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Activation of the KEAP1?NRF2?ARE signaling pathway reduces oxidative stress in Hep2 cells.

ABSTRACT: At present there are no studies investigating the effects of the kelch?like ECH?associated protein 1 (KEAP1)?nuclear factor erythroid 2?related factor 2 (NRF2)?antioxidant response element (ARE) signaling pathway on Hep2 cell line. The present study aimed to investigate this topic through knockdown of the KEAP1 gene. A stable Hep2 cell line specifically silencing the human KEAP1 gene was initially constructed. Hydrogen peroxide (H2O2) was added to the culture medium at various concentrations for various durations to interact with the short hairpin (sh)KEAP1?transfected Hep2 cells. Subsequently, the gene and protein expression levels of KEAP1, NRF2, NAD(P)H quinone oxidoreductase1 (NQO1) and heme oxygenase 1 (HO1) in experimental and control cells were measured by reverse transcription?quantitative polymerase chain reaction and western blotting, respectively. Furthermore, the viability and apoptotic rate of the shKEAP1?transfected Hep2 cells were detected by a Cell Counting?Kit 8 assay and flow cytometry, respectively. In the shKEAP1 Hep2 cell line, the mRNA and protein expression levels of NRF2, NQO1 and HO1 were markedly higher compared with the scramble control?transfected Hep2 and parent Hep2 cell lines. Immunofluorescence staining indicated that NRF2 was primarily located in the cytoplasm of scHep2 and parent Hep2 cell lines, but was present in the nuclei and cytoplasm of the shKEAP1 Hep2 cell line, where it translocates into the nuclei in response to H2O2. Following knockdown of the KEAP1 gene Hep2 cells, the apoptosis rates were 31.8 and 45.3% in scHep2 cells at 0.1 and 0.25 mmol/l H2O2 respectively and 14.1 and 27.9% in shKEAP1 cells. The present study indicated that the KEAP1?NRF2?ARE signaling pathway may exhibit an antioxidative effect within Hep2 cells and may be used for clinical treatment of cancer.

SUBMITTER: Li C

PROVIDER: S-EPMC6102741 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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Activation of the KEAP1‑NRF2‑ARE signaling pathway reduces oxidative stress in Hep2 cells.

Li Changjiang C Cheng Lei L Wu Haitao H He Peijie P Zhang Yanping Y Yang Yue Y Chen Jian J Chen Min M

Molecular medicine reports 20180716 3

At present there are no studies investigating the effects of the kelch‑like ECH‑associated protein 1 (KEAP1)‑nuclear factor erythroid 2‑related factor 2 (NRF2)‑antioxidant response element (ARE) signaling pathway on Hep2 cell line. The present study aimed to investigate this topic through knockdown of the KEAP1 gene. A stable Hep2 cell line specifically silencing the human KEAP1 gene was initially constructed. Hydrogen peroxide (H2O2) was added to the culture medium at various concentrations for ...[more]

PMID: 30015918

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Project description:BACKGROUND:Exposure to cold weather is associated with infaust cardiovascular responses, including myocardial infarction and arrhythmias. However, the exact mechanisms of these adverse changes in the myocardium under cold stress are unknown. This study was designed to investigate the mechanisms of cardiac injury induced by cold stress in mice. METHODS:The mice were randomly divided into three groups, normal control (no handling), 1-week cold stress and 2-week cold stress. We observed physiological changes of the mice and morphological changes of myocardium tissues, and we measured the changes of 3'-nitrotyrosine and 4-hydroxynonenal, the expression levels of superoxide dismutase-1, superoxide dismutase-2, Bax, Bad, Bcl-2, Nuclear factor erythroid-derived 2-like 2 (Nrf2) and Kelch like-ECH-associated protein 1 (Keap1) in myocardium by western blot. Besides, we detected mRNA of superoxide dismutase-1, superoxide dismutase-2, Bax, Bad, Bcl-2, Nrf2 and Keap1 by real-time PCR. One-way analysis of variance, followed by LSD-t test, was used to compare each variable for differences among the groups. RESULTS:Echocardiography analyses demonstrated left ventricle dysfunction in the groups receiving cold stress. Histological analyses witnessed inflammation, vacuolar and eosinophilic degeneration occurred in left ventricle tissues. Western blotting results showed increased 3'-nitrotyrosine and 4-hydroxynonenal and decreased antioxidant enzymes (superoxide dismutase-1 and superoxide dismutase-2) in the myocardium. Expression of Nrf2 and Keap1 followed a downward trend under cold exposure, as indicated by western blotting and real-time PCR. Expression of anti-apoptotic protein Bcl-2 also showed the same trend. In contrast, expression of pro-apoptotic proteins Bax and Bad followed an upward trend under cold exposure. The results of real-time PCR were consistent with those of western blotting. CONCLUSIONS:These findings were very significant, showing that cold exposure induced cardiac injury by inhibiting the Nrf2-Keap1 signaling pathway.

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Activation of the KEAP1?NRF2?ARE signaling pathway reduces oxidative stress in Hep2 cells.

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Activation of the KEAP1‑NRF2‑ARE signaling pathway reduces oxidative stress in Hep2 cells.

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