Protective effect of recombinant Lactobacillus plantarum against H2O2-induced oxidative stress in HUVEC cells

重组植物乳杆菌对H2O2诱导HUVEC细胞产生氧化应激损伤的保护作用

Abstract

This study probed the protective effect of recombinant Lactobacillus plantarum against hydrogen peroxide (H2O2)-induced oxidative stress in human umbilical vein endothelial cells (HUVECs). We constructed a new functional L. plantarum (NC8-pSIP409-alr-angiotensin-converting enzyme inhibitory peptide (ACEIP)) with a double-gene-labeled non-resistant screen as an expression vector. A 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) colorimetric assay was carried out to determine the cell viability of HUVEC cells following pretreatment with NC8-pSIP409-alr-ACEIP. Flow cytometry (FCM) was used to determine the apoptosis rate of HUVEC cells. Cysteinyl aspartate specific proteinase (caspase)-3/8/9 activity was also assayed and western blotting was used to determine protein expression of B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), inducible nitric oxide synthase (iNOS), nicotinamide adenine dinucleotide phosphate oxidase 2 (gp91phox), angiotensin II (AngII), and angiotensin-converting enzyme 2 (ACE2), as well as corresponding indicators of oxidative stress, such as reactive oxygen species (ROS), mitochondrial membrane potential (MMP), malondialdehyde (MDA), and superoxide dismutase (SOD). NC8-pSIP409-alr-ACEIP attenuated H2O2-induced cell death, as determined by the MTT assay. NC8-pSIP409-alr-ACEIP reduced apoptosis of HUVEC cells by FCM. In addition, compared to the positive control, the oxidative stress index of the H2O2-induced HUVEC (Hy-HUVEC), which was pretreated by NC8-pSIP409-alr-ACEIP, iNOS, gp91phox, MDA, and ROS, was decreased obviously; SOD expression level was increased; caspase-3 or -9 was decreased, but caspase-8 did not change; Bcl-2/Bax ratio was increased; permeability changes of mitochondria were inhibited; and loss of transmembrane potential was prevented. Expression of the hypertension-related protein (AngII protein) in HUVEC cells protected by NC8-pSIP409-alr-ACEIP decreased and expression of ACE2 protein increased. These plantarum results suggested that NC8-pSIP409-alr-ACEIP protects against H2O2-induced injury in HUVEC cells. The mechanism for this effect is related to enhancement of antioxidant capacity and apoptosis.

概要

目的

本文研究新功能重组植物乳杆菌NC8-pSIP409-alr-ACEIP保护血管内皮细胞损伤的初步作用机制。

创新点

成功构建非抗性筛选标记的新功能植物乳杆菌NC8-pSIP409-alr-ACEIP, 其具有安全, 方便, 无毒副作用, 且以食疗方式达到降血压的优点。研究NC8-pSIP409-alr-ACEIP对相关蛋白血管紧张素II(AngII), 血管紧张素转化酶2(ACE2), B淋巴细胞瘤-2(Bcl-2), BCL2关联X蛋白(Bax)与氧化应激相关因子的变化情况, 为阐明NC8-pSIP409-alr-ACEIP保护血管内皮细胞损伤的作用机制提供了基础的理论依据。

方法

首次构建以双基因标记的非抗性筛选锚定为表达载体的新功能型植物乳杆菌(NC8-pSIP409-alr-ACEIP), 使用不同浓度的过氧化氢(H2O2)诱导人脐静脉内皮细胞(HUVEC)细胞, 构建氧化应激损伤细胞模型(Hy-HUVEC), 通过MTT, 流式细胞术等方法检测Hy-HUVEC的凋亡, 通过蛋白质印迹法(western blot)检测AngII, ACE2, 诱导型一氧化氮合酶(iNOS), 尼克酰胺腺嘌呤二核苷酸磷酸氧化酶(gp91phox), Bcl-2和Bax的蛋白表达情况, 同时检测氧化应激相应指标活性氧(ROS), 线粒体膜电位(MMP), 丙二醛(MDA)和超氧化物歧化酶(SOD)的变化情况。

结论

NC8-pSIP409-alr-ACEIP对H2O2诱导的HUVEC细胞氧化应激损伤具有保护作用, 其作用机制与增强抗氧化能力和降低由线粒体凋亡通路引发的细胞凋亡有关。

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Acknowledgments

This work is supported the National Key Research and Development Program of China (Nos. 2017YFD0501000 and 2017YFD0501200), the National Natural Science Foundation of China (Nos. 31672528, 31941018, and 32072888), the Science and Technology Project of Jilin Provincial Department of Education (No. JJKH20190942KJ), and the Science and Technology Development Program of Jilin Province (Nos. 20180201040NY and 20190301042NY), China.

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Corresponding authors

Correspondence to Guilian Yang or Chunfeng Wang.

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Author contributions

Guan WANG and Mingyue HAO performed the experimental research and data analysis, and wrote and edited the manuscript. Mingyue HAO performed the establishment of cell models. Qiong LIU, Yanlong JIANG, and Haibin HUANG contributed to the study design, data analysis, and writing and editing of the manuscript. Guilian YANG and Chunfeng WANG performed experimental design. All authors have read and approved the final manuscript and, therefore, have full access to all the data in the study and take responsibility for the integrity and security of the data.

Compliance with ethics guidelines

Guan WANG, Mingyue HAO, Qiong LIU, Yanlong JIANG, Haibin HUANG, Guilian YANG, and Chunfeng WANG declare that they have no conflict of interest.

This article does not contain any studies with human or animal subjects performed by any of the authors.

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Wang, G., Hao, M., Liu, Q. et al. Protective effect of recombinant Lactobacillus plantarum against H2O2-induced oxidative stress in HUVEC cells. J. Zhejiang Univ. Sci. B 22, 348–365 (2021). https://doi.org/10.1631/jzus.B2000441

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Key words

  • Oxidative stress
  • Apoptosis
  • Human umbilical vein endothelial cell (HUVEC)
  • Hydrogen peroxide (H2O2)

关键词

  • 氧化应激
  • 细胞凋亡
  • 人脐静脉内皮细胞(HUVEC)
  • 过氧化氢(H2O2)