CKLF1, transcriptionally activated by FOXC1, promotes hypoxia/reoxygenation‑induced oxidative stress and inflammation in H9c2 cells by NLRP3 inflammasome activation

Jia,Yinfeng; Pan,Jiansheng

doi:10.3892/etm.2023.12347

CKLF1, transcriptionally activated by FOXC1, promotes hypoxia/reoxygenation‑induced oxidative stress and inflammation in H9c2 cells by NLRP3 inflammasome activation

Authors:
- Yinfeng Jia
- Jiansheng Pan
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Affiliations: Department of Cardiovascular Medicine, The Second People's Hospital of Yueqing, Wenzhou, Zhejiang 325608, P.R. China
Published online on: December 11, 2023 https://doi.org/10.3892/etm.2023.12347
Article Number: 59
Copyright: © Jia et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Myocardial ischemia/reperfusion (I/R) injury is a clinical challenge in the treatment of ischemic heart disease. The present study aimed to establish a hypoxia/reoxygenation (H/R)‑induced H9c2 cell model to explore the role and mechanism of chemokine‑like factor 1 (CKLF1) in myocardial I/R injury. First, CKLF1 expression was measured in H/R‑induced H9c2 cells by reverse transcription‑quantitative PCR and western blotting. Subsequently, after CKLF1 silencing, cell viability and apoptosis were evaluated by Cell Counting Kit‑8 assay and flow cytometry. In addition, 2,7‑dichlorodihydrofluorescein diacetate staining was used to assess the levels of cellular reactive oxygen species. Additionally, the levels of superoxide dismutase, glutathione peroxidase and malondialdehyde, and the contents of inflammatory factors IL‑6, IL‑1β and TNF‑α were detected using corresponding commercially available kits. Western blotting was used to examine the expression levels of proteins involved in the NOD‑like receptor family, pyrin domain containing 3 (NLRP3) inflammasome. The JASPAR database predicted that forkhead box protein C1 (FOXC1) would bind to the CKLF1 promoter region, and dual luciferase and chromatin immunoprecipitation assays were performed to verify it. Subsequently, FOXC1 overexpression and CKLF1 silencing were used to clarify the regulatory mechanism of FOXC1 on CKLF1 in H/R‑induced H9c2 cells. The results revealed that CKLF1 expression was markedly enhanced in H/R‑stimulated H9c2 cells. CKLF1 knockdown enhanced the viability and inhibited the apoptosis of H9c2 cells exposed to H/R. Moreover, the oxidative stress and inflammation induced by H/R were alleviated following CKLF1 silencing. CKLF1 knockdown also inhibited NLRP3 inflammasome activation. Furthermore, FOXC1 bound to the CKLF1 promoter region to upregulate CKLF1 expression, and FOXC1 overexpression alleviated the effects of CKLF1 knockdown on H9c2 cell damage induced by H/R via activation of the NLRP3 inflammasome. In conclusion, CKLF1 transcriptionally activated by FOXC1 may promote H/R‑induced oxidative stress and inflammation in H9c2 cells via NLRP3 inflammasome activation.

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