NF-κB Decoy ODN Mediated By Liposome Influence On Severe Acute Pancreatitis In Rat

【Objective】:The decoy ODNs with 5'-GGG ACT TTC C-3' that were synthetized by man-made and have specific congeniality were transfected in severe acute pancreatitic rat to get the message: 1,The transfection efficiency and the distribution of decoy ODNs in severe acute pancreatitic rat; 2,The effect that the decoy ODNs suppresed the activity of NF-κB; 3,The expression of inflammatory factor and adherence factor, after the activity of NF-κB was suppressed; 4,The influence on injury of lungs,hepatic injury and injury of pancreas at earlier period of severe acute pancreatitic in rat, after the activity of NF-κB was suppressed.【Methods】:1. NF-κB decoy ODN mediated by liposome expressed and distributeed on lungs,liver and pancreas severe acute pancreatitic rat:1.1 Severe acute pancreatitis was induced with sodium taurocholate in rat.1.2 Sprague Dawley rats were divided into groups: Sham-operation group was normal control group(n=20). After 1h and 4h, rats were injected by i. v. with naked ODN(n=20) or liposome/decoy ODN complexes(n=20) or liposome/scrambled ODN complexes(n=20) or sodium(n=20), all ODN were labeled with fluorescein isothiocyanate(FITC).1.3 The intensive fluorescence at frozen section of pneumono,hepatic and pancreatic tissue was observed by fluorescence microscope and the transfection efficiency of FITC-ODN was calculated.1.4 The NF-κB activation of pneumono,hepatic and pancreatic tissue was analyzed by electrophoretic mobility shift assay(EMSA).1.5 The specificity of NF-κB decoy ODN that conjugate NF-κB was measured by Electrophoretic Mobility Shift Assay to compare with specific probe.Electrophoretic Mobility Shift Assay experiment were divided into groups: 1,Negative control groups (without nuclear protein) (n=10); 2,Nuclear protein+saline groups (n=10); 3,Nuclear protein+100 times specific probe with label to NF-κB groups (n=10); 4,Nuclear protein+100 times unspecific probe SP1 without label to NF-κB groups (n=10); 5,Nuclear protein+ NF-κB decoy ODN groups (n=10); 6,Nuclear protein+ NF-κB scrambled ODN groups (n=10). All nuclear protein were extracted from sodium groups.2. The NF-κB decoy ODN mediated by liposome influenced on expression of inflammatory factor and and adherence factor and injury of lungs,hepatic injury and injury of pancreas at earlier period of severe acute pancreatitic in rat:2.1 Severe acute pancreatitis was induced with sodium taurocholate in rat.2.2 Sprague Dawley rats were divided into groups: Sham-operation group was normal control group(n=10). After 1h, rats were injected by i. v. with naked ODN(n=10) or liposome/decoy ODN complexes(n=10) or liposome/scrambled ODN complexes(n=10) or sodium(n=10).2.3 ALT,AST,PO2,amylase of blood and Myeloperoxidase(MPO) of pneumono,hepatic and pancreatic tissue were determined.2.4 The NF-κB activation of pneumono, hepatic and pancreatic tissue was analyzed by electrophoretic mobility shift assay(EMSA).2.5 The specificity of NF-κB decoy ODN that conjugate NF-κB was measured by Electrophoretic Mobility Shift Assay to compare with specific probe.Electrophoretic Mobility Shift Assay experiment were divided into groups: 1,Negative control groups (without nuclear protein) (n=10); 2,Nuclear protein+saline groups (n=10); 3,Nuclear protein+ 100 times specific probe with label to NF-κB groups (n=10); 4,Nuclear protein+ 100 times unspecific probe SP1 without label to NF-κB groups (n=10); 5,Nuclear protein+ NF-κB decoy ODN groups (n=10); 6,Nuclear protein+ NF-κB scrambled ODN groups (n=10). All nuclear protein were extracted from sodium groups.2.6 The expression oflL-1α,IL-2,TNF-α,ICAM-land VCAM-1 mRNA of the pneumono,hepatic and pancreatic tissue was detected by RT-PCR.【Results】:1.1 The intensive fluorescence of the FITC-ODN of pneumono,hepatic and pancreatic tissue was seen in cellular nucleus, not in cytoplasm in liposome/decoy ODN complexes group, and it was only observed in cytoplasm in liposome/scrambled ODN complexes group. There was a little intensive fluorescence in cellular nucleus and cytoplasm in naked ODN complexes group, but it was not seen in cellular nucleus and cytoplasm in normal sodium group and normal control group. The intensive fluorescence of the DAPI was seen in cellular nucleus, not in cytoplasm in liposome/decoy ODN complexes group,liposome/scrambled ODN complexes group and naked ODN complexes group.1.2 The transfection efficiency in liposome/decoy ODN complexes group compare with the naked ODN complexes group, there was significantly difference(P＜0.05), but there was not significantly difference between the 1h group and the 4h group(P＞0.05). The FITC-ODN in naked ODN complexes group distributed in lung,liver and pancreas has not significantly difference(P＞0.05), but has significantly difference in liposome/decoy ODN complexes group(P＜0.05). The intensive fluorescence was only observed in cytoplasm in liposome/scrambled ODN complexes group and there were not the FITC-ODN in normal sodium group and normal control group, so they have not the transfection efficiency.1.3 The NF-κB activation was observably inhibited in liposome/decoy ODN complexes group, not in normal sodium group and liposome/scrambled ODN complexes group(P＜0.05), there was not markedly difference on the NF-κB activation betweem 1h and 4h(P＞0.05), but there was markedly difference on the NF-κB activation betweem in liposome/decoy ODN complexes group and in naked ODN complexes group(P＜0.05).1.4 The NF-κB activation was observably inhibited in decoy ODN group and specific probe group, there was not markedly difference betweem in decoy ODN group and specific probe group (P＞0.05). The NF-κB activation was not observably inhibited in scrambled ODN group and unspecific probe SP1 probe group, there was not markedly difference betweem in scrambled ODN group and unspecific probe SP1 probe group (P＞0.05).2.1.1 Compared to normal, sodium group,liposome/scrambled ODN group and naked ODN group, the wet weight-dry weight ratio of the lung and the myeloperoxidase(MPO) of lung tissue were remarkably decreased and the blood gas assay was significantly increased in liposome/decoy ODN group(P＜0.05)。2.1.2 Compared to normal sodium group,liposome/scrambled ODN group and naked ODN group, ALT and AST were remarkably decreased in liposome/decoy ODN group(P＜0.05).2.1.3 Compared with normal sodium group,liposome/scrambled complexes ODN group and naked ODN group, the AMY,the wet weight-dry weight ratio of the pancreas and the (MPO) of pancreas tissue were remarkably decreased in liposome/decoy ODN group(P＜0.05). 2.2 The NF-κB activation was observably inhibited in liposome/decoy ODN complexes group, not in normal sodium group and liposome/scrambled ODN complexes group(P＜0.05), there was not markedly difference on the NF-κB activation betweem 1h and 4h(P＞0.05), but there was markedly difference on the NF-κB activation betweem in liposome/decoy ODN complexes group and in naked ODN complexes group(P＜0.05).2.3 The NF-κB activation was observably inhibited in decoy ODN group and specific probe group, there was not markedly difference betweem in decoy ODN group and specific probe group (P＞0.05). The NF-κB activation was not observably inhibited in scrambled ODN group and unspecific probe SP1 probe group, there was not markedly difference betweem in scrambled ODN group and unspecific probe SP1 probe group (P＞0.05).2.4.1 The expression of ICAM-1,TNF-αand VCAM-1 mRNA was significantly suppressed by NF-κB decoy ODN in liposome/decoy ODN group(P＜0.05), compare with naked ODN complexes group,normal sodium group and liposome/scrambled ODN complexes group. The expression of IL-1αand IL-2 mRNA were upgraded, there was markedly difference betweem liposome/decoy ODN complexes group and naked ODN complexes group(P＜0.05).2.4.2 The expression of ICAM-1,TNF-αand VCAM-1 mRNA was significantly suppressed by NF-κB decoy ODN in liposome/decoy ODN group(P＜0.05), compare with naked ODN complexes group,normal sodium group and liposome/scrambled ODN complexes group. The expression of IL-1αand IL-2 mRNA were upgraded, there was markedly difference betweem liposome/decoy ODN complexes group and naked ODN complexes group(P＜0.05).2.4.3 The expression of ICAM-1,TNF-αand VCAM-1 mRNA was significantly suppressed by NF-κB decoy ODN in liposome/decoy ODN group(P＜0.05), compare with naked ODN complexes group,normal sodium group and liposome/scrambled ODN complexes group. The expression of IL-1αand IL-2 mRNA were upgraded, there was markedly difference betweem liposome/decoy ODN complexes group and naked ODN complexes group(P＜0.05).【Conclusion】:1,NF-κB decoy ODN can be transfected by liposome in some certain degree in severe acute pancreatitic rat, but liposome will change the target of NF-κB decoy ODN.2,NF-κB decoy ODN must draw assistance from carrier to elevate the concentration of NF-κB decoy ODN in target cell, but the target of NF-κB decoy ODN will be changed by carrier.3,The NF-κB decoy ODN have very fortis congeniality to combine with NF-κB and markedly suppressed The NF-κB activation.4,Activated or suppressed, The NF-κB will have influence up-regulation or down regulation on expression of inflammatory factor at severe acute pancreatitic in rat.5,The NF-κB decoy ODN depress expression of inflammatory factor, At the same time the injury of lungs,hepatic injury and injury of pancreas were amendmented at earlier period of severe acute pancreatitic in rat.