A Primary Research On The Mechanism Of Short-term Effect Caspase-3SiRNAResulting In More Severe Injury After Cold Storage In A Porcine Auto-Renal Transplantation Model

Object This study aims to investigate the reason why the result in animal model didn’t go along with cell experiment or ex vivo experiment, and investigate the mechanism of the phenomenon, further to explore the effect of Caspase-3siRNA on the systemic innate immune system and finally to briefly tell the mechanism of the damage effect of Caspase-3siRNA on renal tissue, thus providing evidences to the clinical conversion of Caspase-3siRNA.MethodsChoose25to30kg12male Guangxi Bama miniature pigs, randomly divided into six I/R group (I/R), Caspase3siRNA group6(siRNA). Small pig orthotopic autologous renal transplantation model is established, based on previous studies, taking siRNA group after application of the left kidney the dock planner40ml UW organ preservation solution+0.3mg short-acting Caspase3siRNA (synthetic sequence5’-GGGAGACCUUCACAAACUUtt-3’ or5’-AAGUUUGUGAAGGUCUCCCtg-3’) pressurized infusion, I/R group applied UW organ preservation solution also pressor perfusion, frozen for24hours, before transplantation to return frozen kidney biopsy specimens, again to the right kidney transplantation in situ. The auto-transplant kidneys in each group were resected48h after transplantation. We use real time PCR to detect Caspase-3mRNA level in each kidney tissue. And we use western blot to detect various experimental miniature pigs precursor Caspase3in the kidney tissues, activated Caspase3expression level. Finally we use western blot method to detect TLR-3, TLR-7, RIG1, MyD88, PKR, RIG-1and HMGB1expression level in renal tissue of each group.Results48hours after auto-renal transplantation, the TLR-3, TLR-7, MyD88, TRIF, PKR protein level in kidney tissues of IR+siRNA group were significantly higher compared with IR group, semi-quantitative results statistically differences; IR+siRNA group of kidney tissue, mRNA levels of downstream inflammatory cytokine related to Toll-like receptor signaling pathways have increased significantly compared with IR group, the results have statistical difference; Western blot results show that concerned with Caspase3protein, Caspase-3protein loads in IR+siRNA kidney significantly reduced compared with I/R group before transplantation(absorbance OD value of1.02+/-0.24vs.2.32+/-0.28, p<0.01), but in the tissue of48h after transplantation, siRNA compared with I/R group of Caspase3activity of17kd fragmentation increased markedly (OD value:0.22+/-0.04vs.0.06+/-0.01, p<0.05). The HMGB1protein level in renal tissue of the IR+siRNA group were significantly higher than that in IR group, the semi-quantitative results show statistical difference.ConclusionThe synthetic siRNA implicated in this study effectively silenced local Caspase-3mRNA transcription, which effectively reduced apoptosis during cold storage, but48hours after transplantation the kidney tissue in IR+siRNA group endured more severe damage, and the number of apoptotic cells also increased, activation fragment of Caspase-3protein level rise; The main reason is that the system identification of exogenous siRNA by toll-like receptors, triggering the systemic inflammation, strengthens the local tissue inflammatory injury and cell apoptosis; This damage can be sustained to48hours, which is most possible mechanism for the negative feedback loop and HMGB1protein involved in the subsequent loop cascade amplification inflammatory response and apoptosis process; SiRNA treatment, as a new means of intervention, we still need to further eliminate the systemic side effects before clinical application.