Wnt/β-catenin/VEGF通路在副猪嗜血杆菌引起猪内皮细胞损伤中的作用
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作者:
作者单位:

1.华中农业大学动物科学技术学院、动物医学院,武汉430070;2.武汉市动物疫病预防控制中心,武汉 430016

作者简介:

滑坷鑫,E-mail:huakexin@mail.hzau.edu.cn

通讯作者:

金卉,E-mail:jinhui@mail.hzau.edu.cn

中图分类号:

S852.61

基金项目:

湖北省自然科学基金杰出青年人才项目(2020CFA060);湖北省自然科学基金创新群体项目(2021CFA016);国家自然科学基金面上项目(31972643; 31572486)


Role of Wnt/β-catenin/VEGF pathway in endothelial cell damage caused by Haemophilus parasuis in pigs
Author:
Affiliation:

1.College of Animal Science and College of Veterinary Medicine,Huazhong Agricultural University, Wuhan 430070,China;2.Wuhan Animal Disease Control Center,Wuhan 430016,China

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    摘要:

    为探究副猪嗜血杆菌(Haemophilus parasuis,HPS)引起猪渗出性炎症的过程中血管内皮屏障的变化情况,以HPS感染原代猪血管内皮细胞,通过Western blot和荧光定量PCR检测HPS对Wnt/β-catenin通路及其下游靶基因表达的影响,并通过间接免疫荧光和跨内皮电阻测定检测细胞间黏附连接结构的改变。结果显示:HPS感染激活猪内皮细胞中的Wnt/β-catenin信号通路,促使胞质中的β-catenin蛋白发生核转位,导致胞膜处由β-catenin和血管内皮钙黏蛋白(VE-cadherin)构成的黏附连接结构被破坏,细胞通透性增加。抑制Wnt/β-catenin通路下游血管内皮生长因子(VEGF)基因的表达,可以明显抑制β-catenin的核转位,显著恢复内皮细胞间β-catenin和VE-cadherin构成的黏附连接结构,恢复内皮细胞通透性。以上研究结果表明,HPS通过激活猪内皮细胞中Wnt/β-catenin通路,破坏细胞间黏附连接,增加内皮细胞通透性;同时,Wnt/β-catenin通路下游VEGF基因通过正反馈调节效应放大了HPS感染引起的Wnt/β-catenin信号通路的激活效应和细胞结构的损伤。

    Abstract:

    To investigate the changes of vascular endothelial barrier during exudative inflammation caused by Haemophilus parasuis (HPS) in pigs,primary pig vascular endothelial cells were infected with HPS.The effects on the expression of the Wnt/β-catenin pathway and its downstream target genes were detected by Western blot and fluorescence quantitative PCR,and the alterations in the structure of intercellular adhesion junctions were detected by indirect immunofluorescence and transendothelial electrical resistance assay.The results showed that HPS infection activated Wnt/β-catenin pathway in porcine endothelial cells,and induced nuclear translocation of β-catenin protein in the cytoplasm,resulting in the destruction of the adhesive junction structure composed of β-catenin and VE-cadherin at the cell membrane and the increase of cell permeability.Moreover,inhibition of the expression of VEGF gene downstream of Wnt/β- catenin pathway significantly suppressed the nuclear translocation of β-catenin and markedly restored the intercellular adherens junctions composed of β-catenin and VE-cadherin,which benefits the recovery of endothelial cell permeability.These results suggested that HPS infection disrupted intercellular adherens junctions and increased endothelial cell permeability by activating the Wnt/β-catenin pathway in porcine endothelial cells; meanwhile,the VEGF gene downstream of the Wnt/β-catenin pathway amplifies the Wnt/β- catenin pathway activity and cell damage induced by HPS infection through a positive feedback.

    表 1 本研究所用的荧光定量引物序列Table 1 The sequences of primers used in this study for qRT-PCR
    图1 HPS感染的PAEC中Wnt/β-catenin通路活性(A)及下游靶基因表达水平(B)变化Fig.1 Changes in Wnt/β-catenin pathway activity (A) and expression levels of downstream target genes (B) in HPS-infected PAEC
    图2 Western blot (A)和间接免疫荧光(B)检测HPS感染的PAEC中β-catenin的核转位效应Fig.2 Western blot (A) and indirect immunofluorescence (B) detection of nuclear translocation of β-catenin in HPS-infected PAEC
    图3 HPS感染的PAEC中黏附连接结构(A)及细胞通透性(B)的变化Fig.3 Changes in the structure of adherens junctions (A) and cell permeability (B) of HPS-infected PAEC
    图4 HPS感染的PAEC中VEGF对Wnt/β-catenin通路活性的调控作用Fig.4 Regulatory effect of VEGF on the activity of Wnt/β-catenin pathway in PAECs infected with HPS
    图5 干扰VEGF对HPS感染的PAEC中VEGF介导的黏附连接结构(A)及细胞通透性(B)的调控作用Fig.5 Regulation of VEGF-mediated adhesion structure (A) and cell permeability (B) in PAEC with interference of VEGF in HPS infection
    表 2 本研究中所用的干扰因子序列Table 2 The sequences of siRNA used in this study
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滑坷鑫,彭宇娜,孙煜,张俐文,张四化,万云,金卉. Wnt/β-catenin/VEGF通路在副猪嗜血杆菌引起猪内皮细胞损伤中的作用[J].华中农业大学学报,2024,43(5):186-192

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  • 收稿日期:2023-05-08
  • 在线发布日期: 2024-10-08
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