The Experimental Study Of The Treatment Of The Decoy Double-strand Oligonucleotides For NF-kB In Rat Heart Transplantation

Objectives: By using a rat heterotopical heart transplantation model, we investigate the effect of the lipofectin-mediated delivery double-strand oligonuide(ds ODN) decoy to NF-kB binding site in the heart transplantation.Methods: First, we developed a new heterotopic heart transplantation model by using cuff technique. Then, the donor hearts were treated with 1 OD decoy, 1OD decoy Iipofectamine2000 compound or 5 OD decoy Iipofectamine2000 compound. Transfection efficiency was determined with FITC-labeled ODN and fluorescent microscopy. Reperfusion injury was assessed by Langendorff-Neeley reperfusion system and the content of MDA, MPO in heart tissure and NO in right atrial. Reverse transcription-PCR was used to analyze intercellular adhesion molecule 1, vascular celluar adhesion molecule and inducible NOS mRNA expression. Acute rejection was determined by daily palpation. Histopathological and immunohistochemical study for measurment ICAM-1 were examined after 5 days of transplantation.Results: The heterotopic heart transplantation using cuff technique got the similar results as classical heterotopic heart transplantation. Without Lipofectamine2000, the ds ODN can not transfected into the donor heart cell. The transfected 5 OD ds ODN can lasted 14 days after heart transplantation. In the reperfusion experiment, the 5 OD ds ODN treated group had better donor heart function, less tissure MDA, myeloperoxidase(MPO) content and blood nitric oxide (NO) concentration (P<0.01 vs the control group). And the 10 OD ds ODN treated group had more efficient heart preservation effect when compared to the 5 OD treated group( P<0.01 ). The NF-kB decoy(5 OD and 10 OD), not the scramble ds ODN inhibited the donor heart ICAM-1, VCAM, iNOS mRNA expression in vivo after transplantation as assessed by semi-quantitative RT-PCR analysis(P< 0.01 vs controls). The 10 OD ds ODN treatment also prolonged donor heart survival over the controls (10.88 ± 1.55 vs. 6.75 ± 089 080.01), and had the similar results as the low-dose cyclosporine A (2.5 mg/kg) treatment.When decoy employment combined with the the low-dose cyclosporine A treatment can further prolonged the allografts survival. The histopathology showed less rejection scores after 5 days of transplantation while treated with the NF-kB decoy. The immunohistochemical study also showed decoy ds ODN can alleviated the expression of ICAM-1 in allograft rejection tissure .Conclusion: Our results suggested that Lipofectamine2000 could effectively deliver the ds ODN into the donor heart during preservation period. The NF-kB decoy ds ODN treatment can reduce the reperfusion injury and block the donor heart ICAM-1, VCAM, iNOS expression after transplantation. And the NF-kB decoy can also attenuate the acute rejection of the donor heart, the possible mechanism may contribute to the suppression of ICAM-1 expression. This highly targeted therapy may be a clinically viable strategy for the heart transplantation.