NF-?B P65 Targeting SiRNA Transfection Down-regulates Expressions Of Coagulation And Fibrinolysis Factors In LPS-stimulated Alveolar Epithelial Cells Type ?

Objective: We transfected alveolar type ? epithelial cells(AECII)with small interfering RNA(siRNA)targeted to the NF-?B p65 gene in order to achieve the purpose of silencing the nuclear factor-?B(NF-?B)p65 gene in alveolar type ? epithelial cells.On this basis,we observed changes in coagulation and fibrinolysis-related factors of alveolar type ? epithelial cells stimulated by lipopolysaccharide(LPS)after silencing NF-?B p65.Methods: We transfected cells with NF-?B p65 gene-specific si RNA and pre-silenced the NF-?B p65 gene in the cells.On this basis,LPS-stimulated rat alveolar type II epithelial cell lines(RLE-6TN cells)were cultured for 24 hours.Real-time fluorescence quantitative reverse transcription-polymerase chain reaction(RT-PCR)was used to detect the level of NF-?B p65(p65),tissue factor(TF),and plasminogen activator inhibitor-1(PAI-1)mRNA in cells.Changes;Western Blot was used to detect the level of phosphorylated I?B(p-I?B),p65,phosphorylated p65(p-p65)protein,and the changes of TF and PAI-1 protein levels in cells;enzyme-linked immunosorbent assay(ELISA)detection The protein contents of TF,PAI-1 and activated protein C(APC)in the cell culture supernatants were measured by immunofluorescence.Results: After stimulating AEC? TF LPS injury,PAI-1 mRNA and protein were significantly increased,p65 mRNA expression was significantly upregulated(relative to the normal control group,TF Protein: 0.89 ± 0.05 vs 0.37 ± 0.02;PAI-1 Protein: 0.95 ± 0.05 vs 0.49 ± 0.02,P<0.05;normal control group 1,the relative expression of TF mRNA in an amount of8.51 ± 0.53,the relative expression of PAI-1 mRNA in an amount of 8.14 ± 0.36,p65 mRNA relative expression was 5.71 ± 0.19,P <0.01),Cell p-I?B protein(0.74 ± 0.03 vs 0.35 ± 0.02 compared to the normal control group),intracellular p65,p-p65 protein levels were increased(compared with the normal control group,cytoplasmic p65:0.97 ± 0.04 vs 0.64 ± 0.03,p-p65: 0.27 ± 0.01 vs 0.18 ± 0.01,P <0.05),the cell culture supernatant TF,PAI-1 protein relative to normal control group increased significantly,APC protein content Decreased(P<0.05),cellular immunofluorescence showed that the expression of p65 fluorescence in the nucleus was significantly enhanced under LPS stimulation.After transfection efficiency reached 70%,the cells were transfected with p65-specific siRNA and then stimulated with LPS.We detected that the expression of TF and PAI-1 mRNA and protein in cells was slightly higher than that in LPS group,but increased rate of less than non-transfected cells stimulated with LPS effect(as compared with LPS group,TF mRNA relative expression was3.53 ± 0.11 vs 8.51 ± 0.53,TF protein: 0.61 ± 0.04 vs 0.89 ± 0.04;relative expression of PAI-1 mRNA in an amount of 4.94 ± 0.44 vs 8.14 ± 0.36,PAI-1 protein: 0.56 ±0.02 vs 0.95 ± 0.05,P <0.05);cell culture supernatants Elisa results suggest that TF,PAI-1 expression increased,APC protein levels slightly The levels of p-I?B protein,p65,and p-p65 protein levels were lower than those of LPS-stimulated group(P<0.05).Immunofluorescence results showed that transfection of siRNA could reduce the expression of p65 protein in nuclei.,but still higher than the normal control group.Conclusion:(1)NF-?B p65 gene-specific siRNA transfection significantly inhibited the expression of NF-?B p65 gene in rat alveolar type ?epithelial cells;(2)NF-?B signaling pathway is involved in the regulation of LPS-induced pulmonary coagulation and fibrinolysis-related factors in rat alveolar type ? epithelial cells;NF-?B p65 molecule is expected to be a new target for effective prevention and treatment of ARDS alveolar coagulation and fibrinolysis abnormalities.