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

Guan Wang, Mingyue Hao, Qiong Liu, Yanlong Jiang, Haibin Huang, Guilian Yang, Chunfeng Wang

Journal of Zhejiang University. Science. B 2021 May 15

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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.

Citation

Guan Wang, Mingyue Hao, Qiong Liu, Yanlong Jiang, Haibin Huang, Guilian Yang, Chunfeng Wang. Protective effect of recombinant Lactobacillus plantarum against H2O2-induced oxidative stress in HUVEC cells. Journal of Zhejiang University. Science. B. 2021 May 15;22(5):348-365