Metformin ameliorates ferroptosis in cardiac ischemia and reperfusion by reducing NOX4 expression via promoting AMPKα

Zhenhua Wu; Yunpeng Bai; Yujuan Qi; Chao Chang; Yan Jiao; Yaobang Bai; Zhigang Guo

doi:10.1080/13880209.2023.2212700

Metformin ameliorates ferroptosis in cardiac ischemia and reperfusion by reducing NOX4 expression via promoting AMPKα

Pharm Biol. 2023 Dec;61(1):886-896. doi: 10.1080/13880209.2023.2212700.

Authors

Zhenhua Wu^{1

2}, Yunpeng Bai³, Yujuan Qi², Chao Chang², Yan Jiao², Yaobang Bai², Zhigang Guo^{1

3}

Affiliations

¹ Academy of Medical Engineering and Translational Medicine, Tianjin University, Tianjin, China.
² Department of Cardiac Surgery, ICU, Tianjin Chest Hospital, Tianjin, China.
³ Department of Cardiac Surgery, Tianjin Chest Hospital, Tianjin, China.

Abstract

Context: Metformin (Met) has a protective effect against cardiac ischemia and reperfusion (I/R) injury.

Objective: This study uncovered the Met effect on ferroptosis in cardiac I/R.

Materials and methods: Sprague-Dawley rats underwent cardiac I/R treatment (ischaemia 30 min; reperfusion 24 h) (I/R group), and administered intravenously with Met (200 mg/kg) (I/R + Met group). Haematoxylin-eosin staining, Prussian blue staining, immunohistochemistry and transmission electron microscope were conducted on cardiac tissues. H9c2 cells underwent oxygen-glucose deprivation/reoxygenation (OGD/R group) and treated by Met (0.1 mM) (OGD/R + Met group). Adenosine monophosphate-activated protein kinase α (AMPKα) siRNA was transfected into OGD/R-induced H9c2 cells. Cell counting kit-8 (CCK-8) assay, dichloro-dihydro-fluorescein diacetate (DCFH-DA) and JC-1 staining were conducted on H9c2 cells. Ferroptosis-related indicators and gene expression were detected by enzyme-linked immunosorbent assay (ELISA), quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot.

Results: In cardiac I/R rat, Met decreased heart and serum MDA, cardiac and serum non-heme iron, and serum CK-MB and LDH (inhibition rate: 50.0%, 48.8%, 47.6%, 29.5%, 30.6% and 34.7%, respectively), relieved cardiac tissue ferroptosis and mitochondria damage, increased fraction shortening and ejection fraction (157.5% and 146.2% on day 28, respectively), up-regulated AMPKα and down-regulated NOX4 in cardiac tissues. In OGD/R-induced H9c2 cells, Met (0.1 mM) increased cell viability (promotion rate: 170.0%), decreased non-heme iron and MDA (inhibition rate: 30.1% and 47.9%, respectively), relieved ferroptosis, up-regulated AMPKα and down-regulated NOX4. AMPKα silencing abrogated these effects of Met on the OGD/R-induced H9c2 cells.

Discussion and conclusions: Met shows effectiveness in relieving ferroptosis in cardiac I/R. In the future, Met may be an effective drug for relieving ferroptosis in cardiac I/R patients clinically.

Keywords: LDH; MDA; Mitochondrial damage; ROS; non-heme iron.

MeSH terms

AMP-Activated Protein Kinases / metabolism
Animals
Apoptosis
Cell Line
Ferroptosis*
Iron / metabolism
Ischemia
Metformin* / pharmacology
Myocardial Ischemia* / metabolism
Myocytes, Cardiac
Rats
Rats, Sprague-Dawley
Reperfusion
Reperfusion Injury* / drug therapy
Reperfusion Injury* / metabolism
Reperfusion Injury* / prevention & control

Substances

Metformin
AMP-Activated Protein Kinases
Iron

Grants and funding

This work was supported by Tianjin Health Technology Project (ZC20147) and Tianjin Education Commission scientific research project (2022YGYB10) to Zhenhua Wu.