CN110787207A - 柿叶黄酮提取物在制备药物中的应用 - Google Patents
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Abstract
柿叶黄酮提取物在制备药物中的应用，包括在制备抑制破骨细胞分化药物中的应用、制备防治溶骨性骨质疏松药物中的应用、制备预防溶骨性骨质疏松保健食品中的应用；所述的柿叶黄酮提取物能够降低破骨细胞分化与成熟标志性因子mRNA的水平；能够促进线粒体复合物complex I的表达从而降低破骨细胞分化与成熟过程中ROS的产生；通过抑制PI3K‑AKT‑mTOR、JNK、p38信号通路的活化从而抑制破骨细胞的分化；本发明的优势在于，开发了一种治疗骨质疏松的天然产物类药物柿叶黄酮，在破骨细胞分化的过程中加入一定量的柿叶黄酮进行干预，可抑制破骨细胞的分化，减少骨质流失，从而防治骨质疏松；本发明的原材料资源丰富，价格低廉，且毒副作用小，具有很好的应用价值。
Description
技术领域
本发明属于医药技术领域，特别涉及柿叶黄酮提取物在制备药物中的应用。
背景技术
我国骨质疏松患者逐年增多。全国性大规模流行病学调查研究显示，国内骨质疏松症总患病率为12.4％，总人数已超过1.6亿，是全球骨质疏松患者最多的国家。此外骨质疏松发病人群存在明显的“年轻化”趋势，预计到2020年我国骨质疏松患者和低骨量患者人数将增至2.8亿。因此早期诊断、预防和治疗骨质疏松变得尤为重要。
临床研究发现，当破骨细胞对骨的吸收大于成骨细胞骨重建时，往往容易引发骨质疏松。破骨细胞是一种巨大的多核细胞，起源于单核巨噬细胞、单核造血前体细胞，其主要由巨噬细胞克隆刺激因子(M-CSF)、核因子NF-κB配体激活受体(RANKL)刺激分化产生。研究表明，当肿瘤坏死因子相关受体因子6(TRAF6)激活其下游的破骨细胞生成信号通路(包括：NF-κΒ、原癌基因酪氨酸蛋白激酶C-Src、MAPK(p38,JNK)、细胞外基质相关激酶(ERK))后容易导致破骨细胞分化与成熟。此外，氧化应激也会推动骨质疏松的发生与发展，增加活性氧(ROS)可阻遏成骨细胞的分化并诱导其凋亡，同时加速破骨细胞的分化及活性。总之，阻遏破骨细胞的分化及成熟是防治骨质疏松的一个靶点。
目前，治疗骨质疏松的药物主要是以化学药物为主，其作用主要是刺激骨形成并抑制骨吸收。代表药物有双膦酸盐类，维生素D类制剂，降钙素，选择性雌激素受体调节剂等。这些药物不仅价格昂贵，并存在潜在的副作用，例如长期服用双膦酸盐，会出现颌骨坏死，房颤，肿瘤等病症；长期使用雌激素会导致机体其他系统不良反应，如患乳腺癌，子宫内膜癌等风险增加。因此开发低廉，毒副作用小的新型抗骨质疏松药物具有重要意义。
发明内容
为了克服上述现有技术的缺陷，本发明的目的在于提供柿叶黄酮提取物在制备药物中的应用，柿叶黄酮提取物(简称FPL)可明显抑制由RANKL诱导的前体破骨细胞RAW264.7的分化与成熟，FPL能够通过调控PI3K-AKT-mTOR，MAPK(p38,JNK)信号通路及通过改善氧化应激状态来实现抑制破骨细胞的分化及成熟。
为了达到上述目的，本发明是通过以下技术方案来实现：
柿叶黄酮提取物在制备药物中的应用，其特征在于，柿叶黄酮FPL在制备抑制破骨细胞分化药物中的应用。
柿叶黄酮提取物在制备药物中的应用，其特征在于，柿叶黄酮FPL在制备防治溶骨性骨质疏松药物中的应用。
柿叶黄酮提取物在制备药物中的应用，其特征在于，柿叶黄酮FPL在制备预防溶骨性骨质疏松保健食品中的应用。
所述的柿叶黄酮提取物能够降低破骨细胞分化与成熟标志性因子mRNA的水平。
所述的柿叶黄酮提取物够促进线粒体复合物complex I的表达从而降低破骨细胞分化与成熟过程中ROS的产生。
所述的柿叶黄酮提取物通过抑制PI3K-AKT-mTOR、JNK、p38信号通路的活化从而抑制破骨细胞的分化。
与现有技术相比，本发明具有以下有益的技术效果：
柿叶黄酮对人机体代谢无毒副作用，对成骨细胞及破骨细胞无毒害，本发明首次指出柿叶黄酮能够改善破骨细胞分化过程中的氧化应激状态，具有清除ROS的功效；柿叶黄酮能够明显的抑制由RANKL刺激的PI3K-AKT-mTOR信号通路的活化，从而抑制相应分化标志物的表达，同时FPL能够上调线粒体复合物complex I的表达，减少破骨细胞内ROS的产生，抑制破骨细胞的分化与成熟。
柿叶黄酮能够有效的降低由RANKL诱导的破骨细胞分化marker如(TRAP,c-src,c-fos,mmp9,RANK,TRAF6,Nfatc1)的mRNA水平；同时能够有效抑制PI3K-AKT-mTOR、p38、JNK信号通路的活化抑制破骨细胞的分化；此外FPL能够促进线粒体复合物complex I的表达、减少细胞内ROS的积累从而抑制破骨细胞分化。充分表明FPL在防治骨质疏松病症中有良好的应用前景。
附图说明
图1是RANKL诱导RAW 264.7分化4天后相关标志性分子mRNA检测结果及破骨细胞分化程度的TRAP染色结果。(A)为mRNA检测结果，(B)TRAP染色结果。
图2是在RANKL诱导分化的同是用FPL进行干预4天后相关标志性分子mRNA检测结果及破骨细胞分化程度的TRAP染色结果。，(A)为mRNA检测结果，(B)TRAP染色结果。
图3是FPL作用后，对PI3K-AKT-mTOR、p38、JNK信号通路相关蛋白westernblotting的检测结果。
图4(A)是FPL作用后细胞内ROS的检测结果，发现FPL作用后细胞内的ROS有一定程度的降低；图(B)是线粒体复合物complex I蛋白western blotting检测结果；图(C)是RANKL诱导分化同时加入NAC进行干预细胞内ROS的检测结果；图(D)是NAC干预后RAW264.7细胞相关分化marker检测的结果。
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。
1.实验材料
柿叶黄酮是通过水提醇沉、石油醚脱色，氯仿脱蛋白等步骤得到的。TRIzol试剂从Invitrogen公司购买；RNA反转录试剂盒，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公司订购。
2.实验细胞培养及模型建立
小鼠单核巨噬细胞RAW264.7来源于西安交通大学生命科学与技术实验室，RANKL购买于ProTech。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。实验分为四组，分别为(1)Normal组；(2)FPL(10μg/ml)处理+Normal组；(3)RANKL(100ng/ml)组；(4)FPL(10μg/ml)+RANKL组。
3.实验方法
(1)RANKL诱导RAW264.7细胞分化
配置母液为100μg/ml的RANKL，稀释1000倍使其终浓度为100ng/ml进行诱导，诱导4天即可，中间换液一次。
(2)FPL干预RANKL诱导的分化实验
配置母液为10mg/ml的柿叶黄酮(FPL)，稀释1000倍使其终浓度为10μg/ml处理RAW264.7细胞24h后，用含有RANKL(100ng/ml)及FPL(10μg/ml)的培养基处理细胞4天，必要时中间换液一次。
(3)RAW 264.7细胞分化marker的检测
分化marker的检测主要是采用逆转录RNA-实时荧光定量PCR的方法进行检测，具体步骤如下：
1)RNA提取
将TRIzol试剂加入处理后的12孔板细胞培养皿中，每孔500μl，室温摇床孵育5min，收集至无菌无酶的1.5mlep管中，加入200μl氯仿(1/5TRIzol体积)抽提蛋白，涡旋混匀30s，室温放置5min后，12000g，4℃离心10min。将上层水相转移至另一干净的无菌无酶ep管中，加入等体积的异丙醇，温和混匀40～60次，室温静置25min后，12000g，4℃离心10min，弃上清。加入1ml预冷的75％乙醇，混合颠倒数次，12000g，4℃离心10min。弃上清，离心1min，弃掉余下的液体，静置片刻，待乙醇挥发完全后，溶于10μl DEPC水中，用于反转录。
2)反转录
反转录体积为10μl，加入1μgRNA，加入上下游引物各0.5μg.加入2μl的5X MasterMix，用DEPC水补充至10μl体系，用PCR仪37℃反应60min，85℃反应15s，保存于-20℃备用。
3)实时荧光定量PCR(Real-Time PCR)
用SYBR Green法进行，反应体系共10μl包括1μlcDNA,5μl2xSYBR○RPremix EXTaqTM II，各0.5μl上下游引物(10μm)，加入灭菌水3μl。反应程序依照：95℃解链10min，进行40个循环PCR(每个循环包括95℃30S，55℃30S，72℃20s)，最后监测融解曲线(95℃15s，60℃15s,95℃15s)其中β-actin作为内参。实验所用引物序列为：
cathepsin K
forward:5’-ccgaataaatctagcacccttagt-3’
reverse:5’-gaaacttgaacacccacatcc-3’
c-src
forward:5’–tgagccaggatctgaacca-3’
reverse:5’-Tcctgctccgtgtcccta-3’
RANK
forward:5’-caggacagggctgatgagag-3’
reverse:5’-ttactgtttccagtcacgttcc-3’
MMP9:
forward:5’-agacgacatagacggcatcc-3’
reverse:5’-tcggctgtggttcagttgt-3’
TRAF6：
forward:5’-ttgcacattcagtgtttttgg-3’
reverse:5’-tgcaagtgtcgtgccaag-3’
Nfatc1：
forward:5’-tccaaagtcattttcgtgga-3’
reverse:5’-ctttgcttccatctcccaga-3’
c-fos：
forward:5’-gggacagcctttcctactacc-3’
reverse:5’-agatctgcgcaaaagtcctg-3’
Fra-2：
forward:5’-acgccgagtcctactccag-3’
reverse:5’-caggcatatctacccggaac-3’
TRAP:
forward:5’-gagtcagactaatgtcatctgtggtt-3’
reverse:5’-accccgaaaatggtgatg-3’
β-actin：
forward:5’-cgctgtcaaccccaagtt-3’
reverse:5’-ggcacgttcttgtctactcgt-3’
(4)蛋白检测
1)蛋白提取
12孔板，每孔加入150μl的裂解液，用细胞刮刀刮取细胞,4℃涡旋10min后，4℃，12000g离心10min，取上清，用BCA方法进行测定蛋白浓度，加入5X loading buffer和β巯基乙醇，95℃反应10min，-80℃保存，待用。
2)Western blotting
使用10％、8％的SDS-PAGE丙烯酰胺凝胶电泳分离蛋白，上样量为10μg左右，用硝酸纤维素膜(NC)蛋白印迹，室温封闭1h，一抗孵育4℃过液，清洗一抗，室温孵育二抗1h，清洗二抗，化学发光。
(5)TRAP染色
TRAP试剂盒购买于Sigma，染色步骤依照试剂盒使用说明操作，主要步骤是：无水乙醇脱水、戊二醛：丙醇(1:1)固定-37℃避光染色>1h，倒置显微镜进行拍照。
(6)ROS测定
细胞内ROS的测定通过H2DCF-DA检测，配制母液10mmol/L的储备液，避光保存于-20℃备用。使用时用无血清培养基稀释1000倍，将其加入处理好的细胞中37℃避光孵育30min，细胞裂解液裂解细胞，4℃13000g离心取上清检测。
(7)统计学分析
结果以Mean±S.E.M形式表示，数据分析使用双尾t-test和ANOVA分析方法，显著统计学意义为*vs.CON,p<0.05；**vs.CON p<0.01；#vs.CON+RANKL,p<0.05；##vs.CON+RANKL,p<0.01。
4、RANKL可诱导RAW 264.7细胞分化
100ng/ml RANKL刺激RAW 264.7细胞4天，检测相关分化marker的mRNA水平及TRAP染色，参照图(1)所示：破骨细胞分化标志性分子Fra-2,Nfatc1,c-fos,c-src,traf6,MMP9,TRAP,RANK表达量均显著升高；抗酒石酸酸性磷酸酶(TRAP)为破骨细胞的标志酶，特异性的分布于破骨细胞胞浆中。通过染色发现RANKL诱导后细胞聚团明显，染色加深，同时出现多核细胞。
5、FPL抑制由RANKL诱导RAW 264.7细胞的分化过程
参照图(2)所示，FPL(10μg/ml)预处理24h后，RANKL诱导的同时FPL进行干预4天后，RT-PCR检测发现，破骨细胞分化相关marker的表达量显著降低，同样进行TRAP染色发现，FPL干预后RAW 264.7的分化程度显著降低。
6、FPL通过抑制PI3K/AKT、p38及JNK信号通路抑制破骨细胞的分化
RANKL与RANK结合后，RANK受体胞质端的Motif基序与TRAF6结合，可刺激PI3K-AKT-mTOR、JNK、p38信号通路活化，活化的PI3K-AKT可引起Nfatc1的表达增加及核内富集，活化的p38、JNK信号通路，可上调beclin-1的表达，进一步使Nfatc1表达增加，而Nfatc1的激活是破骨细胞最终形成的标志性事件。参照图(3)所示western blotting蛋白检测发现RANKL诱导后RAW264.7细胞内p-AKT、p-mTOR、p-p38、p-JNK的表达量显著上升，而FPL干预后，细胞内相应分子的表达量显著降低，基本恢复正常水平，表明FPL可通过抑制PI3K-AKT-mTOR、JNK、P38信号通路抑制破骨细胞的分化。
7、FPL能够通过促进促进线粒体复合物complex I的表达降低RANKL诱导的ROS的产生
研究发现RANKL诱导刺激后，前体破骨细胞会产生大量的ROS，而ROS可作为细胞内的第二信使刺激多条信号通路的活化，包括：PI3K-AKT-mTOR、JNK、p38信号通路，从而促进破骨细胞分化。参照图(4)所示，我们发现FPL干预后细胞内的ROS有一定程度的降低。而细胞内的ROS主要来自线粒体氧化呼吸，通过对线粒体氧化呼吸链相关蛋白检测发现，RANKL诱导分化的过程中线粒体复合物complex I的表达量显著降低，而FPL干预后，线粒体复合物complex I的表达量基本恢复正常水平。此外，我们在RANKL诱导的同时加入NAC清除细胞内部分ROS，发现部分标志性分化marker的mRNA显著降低其中包括(cathepsinK,mmp9,c-fos,TRAP,RANK,Nfatc1,TRAF6),表明ROS作为RANKL下游诱导因子可以促进RAW264.7细胞的分化，而FPL作为抗氧化剂在清除ROS的同时抑制前体破骨细胞的分化与成熟。
通过以上实验结果证明：柿叶黄酮(FPL)能够有效的抑制由RANKL诱导的破骨细胞的分化与成熟，从而防治骨质疏松的发生。
可进行以下应用：
FPL在制备抑制破骨细胞分化药物中的应用。
进一步的，所述的药物是对RANKL诱导刺激破骨细胞分化过程有抑制作用的药物。
所述的药物是降低破骨细胞分化标志性分子：Fra-2,Nfatc1,c-fos,c-src,TRAF6,MMP9,TRAP,RANK的mRNA水平。
进一步的，所述的药物能够通过抑制PI3K-AKT-mTOR、JNK、p38信号通路的活化从而抑制破骨细胞的分化。
所述的药物是能够促进线粒体复合物complex I的表达从而降低破骨细胞分化与成熟过程中ROS的产生的药物。
正常生理条件下，破骨细胞调节骨吸收与成骨细胞调控骨分化处于动态平衡，而破骨细胞的异常活化会造成溶骨性骨质疏松，氧化应激及核因子受体活化因子配体-核因子受体活化因子-骨保护素(RANKL-RANK-OPG)信号是刺激破骨细胞分化的重要调控因素。柿叶黄酮(FPL)在以上的RANKL诱导分化实验中表现出良好的抑制破骨细胞分化的功效。因此，FPL在防治溶骨性骨质疏松中有良好的应用前景，为防治由于过度激活破骨细胞造成的溶骨性疾病开辟了新的治疗手段。
可进行以下应用：
柿叶黄酮(FPL)在制备抑制破骨细胞分化药物中的应用。
柿叶黄酮(FPL)在制备防治溶骨性骨质疏松药物中的应用。
柿叶黄酮(FPL)在制备预防溶骨性骨质疏松保健食品中的应用。
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 (6)
1.柿叶黄酮提取物在制备药物中的应用，其特征在于，柿叶黄酮FPL在制备抑制破骨细胞分化药物中的应用。
2.柿叶黄酮提取物在制备药物中的应用，其特征在于，柿叶黄酮FPL在制备防治溶骨性骨质疏松药物中的应用。
3.柿叶黄酮提取物在制备药物中的应用，其特征在于，柿叶黄酮FPL在制备预防溶骨性骨质疏松保健食品中的应用。
4.根据权利要求1至3任意一项所述柿叶黄酮提取物在制备药物中的应用，其特征在于，所述的柿叶黄酮提取物能够降低破骨细胞分化与成熟标志性因子mRNA的水平。
5.根据权利要求1至3任意一项所述柿叶黄酮提取物在制备药物中的应用，其特征在于，所述的柿叶黄酮提取物够促进线粒体复合物complex I的表达从而降低破骨细胞分化与成熟过程中ROS的产生。
6.根据权利要求1至3任意一项所述柿叶黄酮提取物在制备药物中的应用，其特征在于，所述的柿叶黄酮提取物通过抑制PI3K-AKT-mTOR、JNK、p38信号通路的活化从而抑制破骨细胞的分化。
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111956805A (zh) * | 2020-08-26 | 2020-11-20 | 中国人民解放军海军军医大学第一附属医院 | L-plastin蛋白抑制剂在抑制破骨前体细胞融合中的应用 |
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