Hydrodynamics-based Transfection Of Plasmid Encoding Rank-fc Protects Against Hepatic Ischemia/reperfusion Injury In Mice

Objective:Hepatic ischemia/reperfusion (I/R) injury is very important in transplant surgery. This study sought to investigate the potential of receptor activator for nuclear factor kappa B–fragment crystallizable (RANK-Fc) to protect against hepatic ischemia/reperfusion injury (HIRI) and clarify the possible mechanism for using this procedure.Methods:Ninety male BALB/c mice were randomly divided into 3 groups: a phosphate-buffered saline (PBS)sham group; a 15μg pLNCX2-IRES-eGFP + I/R negative-control group (where IRES means internal ribosome entry site and eGFP means enhanced green fluorescent protein ); and a 15μg pLNCX2-RANK-Fc-IRES-eGFP + I/R (RANK-Fc) group. All mice were injected with 2.5 ml PBS (with or without plasmids) within 6 sec, via the tail vein. After 3 days, hepatic I/R was induced under warm conditions by partial occlusion of the left and media lobes for 90 min, followed by varying periods of reperfusion in the transgene groups. Sham group accepted the same procedure without the deprivation of blood supply .Blood and liver samples were harvested at diverse time points(1,3,6 and 24 h)(n = 5, in each group) . Hepatic injury was assessed by liver transaminases and histopathology, Tumor necrosis factor alpha, interleukin 6, and interleukin 1βwere measured by enzyme-linked immunosorbent assay, whereas RANK-Fc, phospho-c-Jun, c-Jun N-terminal kinase (JNK), hypoxia inducible factor 1 alpha (HIF-1α), nuclear p65, and total p65 were assessed by western blotting. Apoptosis was identified by terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling.Results:RANK-Fc and eGFP were successfully expressed in liver owing to Hydrodynamics-based transfection. In comparison with the negative-control group, RANK-Fc reduced nuclear factor kappa B (NF-κB) p65 nuclear translocation, JNK phosphorylation, and HIF-1αexpression during I/R(P <0.01). RANK-Fc effectively suppressed proinflammatory cytokine expression(P <0.01). The liver function and the morphological injuries in the RANF-Fc group were relieved more markedly(P <0.01). Apoptosis and cleaved-caspase-3 level were high in the negative control group, but very low in the RANK-Fc group after I/R(P <0.01).Conclusions:The results indicated that RANK-Fc could protect against hepatic I/R injury in mice at least in part via the inhibition of the proinflammatory NF-κB pathway as well as proapoptotic JNK and HIF-1αpathway activation.