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
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.