Investigation of metformin preconditioning on mitochondrial unfolded protein response in hepatic ischemia-reperfusion injury in rats

Hepatic ischemia-reperfusion injury (IRI) is defined as the anomalies in which blood flow is blocked at first and then harshly restart in the liver after transplantation. Hepatic IRI, widely affect mitochondria at the cellular level. Hence, evaluation of the mitochondrial process in the IRI models is critical in terms of identification of tissue rejection and development of putative therapy strategies. Mitochondria induces diverse cellular responses including regulation of cell death and energy mechanisms depending on the stress conditions. Nonetheless, mitochondria also trigger cell survival signals to adapt the stress conditions. Unfolded protein response (UPR) is one of the major stress adaptation pathways. Mitochondrial UPR (UPRmt) involves the prevention of mitochondrial protein synthesis and elimination of unfolded proteins during stress conditions. Nevertheless, the role of UPRmt in hepatic IRI has been limitedly reported. The present study aims to identify molecular mechanisms of UPRmt in rat hepatic IRI models and questions the role of metformin pre-conditioning in those mechanisms. For this aim, 30 Wistar albino rats were divided into 3 groups for optimal IRI modelling and metformin pre-conditioning. Regarding the results of the previous studies of our research group, optimal modelling durations were clarified as 45 min for ischemia, 6 h for reperfusion and 7 days for metformin pre-conditioning. After modellings, hypoxic and inflammatory processes were followed by biochemical and morphological methods. Accordingly, AST (p<0.0001), ALT (p<0.0001), TNFα (p=0.02), ROS (p<0.0001), TOS (p=0.004) and MPO (p=0.016) levels significantly increased in IRI models compared to control group. Nonetheless, GSH (p=0.0009) and ATP (p<0.0001) levels significantly decreased in IRI group. Moreover, metformin pre-conditioning significantly decreased the levels of AST (p<0.0001), ALT (p<0.0001), ROS (p=0.0045) and TOS (p=0.019) compared to IRI alone group. Protein analyses by Western blotting showed that IRI modelling increased the levels of hypoxia marker, HIF1A (p=0.016), speculating that the effects of IRI modelling could be a result of increase in the expression of HIF1A. However, metformin pre-conditioning reversed the effects of IRI modelling putatively by downregulating HIF1A levels (p=0.042). Furthermore, IRI modelling increased the levels of UPRmt markers, ATF5 (p=0,015) and CHOP (p=0,0008). Upregulation of those markers further elevated the levels of proteases and chaperons, CLPP (p=0.0009), HSP10 (p=0.016), HSP60 (p=0.001) and HSP70 (p=0.005), demonstrating the initiation of UPRmt. Metformin pre-conditioning, however, reduced the expression of these proteins, CHOP (p=0.0005), CLPP (p=0.006), HSP10 (p=0.029), HSP60 (p=0.005) and HSP70 (p=0.043) without altering that of ATF5. Hence, the results underlined that metformin pre-conditioning blocked UPRmt activated by IRI. Therefore, metformin pre-treatment in the recipients before transplantation may prevent tissue rejection. Keywords: Liver transplantation, Mitochondrial unfolded protein response, Metformin pre-conditioning