miR-29a-3p in Exosomes from Heme Oxygenase-1 Modified Bone Marrow Mesenchymal Stem Cells Alleviates Steatotic Liver Ischemia-Reperfusion Injury in Rats by Suppressing Ferroptosis via Iron Responsive Element Binding Protein 2

Abstract

Hepatic ischemia-reperfusion injury (IRI) is an inevitable result of liver surgery. Steatotic livers are extremely sensitive to IRI and have worse tolerance. Ferroptosis is considered to be one of the main factors of organ IRI. This study is aimed at exploring the role of ferroptosis in the effect of heme oxygenase-1-modified bone marrow mesenchymal stem cells (HO-1/BMMSCs) on steatotic liver IRI and its mechanism. An IRI model of a steatotic liver and a hypoxia reoxygenation (HR) model of steatotic hepatocytes (SHPs) were established. Rat BMMSCs were extracted and transfected with the Ho1 gene to establish HO-1/BMMSCs, and their exosomes were extracted by ultracentrifugation. Ireb2 was knocked down to verify its role in ferroptosis and cell injury in SHP-HR. Public database screening combined with quantitative real-time reverse transcription PCR identified microRNAs (miRNAs) targeting Ireb2 in HO-1/BMMSCs exosomes. miR-29a-3p mimic and inhibitor were used for functional verification experiments. Liver function, histopathology, terminal deoxynulceotidyl transferase nick-end-labeling staining, cell viability, mitochondrial membrane potential, and cell death were measured to evaluate liver tissue and hepatocyte injury. Ferroptosis was assessed by detecting the levels of IREB2, Fe2+, malondialdehyde, glutathione, lipid reactive oxygen species, glutathione peroxidase 4, prostaglandin-endoperoxide synthase 2 mRNA, and mitochondrial morphology. The results revealed that HO-1/BMMSCs improved liver tissue and hepatocyte injury and suppressed ferroptosis in vivo and in vitro. The expression of IREB2 was increased in steatotic liver IRI and SHP-HR. Knocking down Ireb2 reduced the level of Fe2+ and inhibited ferroptosis. HO-1/BMMSC exosomes reduced the expression of IREB2 and inhibited ferroptosis and cell damage. Furthermore, we confirmed high levels of miR-29a-3p in HO-1/BMMSCs exosomes. Overexpression of miR-29a-3p downregulated the expression of Ireb2 and inhibited ferroptosis. Downregulation of miR-29a-3p blocked the protective effect of HO-1/BMMSC exosomes on SHP-HR cell injury. In conclusion, ferroptosis plays an important role in HO-1/BMMSC-mediated alleviation of steatotic liver IRI. HO-1/BMMSCs could suppress ferroptosis by targeting Ireb2 via the exosomal transfer of miR-29a-3p.

Ferroptosis as an emerging therapeutic target in liver diseases

“…Lipophilic antioxidants, such as Fer-1, vitamin E, and Lip-1, act as radical scavengers to decrease lipid peroxides and are beneficial in abolishing ferroptosis to impede the progression of liver disease ( Dixon et al, 2012 ). Fer-1 therapy was found to increase cell survival in primary mouse hepatocytes with APAP incubation, and it also showed a protective effect in other ALFs, including hepatic IRI ( Lőrincz et al, 2015 ; Li et al, 2022 ). Additionally, in concanavalin A- (Con A)-induced AIH, pretreatment with Fer-1 reduced the severity of Con A-induced liver lesions and the occurrence of ferroptosis events, including the elevation of hepatic GSH, GPX4, and system Xc-expressions and downregulation of ferrous iron levels in liver tissues ( Zeng et al, 2020 ).…”

Ferroptosis: a promising candidate for exosome-mediated regulation in different diseases

“…They found that the expression of IREB2 was increased in steatotic liver IRI and SHP-HR, which led to ferroptosis of hepatocytes and further aggravated liver tissue damage. HO-1/BMMSC-derived exosomes enriched with miR-29a-3p, which downregulates IREB2, ameliorated liver tissue and hepatocyte injury and inhibited ferroptosis in vitro and in vivo [ 108 ]. In the same year, the team used the rat liver transplantation model with severe steatotic donor liver as the donor and the SHP-HR model to verify again that HO-1/BMMSC-derived exosomes were involved in the regulation of hepatocyte ferroptosis.…”

Functional micro-RNA drugs acting as a fate manipulator in the regulation of osteoblastic death

“…130 Iron response element-binding protein 2 (IREB2) is a significant regulator of cellular iron homeostasis, and Li et al reported that miR-29-3p could reduce the level of ferroptosis through the targeted downregulation of (IREB2) expression. 131 The transferrin receptor protein (TFRC) encodes a cell surface receptor necessary for cellular iron uptake. Wang et al reported that miR-370 targets TFRC and inhibits ferroptosis.…”