The Effects Of TRAF6 Gene Silencing On LPS-induced Inflammation

Part I Effects of LPS on Expression of TRAF6 gene in RAW264.7 cellsObjective:To study the effects of LPS on the expression of TRAF6 mRNA in RAW264.7 cells.Methods:RAW264.7 cells were divided into five groups, lipopolysaccharide (LPS) group, dexamethasone (DM) group, curcumin (Cur) group, experiment control group and normal control group. Cells in LPS group were added LPS (100μg/L) in cell culture. Cells in Cur group or DM group were pretreated with dexamethasone (0.5 mg/L) or curcumin (20μmol/L) for 8 hours. Cells in experiment control group were treated with DMEM culture media with DMSO (<0.1%). No treatment was done for the normal control group. The expression of TRAF6 was determined by immunocytochemical staining and realtime-PCR at 2,4,8 and 16 hours after LPS stimulation. TRAF6 protein expression was determined by Western blotting at 8 h after LPS stimulation. In order to elucidate the relation between LPS and expression of TRAF6 gene, our studies revealed the effects of LPS on expression of TRAF6 gene both at the mRNA and protein levels.Results:The expressed mRNA of TRAF6 gene was detected by realtime-PCR. There was the low expression of TRAF6 mRNA in RAW264.7 cells. When RAW264.7 cells were continually stimulated with LPS (100μg/L), the number of expression increased markedly, and peaked at 16h. In RAW264.7 cells, the expressions of TRAF6 mRNA were much lower than those in LPS group at the corresponding time points (P<0.05). Howere, the expression of TRAF6 mRNA was still higher than normal level. Expressions of TRAF6 mRNA in DM group shown no significant difference as compared with those in Cur group at the corresponding time points (P>0.05). Levels of TRAF6 mRNA were unobviously changed in the experiment and normal control group (P>0.05). The expressed protein of TRAF6 gene was detected with Western blot. The statistics show that there are a lot of similarities between realtime-PCR and Western blotting/Immunocytochemical staining.Conclusions:There are tight relations between LPS and expression of TRAF6 gene. Part II The Construction and Screening of TRAF6-shRNA expressing Plasmid The first experiment:Construction and identification of expression plasmid for mouse TRAF6Objective:To construct the eukaryotic expression plasmid expressing shRNA targeting TNF receptor-associated factor-6 (TRAF6) gene as a tool for following experiments.Methods:TRAF6 mRNA sequence of mouse was retrieved in nucleotide library of NCBI. According to guidelines for shRNA design, four pairs of oligos for hairpin RNA which targeted mice TRAF6 gene were chemically synthesized. Coding sequences of short hairpin RNA DNA single-strands were synthesized in vitro. The annealed oligos were inserted into the down stream of U6 promoter of linearized pGCsi-U6/GFP/Hygro vector to construct RNA interference (RNAi) plasmid (pGCsi-TRAF6-shRNA) respectively.Results:The shRNA expression plasmid was constructed, and the inserted sequence was confirmed by restrict endonuclease digestion and DNA sequencing.Conclusions:The siRNA plasmid targeting mice TRAF6 gene was successfully constructeded and can be applied to study the function of TRAF6 on LPS-induced inflammatory reaction in vitro and in vivo.The second experiment:Identification and Screening of TRAF6 Gene silencing optimization sequencesObjective:To select TRAF6 (TNF receptor-associated factor-6) shRNA (short hairpin RNA) eukaryotic expression plasmid that can inhibit TRAF6 gene expression in Raw 264.7 cells.Methods:To get most effective and optimal dosage pTRAF6-shRNA, the four vectors were transfected into Raw 264.7 cell with different ratio between plasmid (g) and Trans Fectin (L), the expression of fluorescence and efficiency of transfection were detected by fluorescence microscopy. Forty-eight hours after pTRAF6-shRNA transfection with the best ratio of plasmid (g) and Trans Fectin (L), proliferation of transfected cells was measured with MTT assay, and the levels of TRAF6 mRNA and TRAF6 protein were detected by realtime-PCR and Western blot, respectively.Results:The ratio of 2:5 between plasmid (g) and Trans Fectin (L) were considered as the most ratio. The results of MTT in transfected cells were significantly decreased compared with that of. The results of realtime-PCR revealed that pTRAF6-shRNA1 can markedly inhibit the expression of TRAF6 mRNA (the inhibition rate was 75.25%), comparing with the normal control group (P<0.05). The results of Western blotting indicated that pTRAF6-shRNAl can markedly inhibit the expression of TRAF6 protein (the inhibition rate was 70.07%), comparing with the normal control group (P<0.05).Conclusions:The construct of pTRAF6shRNA1 successfully interfered in the TRAF6 gene expression in vitro, and it can be applied to study the function of TRAF6 on LPS-induced inflammatory reaction.PartⅢInfluence of TRAF6 siRNA on LPS-induced inflammation in vitroObjective:To investigate the influences of silencing TRAF6 gene by RNA interference (RNAi) on LPS-induced inflammation in vitro.Methods:Cells were divided into five groups:(A) pTRAF6-shRNA1 (pGCsi-TRAF6-shRNA1)group, pTRAF6-shRNA1+LPS; (B) positive control group, curcumin (20μmol/L) +LPS; (C) negative control group, blank plasmid+LPS; (D) blank control group, stimulated with LPS(100μg/L); (E) normal control group, maintained in DMEM without any intervention. siRNA recombinant expression vector targeting TRAF6 gene was transfected into RAW264.7. The effect of siRNA recombinant expression vector was detected by Realtime PCR and Western blot. The cells were cultured for 48h after the second pTRAF6-shRNA transfection, and stimulated by LPS (100μg/L), and then supernatants were harvested after 0h,4h,8h and 16h. TNF-α, IL-1βand TGF-β1 were assayed by ELISA. TRAF6, IL-6, COX-2 mRNA were detected by real-time PCR, and intranuclear NF-κB P65 protein level were examined by Western blotting.Results:ELISA indicated that the secretions of TNF-α, IL-1βand TGF-β1 were markedly higher than those of normal cells (P<0.01) at 4h,8h and 16h after LPS stimulation, and peaked in a short time, TNF-α, IL-1βat 8h, TGF-β1 at 16h respectively. After LPS stimulation with TRAF6 gene knocked down, the rates of increase of those cytokines were significantly inhibited than that in single LPS stimulation (P<0.01). The experiment indicated that TRAF6-shRNA could down regulate the expression of IL-6, COX-2 mRNA. In the same way, it could significantly inhibit the activation of NF-κB.Conclusions:pTRAF6-shRNA1 inhibited inflammatory reaction which stimulated by LPS resulting from inflammatory cytokines and mediators suppression through NF-κB levels down-regulation in RAW264.7 cell.PartⅣTNF Receptor-associated Factor 6 Gene Silencing Ameliorates Acute Liver Failure in MiceThe first experiment:Comparison of Three Nonviral Transfection Methods for GFP Plasmid Expression in Liver of MiceObjective:To compare the transfection efficiency and the expression intensity of a green fluorescent protein (GFP) reporter gene in liver of mice by using three nonviral transfection methods, i.e., hydrodynamic injection, portal vein injection and peritoneal injection.Methods:The naked GFP plasmid or liposome encapsulated plasmid were injected via the tail vein or portal vein or abdominal cavity of different mice with homogeneity. The blood and liver were harvested 48 h after injection, and the contents of alanine aminotransferase (ALT) and total bilirubin (TB) were detected in serum. HE staining was used to observe the pathological changes of liver tissues. Meanwhile, the expression intensity of GFP was evaluated by fluorescence microscopy.Results:The levels of ALT and TB in peritoneal injection (PI) group were little higher than that in normal group at 48h after injection, and there is no significant difference comparing with hydrodynamic injection (HI) group (P>0.05). Furthermore, there was no statistically significant difference between liposome-DNA compound and naked plasmid DNA in same group (P>0.05). HE staining of liver indicated that dropsy in hepatocytes could be seen in HI and portal vein (PV) groups. Of the three transfection methods employed, hydrodynamic gene delivery and portal vein injection conferred the stronger expression of GFP with similar transfection efficiency (P>0.05). However, after encapsulated by liposome, the expression levels of GFP are significantly higher than that of naked plasmid DNA (P<0.05).Conclusions:High level gene expression in mouse liver can be achieved by hydrodynamic injection or portal vein injection of liposome encapsulated plasmid DNA, and hepatic delivery of foreign gene can be accomplished by hydrodynamics-based injection.The second experiment:TNF Receptor-associated Factor 6 Gene Silencing Ameliorates Acute Liver Failure in MiceObjective:To investigate the influence and protective mechanism of tumor necrosis factor (TNF) receptor-associated factor-6 (TRAF6) gene silencing on inflamation in LPS/D-galactosamine (D-GalN)-induced acute liver failure (ALF) mice.Methods:BALB/c mice were divided into five groups, normal control group, ALF model group, positive control (curcumin, Cur) group, negative control (blank plasmid) group and RNAi (pTRAF6-shRNA1) group. Specific expressing plasmid pTRAF6-shRNAl was delivered into BALB/c mice repeatedly by hydrodynamics-based gene transfection. LPS (20μg/kg) plus D-GalN (600 mg/kg) were injected intraperitoneally into mice after the first injection of plasmid DNA for 48 hours. The survival of mice was examined and blood samples were collected from the retro-orbital plexus each mouse, at 4, 8 and 16h after LPS/D-GalN injection. Serum [TNF-a, interleukin (IL)-1β, transforming growth factor (TGF)-β1] were measured by ELISA kits respectively. Sixteen hours after the injection of LPS/D-GalN, serum were gathered from the eyeball to measure aminotransferase (ALT) and aspartate aminotransferase (AST) using a colorimetric analyzer. TRAF6, IL-6, cyclooxygenease (COX)-2 and NF-κB p65 mRNA levels were detected by realtime-PCR. Expression features of inducible nitric oxide synthase (iNOS) and NF-κB in liver tissue were detected in immunohistochemistry. NF-κB p65 in the nuclear extracts were assessed by Western blotting. Results:Results bright green fluorescence of the transfected cells in mice liver sections can be observed under fluorescent microscopesafter transfection. The recombinant plasmid pTRAF6-shRNA1 could be expressed in liver tissues of mice. Our results show that TRAF6 mRNA expression was remarkably reduced by 60.13% in mice liver during the experiment period. Concomitantly, pTRAF6-shRNA1 decreased serum transaminases levels and increased survival of mice. The amounts of mRNA of interleukin (IL)-6 and cyclooxygenease-2(COX-2), the productions of inflammatory cytokines [TNF-α, IL-1βand transforming growth factor (TGF)-β1], and the expression of inducible nitric oxide synthase (iNOS) in liver were significantly lower in pTRAF6-shRNAl group than that in pNi-shRNA control group. Moreover, NF-κB p65 levels in the total cellular and the nuclear extracts were effectively inhibited by pTRAF6-shRNA1. This may due to the inhibition of NF-κB activity specified with decreased expression of inflammatory cytokines and inflammatory mediators.Conclusions:pTRAF6-shRNA1 inhibited inflammation resulting from inflammatory cytokines and mediators suppression through levels down-regulation in LPS/D-GalN-stimulated mice. pTRAF6-shRNA1 successful alleviated LPS/D-GalN induced acute liver injury in vivo, which has implications for our understanding of the role of TRAF6 gene silencing in LPS/D-GalN-mediated liver injury and for the development of new therapies for human ALF.