| Objective Over-inflammatory response severely influence the life quality of patients suffered from chronic diseases, and extensive and severe insult on the tissue and the physical function induced by over-inflammatory response imperil patients' lives. Therefore, besides treatment for primary diseases, anti-inflammation is the basic strategy in clinic. Considering nuclear factor-κB (NF-κB) is the key point to control gene expression of inflammatory factors, decoy-ODNs in dumbbell shape with the nuclear sequence similar to NF-κB cis-elements were synthetized in this study. To measure the anti-inflammatory action of decoy-ODNs targeting NF-κB, decoy-ODNs wrapped up by cationic liposomes were transfected into pMφ cells from rats. Then the effects of decoy-ODNs on expression of inflammatory mediators (TNFαand IL-6) and anti-inflammatory mediators (IL-10) in protein and gene levels were observed with enzyme-linked immunoadsorbent assay (ELISA) and reverse transcription polymerase chain reaction (RT-PCR).Materials and Methods 1. Using cationic liposomes as carriers, decoy-ODNs (0.2 μg/ml) labeled with fluorescein were transfected into the purified pMφ cells from rats, and the dynamic intracellular changes of decoy-ODNs were tested at 30 minutes, 1, 3, 6, 9, 12, 18 and 24 hours after transfection by fluorescent microscope and confocal laser scanning microscope. 2. The changes of NF-κB p65 in pMφ cells were measured at 1 hour before and after LPS stimulation, respectively, with immunohistochemistry method. 3.The changes of TNFα, IL-6 and IL-10 in protein and gene levels were measured before LPS stimulation and at 1, 2, 4, 6, 12, 18 and 24 hours after LPS stimulation. 4. With carriers of cationic liposomes, decoy-ODNs were transfected into pM cells from rats. The pMφ transfected with ODNs of unrelated sequence or stimulated by LPS only to be as control. Then the inhibiting effects of the decoy-ODNs with different concentrations on protein and gene of TNFα, IL-6 and IL-10 were tested at the different intervals after stimulation of LPS (10 ug/ml).Results 1. Decoy-ODNs labeled with fluorescein could be observed in cells under the fluorescent microscope at 30 minutes after transfection. With time passing by, the intracellular fluorescent light became brighter and brighter, reached to the brightest at 6-12 hours after transfection. The confocal laser scanning microscope showed decoy-ODNs was distributed mainly in plasma at 30 minutes after transfection. With time passing by, the intracellular especially the nuclear fluorescent light became brighter and brighter, and it also reached the brightest at 6-12 hours after transfection. 2. Before stimulation of LPS, NF-κBp65 mainly existed in nucleus, and cells showed "vacuole" phenomenon. After stimulation, NF-κBp65 mainly existed in nucleus and cells showed "solid" phenomenon. 3. After stimulation of LPS, the expressions of TNFα, IL-6 and IL-10 were higher significantly compared with that before stimulation (P < 0.01), especially at 2-6, 12-18 and 4-12 hours after stimulation, respectively. 4. The gene expressions of TNFα, IL-6 and IL-10 were higher significantly compared with that before stimulation (P < 0.01), especially at 2, 12 and 6 hours after stimulation, respectively. 5. Decoy-ODNs could decrease the expression of TNFα and IL-6 by dose-dependent fashion. There existed statistical inhibiting significance in IL-10 expression and gene expression when only 0.8μg/ml decoy-ODNs was used. And the inhibiting effect increased with time passing by.Conclusions 1. With carriers of cationic liposomes, Decoy-ODNs in dumbbell shape can be transfected into the pMφ cells from rats and distributed in plasma and nucleus. 2. LPS can activate NF-κB and significantly increased the expressions of TNFα, IL-6 and IL-10 in protein and gene levels. 3. Decoy-ODNs in dumbbell shape can significantly decrease the expressions of TNFαand IL-6 in protein and gene levels, but have weaker inhibiting effect on IL-10.
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