CN113679732A - 女贞苷在制备防治钙化性主动脉瓣疾病药物中的应用 - Google Patents
女贞苷在制备防治钙化性主动脉瓣疾病药物中的应用 Download PDFInfo
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Abstract
本发明涉及女贞苷在制备治疗钙化性主动脉瓣疾病药物中的应用。本发明经研究发现女贞苷可以有效下调瓣膜间质细胞中成骨分化标志基因RUNX2和Osterix的表达，抑制瓣膜间质细胞成骨分化，从而延缓或者抑制瓣膜钙化，并提供了一种通过女贞苷处理后可显著改善瓣膜间质细胞的体外成骨分化的方法，进而为钙化性主动脉瓣疾病的预防和治疗提供了新的药物选择。
Description
技术领域：
本发明涉及生物医药领域，具体涉及女贞苷在制备防治钙化性主动脉瓣疾病药物中的应用。
背景技术：
钙化主动脉瓣疾病(calcific aortic valve disease，CAVD)是一种高发病率和高死亡率的老年性进行性疾病，主要病理生理改变为主动脉瓣瓣叶纤维增生钙化从而导致瓣膜变硬并引起血流动力学改变，从而影响心脏功能。以往观点均认为CAVD是一种与年龄相关的退行性病变，是随着年龄增长，瓣膜组织的退化硬化过程。然而近年来的基础研究表明，CAVD是一种涉及内皮损伤，炎症细胞浸润、细胞外基质重塑以及瓣膜间质细胞成骨样分化等复杂病理改变的主动进展过程。瓣膜组织中的细胞主要包括瓣膜内皮细胞(Valvularendothelial cells,VEC)、瓣膜间质细胞(Valvular interstitial cells,VICs)、瓣膜前体细胞等，已有研究证实瓣膜间质细胞成骨样分化所导致的钙盐增多可能是瓣膜钙化发生的重要始动因素。目前，CAVD缺乏有效的临床可用药物治疗方案，其主要治疗手段为主动脉瓣置换手术。然而，接受外科手术的患者必然会承担较高的医疗手术风险及经济负担。因此，探究CAVD具体发病机制，采用非手术方法有效预防和/或治疗钙化性主动脉瓣疾病是目前临床治疗CAVD的迫切需求。
钙敏感受体(calcium-sensing receptor，CasR),是G蛋白偶联受体家族的一员，该受体参与调节甲状旁腺激素的合成和分泌，并广泛分布于甲状旁腺，胃肠道，神经系统及骨组织，能够感知并影响细胞外钙离子浓度变化。研究发现，瓣膜间质细胞中CasR的激活可能对CAVD的进展起到关键的促进作用，这一结果提示通过药物调节CasR表达或者活性而改善钙化性主动脉瓣疾病的可能性。
女贞苷是中药女贞子的专属性成分，药理活性明确，在维持钙代谢平衡，预防老年性骨质疏松症中作用明显。专利申请号为CN201910418873.7的专利提供了阿克苷和/或紫茎女贞苷A在制备防治血管内皮细胞炎性损伤药物中的应用。最新研究也表明，女贞苷是一种潜在的CasR拮抗剂(Feng R,et al.Protective Effects of Ligustroflavone,anActive Compound from Ligustrum lucidum,on Diabetes-Induced Osteoporosis inMice:A Potential Candidate as Calcium-Sensing Receptor Antagonist.Am J ChinMed.2019；47(2):457-476)。但是，对于女贞苷与钙化性主动脉瓣疾病之间的研究，目前尚未见报道。
发明内容：
(一)解决的技术问题
针对上述背景，本发明通过研究发现女贞苷可以有效下调瓣膜间质细胞中成骨分化标志基因RUNX2和Osterix的表达，抑制瓣膜间质细胞成骨分化，从而延缓或者抑制瓣膜钙化，提出了女贞苷在制备防治钙化性主动脉瓣疾病药物中的应用，并提出一种通过女贞苷处理后可显著改善瓣膜间质细胞的体外成骨分化的方法，进而为钙化性主动脉瓣疾病的预防和治疗提供新的药物选择。
(二)技术方案
为达到上述目的，本发明采用如下技术方案：
本发明公开第一方面，提供女贞苷在制备防治钙化性主动脉瓣疾病药物中的应用，本发明中，女贞苷是指CAS号为260413-62-5；分子式为C33H40O18的物质，其化学结构如图1A所示。
具体地，所述女贞苷用于制备干预瓣膜间质细胞的成骨样分化的药物。
进一步地，所述女贞苷用于制备调节CasR表达或活性的药物。
进一步地，所述女贞苷的浓度为10μM-50μM。
进一步地，所述女贞苷的浓度为25μM。
女贞苷是CasR的一个潜在拮抗剂，在成骨诱导的体外成骨分化细胞模型下，显示出女贞苷可以抑制瓣膜间质细胞成骨分化，并证明其通过抑制钙敏感受体CasR表达，下调成骨分化标志基因RUNX2和Osterix表达，发挥抗钙化作用。
本发明公开第二方面，提供一种抑制瓣膜间质细胞成骨分化的细胞培养方法，利用含有女贞苷的钙化培养基诱导培养人瓣膜间质细胞7-14天，所述含有女贞苷的钙化培养基组成如下：25μM女贞苷，50mg/mL维生素C，5mmol/Lβ-甘油磷酸,100nmol/L地塞米松，2％胎牛血清，高糖培养基(DMEM)。
具体的，所述诱导培养在二氧化碳体积浓度5％，氧气浓度95％的混合气体中进行，培养温度为37℃恒温。
(三)有益效果
本发明产生的有益效果是：本发明提供了女贞苷在制备防治钙化性主动脉瓣疾病药物中的应用，女贞苷可以有效抑制CasR表达，下调瓣膜间质细胞成骨分化标志基因RUNX2和Osterix的表达，从而抑制瓣膜间质细胞成骨分化,发挥抗主动脉瓣钙化效果，本发明还提供了一种通过女贞苷处理后可明显减弱瓣膜间质细胞体外成骨分化表型转化的方法，进而为钙化性主动脉瓣疾病提供了新的药物选择。
附图说明：
为了更清楚地说明本发明实施例中的技术方案，下面将对实施例中所需要使用的附图作简单地介绍。
图1A为女贞苷的化学结构；
图1B为25μM浓度女贞苷处理改善人瓣膜间质细胞在体外钙化诱导培养条件下的CasR蛋白水平变化图：对照组代表常规非钙化诱导培养基培养条件，OM代表钙化诱导培养基培养条件，OM+女贞苷代表女贞苷处理组。
图2为25μM浓度女贞苷处理改善人瓣膜间质细胞在体外钙化诱导培养条件下的CasR蛋白免疫荧光水平变化图：对照组代表常规非钙化诱导培养基培养条件，OM代表钙化诱导培养基培养条件，OM+女贞苷代表女贞苷处理组。
图3A为25μM浓度女贞苷处理改善人瓣膜间质细胞在体外钙化诱导培养条件下的钙化标志基因蛋白水平变化图：对照组代表常规非钙化诱导培养基培养条件，OM代表钙化诱导培养基培养条件，OM+女贞苷代表女贞苷处理组。
图3B为25μM浓度女贞苷处理改善人瓣膜间质细胞在体外钙化诱导培养条件下的碱性磷酸酶活性变化图：对照组代表常规非钙化诱导培养基培养条件，OM代表钙化诱导培养基培养条件，OM+女贞苷代表女贞苷处理组。
图4A为25μM浓度女贞苷处理改善人瓣膜间质细胞在体外钙化诱导培养条件下的钙结节形成图：对照组代表常规非钙化诱导培养基培养条件，OM代表钙化诱导培养基培养条件，OM+女贞苷代表女贞苷处理组。
图4B为25μM浓度女贞苷处理改善人瓣膜间质细胞在体外钙化诱导培养条件下的钙含量变化图：对照组代表常规非钙化诱导培养基培养条件，OM代表钙化诱导培养基培养条件，OM+女贞苷代表女贞苷处理组。
图5A为25μM浓度女贞苷处理改善人瓣膜间质细胞在体外钙化诱导培养条件下CasR过表达引起的钙化标志基因蛋白水平变化图：OM代表钙化诱导培养基培养条件，OM+CasR代表CasR过表达组，OM+CasR+女贞苷代表女贞苷处理组。
图5B为25μM浓度女贞苷处理改善人瓣膜间质细胞在体外钙化诱导培养条件下CasR过表达引起的钙结节变化图：OM代表钙化诱导培养基培养条件，OM+CasR代表CasR过表达组，OM+CasR+女贞苷代表女贞苷处理组。
图5C为25μM女贞苷处理改善人瓣膜间质细胞在体外钙化诱导培养条件下CasR过表达引起的钙定量变化图：OM代表钙化诱导培养基培养条件，OM+CasR代表CasR过表达组，OM+CasR+女贞苷代表女贞苷处理组。
具体实施方式：
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述。
步骤一、钙化诱导培养基诱导人瓣膜间质细胞细胞成骨分化细胞模型的构建。
取培养密度70％左右的人瓣膜间质细胞，用含2％胎牛血清的DMEM高糖培养基饥饿过夜，第二日以新配置成骨诱导培养基(50mg/mL维生素C，5mmol/Lβ-甘油磷酸,100nmol/L地塞米松，溶剂为含2％胎牛血清的高糖DMEM培养基)对人瓣膜间质细胞进行成骨分化诱导，诱导7天后检测不同处理组CasR及钙化标志基因RUNX2、Osterix蛋白表达变化。诱导21天后考察不同处理对瓣膜间质细胞成骨分化的影响。
步骤二、女贞苷抑制人瓣膜间质细胞细胞成骨分化表型。
采用如上步骤一构建的人瓣膜间质细胞成骨分化模型，将人瓣膜间质细胞分为3个不同处理组，分别为对照组(正常培养基)，OM组(成骨诱导培养基)以及OM+女贞苷组(成骨诱导培养基+女贞苷)，女贞苷购自MedChemExpress公司(https://www.medchemexpress.cn/ligustroflavone.html)，产品目录HY-N0546，浓度为在培养基中的终浓度。各组同时处理7天后提取蛋白检测CasR及钙化标志基因RUNX2、Osterix蛋白表达变化；免疫荧光染色观察CasR表达变化；处理21天后各组进行茜素红染色，观察成骨分化表型变化。
由图1B和图2可知使用OM处理人瓣膜间质细胞7天后，CasR蛋白表达量与对照组相比显著上调(P＜0.05)。而向OM处理组中加入浓度为25μM女贞苷后，则显著抑制了OM培养基诱导的CasR蛋白表达上调效应(P＜0.05)。
由图3A和图3B可知，可知使用OM处理人瓣膜间质细胞7天后，RUNX2、Osterix(成骨分化标志基因)蛋白表达量与对照组相比显著上调(P＜0.05)。而向OM处理组中加入浓度为25μM女贞苷后，则显著抑制了OM培养基诱导的RUNX2、Osterix蛋白表达上调效应(P＜0.05)。使用OM处理人瓣膜间质细胞7天后，细胞碱性磷酸酶活性与对照组相比显著升高(P＜0.05)。而向OM处理组中加入浓度为25μM女贞苷后，则显著抑制了OM培养基诱导的细胞碱性磷酸酶活性升高效应(P＜0.05)。
由图4A和图4B可知，在各组处理21天后，对各组人瓣膜间质细胞进行茜素红染色及钙定量分析，结果显示使用OM处理人瓣膜间质细胞7天后，细胞茜素红阳性染色及钙含量与对照组相比显著增多(P＜0.05)。而向OM处理组中加入浓度为25μM女贞苷后，则显著抑制了OM培养基诱导的细胞茜素红阳性染色及钙含量增多效应(P＜0.05)。
以上结果表明，女贞苷可以抑制人瓣膜间质细胞体外成骨分化表型转变。
步骤三、女贞苷可抑制由CasR过表达引起的人瓣膜间质细胞成骨分化表型增强效应。
通过分离培养瓣膜间质细胞,构建了过表达腺病毒。利用过表达腺病毒转染瓣膜间质细胞72小时后,利用成骨分化诱导培养基诱导瓣膜间质细胞7天构建体外成骨分化模型。利用茜素红染色、钙定量分析以及检测RUNX2、Osterix(成骨分化标志基因)蛋白表达水平评估瓣膜间质细胞钙化程度。茜素红染色及钙定量分析显示与对照组相比,过表达CasR后瓣膜间质细胞钙盐结节及钙盐沉积明显增多(P＜0.05)。而女贞苷处理可抑制由CasR过表达引起的人瓣膜间质细胞钙盐结节及钙盐沉积增多效应(P＜0.05)，见图5A和图5B。
RUNX2、Osterix蛋白表达水平检测显示显示与对照组相比,过表达CasR后瓣膜间质细胞RUNX2、Osterix蛋白表达水平明显上调(P＜0.05)。而女贞苷处理可抑制由CasR过表达引起的人瓣膜间质细胞RUNX2、Osterix蛋白表达水平上调效应(P＜0.05)，见图5C。
以上结果表明女贞苷可通过抑制CasR表达从而抑制人瓣膜间质细胞成骨分化表型转变。
综上所述，女贞苷可以有效下调瓣膜间质细胞中成骨分化标志基因RUNX2和Osterix的表达，抑制瓣膜间质细胞成骨分化，从而延缓或者抑制瓣膜钙化，为钙化性主动脉瓣疾病的预防和治疗提供新的药物选择。
最后需要说明的是，以上实施例仅用于说明本发明而非限制本发明的保护范围。另外，在阅读了本发明的技术内容之后，本领域技术人员可以对本发明作各种改动、修改或变型，所有的这些等价形式同样属于本申请所要求限定的保护范围之内。
Claims (8)
1.女贞苷在制备防治钙化性主动脉瓣疾病药物中的应用。
2.根据权利要求1所述的女贞苷在制备防治钙化性主动脉瓣疾病药物中的应用，其特征在于，所述女贞苷为从木犀科女贞属植物女贞子的叶中提取得到的化合物，CAS号为260413-62-5，分子式为C33H40O18。
3.根据权利要求2中所述的药物，其特征在于，所述女贞苷的浓度为10μM-50μM。
4.根据权利要求3中所述的药物，其特征在于，所述女贞苷的浓度为25μM。
5.根据权利要求1所述的女贞苷在制备防治钙化性主动脉瓣疾病药物中的应用，其特征在于，所述女贞苷用于制备干预瓣膜间质细胞的成骨样分化的药物。
6.根据权利要求1所述的女贞苷在制备防治钙化性主动脉瓣疾病药物中的应用，其特征在于，所述女贞苷用于制备调节CasR表达或活性的药物。
7.一种抑制瓣膜间质细胞成骨分化的细胞培养方法，其特征在于，利用含有女贞苷的钙化培养基诱导培养人瓣膜间质细胞7-14天，含有女贞苷的钙化培养基组成如下：25μM女贞苷，50mg/mL维生素C，5mmol/Lβ-甘油磷酸,100nmol/L地塞米松，2％胎牛血清，高糖培养基(DMEM)。
8.根据权利要求7所述的一种抑制瓣膜间质细胞成骨分化的细胞培养方法，其特征在于，诱导培养在二氧化碳体积浓度5％，氧气浓度95％的混合气体中进行，培养温度为37℃恒温。
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