The present study aimed to explore how semaglutide can help protect the heart from injury caused by hypoxia/reoxygenation (H/R) and to reveal the underlying mechanism. Briefly, AC16 cardiomyocytes were subjected to 8 h of hypoxia followed by 12 h of reoxygenation to simulate H/R. The cells were divided into the following five groups: Normoxia, H/R, H/R + semaglutide, H/R + semaglutide + rapamycin (autophagy inducer), and H/R + semaglutide + 3‑methyladenine (3‑MA; autophagy inhibitor) groups. Cell viability was examined using a Cell Counting Kit‑8 assay, ATP levels were examined using a bioluminescent detection kit, reactive oxygen species (ROS) production was detected using a ROS Assay Kit, and monomeric red fluorescent protein (mRFP)‑green fluorescent protein (GFP)‑LC3 was assessed using tandem mRFP‑GFP fluorescence microscopy, while autophagosomes were observed using transmission electron microscopy. Furthermore, the protein expression levels of autophagy markers (LC3, p62 and Beclin1) and regulators of mitochondrial autophagy [PTEN‑induced putative kinase protein‑1 (PINK1) and Parkin] were examined using western blot analysis. In AC16 cells, exposure to hypoxia followed by reoxygenation led to an increase in oxidative stress. This condition also induced an increase in autophagy activity, as evidenced by an increase in the number of autophagosomes, elevated LC3‑II/LC3‑I ratio, and upregulation of p62, Beclin1, PINK1 and Parkin expression compared with those in cells cultured under normoxia. Notably, treatment with semaglutide or rapamycin effectively reversed the H/R‑induced oxidative stress, enhanced the changes in autophagy activity, autophagosome levels and elevated LC3BII/LC3BI ratio, and increased the expression levels of Beclin1, PINK1, Parkin and p62 expression. Notably, the use of 3‑MA exhibited distinct effects under the same conditions; it exacerbated oxidative stress, decreased autophagy activity and reduced the LC3BII/LC3BI ratio. In conclusion, semaglutide was found to reduce oxidative stress caused by H/R and to increase autophagy via the ROS/PINK1/Parkin/p62 pathway. The present study offers a novel understanding of how semaglutide may protect the heart, and suggests its potential use in the treatment of myocardial ischemia/reperfusion injury.

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