CN114480264B - 一种大豆肽在促进成骨活性中的应用 - Google Patents
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Abstract
本发明提供一种大豆肽在促进成骨活性中的应用，通过检测成骨细胞增殖水平和分化前期标志物ALP活性以及分化后期标志物OCN，在生化水平上检测大豆肽CBP促进成骨细胞增殖与分化；在大豆肽CBP促进成骨细胞分化过程中，MAPK信号通路的三条经典信号途径—ERK，JNK,p38旁路参与其中以及关键信号传导步骤；大豆肽CBP促进成骨细胞内MAPK信号通路关键因子Runx2的基因表达和磷酸化水平。
Description
技术领域
本发明涉及大豆肽促成骨活性中的应用，属于生物医药领域。
背景技术
大豆蛋白作为一种来源于大豆的天然活性蛋白，一直以来都是营养学领域研究者们关注的热点，已有众多文献报道了大豆蛋白的功能活性，如：调节免疫、促进肠道菌群平衡、抗氧化、抗癌作用等。但是对于小分子大豆肽的研究相对滞后，限制了大豆的进一步开发和应用，因此亟待从细胞水平和分子水平阐明大豆肽的成骨机制。
在体外实验方面，2014年Kyungho等人报道了大豆提取物可以通过调节成骨细胞产生的破骨细胞因子来间接抑制破骨细胞分化，0.001mg/mL的大豆提取物显著提高了OPG/RANKL的比率。在体内实验方面，2011年Chiagn等人通过给去卵巢小鼠饲喂乳酸菌发酵后的豆乳，发现其对雌激素缺乏后的骨小梁和皮质骨丢失具有明显的调节作用。以上结果表明大豆蛋白具有体内、体外的成骨活性，是一种具有治疗骨质疏松症潜力的天然活性物质。
丝裂素活化蛋白激酶(Mitogen Activated Protein Kinase,MAPK)信号通路是骨代谢机理研究报道最多的一条途径，它参与了成骨细胞增殖、定向、分化、凋亡等许多生理过程，对骨代谢起着极为重要的作用。一些已知的治疗骨质疏松症的药物和骨代谢调节因子，如雷尼酸锶、雌激素、骨形态发生蛋白(Bone morphogenetic protein,BMP)、转化生长因子(TGF-β)、甲状旁腺激素相关肽(PTHγP)等已被证明可以通过MAPK途径促进骨合成代谢，并可显著提高相关成骨标志物的表达量。不仅如此，许多新发现的成骨活性物质同样通过激活MAPK途径发挥作用。其中研究较多的是脂联素和他汀类：脂联素可以激活成骨细胞JNK(C-jun N-terminal kinase or stress activated protein kinase)、p38(p38kinase)途径，且这两条途径在脂联素调节成骨细胞的功能中处于不同地位：SB203580(p38抑制剂)可以显著降低成骨分化早期标志物碱性磷酸酶(Alkaline phosphatase,ALP)活性，而SB600125(JNK抑制剂)则对成骨细胞的增殖起明显抑制作用。与脂联素不同，他汀不仅可以激活ERK1/2(Extracellular signal-regulated kinase1/2)，还通过PI3K/AKT途径促进BMP的表达量，从而刺激成骨细胞分化。此外，蛇床子素、香叶木素、镧也有类似研究报道。以上研究均显示，MAPK三条信号通路对于外源物质刺激成骨细胞后的信号传递具有重要作用，并且可以被选择性激活，通过不同通路间的交叉通话产生不同的生物学效应。
目前，大豆制品的潜在健康益处已被广泛报道。流行病学研究和临床试验表明，大豆对绝经后症状、心血管疾病、骨健康问题以及乳腺癌、前列腺癌和结肠癌有保护作用。本课题组在之前的研究中已发现发酵酸豆乳在体外成骨细胞实验中具有成骨活性，并利用超滤及金属离子螯合亲和层析的方法分离纯化具有和钙离子结合能力的肽，通过LC-MS/MS鉴定其氨基酸组成为DEDEQIPSHPPR，但目前尚不清楚其成骨机制，因此，本项目旨在通过评价大豆肽在斑马鱼幼鱼及前成骨细胞MC3T3-E1中的成骨活性和成骨机制，为大豆肽作为一种潜在的成骨成分应用于功能性食品中提供科学依据和理论基础。
发明内容
为填补现有技术中的空白，本发明将发明人前期鉴定分离的大豆肽DEDEQIPSHPPR(下称CBP)对成骨细胞的作用机制进行了研究，明确其作用机理，为制备促进成骨细胞增殖、分化以及矿化的制剂提供应用基础。
本发明的第一个方面是提供大豆肽CBP在促进成骨细胞增殖中的应用，所述的应用为非治疗目的的；在一个具体的实施例中，所述的大豆肽CBP是通过促进转录因子Runx2的表达，从而促进成骨细胞的增殖；进一步的，所述的大豆肽CBP是通过提高转录因子Runx2表达，介导p38-MAPK通路的磷酸化促进成骨细胞增殖。
本发明的第二个方面是提供大豆肽CBP大豆肽CBP制备促进成骨细胞增殖制剂中的应用；在一个具体的实施例中，所述的大豆肽CBP是通过促进转录因子Runx2的表达，从而促进成骨细胞的增殖；进一步的，所述的大豆肽CBP是通过提高转录因子Runx2表达，介导p38-MAPK通路的磷酸化促进成骨细胞增殖。
本发明的第三个方面是提供大豆肽CBP在促进成骨细胞分化中的应用，所述的应用为非治疗目的的；在一个具体的实施例中，所述的大豆肽CBP是通过促进转录因子Runx2的表达，从而促进成骨细胞的分化；进一步的，所述的大豆肽CBP是通过提高转录因子Runx2表达，介导p38-MAPK通路的磷酸化促进成骨细胞分化。
本发明的第四个方面是提供大豆肽CBP在制备促进成骨细胞分化试剂中的应用；在一个具体的实施例中，所述的大豆肽CBP是通过促进转录因子Runx2的表达，从而促进成骨细胞的分化；进一步的，所述的大豆肽CBP是通过提高转录因子Runx2表达，介导p38-MAPK通路的磷酸化促进成骨细胞分化。
本发明的第五个方面是提供大豆肽CBP在促进成骨细胞矿化中的应用，所述的应用为非治疗目的的；在一个具体的实施例中，所述的大豆肽CBP是通过促进转录因子Runx2的表达，从而促进成骨细胞矿化；进一步的，所述的大豆肽CBP是通过提高转录因子Runx2表达，介导p38-MAPK通路的磷酸化促进成骨细胞矿化。
本发明的第六个方面是提供大豆肽CBP在制备促进成骨细胞矿化试剂中的应用；在一个具体的实施例中，所述的大豆肽CBP是通过促进转录因子Runx2的表达，从而促进成骨细胞的矿化；进一步的，所述的大豆肽CBP是通过提高转录因子Runx2表达，介导p38-MAPK通路的磷酸化促进成骨细胞矿化。
本发明的第七个方面是提供大豆肽CBP在促进转录因子Runx2表达方面的应用，所述的应用为非治疗目的的；在一个具体的实施例中，所述的大豆肽CBP在促进成骨细胞转录因子Runx2表达。
本发明具有的技术效果为：
1)通过检测成骨细胞增殖水平和分化前期标志物ALP活性以及分化后期标志物OCN，在生化水平上检测大豆肽CBP促进成骨细胞增殖与分化；
2)揭示在大豆肽CBP促进成骨细胞分化过程中，MAPK信号通路的三条经典信号途径—ERK，JNK,p38旁路参与其中以及关键信号传导步骤；大豆肽CBP促进成骨细胞内MAPK信号通路关键因子Runx2的基因表达和磷酸化水平。
附图说明
图1大豆肽CBP对成骨细胞增殖的影响：测试了浓度为0.7、7和70μM的CBP在24、48和72小时诱导细胞增殖的情况；通过酶标仪测量570nm处的吸光度来量化MTT测定。n＝5，数据表示为平均值±SEM，并通过单向ANOVA分析，然后进行Tukey多重比较测试。*p<0.05，**p<0.01，和***p<0.001与对照组比较。
图2大豆肽CBP对成骨细胞分化和矿化的影响：(A)CBP处理(0.7,7,70μM)24小时增加MC3T3-E1中碱性磷酸酶(ALP)的活性；(B)CBP处理(0.7,7,70μM)24小时增加MC3T3-E1中骨钙素(OCN)的活性；(C、D)用含有抗坏血酸和β-甘油磷酸盐的培养基，用不同浓度(0.7、7和70μM)的CBP处理细胞不同天数(7、14、21和28天)，然后，用茜素红对细胞进行染色，并拍摄图像；用氯化十六烷基吡啶对细胞进行脱色后测量490nm处的吸光度；数据是来自3个独立实验的平均值±SEM。*p<0.05，**p<0.01，和***p<0.001与对照组比较。
图3CBP对MC3T3-E1细胞中骨形成标志物(ALP、OCN、Col-1和RUNX2)mRNA表达的影响：MC3T3-E1细胞用0–70μM CBP处理7天。n＝3，数据表示为平均值±SEM。*p<0.05，**p<0.01，和***p<0.001与对照组比较。
图4大豆肽CBP对成骨细胞内MAPK信号通路激活的影响:(A)暴露于70μM CBP 0-24小时的MC3T3-E1细胞中ERK信号通路的磷酸化水平；(B)暴露于70μM CBP 0-24小时的MC3T3-E1细胞中JNK信号通路的磷酸化水平。(C)暴露于70μM CBP 0-24小时的MC3T3-E1细胞中p38信号通路的磷酸化水平。n＝3，数据表示为平均值±SEM。***p<0.001与对照组比较。
图5MAPK信号通路在大豆肽促进成骨细胞分化过程中的作用：CBP通过RUNX2通路的p38-MAPK介导促进成骨细胞分化：(A)CBP处理(0.7,7,70μM)24h增加了RUNX2的表达；(B)MAPK抑制剂(SB203580、SP600125和U0126)降低了CBP处理(70μM)24小时MC3T3-E1的OCN活性；(C)p38通路抑制剂(SB203580)降低了CBP处理(70μM)24小时MC3T3-E1的OCN活性；n＝3中，数据表示为平均值±SEM。*p<0.05，**p<0.01，和***p<0.001与对照组比较，ns，不显著。
具体实施方式
以下通过参考示范性实施例，本发明的目的和功能以及用于实现这些目的和功能的方法将得以阐明。然而，本发明并不受限于以下所公开的示范性实施例；可以通过不同形式来对其加以实现。说明书的实质仅仅是帮助相关领域技术人员综合理解本发明的具体细节。
本发明中选择目前应用于成骨细胞信号通路研究中最广泛的一种细胞：MC3T3-E1成骨前体细胞系，以此作为体外实验体系，选择斑马鱼幼鱼作为动物实验模型，针对MAPK三条主要旁路途径，开展以下几部分的研究：
实施例1大豆肽CBP对成骨细胞增殖的影响
本研究通过MTT法，将MC3T3-E1以2×104/cm2的密度接种于96孔板，分别测量1、10、100μg/mL的大豆肽与成骨细胞共孵育24、48、72h条件下的增殖情况，分析大豆肽是否对成骨细胞具有增殖作用。
结果见图1所示，途中可以看出，大豆肽CBP可以剂量和时间依赖促进成骨细胞的增殖。
实施例2大豆肽CBP对成骨细胞分化的影响
成骨细胞的分化对骨的合成代谢至关重要，在成骨细胞分化过程中，碱性磷酸酶(ALP)和骨钙蛋白(OCN)分别是分化早期和分化末期特征性的表形标志物，我们利用ELISA试剂盒测定分化过程中碱性磷酸酶的活性和骨钙蛋白的分泌量。
1)大豆肽CBP对碱性磷酸酶ALP的影响
将MC3T3-E1细胞以2×104/cm2的密度接种于24孔板，在完全培养基中培养2天后，换分化培养基继续培养4天，在第7天换无血清培养基，同时加入100μg/mL的大豆肽与成骨细胞共孵育72h后破碎细胞，并离心提取蛋白，通过BCA蛋白浓度试剂盒测定蛋白浓度，通过碱性磷酸酶试剂盒测定碱性磷酸酶活性，分析大豆肽对成骨细胞碱性磷酸酶活性的影响。
结果见图2A，图中显示，大豆肽CBP可以显著促进成骨细胞中ALP的活性，且随着剂量的增加而提升。
2)大豆肽CBP对骨钙蛋白OCN的影响
将MC3T3-E1细胞以2×104/cm2的密度接种于24孔板，在完全培养基中培养48h后，换分化培养基继续培养11天，在第14天换无血清培养基，在无血清的条件下将100μg/mL的大豆肽CBP与成骨细胞共孵育72h后将培养基离心并收集上清液，通过小鼠骨钙素酶联免疫试剂盒测定骨钙素含量，并参考蛋白浓度矫正结果，作为骨钙素的相对量，从而分析大豆肽对成骨细胞骨钙素的影响。
结果见图2B，途中显示，大豆肽CBP可以显著提升OCN的活性，且随着剂量的增加而提升。
实施例3大豆肽对成骨细胞矿化的影响
通过分化培养基诱导成骨细胞分化35天，其间每两天更换一次分化培养基，分化其间同时加入100μg/mL的大豆肽，通过茜红素染色液对成骨细胞特异性染色，在显微镜下观察染色情况并拍照，根据染色面积分析大豆肽对成骨细胞矿化的影响。具体步骤为：
对成骨细胞进行茜红素染色(Alizarin-S stain),测定基质矿化水平，全面衡量成骨细胞的分化水平。用含有抗坏血酸和β-甘油磷酸盐的培养基，用不同浓度(0.7、7和70μM)的CBP处理细胞不同天数(7、14、21和28天)，然后，用茜素红对细胞进行染色，并拍摄图像。用氯化十六烷基吡啶对细胞进行脱色后测量490nm处的吸光度。结果如图2C-2D，图中可以看出，随着处理时间的延长，大豆肽可以显著促进成骨细胞的矿化，且随着剂量的增加，效果更加突出。
实施例4成骨细胞分化相关基因表达的调控
通过分化培养基诱导成骨细胞分化，在无血清的条件下将100μg/mL的大豆肽与成骨细胞共孵育72h后通过TriZol试剂提取总RNA，设计成骨分化相关基因引物，利用RT-PCR技术分析大豆肽对成骨细胞分化相关基因：ALP、OCN、Col-1和Runx2(Runt-relatedtranscription factor 2)表达的调控。
设计合成ALP、OCN、Col-1、Runx2引物序列，
ALP forward:5’-AACCCAGACACAAGCATTCC-3’(SEQ ID NO:1),
reverse:5’-GAGAGCGAAGGGTCAGTCAG-3’(SEQ ID NO:2)；
Col-1forward:5’-AGAGCATGACCGATGGATTC-3’(SEQ ID NO:3),
reverse:5’-CCTTCTTGAGGTTGCCAGTC-3’(SEQ ID NO:4)；
OCN forward:5’-CCGGGAGCAGTGTGAGCTTA-3’(SEQ ID NO:5),
reverse:5’-TAGATGCGTTTGTAGGCGGTC-3’(SEQ ID NO:6)，
RUNX2 forward:5’-AAGTGCGGTGCAAACTTTCT-3’(SEQ ID NO:7),
reverse:5’-TCTCGGTGGCTGGTAG-3’(SEQ ID NO:8)。
TGA-3’；提取成骨细胞总RNA，利用RT-PCR方法分析ALP、OCN、Col-1、Runx2在转录水平的表达量，具体方法参考《分子克隆实验指南》第二版的方法。
结果如图3所示，图中可以看出，大豆肽可以促进ALP、OCN、BMP以及RunX2的表达，且随着剂量提升，促进表达的效果越显著。
实施例5成骨细胞转录因子Runx2的影响
通过大豆肽与成骨细胞共同培养，通过Western blot技术检验大豆肽能否激活成骨细胞内骨核心转录因子，Runx2是否是大豆肽促成骨细胞分化的关键下游靶点。结果见图5A，图中可以看出CBP处理(0.7,7,70μM)24h增加了RUNX2的表达，且促进作用具有剂量依赖的特征，即随着剂量增加，RunX2的表达量增加显著。
实施例6大豆肽对成骨细胞内MAPK信号通路激活的影响
利用ERK、JNK、p38三种激酶的特征性抑制剂U0126、SP600125、SB203580，从激活和抑制两方面研究ERK、JNK、p38三条MAPK旁路在大豆肽作用过程中的作用，同时利用RT-PCR和Western blot技术在转录、翻译水平验证MAPK信号通路的关键激酶的激活情况和各通路对成骨细胞功能的不同影响。根据实验结果，对ERK、JNK、p38中参与介导胞外大豆肽刺激信号的通路进行进一步研究，分析核心激酶上游关键信号的传递过程和下游转录因子的激活情况。
1)利用ERK、JNK、p38的苏氨酸、酪氨酸双位点磷酸化抗体，检测了大豆肽中MAPK的激活及表达情况。100μg/mL大豆肽刺激成骨细胞后，分别在0、3、6、12、24h提取了总蛋白样品，进行western blot分析。
结果如图4所示，暴露于70μM CBP 0-24小时的MC3T3-E1细胞中ERK信号通路的磷酸化水平(A)、JNK信号通路的磷酸化水平(B)以及p38信号通路的磷酸化水平(C)均有所提升，且在暴露3小时时达到顶峰，随后有所下降，总体在24小时内均促进了三条信号通路的磷酸化水平。
2)利用MAPK三条信号通路的选择性抑制剂，阻断细胞内的信号转导，然后再用大豆肽刺激细胞，观察分化标志物ALP活性的变化，判断MAPK三条通路在大豆肽促成骨细胞分化过程中的作用。
结果如图5B-C所示，图中可以看出，MAPK抑制剂(SB203580、SP600125和U0126)降低了CBP处理(70μM)24小时MC3T3-E1的OCN活性(B)；而p38通路抑制剂(SB203580)降低了CBP处理(70μM)24小时MC3T3-E1的OCN活性(C)。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明技术原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
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<213> 人工序列(Artificial Sequence)
<400> 6
tagatgcgtt tgtaggcggt c 21
<210> 7
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
aagtgcggtg caaactttct 20
<210> 8
<211> 16
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
tctcggtggc tggtag 16
<210> 9
<211> 12
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 9
Asp Glu Asp Glu Gln Ile Pro Ser His Pro Pro Arg
1 5 10
Claims (1)
1.大豆肽CBP在制备用于促进成骨细胞中的转录因子Runx2的表达，介导p38-MAPK通路的磷酸化的非治疗目的的试剂中的应用，其特征在于，所述的大豆肽CBP的氨基酸序列为DEDEQIPSHPPR(SEQ ID NO:9)。
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20140057709A (ko) * | 2012-10-31 | 2014-05-14 | (주)메디언스 | 키성장 및 골 강화용 조성물 |
| CN108531532A (zh) * | 2018-04-17 | 2018-09-14 | 南京财经大学 | 一种大豆蛋白肽及其制备方法和应用 |
| CN112812155A (zh) * | 2021-02-08 | 2021-05-18 | 南京财经大学 | 一种促进成骨细胞增殖的小肽 |
| CN112940093A (zh) * | 2021-02-08 | 2021-06-11 | 南京财经大学 | 促进成骨细胞增殖的小肽 |
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2022
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20140057709A (ko) * | 2012-10-31 | 2014-05-14 | (주)메디언스 | 키성장 및 골 강화용 조성물 |
| CN108531532A (zh) * | 2018-04-17 | 2018-09-14 | 南京财经大学 | 一种大豆蛋白肽及其制备方法和应用 |
| CN112812155A (zh) * | 2021-02-08 | 2021-05-18 | 南京财经大学 | 一种促进成骨细胞增殖的小肽 |
| CN112940093A (zh) * | 2021-02-08 | 2021-06-11 | 南京财经大学 | 促进成骨细胞增殖的小肽 |
Non-Patent Citations (1)
| Title |
|---|
| 多组分共存及乳酸菌发酵对豆浆中钙存在形式及体外吸收的影响研究;甘晶;中国博士学位论文;摘要 * |
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