miR-124-3p delivered by exosomes from heme oxygenase-1 modified bone marrow mesenchymal stem cells inhibits ferroptosis to attenuate ischemia-reperfusion injury in steatotic grafts

Background: Steatotic livers tolerate ischemia-reperfusion injury (IRI) poorly, increasing the risk of organ dysfunction. Ferroptosis is considered the initiating factor of organ IRI. Heme oxygenase oxygen-1 (HO-1)-modified bone marrow mesenchymal stem cells (BMMSCs) (HO-1/BMMSCs) can reduce hepatic IRI; however, the role of Ferroptosis in IRI of steatotic grafts and the effect of HO-1/BMMSCs-derived exosomes (HM-exos) on Ferroptosis remain unknown.

Methods: A model of rat liver transplantation (LT) with a severe steatotic donor liver and a model of hypoxia and reoxygenation (H/R) of steatotic hepatocytes were established. Exosomes were obtained by differential centrifugation, and the differentially expressed genes (DEGs) in liver after HM-exo treatment were detected using RNA Sequencing. The expression of Ferroptosis markers was analyzed. MicroRNA (miRNA) Sequencing was used to analyze the miRNA profiles in HM-exos.

Results: We verified the effect of a candidate miRNA on Ferroptosis of H/R treated hepatocytes, and observed the effect of exosomes knockout of the candidate miRNA on hepatocytes Ferroptosis. In vitro, HM-exo treatment reduced the IRI in steatotic grafts, and enrichment analysis of DEGs suggested that HM-exos were involved in the regulation of the Ferroptosis pathway. In vitro, inhibition of Ferroptosis by HM-exos reduced hepatocyte injury. HM-exos contained more abundant miR-124-3p, which reduced Ferroptosis of H/R-treated cells by inhibiting prostate six transmembrane epithelial antigen 3 (STEAP3), while overexpression of Steap3 reversed the effect of mir-124-3p. In addition, HM-exos from cell knocked out for miR-124-3p showed a weakened inhibitory effect on Ferroptosis. Similarly, HM-exo treatment increased the content of miR-124-3p in grafts, while decreasing the level of STEAP3 and reducing the degree of hepatic Ferroptosis.

Conclusion: Ferroptosis is involved in the IRI during LT with a severe steatotic donor liver. miR-124-3p in HM-exos downregulates Steap3 expression to inhibit Ferroptosis, thereby attenuating graft IRI, which might be a promising strategy to treat IRI in steatotic grafts.