GHRH‑A可以提高平滑肌细胞中cAMP水平和蛋白激酶A(PKA)活性,降低平滑肌细胞NAPDH氧化酶表达和活性,降低平滑肌细胞中ROS的水平,降低平滑肌细胞中Runx2表达和活性及细胞碱性磷酸酶(ALP)表达和活性,以及降低体内血液中无机磷的含量,从而减少主动脉中钙沉积和磷沉积,减少主动脉中ALP活性,达到治疗或预防血管钙化、动脉硬化的目的。
Description
技术领域
本发明涉及药理学和化学生物学领域,具体涉及促生长激素释放激素激动剂在制备抗血管钙化药物中的应用。
背景技术
血管钙化(Vascular Calcification,VC)是一种以血管内膜及中层钙盐沉积为特征的疾病,与多种疾病如动脉粥样硬化、糖尿病及慢性肾病存在密切联系,严重影响这些疾病的预后。早期的研究认为动脉钙化主要是被动的钙、磷酸盐沉着。然而,随着研究的深入,人们发现动脉钙化过程与骨矿化之间存在相似性,不只是被动地钙盐沉积,更多的是平滑肌细胞在应激环境下向成骨样细胞分化,导致钙化的持续发展。同时,目前依旧缺乏有效治疗动脉钙化的药物。
促生长激素释放激素(Growth Hormone Release Hormone,GHRH)是一种神经多肽,由下丘脑分泌,经垂体门脉***进入后与垂体细胞表面促生长激素释放激素受体结合,促进生长激素释放。近年来,研究者发现该受体不仅仅表达于垂体,同时也表达在诸如心肌细胞、成纤维细胞、间充质干细胞表面。人工合成的促生长激素释放激素激动剂(GHRH-A)比天然的GHRH更加稳定,因此已经在多种疾病模型中被证明有较好的治疗效果,如心肌梗死、糖尿病及肺损伤等。此外,用GHRH-A预处理骨髓间充质干细胞可以显著促进骨髓间充质干细胞的治疗下肢缺血能力。然而,关于GHRH-A的用途仍有待于进一步研究。
发明内容
本发明的目的在于克服现有技术中存在的缺陷与不足,提供促生长激素释放激素激动剂(GHRH-A)在医学上的一种新用途,即提供促生长激素释放激素激动剂在制备抗血管钙化药物中的应用,所述促生长激素释放激素激动剂能提高平滑肌细胞中cAMP水平和蛋白激酶A活性,降低平滑肌细胞NAPDH氧化酶表达和活性,降低平滑肌细胞中ROS的水平,减少主动脉中钙沉积和磷沉积,和减少主动脉中ALP活性。
GHRH-A以hGHRH的1-29位氨基酸(人GHRH 1-44位氨基酸如SEQ ID NO.1所示)为骨架,进行特定位点氨基酸的替换或者修饰,GHRH-A的序列如表1所示。其中,MR-326/327/502/502包含30个氨基酸,而MR-702则含有31个氨基酸。首行中的数字代表对应的氨基酸位点,“—”表示该氨基酸位点与hGHRH中的相同。
表1:GHRH-A的序列:
其中,N-Me-Tyr指酪氨酸N端存在甲基化,本发明中的GHRH-A还包括其药学上可接受的盐、酯、溶剂合物。
本发明所提供的GHRH-A的新用途是GHRH-A或其药学上可接受的盐、酯、溶剂合物等药物组合在治疗或预防血管钙化中的应用。
实验结果表明,GHRH-A可以提高平滑肌细胞中环磷酸腺苷(cAMP)水平和平滑肌细胞蛋白激酶A(PKA)活性,降低平滑肌细胞NAPDH氧化酶表达和活性,降低平滑肌细胞中活性氧簇(reactive oxygen species,ROS)的水平,降低平滑肌细胞中Runx2表达和活性及细胞碱性磷酸酶(ALP)表达和活性,以及降低体内血液中无机磷的含量,从而减少主动脉中钙沉积和磷沉积,减少主动脉中ALP活性,达到治疗或预防血管钙化、动脉硬化的目的。
所述药物为冻干粉针、注射液、片剂、丸剂、颗粒剂、胶囊、糖浆、软胶囊、药膏或贴剂。
所述药物可通过注射、渗透、吸收、物理或化学介导的方法导入机体如肌肉、皮内、皮下、粘膜组织;或是被其他物质混合或包裹后导入机体。必要时在上述药物中还可以加入一种或多种药学上可接受的载体。所述载体包括药学领域常规的稀释剂、赋形剂、填充剂、粘合剂、湿润剂、崩解剂、吸收促进剂、表面活性剂、吸附载体、润滑剂等。
所述血管钙化包括所有涉及钙盐在血管壁及心脏瓣膜的沉积为特征的血管及瓣膜病变。
本发明GHRH-A治疗心血管疾病(如血管钙化、动脉硬化、心肌病等)药物的药理机制,包括1)GHRH-A可以治疗或预防血管钙化。2)GHRH-A可以治疗或预防动脉硬化。3)GHRH-A可以治疗或预防平滑肌细胞钙化。4)GHRH-A可以提高平滑肌细胞中cAMP水平。5)GHRH-A可以提高平滑肌细胞PKA活性。6)GHRH-A可以降低平滑肌细胞NAPDH氧化酶表达和活性。7)GHRH-A可以降低平滑肌细胞中ROS的水平。8)GHRH-A可以降低平滑肌细胞Runx2表达和活性。9)GHRH-A可以降低平滑肌细胞碱性磷酸酶(ALP)表达和活性。10)GHRH-A可以降低体内血液中无机磷的含量。11)GHRH-A可以减少主动脉中钙沉积。12)GHRH-A可以减少主动脉中磷沉积。13)GHRH-A可以减少主动脉中ALP活性。
本发明利用体外细胞培养钙化模型及体内转基因小鼠(OPG-/-,自发动脉钙化小鼠)模型检测GHRH-A,MR409,证实了促生长激素释放激素激动剂在抑制钙化中的作用。
实验证明,GHRH-A(MR409)可以显著抑制平滑肌细胞在体外的钙化过程,抑制成骨相关基因的表达。同时,体内实验进一步证实GHRH-A降低血磷水平,减少动脉钙化面积。
如图1所示,是GHRH-A抑制平滑肌细胞体外钙化示意图。其中,A)小鼠主动脉平滑肌细胞体外β-GP诱导钙化后行茜素红染色;B)钙化统计图,纵坐标为A中染料萃取后的吸光度值。C)人主动脉平滑肌细胞体外钙化诱导;D)钙化统计图,纵坐标为C中染料萃取后的吸光度值。Pi:β-甘油磷酸(β-glycerophosphoric acid),MR409即GHRH-A,MIA602即GHRH拮抗剂(GHRH Antagonist)。从图中可以得出:GHRH-A(MR409),可以抑制β-磷酸甘油(β-GP,Pi)诱导的平滑肌细胞钙化。
如图2所示,是GHRH-A抑制平滑肌细胞成骨相关蛋白的表达及活性示意图。,钙化过程中钙盐沉积(A)减少,ALP活性(B)增强,Runx2的表达增加,包括mRNA水平(E)和蛋白水平(C,D),可被GHRH-A抑制。从图中可得出:GHRH-A可显著抑制成骨相关蛋白Runx2表达及碱性磷酸酶(ALP)活性。
图3是高磷诱导平滑肌细胞氧化应激示意图。平滑肌细胞处于高磷环境时,细胞内ROS含量显著增加(图4A),流式检测荧光强度显示MR409抑制高磷诱导的ROS增加。A)DHE染色检测ROS情况,红色越浓提示ROS含量越高。B)将A中细胞消化后进行流式检测,定量分析荧光强度。NC:空白对照组;Pi:添加有10mMβ-磷酸甘油;Pi+MR409:10mMβ-磷酸甘油+10nM的GHRH-A;Pi+MR409+MIA602:10mMβ-磷酸甘油+10nM的GHRH-A+100nM的GHRH-Antagonist。从图中可得出:GHRH-A可抑制高磷诱导平滑肌细胞ROS含量增加。
图4是GHRH-A抑制OPG-/-小鼠主动脉钙化示意图。A)GHRH-A干预4周后收集主动脉,行Micro-CT扫描,GHRH-A可显著抑制钙盐在主动脉的沉积。B)Micro-CT统计结果。C)主动片石蜡切片后茜素红染色,发现GHRH-A减少主动脉环上的钙结节;D)染色阳性面积占总面积的比例。WT:野生型小鼠;OPG-NC:OPG-/-小鼠溶剂对照组;OPG-MR409:OPG-/-小鼠+GHRH-A。从图中可得出:GHRH-A可抑制OPG-/-小鼠动脉钙化程度。
图5是GHRH-A减少OPG-/-小鼠主动脉中的钙磷沉积、ALP活性及血磷水平示意图。A)主动脉钙含量;B)主动脉磷含量;C)主动脉ALP活性;E)血磷水平。从图中可得出:GHRH-A降低OPG-/-小鼠血磷浓度,减少主动脉中的钙磷沉积及ALP活性。
图6是GHRH激动剂预处理SMCs(平滑肌细胞)示意图。提高SMCs中cAMP浓度(A),降低NAPDH氧化酶活性(C,D),和ROS水平(B)。MR409的效果都会被PKA抑制剂H89消除,表明GHRH-A降低NAPDH氧化酶活性及ROS水平都与PKA活性相关。从图中可得出:GHRH-A可以提高平滑肌细胞中cAMP水平,降低NAPDH氧化酶表达和活性,ROS水平。
在一个优选的实施方式中,本发明药物形式推荐冻干粉针,以0.1%DMSO+10%丙二醇作为溶剂,使用方式为皮下注射。在该注射浓度下,促生长激素释放激素激动剂表现出了抗血管钙化的特性,实现了对血管钙化病理进程的干预和病理症状的改善。
附图说明
下面结合附图对本发明的具体实施方式作进一步详细说明。
图1是GHRH-A抑制平滑肌细胞体外钙化示意图。
图2是GHRH-A抑制平滑肌细胞成骨相关蛋白的表达及活性示意图。
图3是高磷诱导平滑肌细胞氧化应激示意图。
图4是GHRH-A抑制OPG-/-小鼠主动脉钙化示意图。
图5是GHRH-A减少OPG-/-小鼠主动脉中的钙磷沉积、ALP活性及血磷水平示意图。
图6是GHRH激动剂预处理SMCs(平滑肌细胞)示意图。
具体实施方式
下面通过具体实施例对本发明进行说明,但本发明并不局限于此。
下述实施例中所述实验方法,如无特殊说明,均为常规方法;所述试剂和材料,如无特殊说明,均可从商业途径获得。其中促生长激素释放激素激动剂(GHRH-A,MR409)及拮抗剂(GHRH-Anta,MIA602)由University of Miami,Andrew V.Schally提供。
MR409氨基酸序列:N-Me-Tyr—D-Ala—Asp—Ala—Ile—Phe—Thr—Asn—Ser—Tyr—Arg—Orn—Val—Leu—Abu—Gln—Leu—Ser—Ala—Arg—Orn—Leu—Leu—Gln—Asp—Ile—Nle—Asp—Arg-NHCH3。(N-甲基酪氨酸—D-丙氨酸—天冬氨酸—丙氨酸—异亮氨酸—苯丙氨酸—苏氨酸—天冬酰胺—丝氨酸—酪氨酸—精氨酸—鸟氨酸—缬氨酸–亮氨酸—2-氨基丁酸—谷氨酰胺—亮氨酸—丝氨酸—丙氨酸—精氨酸—鸟氨酸—亮氨酸—亮氨酸—谷氨酰胺—天冬氨酸—异亮氨酸—正亮氨酸—天冬氨酸—精氨酸-NHCH3)。
实施例1:检测GHRH-A在平滑肌细胞体外钙化的作用
实施流程:6-8周龄C57BL/6雄性小鼠(购于上海斯莱克动物实验动物有限公司)异氟烷麻醉后处死获取胸主动脉,经胶原酶及弹力酶联合消化后铺板培养,选取3-5代平滑肌细胞作为实验对象。在12孔板中进行诱导,分组为:(A)对照组,(B)成骨培养基组,(C)成骨培养基+GHRH-A组,(D)成骨培养基+GHRH-A——GHRH-Anta。GHRH-A及GHRH-Anta溶解于DMSO(Sigma,USA)中,以1000x母液储存。GHRH-A工作浓度为10nM,GHRH-Anta工作浓度为100nM。诱导14天后行钙化染色,即茜素红(赛业生物科技,中国)染色,并定量。在加入GHRH-A后,平滑肌钙化诱导后的钙结节显著减少,定量分析显示,茜素红OD值显著降低,从成骨培养基组的0.92±0.05降低到0.53±0.02。如图1所示。
免疫印迹法检测细胞内Runx2蛋白表达水平。同前述诱导方案,收集D14蛋白,20μg蛋白样品在10~15%(v/v)SDS-PAGE梯度胶用Mini-Gel system(Bio-Rad,Piscataway,USA)电泳分离,用Hoefer Transfer Tank(Bio-Rad,Piscataway,USA)将蛋白转移至PVDF膜(Bio-Rad,USA)上,膜置于缓冲液[Tris缓冲盐溶液,含0.1%(v/v)Tween-20(TBS-T),7%(w/w)牛奶,pH 7.6]室温下封闭2h,加入相应一抗(1:1000,Cell Signaling,USA)于4℃条件下孵育过夜,Anti-GAPDH(KC-5G5,KANGCHEN,China)作为蛋白上样对照;膜用0.5%(v/v)TBS-T洗涤后室温下与结合辣根过氧化物酶的抗兔IgG(Santa Cruz Technology,USA)孵育2小时,最后TBS-T及TBS洗涤后用ECL溶液显色(Bio-Rad,USA),扫描后用Image pro5.2软件分析信号。结果显示:GHRH-A可以抑制平滑肌细胞在成骨转录因子的表达,且这种抑制作用可以被GHRH-Anta阻断(如图2所示)。
实施例2:检测GHRH-A对体外主动脉钙化的作用
实施流程:8周龄C57BL/6野生型小鼠及同周龄OPG-/-小鼠(浙江大学医学院附属第二医院单鹏飞课题组赠予)进行如下分组:(A)野生型小鼠,(B)OPG-/-小鼠,(C)OPG-/-小鼠+GHRH-A。GHRH-A用0.1%DMSO+10%丙二醇溶解。GHRH-A的使用剂量为5μg/d,皮下注射,连续干预4周。4周后安乐死小鼠,留取血液样本及主动脉。主动脉用10%多聚甲醛浸泡24小时后用D-PBS洗净,进行Micro-CT扫描检测(YUEBO Biotechnology,China)。结果显示:野生型小鼠主动脉几乎无钙化灶,而OPG-/-小鼠可见显著的钙化结节,而在使用GHRH-A组,主动脉钙化结节减少。如图3所示。
小鼠全血经5000rpm*10min离心后,收集血清,测定血磷水平。结果显示,OPG-/-小鼠外周血中磷离子浓度显著升高,而在GHRH-A处理后的OPG-/-小鼠血清中,磷离子浓度降至几乎与野生型小鼠同一水平。如图4所示。
序列表
<110> 余红
<120> 促生长激素释放激素激动剂在制备抗血管钙化药物中的应用
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<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 44
<212> PRT
<213> GHRH
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Tyr Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly
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Gln Leu Ser Ala Arg Lys Leu Leu Gln Asp Ile Met Ser Arg Gln
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Gln Gly Glu Ser Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu
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<212> PRT
<213> MR409
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Tyr Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Orn Val Leu Abu
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Gln Leu Ser Ala Arg Orn Leu Leu Gln Asp Ile Nle Asp Arg
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Claims (5)
1.促生长激素释放激素激动剂在制备抗血管钙化药物中的应用,所述促生长激素释放激素激动剂能提高平滑肌细胞中cAMP水平和蛋白激酶A活性,降低平滑肌细胞NAPDH氧化酶表达和活性,降低平滑肌细胞中ROS的水平,减少主动脉中钙沉积和磷沉积,和减少主动脉中ALP活性。
2.如权利要求1所述的应用,其特征在于,所述药物以促生长激素释放激素激动剂或其药学上可接受的盐、酯、溶剂合物为主要成分。
3.如权利要求2所述的应用,其特征在于,所述药物为冻干粉针、注射液、片剂、丸剂、颗粒剂、胶囊、糖浆、软胶囊、药膏或贴剂。
4.如权利要求3所述的应用,其特征在于,所述药物为冻干粉针,以0.1%DMSO+10%丙二醇作为溶剂,使用方式为皮下注射。
5.如权利要求1所述的应用,其特征在于,所述血管钙化是所有涉及钙盐在血管壁及心脏瓣膜的沉积为特征血管及瓣膜病变。
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