基于体外HUVECs细胞模型的条斑紫菜多糖促血管生成活性  

In Vitro Pro-angiogenic Activity of Porphyran Prepared from Porphyra yezoensis in Human Umbilical Vein Endothelial Cells(HUVECs) Model

作　　者：冯丰珍 郑明静 洪涛 李志朋 杜希萍[1,2,3] 朱艳冰 倪辉[1,2,3] 姜泽东 FENG Fengzhen;ZHENG Mingjing;HONG Tao;LI Zhipeng;DU Xiping;ZHU Yanbing;NI Hui;JIANG Zedong(College of Ocean Food and Biological Engineering,Jimei University,Xiamen 361021,China;Fujian Provincial Key Laboratory of Food Microbiology and Enzyme Engineering,Xiamen 361021,China;Research Center of Food Biotechnology of Xiamen City,Xiamen 361021,China)

机构地区：[1]集美大学海洋食品与生物工程学院,福建厦门361021 [2]福建省食品微生物与酶工程重点实验室,福建厦门361021 [3]厦门市食品生物工程技术研究中心,福建厦门361021

出　　处：《食品科学》2023年第7期1-9,共9页Food Science

基　　金：国家自然科学基金面上项目(31972081);福建省自然科学基金面上项目(2021J01833);厦门市自然科学基金面上项目(2022CXY0303)。

摘　　要：从条斑紫菜中提取条斑紫菜多糖,对其总糖质量分数、蛋白质量分数、3,6-内醚半乳糖质量分数、硫酸基质量分数、分子质量和单糖组成进行分析,并基于人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)的体外血管生成模型,研究条斑紫菜多糖对HUVECs的血管生成活性以及作用机制。采用噻唑蓝法测定条斑紫菜多糖对HUVECs细胞活力的影响,黏附实验和细胞划痕实验测定其细胞黏附和细胞迁移能力,体外血管形成实验检测HUVECs的管腔形成数目,Western Blotting检测经多糖处理的HUVECs中E-钙黏蛋白和N-钙黏蛋白的表达水平,并通过丝裂原活化蛋白激酶(mitogen-activated protein kinases,MAPKs)特异性抑制剂和血管内皮生长因子(vascular endothelial growth factor,VEGF)受体酪氨酸激酶抑制剂探究其细胞迁移的作用机制。结果表明,条斑紫菜多糖的总糖质量分数为(77.48±2.62)%,蛋白质量分数为(3.24±0.58)%,3,6-内醚半乳糖质量分数为(20.30±1.06)%,硫酸基质量分数为(20.78±3.31)%。多糖的分子质量为39.81 kDa,主要由半乳糖、3,6-内醚半乳糖和硫酸基组成,是一种硫酸化半乳聚糖。条斑紫菜多糖在0~200μg/mL质量浓度范围内对HUVECs无直接毒性且能够促进HUVECs模型中细胞增殖、黏附、迁移和血管网络的形成;进一步研究表明,条斑紫菜多糖主要通过c-Jun氨基末端激酶(c-Jun N-terminal kinase,JNK)MAPK和VEGF受体信号通路介导E-钙黏蛋白表达下调、N-钙黏蛋白表达上调,促进细胞血管生成。综上所述,条斑紫菜多糖能够促进HUVECs血管生成,为其开发新型的促血管生成营养因子和以精准营养为导向的条斑紫菜精深加工和高值利用奠定科学理论基础。In this study,the total sugar,protein,3,6-anhydro galactose and sulfate contents,apparent molecular mass and monosaccharide composition of porphyran isolated from Porphyra yezoensis were analyzed.The pro-angiogenic activity and underlying mechanism of porphyran were further investigated using human umbilical vein endothelial cells(HUVECs) as an in vitro angiogenesis model.The effect of porphyran on the cell viability of HUVECs was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay.Cell adhesion and migration capacity were evaluated using cell adhesion and wound healing assays.The number of lumens formed by HUVECs was analyzed using in vitro tube formation assay.The expression levels of E-cadherin and N-cadherin in HUVECs were analyzed by Western Blotting,and the pro-angiogenic mechanism of porphyran in HUVECs was explored using specific inhibitors of mitogen-activated protein kinases(MAPKs) and vascular endothelial growth factor(VEGF) receptor tyrosine kinase inhibitors.The results showed that porphyran contained(77.48 ± 2.62)% total sugar,(3.24 ± 0.58)% protein,and(20.30 ± 1.06)% 3,6-anhydrogalactose as well as(20.78 ± 3.31)% sulfate groups.The apparent molecular mass of porphyran was 39.81 kDa.Porphyran was mainly composed of galactose,3,6-anhydrogalactose and sulfate groups,suggesting it to be a sulfated galactan.Furthermore,porphyran treatment in the concentration range of 0–200 μg/mL had no significant toxicity no HUVECs,but instead promote cell proliferation,adhesion,migration,and vascular network formation.Further studies demonstrated that porphyran exerted its pro-angiogenic effect primarily by mediating the down-regulation of E-cadherin expression and the up-regulation of N-cadherin expression through the c-Jun N-terminal kinase(JNK) MAPK and VEGF receptor signaling pathways.In conclusion,porphyran has pro-angiogenic potential,which will provide a scientific basis for the development of porphyran as a novel pro-angiogenic factor and the precision nutrition-guided

关 键 词：条斑紫菜多糖 人脐静脉内皮细胞 细胞迁移 促血管生成活性 

分 类 号：TS201.4[轻工技术与工程—食品科学]