Hypoxia-preconditioning human bone marrow-derived mesenchymal stem cells induce high-quality mitochondrial transfer through gap junctions to alleviate ischemia-reperfusion injury in liver graft

Background: Amid the widespread scarcity of donor livers, mitigating ischemia-reperfusion injury (IRI) of liver grafts is vital for ensuring early recovery of post-transplant liver function. Human bone marrow-derived mesenchymal stem cells (hBMSCs) have shown potential in alleviating IRI damage by regulating mitochondrial function. Hypoxia-preconditioning hBMSCs (hypo-hBMSCs) have shown considerable promise in enhancing therapeutic efficacy, yet the underlying mechanism remain to be elucidated. Therefore, this study aims to explore the role of hypo-hBMSCs in alleviating hepatic IRI and uncover their potential mechanisms, with the goal of offering new strategies for the application of hBMSCs in liver protection after transplantation.

Methods: Initially, we investigated the impact of hypoxia preconditioning on the quality of hBMSCs mitochondria and whether hypo-hBMSCs can alleviate IRI damage in liver grafts by transferring mitochondria. Subsequently, by employing the enhancer RA and the inhibitor Gap26 to modulate the function of gap junctions (GJs) in vivo and in vitro, we confirmed their crucial role in the process of hypo-hBMSCs transferring mitochondria to hepatocytes. Ultimately, through bioinformatics analysis, Co-IP, siRNA and overexpression, we demonstrate that the up-regulated Cx43 and Cx32 in hypo-hBMSCs can form homotypic Cx43-GJs and Cx32-GJs with hepatocytes, thereby enhancing the transfer of mitochondria.

Results: The results indicate that hypoxia preconditioning diminishes superoxides accumulation and elevates the mitochondrial membrane potential by inducing Mitophagy in hBMSCs, consequently improving mitochondrial quality. Upon administration via portal vein injection, hypo-hBMSCs significantly mitigate hepatic IRI. Compared with hBMSCs, hypo-hBMSCs are capable of transferring more mitochondria to hepatocytes through GJs. When the function of GJs is modulated by the enhancer RA or the inhibitor Gap26, the efficiency of mitochondrial transfer correspondingly shifts. Further investigation uncovers that hypo-hBMSCs prompts an upsurge in the expression of Cx43 and Cx32 (not Cx26). Nevertheless, these proteins are unable to form heterotypic GJs (Cx43-Cx32-GJs) with hepatocytes; instead, they form homotypic Cx43-GJs and Cx32-GJs, which facilitate the transfer of mitochondria between hypo-hBMSCs and hepatocytes.

Conclusion: Hypo-hBMSCs can enhance mitochondrial quality by inducing Mitophagy. Meanwhile, they can up-regulate Cx43 and Cx32 to form homotypic Cx43-GJs and Cx32-GJs with hepatocytes, thereby transferring more high-quality mitochondria to hepatocytes to exert a protective effect.

Gap junctions; HBMSCs; Hepatic ischemia-reperfusion injury; Hypoxia preconditioning; Mitochondrial transfer.