CN112791091A - 王不留行黄酮苷在改善肠道屏障功能中的应用 - Google Patents
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Abstract
本发明公开了王不留行黄酮苷在改善肠道屏障功能中的应用，属于药物开发技术领域。本发明将王不留行种子的主要活性成分——王不留行黄酮苷，应用于改善肠道屏障功能中。这种天然的物质能够显著改善动物肠道屏障功能，能使屏障功能发生损害的小鼠血清内毒素含量降低、提升肠道碱性磷酸酶水平和紧密连接蛋白表达量、粪便中的短链脂肪酸丙酸和丁酸含量，并能调节肠道菌群平衡，降低厚壁菌门的相对丰度，提高拟杆菌门和变形菌门的相对丰度。
Description
技术领域
本发明涉及王不留行黄酮苷在改善肠道屏障功能中的应用，属于药物开发技术领域。
背景技术
肠道屏障功能主要包括机械屏障、生态屏障和免疫屏障，具体主要包括肠道菌群、黏液层、肠道上皮细胞和细胞间连接以及肠道免疫系统。当肠道屏障被破坏后，进入血液循环的细菌代谢物如内毒素释放增加，肠道通透性增加，导致全身炎症反应，进一步促进慢性疾病的发生发展。
目前比较常用的改善肠道屏障功能的方法有益生菌疗法和药物疗法，益生菌疗法包括移植粪菌或微生态制剂，能与宿主和原驻菌群进行复杂的互作反应，维护微生态系统的平衡，保护机体的正常功能。但是益生菌治疗也存在不足症(产气，腹胀，便秘和头痛等)以及免疫系统低下，静脉插管或急性胰腺炎患者不宜服用益生菌的情况，安全性评估依旧缺乏。目前采取了一些天然的药物治疗方法，但是很多药物的作用效果不好，但是天然药物因为其温和型和安全性，受到越来越多的关注。
发明内容
针对目前在治疗肠道屏障功能中存在的作用效果不好、缺少效果较好天然物质，本发明提出了将王不留行种子的主要活性成分——王不留行黄酮苷，应用于改善肠道屏障功能中。
本发明提供了王不留行黄酮苷在制备预防、缓解或改善肠道屏障的产品方面的应用。
在一种实施方式中，所述产品用于提高机体肠道免疫力，调节肠道物质平衡。
在一种实施方式中，所述产品包括药物或药物组合物，所述药物或药物组合物用于(a)～(e)至少一方面：
(a)增加肠道碱性磷酸酶；
(b)提高肠道紧密连接蛋白表达量；
(c)降低血清内毒素水平；
(d)提高粪便中的短链脂肪酸水平；
(e)调节肠道菌群平衡。
在一种实施方式中，所述肠道紧密连接蛋白包括ZO-1、Claudin-1、Occludin，表达量包括转录和蛋白水平的表达量。
在一种实施方式中，所述短链脂肪酸为丙酸和丁酸。
在一种实施方式中，所述调节肠道菌群平衡为降低厚壁菌门的相对丰度，提高拟杆菌门和变形菌门的相对丰度。
在一种实施方式中，所述药物或药物组合物还包括药学上可接受的赋型剂；所述药学上可接受的赋型剂是指任何可用于药学领域的稀释剂、辅助剂和/或载体。
在一种实施方式中，所述产品包括但不限于食品、保健品饮品、肠内营养制剂、膳食补充剂、兽药或者饲料添加剂。
在一种实施方式中，所述食品、保健品饮品、肠内营养制剂、膳食补充剂、兽药或者饲料添加剂中还含有常规辅料。
在一种实施方式中，所述常规辅料包括填充剂、矫味剂、粘合剂、崩解剂、润滑剂、抗酸剂、以及营养强化剂中的一种或多种。
本发明的有益效果：
本发明发现王不留行黄酮苷具有改善肠道屏障功能障碍的作用：用C57BL/6小鼠制作2型糖尿病小鼠模型并将小鼠分为为空白组、模型组、王不留行黄酮苷腹腔注射组。与模型组相比，王不留行黄酮苷组血清内毒素含量显著降低，降低至正常水平；肠道碱性磷酸酶水平和紧密连接蛋白表达量升高，粪便中的短链脂肪酸丙酸和丁酸含量显著提升，趋向于正常水平。王不留行黄酮苷降低了厚壁菌门的相对丰度，增高了拟杆菌门和变形菌门的相对丰度。在属水平上，王不留行黄酮苷增加了拟杆菌属，脱硫弧菌属，阿克曼氏菌Akkermansia和Muribaculaceae的相对丰度。
附图说明
图1为各组小鼠血清内毒素水平图；
图2为各组小鼠肠道碱性磷酸酶水平图；
图3为ZO-1、Occludin，Claudin-1的基因表达水平图；
图4为ZO-1、Occludin，Claudin-1的蛋白表达水平图；
图5为小鼠粪便中短链脂肪酸含量(乙酸、丙酸、丁酸)图；
图6为小鼠肠道菌群组成的变化图。
具体实施方式
实施例中所用小鼠：C57BL/6小鼠，购自南京大学模式生物研究所。
碱性磷酸酶试剂盒：购自南京建成生物工程研究所。
实施例1：小鼠模型的制备与给药
将小鼠随机分为3组(n＝8)，分别为对照组、模型组、王不留行黄酮苷组。正常组喂普通饲料(14.7％J，13％脂肪)，其余各组饲喂HFD(21.8％J，60％脂肪，D12492)，自由进食。饮食干预8周后，除正常组外，其余两组禁食12h后均给予120mg/kg STZ(10mM柠檬酸缓冲液，pH 4.0溶解)。正常组小鼠以相同的频率给予注射缓冲液。各组继续维持当前饮食，一周后从尾静脉采血测定血糖水平。当空腹血糖(FBG)水平高于11.1mmol/L时，将小鼠作为T2DM模型。
各组继续维持当前饮食，8周后，将模型组每天腹腔注射0.1mL体积的生理盐水，王不留行黄酮苷组按1mg/kg浓度腹腔注射0.1mL。记录小鼠体重、血糖、生活状态等基本情况，共给药6周。
实施例2：王不留行黄酮苷在改善小鼠肠道屏障功能中的应用
6周后，将小鼠取出，注射10％的水合氯醛麻醉后眼球取血并处死取小鼠回肠组织和粪便。
(1)小鼠血清中内毒素的变化情况
小鼠全血低温12000rpm离心后取上清，用内毒素测定试剂盒定量几组小鼠血清中内毒素的表达，如图1和表1所示，模型组的小鼠回肠中的血清内毒素的含量显著提升，但是用王不留行黄酮苷给药后，能使得血清内毒素的含量降低至正常水平。
(2)小鼠回肠组织内碱性磷酸酶(IAP)的变化情况
用碱性磷酸酶试剂盒定量几组小鼠回肠组织内碱性磷酸酶(IAP)的表达，王不留行黄酮苷治疗后恢复了模型组碱性磷酸酶的水平，如图2和表1所示，模型组肠道中的碱性磷酸酶含量显著降低，但使用王不留行黄酮苷给药后，能够使得肠道内碱性磷酸酶含量得到提升。
表1各组小鼠血清内毒素(LPS)含量和肠道碱性磷酸酶含量
(3)ZO-1、Claudin-1、Occludin的表达水平
①ZO-1、Claudin-1、Occludin的基因表达水平
使用Trizol试剂提取回肠组织总RNA，取1ug总RNA合成cDNA。用SYBR Green进行荧光定量PCR(qPCR)，定量表达mRNA。采用2-CT法计算相对基因表达量，以β-actin为参照归一化。结果如图3和表2所示：模型组小鼠肠道组织紧密连接蛋白在转录水平都显著降低，但使用王不留行黄酮苷给药后，能够使得肠道组织紧密连接蛋白在转录水平都显著得到提升。
定量引物：
β-actin-F：AGCTGAGAGGGAAATCGTGC(SEQ ID NO.1)，
β-actin-R：TCCAGGGAGGAAGAGGATGC(SEQ ID NO.2)，
ZO-1-F：CCCACAAGGAGCCATTCCTG(SEQ ID NO.3)，
ZO-1-R：GTCAGGAGTCATGGACGCAC(SEQ ID NO.4)，
Claudin-1-F：AAAGCACCGGGCAGATACAG(SEQ ID NO.5)，
Claudin-1-R：ATCTTCCAGGCACCTCATGC(SEQ ID NO.6)，
Occludin-F：TTCGCTTATCTTGGGAGCCTG(SEQ ID NO.7)，
Occludin-R：ACATGCATCTCTCCGCCATAC(SEQ ID NO.8)。
表2各组小鼠肠道组织紧密连接蛋白的mRNA转录水平
②ZO-1、Claudin-1、Occludin的蛋白表达水平
取回肠组织样品，冰上解冻后粗略称取约30mg，置入冰上预冷的组织匀浆器中，迅速加入300ul预冷含蛋白酶/磷酸酶抑制剂的RIPA溶液，于冰上匀浆。充分匀浆裂解后，转移样品至离心管中以4℃、14000rpm离心10min，然后取上清以BCA法参照试剂盒操作说明对蛋白浓度进行检测，调整各组蛋白浓度至等同后，向样品中加入相应体积的上样缓冲液，混匀后水浴煮沸5min，-20℃冻存。蛋白质提取液经聚丙烯酰胺凝胶电泳(PAGE)分析后转移到PVDF膜上。用一抗(Occludin(Abcam，1:50000),Claudin-1(Abcam，1:2000)and ZO-1(ABclonal，1:500).β-actin(Abcam，1:1000))在4℃孵育过夜后，用辣根过氧化物酶标记的二抗(除β-actin用山羊抗鼠外，其余均用山羊抗兔，稀释比为1:2000)孵育膜。用增强型化学发光检测试剂对信号进行可视化。免疫反应条带的密度测定用Image-J软件进行分析，并归一化为β-actin的量。所有样品采用技术重复运行，实验重复3次。
结果如图4和表3所示，模型组小鼠肠道组织紧密连接蛋白的蛋白质表达水平显著降低，与模型组相比，王不留行黄酮苷可以增加了模型组小鼠ZO-1、Claudin-1、Occludin的表达。
表3各组小鼠肠道组织紧密连接蛋白的蛋白质表达水平(目标蛋白/内参)
(4)小鼠粪便短链脂肪酸变化情况
通过高效液相色谱法测定小鼠粪便短链脂肪酸浓度。取0.3g粪便样品，加入1ml去离子水，1200转/分钟离心10分钟，收集上清，加入100ul浓盐酸，混匀后加入5ml乙醚，充分混匀，常温下萃取20分钟，3500转/分钟离心10分钟，上层有机相转移到另一个管中，加入500ul 1M NaOH，充分混匀，常温下萃取20分钟，3500转/分钟离心10分钟，收集下层水相，加入100ul浓HCL混匀后过滤，通过自动进样器吸取20ul进入高效液相色谱。流动相为0.025％磷酸溶液和乙腈溶液的混合溶液，0.025％磷酸溶液:乙腈溶液＝5％:95％。乙腈在45分钟内以梯度从5％增加到95％，流速1mL/min，柱温30℃，检测波长210nm。
结果如图5和表4所示，与模型组相比，王不留行黄酮苷可显著增加粪便中丙酸和丁酸的含量。
表4各组小鼠肠道短链脂肪酸含量(mmol/g)
(5)粪便微生物群落结构的变化情况
为了评估粪便微生物群落结构，采用高通量测序方法Illumina测序，以16S rRNA基因为靶点。
每组随机抽取3只小鼠，使用DNA提取试剂盒提取样本DNA，检测DNA浓度后进行PCR扩增和文库构建，以20-30ng DNA为模板，使用上游和下游引物扩增V3和V4区域；再使用Illumina MiSeq/NovaSeq(Illumina,San Diego,CA,USA)进行测序。
上游引物：CCTACGGRRBGCASCAGKVRVGAAT(SEQ ID NO.9)；
下游引物：GGACTACNVGGGTWTCTAATCC(SEQ ID NO.10)。
如图6和表5所示，结果表明王不留行黄酮苷干预并未改变模型组小鼠的粪便微生物多样性，但在门水平上，降低了厚壁菌门(Firmicutes)的相对丰度，增高了拟杆菌门(Bacteroidetes)和变形菌门(Proteobacteria)的相对丰度。
在属水平上，王不留行黄酮苷增加了拟杆菌属Bacteroides，脱硫弧菌属Desulfovibrio，阿克曼氏菌Akkermansia和Muribaculaceae_的相对丰度。
表5各组小鼠粪便微生物状况(％)
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。
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Claims (10)
1.王不留行黄酮苷在制备预防、缓解或改善肠道屏障的产品方面的应用。
2.根据权利要求1所述的应用，其特征在于，所述产品用于提高机体肠道免疫力，调节肠道物质平衡。
3.根据权利要求2所述的应用，其特征在于，所述产品包括药物或药物组合物，所述药物或药物组合物用于(a)～(e)至少一方面：
(a)增加肠道碱性磷酸酶；
(b)提高肠道紧密连接蛋白表达量；
(c)降低血清内毒素水平；
(d)提高粪便中的短链脂肪酸水平；
(e)调节肠道菌群平衡。
4.根据权利要求3所述的应用，其特征在于，所述肠道紧密连接蛋白包括ZO-1、Claudin-1、Occludin，表达量包括转录和蛋白水平的表达量。
5.根据权利要求4所述的应用，其特征在于，所述短链脂肪酸为丙酸和丁酸。
6.根据权利要求5所述的应用，其特征在于，所述调节肠道菌群平衡为降低厚壁菌门的相对丰度，提高拟杆菌门和变形菌门的相对丰度。
7.根据权利要求6所述的应用，其特征在于，所述药物或药物组合物还包括药学上可接受的赋型剂；所述药学上可接受的赋型剂是指任何可用于药学领域的稀释剂、辅助剂和/或载体。
8.根据权利要求7所述的应用，其特征在于，所述产品包括但不限于食品、保健品饮品、肠内营养制剂、膳食补充剂、兽药或者饲料添加剂。
9.根据权利要求8所述的应用，其特征在于，所述食品、保健品饮品、肠内营养制剂、膳食补充剂、兽药或者饲料添加剂中还含有常规辅料。
10.根据权利要求9所述的应用，其特征在于，所述常规辅料包括填充剂、矫味剂、粘合剂、崩解剂、润滑剂、抗酸剂、以及营养强化剂中的一种或多种。
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| CN103948619A (zh) * | 2014-05-12 | 2014-07-30 | 江南大学 | 一种具有抗氧化抗高糖损伤的王不留行黄酮苷的应用 |
| CN106668053A (zh) * | 2017-03-03 | 2017-05-17 | 江南大学 | 王不留行黄酮苷在制备抗糖尿病糖脂代谢紊乱药物中的应用 |
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