CN114917210A - 石斛酚在制备用于预防和治疗血管钙化的药物中的应用 - Google Patents
石斛酚在制备用于预防和治疗血管钙化的药物中的应用 Download PDFInfo
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Abstract
Description
技术领域
本发明涉及一种石斛酚在制备用于预防和治疗血管钙化的药物中的应用,属于医药技术领域。
背景技术
血管钙化(vascular calcification)是动脉粥样硬化、高血压、糖尿病血管病变、血管损伤、慢性肾病和衰老等普遍存在的共同的病理表现。根据钙化在血管中的位置,血管钙化分为内膜钙化和中膜钙化。血管钙化主要表现为血管壁弹性、顺应性降低,从而影响心血管血流动力,导致高血压、主动脉狭窄、心肌肥厚、心肌缺血和下肢缺血、充血性心力衰竭和结构完整性受损等发生。主动脉病理切片可见斑块内存在不规则的矿化基质或软骨样的骨质。不规则的矿化基质更加常见,而软骨样的骨质一般由不规则的矿化基质发展而来。动脉粥样硬化斑块处凋亡的细胞、细胞外基质和坏死核心处的物质都能够成为血管钙化的病灶。这些钙化灶的进一步发展扩大将破坏斑块的稳定性。斑块不稳定促进了斑块破裂和恶性心血管事件的发生。钙化是一个复杂、主动、部分类似于骨矿化的过程。钙化形成原因和机制复杂,包括高磷血症、血管平滑肌细胞(vascular smooth muscle cell,VSMC)成骨转化、基质囊泡释放、凋亡、炎症、氧化应激、内质网应激等。
目前,引起钙化的分子机制并不完全清楚,特别是钙化的内源性促进因子尚不清楚。普遍认为血管细胞特别是血管平滑肌细胞向成骨样细胞分化,导致细胞和基质中的钙盐沉积,是血管钙化的根本原因。平滑肌细胞在向成骨样细胞分化过程中,其逐渐丢失收缩型特征基因的表达,进而表达成骨细胞特异性基因。RUNX2(runt-related transcriptionfactor 2)也被称作Cbfa1,是成骨分化过程中关键转录因子。RUNX2缺陷的小鼠成骨细胞功能异常,从而导致软骨肥大和骨矿化发育异常。平滑肌细胞中RUNX2的激活将导致平滑肌细胞中钙化的发生。
目前临床上没有一种有效的药物能够预防或治疗血管钙化。特别的,他汀类药物、华法林等抗凝药有促进钙化的副作用。因此,寻找和开发治疗钙化疾病药物具有重要意义。
石斛酚(Dendrophenol,CAS号108853-14-1),是从环草石斛茎中的分离出来的天然产物,是石斛的药用有效成分之一。石斛具有多种生物学活性,包括增强机体免疫力、帮助胃部消化、抗白内障、调节血糖、抗肿瘤等作用。其中环草石斛的主要成分是联苄基化合物,具有多种生物学效应,包括抗炎、抗氧化、抗癌和抑制视网膜新血管生成。目前我们已知石斛酚(Dendrophenol,CAS号108853-14-1)可用作NF-κB通路的抑制剂,具有较好的抗肿瘤活性。
授权公告号为CN107308141B的发明专利公开了石斛酚及其组合物在制备预防和治疗动脉粥样硬化药物中的应用。迄今为止,尚未见以石斛酚为预防和治疗血管钙化药物的相关报道。
发明内容
本发明的目的在于提供一种石斛酚在制备用于预防和治疗血管钙化的药物中的应用。
为实现上述目的及其他相关目的,本发明提供的技术方案是:石斛酚在制备用于预防血管钙化的药物中的应用。
优选的技术方案为:所述石斛酚的结构式如下:
为实现上述目的及其他相关目的,本发明提供的技术方案是:石斛酚在制备用于治疗血管钙化的药物中的应用。
优选的技术方案为:所述石斛酚的结构式如下:
优选的技术方案为:所述药物的剂型包括口服液、注射剂、片剂、丸剂、分散剂、胶囊剂、滴丸、颗粒剂、悬浮剂、乳剂。
为实现上述目的及其他相关目的,本发明提供的技术方案是:一种用于预防和治疗血管钙化的药物组合物,由石斛酚和药学可接受的辅料组成。
由于上述技术方案运用,本发明与现有技术相比具有的优点是:
1、本发明提供了对血管钙化具有预防和治疗作用的石斛酚剂型(保健品或药物),用于预防和治疗血管钙化。本发明具有如下明显的进步和优点:1、成分明确(石斛酚);2、目标明确(血管钙化);3、调节作用明确(明显改善和治疗作用)。
2、石斛酚物还可以应用于在体外进行血管平滑肌细胞钙化水平试验中。在细胞水平上对石斛酚延缓血管钙化进程这一活性进行了评价和筛选,所涉及的石斛酚具有高活性低毒性、可显著抑制血管钙化。通过钙化动物模型展开药理学实验,研究结果表明,石斛酚能显著降低小鼠主动脉血管钙化水平,该实验结果证实了石斛酚对血管钙化的预防和治疗作用。本发明的药物毒性较小,安全性较高。
附图说明
附图1是实施例一中在正常培养条件下石斛酚0μΜ、2μΜ、5μM对细胞的毒性测定结果,结果表明石斛酚对细胞伤害较低,具有较高安全性。
附图2是实施例二中在钙化培养基(高磷酸盐)中石斛酚1.2μΜ、1.4μΜ、1.6μΜ、1.8μΜ对细胞的毒性测定,结果表明在一定范围内石斛酚对细胞伤害仍较低,较安全。
图3为实例三中石斛酚治疗平滑肌细胞成骨分化的茜素红染色的结果,及其定量后统计结果。结果表明石斛酚在0.5μΜ即能对平滑肌细胞成骨分化产生较好的抑制效果;*p<0.05,**p<0.01,***p<0.001。
图4为实例四中石斛酚治疗平滑肌细胞钙化的RUNX2的mRNA表达水平,结果表明石斛酚能较好的降低RUNX2在mRNA水平的表达。*p<0.05,**p<0.01。
图5为实例五中石斛酚治疗野生型雄性小鼠尼古丁与VD3诱导血管钙化后全主动脉的茜素红染色结果,结果表明石斛酚对小鼠血管钙化具有较好的治疗效果。
图6为实例六中石斛酚治疗野生型雄性小鼠尼古丁与VD3诱导血管钙化后主动脉根部的免疫荧光染色结果,蓝色为细胞核染色,红色为RUNX2染色,结果证明石斛酚能够有效降低小鼠主动脉根部RUNX2的表达量,进一步证明其针对血管钙化有较好的治疗效果。
具体实施方式
以下由特定的具体实施例说明本发明的实施方式,熟悉此技术的人士可由本实施例所揭露的内容轻易地了解本发明的其他优点及功效。
请参阅图1-6。须知,本说明书所附图式所绘示的结构、比例、大小等,均仅用以配合说明书所揭示的内容,以供熟悉此技术的人士了解与阅读,并非用以限定本发明可实施的限定条件,故不具技术上的实质意义,任何结构的修饰、比例关系的改变或大小的调整。提供以下实施例以便更好地理解本发明,而非限制本发明。以下实施例中的实验方法如无特殊说明,均为常规方法。下述实施例中所用的实验材料如无特殊说明,均为常规生化试剂商店购买所得。
本发明要解决的技术问题:提出石斛酚(Dendrophenol,CAS号108853-14-1)在制备预防和治疗血管钙化药物中的应用。
技术方案:经大量实验,本发明确定石斛酚防治由尼古丁及VD3诱导的血管钙化和缓解高磷酸盐引发的血管平滑肌细胞成骨分化。
作为优选,石斛酚可用于制备治疗血管钙化的药物。
所述治疗血管钙化药物以石斛酚(Dendrophenol,CAS号108853-14-1)类为有效成分。
石斛酚可通过注射、喷射、滴鼻、滴眼、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、静脉、粘膜组织;或是被其他物质混合或包裹后导入机体。
需要的时候,在石斛酚中还可以加入一种或多种药学上可接受的载体,以制备成注射液、片剂、粉剂、颗粒剂、胶囊、口服液、膏剂、霜剂等多种形式的药品和/或保健品。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、润湿剂、崩解剂、吸收促进剂、表面活性剂、吸附载体、润滑剂等。各种剂型的药物均可以按照药学领域的常规方法制备。
实施例1:石斛酚在制备用于预防和治疗血管钙化的药物中的应用
所用的石斛酚(Dendrophenol,CAS号108853-14-1)结构式如下:
一、石斛酚对细胞的毒性测定
(1)培养2-10代人主动脉平滑肌细胞,用含10%胎牛血清的培养液制成细胞悬液,以每孔5000个细胞接种到96孔板,每孔细胞悬液体积200μl。
(2)设置3个组别,分别为:正常组、2μM、5μM。药物每组设置6个复孔,药物处理24h。
(3)培养结束后,每孔加MTT溶液(5mg/ml,用双无培养基配制)20μl。孵育4小时之后终止培养,小心吸弃孔内培养基,每孔加150μlDMSO,室温下摇床振荡15min,使结晶物充分融解。
(4)选择550nm波长,在酶联免疫监测仪上检测各孔光吸收值,记录结果。
二、石斛酚在钙化培养基中对细胞的毒性测定
1、钙化培养基的配制
在DMEM中添加相应浓度的无机磷酸盐(Na2HPO4/NaH2PO4,1:2)诱导平滑肌细胞中钙化的发生。诱导过程持续3-7天,每两天更换一次新鲜培养基。
2、石斛酚在钙化培养基中对细胞的毒性测定
(1)培养2-10代人主动脉平滑肌细胞,用含10%胎牛血清的培养液制成细胞悬液,以每孔1000个细胞接种到96孔板,每孔细胞悬液体积200μl。
(2)设置6个组别,分别为:正常组、钙化组、钙化-1.2μM、钙化-1.4μM、钙化-1.6μM、钙化-1.8μM。药物每组设置6个复孔。同一培养条件下培养3天。
(3)培养结束后,每孔加MTT溶液(5mg/ml,用双无培养基配制)20μl。孵育4小时之后终止培养,小心吸弃孔内培养基,每孔加150μlDMSO,室温下摇床振荡15min,使结晶物充分融解。
(4)选择550nm波长,在酶联免疫监测仪上检测各孔光吸收值,记录结果。
三、石斛酚治疗平滑肌细胞成骨分化的茜素红染色
(1)在12孔板中,每个孔加入500个细胞,待细胞贴壁过夜稳定。
(2)按照上述第二部分的钙化培养基的配置配置钙化培养基。在钙化培养基的环境下,按1.2/1.4/1.6/1.8μM的石斛酚浓度对细胞进行处理。
(3)置于培养箱中培养,两天换一次培养基,共培养四天。
(4)培养过后,吸走培养基,加入4%多聚甲醛固定细胞30min,使用PBS洗3×5min。然后加入茜素红染液染色30min,再使用酸性PBS洗3×5min。进行拍照。
四、石斛酚降低细胞中钙化关键转录因子RUNX2的mRNA表达水平
(1)将细胞接种于6孔板中。按照第三部分处理细胞,四天后将细胞用PBS清洗2次。在6孔板中加入预冷的总RNA提取试剂(Trizol),每孔400μL,吹打,移至1.5ml离心管内,按体积比5:1加入三氯甲烷,振荡混匀。使用4℃离心机12000rpm离心10min,收集上清至1.5ml离心管内,加入等体积异丙醇,存放于-20℃冰箱中过夜沉降。
(2)过夜后离心,获得RNA沉淀,依次加入500μl 70%乙醇、100%乙醇,使用4℃离心机12000rpm离心10min,对RNA分别进行清洗去除有机试剂。清洗后RNA置于超净台晾干,随后加入20μl ddH2O溶解RNA,使用超微分光光度仪测RNA浓度。
(3)按计算过浓度的RNA1 mg按照表1进行反转体系预混。
表1
成分 | 用量 |
4×gDNA wiper Mix | 4μL |
模板RNA | 1μg |
RNase-free ddH2O | 16-V<sub>RNA</sub>μL |
向样品中加入4μL 5×HiScript II Select qRTSuperMix II,离心混匀,50℃孵育15min,后85℃孵育2min,即得相应的样品cDNA。
(4)使用罗氏荧光定量PCR仪以表2所示的条件进行混样实验:
表2
成分 | 用量(μL) |
2×SYBR-GREEN MIX | 10 |
上游引物 | 0.5 |
下游引物 | 0.5 |
无菌水 | 7 |
cDNA | 2 |
表3
(5)实验涉及到的引物(实验中所用内参为GAPDH):
RUNX2:上游:TCCTCCCCAAGTAGCTACCT;
下游:AGGACTTGGTGCAGAGTTCA。
GAPDH:上游:ACCCAGAAGACTGTGGATGG;
下游:ACACATTGGGGGTAGGAACA。
引物设计的目的是找到一对有效的核苷酸片段,从而对模板DNA目的序列进行有效扩增,我们首先通过NCBI Gene数据库获得RUNX2及GAPDH的cDNA保守区序列,之后通过NCBI BLAST获得引物序列,并对引物保守性进一步验证,获得具有较高保守性引物序列。设计得到的引物序列由擎科生物提供合成产物。
五、石斛酚对尼古丁与VD3诱导的钙化雄性小鼠全主动脉影响;
(1)通过灌胃尼古丁(25mg/kg)早晚一次及皮下注射VD3(5.5×105IU/kg)建立小鼠钙化模型,之后对小鼠进行腹腔注射石斛酚(10mg/kg*d),注射周期为21天。结束后安乐死小鼠,取出整条主动脉。
(2)小鼠主动脉组织染色前使用4%多聚甲醛固定过夜;
(3)全主动脉茜素红染色使用0.003%的茜素红溶液,溶剂为1%氢氧化钾溶液。
(4)将全主动脉置于染液中过夜。
(5)次日使用2%氢氧化钾溶液冲洗两次,置于体式显微镜下拍照,染色结果见图5。
六|小鼠主动脉根部免疫荧光染色检测主动脉根部RUNX2表达量
(1)取材:心脏处剥离主动脉根部
(2)固定:4%多聚甲醛固定24小时以上(4℃)。
(3)脱水:30%蔗糖溶液脱水至组织块沉底。
(4)冰冻切片:组织从蔗糖溶液取出后直接OCT包埋、常规冰冻切片。切片保存在-40℃备用。
(5)免疫荧光染色:冰冻切片取出后,室温放置30min,PBS浸泡5min。
(6)0.1%Triton x-100浸泡10min破膜,PBS浸泡5min×2次。
(7)滴加2%BSA封闭,室温孵育10-15min,倾去。
(8)滴加一抗抗体,4℃过夜。PBS浸泡冲洗,10min×3次。
(9)滴加荧光标记二抗,37℃孵育45min。PBS浸泡冲洗,10min×3次
(10)滴加DAPI染色液,室温孵育15min,PBS浸泡冲洗,10min×3次。
(11)封片:选用防荧光淬灭封片剂封片,避光水平放置2h以上,晾干后荧光显微镜照相,染色结果见图6。
综上所述,本发明的石斛酚(Dendrophenol,CAS号108853-14-1)可显著治疗由尼古丁及VD3诱导的血管钙化以及高磷诱导的血管细胞钙化。因此在血管钙化的预防、治疗中具有比较广阔的应用前景。
以上所述者仅为用以解释本发明之较佳实施例,并非企图具以对本发明做任何形式上之限制,是以,凡有在相同之发明精神下所作有关本发明之任何修饰或变更,皆仍应包括在本发明意图保护之范畴。
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