CN110433158A - 瑞香素在制备改善主动脉内皮细胞功能的药物中的应用 - Google Patents
瑞香素在制备改善主动脉内皮细胞功能的药物中的应用 Download PDFInfo
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Abstract
本发明公开了瑞香素在制备改善主动脉内皮细胞功能的药物中的应用；瑞香素可明显促进饱和脂肪酸抑制的人主动脉内皮细胞的活力、抑制饱和脂肪酸造成的人主动脉内皮细胞炎症反应及增加血管内皮细胞线粒体和复合物生成，通过抗炎和保护线粒体功能来预防动脉粥样硬化的发生和发展。
Description
技术领域
本发明属于医药学技术领域，特别涉及瑞香素在制备改善主动脉内皮细胞功能的药物中的应用。
背景技术
据2011年世界卫生组织发布的关于心血管疾病的预防与控制的全球数据显示，心血管疾病导致的死亡占全球死亡人数的31％，仅次于其它非传染性疾病的总数。且预计2008年到2030年心血管疾病导致的死亡率明显高于其它疾病，处于领先地位。心血管疾病主要包括心脑血管疾病，如心肌梗死和脑梗死等。而动脉粥样硬化是心血管疾病的病理基础。
动脉粥样硬化的发病机制非常复杂，发生于血管壁中，是一种慢性炎症反应、形成斑块、且伴随有血管内皮细胞损伤。导致动脉粥样硬化的因素主要有高盐、高脂、高能量、吸烟等不健康饮食以及代谢型危险因素，包括三高及肥胖等疾病。
目前动脉粥样硬化有药物和手术等治疗方式，但该类型疾病隐匿性高、致死和致残率都较高，因此提早预防显得尤为重要，有研究报道，天然活性成分如绿原酸、芎芍及木酚素等具有抗动脉粥样硬化的功能，且以此为原料的功能性食品相继问世。挖掘更多效能更好的抗动脉粥样硬化等心血管疾病的天然物质具有重要的意义和前景。
瑞香素是从瑞香属植物长白瑞香(Daphne Korean Nakai)中提取出来的有效成分，化学名7，8-二羟基香豆素(7,8-dihydroxycoumarin)，又名祖师麻甲素，是香豆素类化合物的代表性单体成分，为我国自主研发的天然药物之一，具有抗炎、抗氧化、抗糖尿病、保肝作用的单体分子-瑞香素(Daphnetin)。临床用于血栓闭塞性脉管炎及其他闭塞性血管疾病和冠心病的辅助治疗。
发明内容
本发明解决的问题在于提供瑞香素在制备改善主动脉内皮细胞功能的药物中的应用，瑞香素可明显抑制饱和脂肪酸造成的人主动脉内皮细胞炎症反应及增加血管内皮细胞线粒体膜融合相关蛋白Mfn1的生成，通过抗炎和保护线粒体功能来预防动脉粥样硬化的发生和发展。
本发明是通过以下技术方案来实现：
瑞香素在制备改善主动脉内皮细胞功能的药物中的应用。
所述的瑞香素促进饱和脂肪酸抑制的人主动脉内皮细胞的活力，对饱和脂肪酸造成的主动脉内皮细胞炎症反应有抑制作用。
所述的瑞香素降低主动脉内皮细胞内的IL-6的mRNA水平含量。
所述的瑞香素对饱和脂肪酸造成的主动脉内皮细胞炎症反应所损伤的线粒体有保护作用。
所述的瑞香素增加线粒体膜融合相关蛋白Mfn1的表达。
瑞香素在制备防治心血管疾病的药物中的应用。
所述的瑞香素在治疗动脉粥样硬化药物中的应用。
瑞香素在制备预防动脉粥样硬化发生的药物或保健品中的应用。
与现有技术相比，本发明具有以下有益的技术效果：
瑞香素对饱和脂肪酸抑制人主动脉内皮细胞生长有保护的作用，本发明首次公开指出瑞香素在血管内皮损伤和炎症反应的动脉粥样硬化等血管疾病发生的过程中，瑞香素可明显促进饱和脂肪酸抑制的人主动脉内皮细胞活力、抑制饱和脂肪酸造成的人主动脉内皮细胞炎症反应及增加血管内皮细胞线粒体膜融合蛋白Mfn1的生成，通过抗炎和保护线粒体功能来预防动脉粥样硬化的发生和发展。
瑞香素能够促进饱和脂肪酸抑制的人主动脉内皮细胞的生长，有效降低饱和脂肪酸造成的人主动脉内皮细胞炎性反应，如白介素-6(IL-6)的mRNA水平含量；瑞香素能够有效保护饱和脂肪酸对人主动脉内皮线粒体功能的损伤，如可增加线粒体膜融合相关蛋白Mfn1的表达等；其对于预防血管疾病发生和进展中具有很好的应用前景。
附图说明
图1是瑞香素可促进饱和脂肪酸抑制的人主动脉内皮细胞的活性。其中：横坐标为瑞香素的作用浓度，纵坐标为细胞活性。
图2是瑞香素对棕榈酸诱导人主动脉内皮细胞炎症反应的抑制作用。其中：横坐标为瑞香素的作用浓度，纵坐标为IL-6的mRNA水平含量。
图3A～图3B是瑞香素可上调棕榈酸诱导人主动脉内皮细胞线粒体膜融合相关蛋白Mfn1表达的降低。其中：图3A为Western blot结果图；图3B为Western blot统计图，横坐标为蛋白名称，纵坐标为相对蛋白表达水平含量。
具体实施方式
下面结合具体的实施例对本发明做进一步的详细说明，所述是对本发明的解释而不是限定。
1.实验材料
瑞香素(Daphnetin)购买于南京普怡生物科技有限公司；TRIzol试剂从Invitrogen公司购买；RNA逆转录试剂盒，SYBR荧光染料从大连宝生物公司购买。RNA引物序列从西安擎科泽西生物有限公司订购合成。
2.实验细胞培养及模型建立
人主动脉内皮细胞(Human aortic endothelial cells,HAEC)从上海拜力生物科技有限公司购买，棕榈酸购买于sigma公司。细胞培养于含有95％空气和5％CO2的37℃恒温、湿润、无菌培养箱中。实验分为三组，分别为：(1)control组；(2)500μM棕榈酸处理组；(3)瑞香素保护组。
3.实验方法
(1)MTT检测
用不同浓度的瑞香素作用于HAEC，细胞培养箱培养24h，后用500μM的棕榈酸作用24h后，用PBS清洗细胞一遍，加入0.5mg/ml的MTT，在含有95％空气和CO2的37℃培养箱中孵育4h后，用PBS清洗三遍，再加入DMSO溶解，并在490nm的波长下检测其吸光值。
(2)白介素-6的mRNA含量检测
采用逆转录RNA-实时荧光定量PCR的方法进行检测。具体方法如下：
1)RNA提取
12孔细胞培养板，每孔加入500μL的TRIzol试剂，室温，摇床5min，加入200μl氯仿(1/5总体积)抽提蛋白，剧烈混匀15s，室温放置15min后，12,000g,4℃,离心10分钟。将上层水相转移至另一EP管中，加入等体积异丙醇，混匀后-20℃放置1h后，12,000g,4℃,离心10min，弃上清。加入1mL预冷的75％乙醇，颠倒混匀，12,000g,4℃，离心10min。弃上清，超净工作台放置30min，使乙醇挥发完全挥发掉，溶于10μl DEPC水中。用紫外分光光度计检测测定其浓度，用于反转录。
2)RNA反转录
转录体积为20μl，取出2μg RNA，加入随机引物0.5μg。加入4μl的5X Master Mix，用DEPC水补充至20μl体积，37℃孵育60min，80℃作用15s。置于-20℃备用。
3)实时荧光定量PCR(Real-time PCR)
用SYBR Green法进行，反应体系包括1μl cDNA，5μlPremix ExTaqTMII，0.5μl上下游引物混合液(10μM)，加灭菌水至10μl。反应条件依照说明书，95℃解链10min，进行40个循环PCR(每个循环包括95℃ 30s，55℃ 30s，72℃ 20s)，最后观测融解曲线(95℃ 15s，60℃ 15s，95℃ 15s)。β-actin作为内参，实验所用的引物序列为：
IL-6:
forward：5’-TTTTGTACTCATCTGCACAGC-3’
reverse：5’-GGATTCAATGAGGAGACTTGC-3’
β-actin:
forward:5'-ATCATGTTTGAGACCTTCAA-3'
reverse:5'-AGATGGGCACAGTGTGGGT-3’
(3)蛋白检测
1)蛋白提取
6孔细胞培养板，每孔加入150μL的IP裂解液，用细胞刮刮取细胞，振荡15s，冰浴10min，如此反复三次，保证冰浴时间至少在30min以上，4℃，12000rcf离心10min，取上清，用BCA方法进行蛋白定量，调平，加入5X loading buffer和巯基乙醇，煮沸10min，使蛋白变性，-80℃保存，待用。
2)Western blot
利用10％的丙烯酰胺凝胶进行跑胶，蛋白上样量为10μg，利用PVDF膜转印，后封闭，孵育一抗，4℃过夜，清洗一抗，孵育二抗，室温1h，清洗二抗，进行化学发光。
4.统计分析
结果以Mean±S.E.M形式表示，数据分析使用One Way-ANOVA分析方法，显著统计学意义为*p<0.05,**p<0.01,***p<0.001。
5、瑞香素可促进饱和脂肪酸抑制的人主动脉内皮细胞的活力
MTT检测瑞香素先作用于人内皮细胞24h后再加500μM棕榈酸作用24h，图1显示10μM瑞香素可促进饱和脂肪酸抑制的人主动脉内皮细胞的生长。
6、瑞香素对棕榈酸诱导人血主动脉内皮细胞炎症反应的抑制作用
瑞香素先作用于人内皮细胞24h后再加500μM棕榈酸作用24h，图2显示瑞香素对可以显著抑制棕榈酸损伤人血管内皮细胞产生的炎症反应，500μM的棕榈酸使人主动脉内皮细胞产生炎症反应，如细胞炎症因子白介素IL-6较无棕榈酸处理组有明显升高，在mRNA水平上，IL-6升高了约20倍，模型组炎症水平增高明显，当瑞香素的作用浓度为100μM时产生了明显的抑制炎症反应的效果，提示瑞香素的抗炎作用及抗动脉粥样硬化效果。
7、瑞香素可上调棕榈酸诱导人主动脉内皮细胞线粒体膜融合相关蛋白Mfn1表达的降低
线粒体融合与分裂不仅可以改变线粒体的形态、影响线粒体的功能，而且还影响细胞的生存，线粒体的分裂和融合是由多种蛋白质精确调控完成的。参与线粒体融合的蛋白质主要包括Mfn1,Mfn2及OPA1。图3A～图3B显示棕榈酸可降低人主动脉内皮细胞线粒体膜融合相关蛋白Mfn1和Mfn2蛋白的表达，而瑞香素可明显增加Mfn1的表达量，促进细胞的融合，维持线粒体动态平衡。
通过以上实验结果证明瑞香素可促进饱和脂肪酸抑制的人主动脉内皮细胞的生长、能够有效抑制高脂诱导的人主动脉内皮细胞的炎症反应和线粒体损伤，从而改善人主动脉内皮细胞功能。可进行以下应用：
瑞香素在制备改善主动脉内皮细胞功能的药物中的应用。
进一步的，所述的药物是促进饱和脂肪酸抑制的人主动脉内皮细胞的活力以及对饱和脂肪酸造成的主动脉内皮细胞炎症反应有抑制作用的药物。
所述的药物是降低主动脉内皮细胞内的IL-6的mRNA水平含量。
进一步的，所述的药物是对饱和脂肪酸造成的主动脉内皮细胞炎症反应所损伤的线粒体有保护作用的药物。
所述的药物是增加线粒体膜融合相关蛋白Mfn1的表达的药物。
由于内皮细胞功能受损是动脉粥样硬化的初期表征及发生、发展的基础和原因，动脉粥样硬化是一种慢性炎症反应，炎症反应的发生是该病发生的重要原因之一，且线粒体损伤会造成内皮细胞能量不足和功能下降，被报道将可能也是动脉粥样硬化发生的重要原因之一，瑞香素在以上内皮细胞损伤实验中表现出很好的保护内皮细胞生长、炎症、线粒体损伤的特性。由此，瑞香素在预防高脂引起内皮损伤的动脉粥样硬化等心血管疾病有很好的应用前景，为防治膳食结构失衡所致内皮损伤导致的动脉粥样硬化等心血管疾病系列问题开辟新的医药途径。
可进行以下应用：
瑞香素在制备防治心血管疾病的药物中的应用。
所述的药物是治疗动脉粥样硬化的药物。
瑞香素在制备预防动脉粥样硬化发生的药物或保健品中的应用。
以上给出的实施例是实现本发明较优的例子，本发明不限于上述实施例。本领域的技术人员根据本发明技术方案的技术特征所做出的任何非本质的添加、替换，均属于本发明的保护范围。
Claims (9)
1.瑞香素在制备改善主动脉内皮细胞功能的药物中的应用。
2.根据权利要求1所述的瑞香素在制备改善主动脉内皮细胞功能的药物中的应用，其特征在于，瑞香素在制备改善主动脉内皮细胞功能的药物中的应用。
3.根据权利要求1所述的瑞香素在制备改善主动脉内皮细胞功能的药物中的应用，其特征在于，所述的瑞香素促进饱和脂肪酸抑制的人主动脉内皮细胞的活力，对饱和脂肪酸造成的主动脉内皮细胞炎症反应有抑制作用。
4.根据权利要求1所述的瑞香素在制备改善主动脉内皮细胞功能的药物中的应用，其特征在于，所述的瑞香素降低主动脉内皮细胞内的IL-6的mRNA水平含量。
5.根据权利要求1所述的瑞香素在制备改善主动脉内皮细胞功能的药物中的应用，其特征在于，所述的瑞香素对饱和脂肪酸造成的主动脉内皮细胞炎症反应所损伤的线粒体有保护作用。
6.根据权利要求1所述的瑞香素在制备改善主动脉内皮细胞功能的药物中的应用，其特征在于，所述的瑞香素增加线粒体膜融合相关蛋白Mfn1的表达。
7.瑞香素在制备防治心血管疾病的药物中的应用。
8.根据权利要求6所述的瑞香素在制备改善主动脉内皮细胞功能的药物中的应用，其特征在于，所述的瑞香素在治疗动脉粥样硬化药物中的应用。
9.瑞香素在制备预防动脉粥样硬化发生的药物或保健品中的应用。
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| 姜秀莲等: "瑞香素对血清脂质及脂蛋白胆固醇含量的影响", 《药学学报》 * |
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