| Background and Objective Sepsis is a systemic inflammatory response syndrome (SIRS) caused by infection, and a common complication and the leading cause of deaths in intensive care unit (ICU). Since the costs and consumption of medical resources for sepsis treatment are extremely high, it has already become a great threat to human health and a big burden to social economy. CCAAT enhancer binding proteins (C/EBPs) are a group of proteins belong to the basic region leucine zipper (bZIP) family of transcription factors, consisting of six members:CEBPa, CEBPβ, CEBPy, CEBPδ, CEBPε and CEBPζ. Members of α, β,δ and ε play a key role in the acute phase reaction of various inflammatory diseases, and as a transcription factor they share a common specificity recognition sequence RTTGCGYAAY (R for A or G, Y for C or T). Therefore, in order to search new targets for sepsis prevention and treatment, a dumb-bell decoy oligonucleotides against C/EBPs (C/EBPs decoy ODNs) was first designed and synthesized according to the transcription factor decoy strategy (TFDs), and the effect of C/EBPs decoy ODNs on sepsis and its mechanisms are then investigated.Methods1) In vitro experiment:changes of the expression of C/EBPs and its DNA binding activity in RAW264.7cell after LPS treatment (1μg/mL) were determined by western blot (WB) and electrophoretic mobility shift assay (EMSA); synthesized dumb-bell C/EBPs decoy ODNs and mutant oligonucleotides (C/EBPs mutant ODNs,used as control) were tested by electrophoresis and the effect on DNA binding activity of C/EBPs was validated by EMSA; then both ODNs (FAM marked) were transfected into RAW264.7cells using a cationic transfection reagent TurboFect; the efficiency of transfection was determined by flow cytometry; intracellular distribution of C/EBPs decoy ODNs was determined by fluorescence microscopy after Hoechst dye; cytotoxity of ODNs was tested by alamarBlue assay; the effect of ODNs on expression of C/EBPs protein was determined by WB; the expression of TNF-α, IL-1β and IL-6in medium of RAW264.7cells after LPS stimulation (1μg/mL) was determined by ELISA; mRNA expression of inflammation related genes was detected by Real-time PCR.2) In vivo experiment:a classic model of sepsis was prepared in mice by cecum ligation puncture (CLP), the survival rate of septic mice was observed for7days after intravenous administration of C/EBPs decoy ODNs at different doses or different time points; fluorescence intensity of FAM marked C/EBPs decoy ODNs was measured in the supernatants of tissue homogenate in order to observe the distribution of C/EBP decoy ODNs in the mice organs at6h and24h after tail vein injection; serum biochemical indexes related to organ injury and morphologic changes of multiple organs were examined; expression of TNF-a, IL-1β and IL-6in serum was detected by ELISA; mRNA expression of inflammation related genes in the lung of septic mice was detected by Real-time PCR.Results1) In vitro experiment:the expression of CEBPa was high in RAW264.7cells under basic condition, it reduced at1h,3h and6h after LPS stimulation, then started to rise at12h and returned to the base level at24h; the expression of CEBPβ was low in RAW264.7cells under basic condition, it up-regulated at1h and reached to the peak at12h after LPS stimulation, then reduced but still with a high level at24h; the expression of CEBPδ was low in RAW264.7cells under basic condition, and rised from1h to24h continuously after LPS stimulation; the expression of CEBPε up-regulated at6h after LPS stimulation, and recovered although still with a high level at24h. The changes in expression of CEBPα,β,δ and ε in the nuclear of RAW264.7cells were very similar to those mentioned above except CEBPa reduced at6h and CEBPε reduced at1h then began to rise at3h after LPS stimulation. The DNA binding activity of C/EBPs began to rise at1h, peaked at6h and12h, and kept at a high level until24h after LPS stimulation; the DNA binding activity of CEBPβ and CEBPδ was much higher than that of CEBPa and CEBPε. The quality of dumb-bell ODNs was evaluated by electrophoresis and their inhibitory effect on DNA binding activity of C/EBPs was validated by EMSA; the transfection efficiency of decoy ODNs and mutant ODNs in RAW264.7cells at4h after transfection was87.35%and87.71%respectively; and the decoy ODNs mainly located in the nucleus of RAW264.7cells at4h after transfection; both decoy ODNs and mutant ODNs exhibited no obvious cytotoxicity and no effect on expression of CEBPs in RAW264.7cells. C/EBPs decoy ODNs inhibited expression of TNF-α, IL-1β and IL-6in medium of RAW264.7cells after LPS stimulation; C/EBPs decoy ODNs also decreased mRNA expression of IL-6, IL-1β, IL-18, MCP-1, MIF, caspase-1, NF-κB1, ICAM1, VCAM1and Fga in RAW264.7cells at4h after LPS stimulation.2) In vivo experiment:C/EBPs decoy ODNs intravenous injection (10μg,30μg,90μg) immediately after CLP could improve the7-day survival rate of septic mice with a dose-dependent manner; C/EBPs decoy ODNs treatment intravenous injection (30μg) at0,3,6,12h after CLP could also improve the7-day survival rate of septic mice with a time-dependent manner. C/EBPs decoy ODNs could be detected in the liver, lung and kidney at6h and in the liver, lung but not kidney at24h after injection. C/EBPs decoy ODNs reduced serum levels of BUN, Cr, ALT, AST, LDH and CK-MB, and alleviated tissue damage of heart, liver, lung and kidney as shown by microscopy; C/EBP decoy ODNs inhibited up-regulation of TNF-α, IL-1β and IL-6in the serum of septic mice and blocked several inflammation related genes in the lung of septic mice including IL-6, IL-1, IL-18, MCP-1, MIF, Caspase-1, NF-κB1, ICAM1, VCAM1and Fga.Conclusion C/EBPs played an important role in LPS-induced inflammation and might be used as potential intervention targets for sepsis treatment; C/EBPs decoy ODNs could block expression of multiple inflammation related target genes by inhibiting the DNA binding activitiy of C/EBPs and thereby provided effective protective effect against multiple organ damage in mice with sepsis. |
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