CN112603918B - κ-阿片受体激动剂U50,488H在制备治疗血管钙化的药物中的用途 - Google Patents
κ-阿片受体激动剂U50,488H在制备治疗血管钙化的药物中的用途 Download PDFInfo
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Abstract
本发明属于医药技术领域，涉及κ‑阿片受体激动剂U50,488H的新用途，更具体的，涉及κ‑阿片受体激动剂U50,488H在制备治疗血管钙化的药物中的用途，本发明通过实验证明，其抑制血管钙化的作用机理是：通过降低PFKFB3表达，进而抑制糖酵解水平，从而抑制了高磷诱导的VSMCs的钙化，研究结果为临床治疗血管钙化提供了潜在的药物靶点和治疗策略。
Description
技术领域
本发明属于医药技术领域，涉及κ-阿片受体激动剂U50,488H的新用途，更具体的，涉及κ-阿片受体激动剂U50,488H在制备治疗血管钙化的药物中的用途。
背景技术
血管钙化(Vascular Calcification，VC)是动脉壁间叶细胞，尤其是平滑肌细胞(vascular smooth muscle cell,VSMCs)在各种病理因素作用下转化为成骨细胞表型，导致钙盐异常沉积在血管壁的过程。血管钙化降低血管的顺应性，造成血管局部缺氧，增加血管破裂以及动脉瘤形成的危险，易导致心肌缺血、左心室肥大、心力衰竭和脑卒中等心血管事件的发生。尽管对血管钙化进行了广泛的研究并耗费了巨大的医疗成本，目前还没有任何治疗方法可以阻止或逆转其进程。虽然血管钙化的潜在机制尚未阐明，但缺氧导致的血管局部糖酵解增加被认为是血管钙化的基础因素之一。U50,488H是一种κ-OR选择性激动剂，它可以通过激活心肺组织κ-OR，改善血管功能，减少缺氧引起的细胞凋亡发挥血管保护作用。本发明前期研究证实了κ-OR激活后对大中血管内皮细胞具有保护作用，但对于κ-OR激活是否能够改善血管钙化时的血管重塑，以及逆转 VSMCs的成骨转化及凋亡，改善局部钙磷比例及糖酵解水平等迄今国内外尚无报道。
发明内容
本发明所要解决的技术问题在于针对现有技术的不足，提供κ-阿片受体激动剂U50,488H的新用途。
为解决上述技术问题，本发明采用如下技术方案：
κ-阿片受体激动剂U50,488H在制备治疗血管钙化的药物中的用途。
进一步的，所述κ-阿片受体激动剂U50,488H能够靶向抑制PFKFB3的表达。
更进一步的，所述κ-阿片受体激动剂U50,488H通过靶向抑制PFKFB3的表达，进而抑制糖酵解水平。
更进一步的，所述κ-阿片受体激动剂U50,488H通过靶向抑制PFKFB3的表达，进而抑制糖酵解水平，从而抑制高磷诱导的VSMCs的钙化。
进一步的，所述治疗血管钙化的药物是以κ-阿片受体激动剂U50,488H为活性成分，加上药学上可接受的辅料或辅助性成分制备而成的药物制剂。
与现有技术相比，本发明具有如下有益效果：
本发明首次发现κ-阿片受体激动剂U50,488H在具有良好的抑制血管钙化的作用，并通过研究表明其机理是通过降低PFKFB3表达，进而抑制糖酵解水平，从而抑制了高磷诱导的VSMCs的钙化，研究结果为临床治疗血管钙化提供了潜在的药物靶点和治疗策略。
附图说明
图1为本发明组织贴壁法培养大鼠血管平滑肌细胞(40×视野)，图中，A：平滑肌细胞从组织块爬出B：6-10d后细胞融合C：“峰-谷”现象。
图2为本发明免疫荧光染色鉴定大鼠原代血管平滑肌细胞(400×视野)，图中，A：DAPI B：a-SMA C：Merge。
图3为本发明不同浓度U50,488H对高磷诱导VSMCs钙化的影响(Scale bar＝50μm)，图中，A为RUNX2水平的Western blot典型图，B为RUNX2 水平的定量分析图，C为VSMC茜素红染色典型图，D为钙沉积量的定量分析图；1：对照组；2：高磷诱导组；3-6为高磷诱导+U50,488H组，U50,488H浓度依次为5μmol/L、10μmol/L、20μmol/L、40μmol/L。
图4为本发明浓度为40μmol/L的U50,488H对高磷诱导VSMCs细胞活力的影响，图中，1：对照组；2：对照+U组；3：高磷诱导组；4：高磷诱导+U 组；5：高磷诱导+N(nor-BNI,5μmol/L,一种选择性的κ-阿片受体阻断剂)+U 组。n＝6，与对照组比较，aP<0.05；与高磷诱导组比较，cP<0.05；与高磷诱导 +U组比较，eP<0.05。
图5为本发明浓度为40μmol/L的U50,488H对高磷诱导VSMCs钙化相关蛋白表达的影响，图中，A为RUNX2、BMP-2和SM22a水平的Western blot典型图，B为RUNX2水平的定量分析图，C为BMP-2水平的定量分析图，D为 SM22a水平的定量分析图；1：对照组；2：对照+U组；3：高磷诱导组；4：高磷诱导+U组；5高磷诱导+N+U组；n＝5，与对照组比较，aP<0.05，bP<0.01；与高磷诱导组比较，cP<0.05，dP<0.01；与高磷诱导+U组比较，eP<0.05，fP<0.01。
图6为本发明浓度为40μmol/L的U50,488H对高磷诱导的VSMCs中 PFKFB3表达的影响，图中，A为PFKFB3水平的Western blot典型图，B为 PFKFB3水平的定量分析图；1：对照组；2：对照+U组；3：高磷诱导组；4：高磷诱导+U组；5高磷诱导+N+U组；n＝6，与对照组比较，bP<0.01，与高磷诱导组比较，dP<0.01，与高磷诱导+U组比较，eP<0.05。
图7为3-PO(一种PFKFB3小分子抑制剂,1mol/L),对高磷诱导VSMCs钙化及相关蛋白表达的影响(Scale bar＝50μm)，图中，A为RUNX2、BMP-2和SM22a水平的Western blot典型图，B为RUNX2水平的定量分析图，C为 BMP-2水平的定量分析图，D为SM22a水平的定量分析图，E为VSMC茜素红染色典型图，F为钙沉积量的定量分析图；1：对照组；2：对照+3PO组；3：高磷诱导组；4：高磷诱导+3PO组。n＝6，与对照组比较，aP<0.05，bP<0.01，与高磷诱导组比较，cP<0.01。
具体实施方式
下面结合附图和具体实施例对本发明进行详细说明，但不应理解为本发明的限制。如未特殊说明，下述实施例中所用的技术手段为本领域技术人员所熟知的常规手段，下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1
以下通过实验来说明κ-阿片受体激动剂U50,488H对大鼠血管平滑肌细胞钙化模型的抑制钙化作用及其机制
一、大鼠血管平滑肌细胞钙化模型建立及分组
冰上分离大鼠胸主动脉，去除血管外膜及脂肪组织，纵行剖开血管，轻刮去除内膜细胞，将血管中膜组织剪为1mm3组织块，均匀铺在25cm2培养瓶底部，待6h后组织贴壁牢固后加入足量培养基，待平滑肌细胞爬出后培养传代，实验用6-8代细胞。实验分为对照(Con)组：在常氧培育箱中正常培养，血清浓度10％；对照+U50,488H(Con+U)组:在对照组基础上给予U50,488H(40μmol/L) 共同培养；高磷诱导(β-GP)组：给予β-磷酸甘油(10mmol/L)，持续10d 建立钙化模型；高磷诱导+U50,488H(β-GP+U)组：在高磷诱导组基础上给予 U50,488H(40μmol/L)；高磷诱导+nor-BNI+U50,488H(β-GP+N+U)组在高磷诱导组基础上先给予nor-BNI(5μmol/L)，10min后给予U50,488H(40 μmol/L)；对照+3-PO(Con+3-PO)组：在对照组基础上给予3-PO(1mmol/L)；高磷诱导+3-PO(β-GP+3-PO)组：在高磷诱导组基础上给予3-PO(1mmol/L)。
二、VSMCs细胞活力检测
将密度为1×105细胞悬液接种至96板中(100μL/孔)，置于37℃，5％CO2培养箱培养24h后，每孔加入10μL的CCK-8溶液，将培养板再置于培养箱内孵育3h，采用酶标仪测定其吸光度值(450nm)。
三、VSMCs细胞内钙含量、ALP及糖酵解产物检测
取生长对数期的VSMCs随机分组，按照各检测试剂盒的说明书，在指定的波长检测各指标的吸光度值，再根据标准品浓度计算出样品浓度。
四、钙化相关蛋白及PFKFB3表达的Western blot检测
将各组VSMCs按比例加入裂解液，蛋白酶抑制剂及磷酸酶抑制剂，机械匀浆，11000g，4℃离心后取上清，取10μL进行BCA法测量蛋白浓度，其余加入适量Loading buffer冻存。将等量的蛋白加入10％SDS-聚丙烯酰胺凝胶，电泳后转移到PVDF膜上，使用5％脱脂奶粉封闭1h，与目的蛋白一抗(1:1000) 在4℃孵育过夜，然后与辣根过氧化物酶偶联的抗兔或抗鼠IgG二抗(1:5000) 在室温下孵育1h后进行化学增强发光并记录结果。采用Quantity one软件分析灰度。
五、茜素红染色及钙沉积定量
取生长对数期的VSMCs按1×105密度接种于六孔板中，按上述分组给予处理后用PBS溶液漂洗3次，4％多聚甲醛固定10min，95％乙醇固定20min。之后用1％茜素红溶液对细胞进行染色20min，倒置显微镜下观察钙盐沉积情况并拍照扫描，并用10％氯化十六烷基吡啶脱色30min，并测定其吸光度值(562 nm)，与标准品蛋白相比评估钙化程度。
六、统计学处理
所有值均表示为
通过单因素方差分析比较差异，P<0.05为具有显著统计学差异。所有统计均使用GraphPad Prism软件8.0版进行分析。
七、结果与分析
1、大鼠原代平滑肌细胞的建立及鉴定
分离的大鼠胸主动脉血管组织块进行贴壁接种培养，2-4d后可见细胞从边缘爬出，培养6-10d后细胞生长迅速，呈梭形伸展，并融合成片生长，细胞密度达到80％后进行传代，生长过程中可见平滑肌细胞特征性“峰-谷”现象，见图 1。使用第6代细胞行平滑肌特异蛋白(a-SMA)免疫荧光染色，可见胞质中有大量与细胞长轴平行的肌动蛋白纤维细丝(绿色荧光)，细胞计数法计算VSMCs 阳性率为95％以上，见图2。
2、使用不同浓度的U50,488H(5μmol/L、10μmol/L、20μmol/L、40μmol/L) 分别处理高磷诱导的VSMCs，发现U50,488H以浓度依赖的方式抑制成骨特异性转录因子(RUNX2)的表达，其中40μM U50,488H对血管平滑肌细胞成骨分化的抑制作用最明显(后续实验全部采用该浓度)，见图3A和图3B；实验第10d时茜素红染色结果显示，高磷诱导组的红色钙化沉积明显多于正常对照组(P<0.01)；与高磷诱导组相比，随着U50,488H浓度的增高，红色钙化沉积浓度依赖性的逐渐减少，见图3C和图3D。
3、各组VSMCs活力及钙含量、ALP活性比较
与对照组相比，仅加入U50,488H后VSMCs活力无显著改变；高磷诱导后VSMCs活力明显降低(P<0.05)；与高磷诱导组相比，高磷诱导+U组VSMCs 活力显著升高(P<0.05)，U50,488H降低VSMCs活力的作用可被κ-OR阻断剂nor-BNI所阻断，见图4；细胞内钙含量测定及ALP活性结果与VSMCs活力结果一致，见表1。
表1 U50,488H对高磷诱导VSMCs钙含量及 ALP活性的影响(
n＝6)
与对照组相比较，aP<0.05；与高磷诱导组相比较，cP<0.05；与高磷诱导+U 组相比较，eP<0.05。
4、U50,488H对高磷诱导的VSMCs钙化相关蛋白表达的影响
与对照组相比较，仅加入U50,488H并未改变平滑肌特征蛋白SM22a及成骨细胞特征蛋白RUNX2、BMP-2的表达；高磷诱导后SM22a的表达量较对照组显著降低(P<0.05)，RUNX2、BMP-2的表达量显著增加(P<0.05)，加入 U50,488H可显著缓解高磷诱导的VSMCs成骨转化(P<0.05)，并逆转平滑肌 /成骨特征蛋白表达；U50,488H降低VSMCs成骨转化的作用可被κ-OR阻断剂 nor-BNI所阻断，见图5。
5、U50,488H对高磷诱导的VSMCs中PFKFB3表达及糖酵解相关产物的影响
与对照组相比较，仅加入U50,488H并未改变糖酵解关键酶PFKFB3的表达水平；高磷诱导后PFKFB3的表达量较对照组显著增加(P<0.01)，细胞内乳酸和乳酸脱氢酶(LDH)含量也显著增加(P<0.05)，加入U50,488H后可显著降低PFKFB3蛋白表达(P<0.01)以及乳酸和LDH含量(P<0.01)； U50,488H的这种作用可被κ-OR阻断剂nor-BNI所阻断，见图6及表2。
表2 U50,488H对高磷诱导VSMCs乳酸与乳酸脱氢酶(LDH)含量的影响(
n＝8)
注：与Con组相比较，aP<0.05；bP<0.01；与高磷诱导组相比较，dP<0.01；与高磷诱导+U组相比较，eP<0.05。
6、PFKFB3抑制剂3-PO对高磷诱导的VSMCs钙化的影响
与对照组相比较，仅加入3-PO并未改变平滑肌特征蛋白SM22a及成骨细胞特征蛋白RUNX2、BMP-2的表达；高磷诱导后SM22a的表达量较对照组显著降低(P<0.01)，RUNX2、BMP-2的表达量显著增加(P<0.01)，加入3-PO (剂量)可显著缓解高磷诱导的VSMCs成骨转化，并逆转平滑肌/成骨特征蛋白表达(P<0.05)，见图7A-7D；VSMC茜素红染色结果与成骨转化相关蛋白表达趋势一致，见图7E-7F。
本实验通过检测PFKFB3表达、LDH活力和乳酸生成量，发现在高磷诱导作用下，PFKFB3表达、LDH活力及乳酸生成量显著增加。而通过U50,488H 激活κ-OR后可以使PFKFB3、LDH活力及乳酸生成量表达减少，且这一作用均可被nor-BNI及3-PO所阻断。表明κ-OR激活可以发挥显著的降低VSMCs 钙化作用。
综上所述，本发明结果证实U50,488H激活κ-OR后可通过PFKFB3途径降低糖酵解产物的产生，进而抑制高磷诱导的VSMCs钙化，发挥血管保护作用。本发明为治疗血管钙化药物的制备提供了一种新的选择。
尽管已描述了本发明的优选实施例，但本领域内的技术人员一旦得知了基本创造性概念，则可对这些实施例作出另外的变更和修改。所以，所附权利要求意欲解释为包括优选实施例以及落入本发明范围的所有变更和修改。
显然，本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样，倘若本发明的这些修改和变型属于本发明权利要求及其等同技术的范围之内，则本发明也意图包含这些改动和变型在内。
Claims (5)
1.κ-阿片受体激动剂U50,488H在制备治疗血管钙化的药物中的用途。
2.根据权利要求1所述的用途，其特征在于，所述κ-阿片受体激动剂U50,488H能够靶向抑制PFKFB3的表达。
3.根据权利要求2所述的用途，其特征在于，所述κ-阿片受体激动剂U50,488H通过靶向抑制PFKFB3的表达，进而抑制糖酵解水平。
4.根据权利要求3所述的用途，其特征在于，所述κ-阿片受体激动剂U50,488H通过靶向抑制PFKFB3的表达，进而抑制糖酵解水平，从而抑制高磷诱导的VSMCs的钙化。
5.根据权利要求1所述的用途，其特征在于，所述治疗血管钙化的药物是以κ-阿片受体激动剂U50,488H为活性成分，加上药学上可接受的辅料或辅助性成分制备而成的药物制剂。
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