The Experimental Research On NF-κB P65 SiRNA Transfection To Inhibit VSMC Proliferaion In Vitro And To Prevent Vein Graft Neointimal Hyperplasia In Animal Model

Background:Coronary artery bypass graft surgery(CABG) is an important treatment for coronary atherosclerotic, autologous saphenous vein is widely used for bypass graft vascular materials, but its stenosis or failure are important long-term constraints. Inflammatory and vascular smooth muscle cells (VSMC) proliferaion were highly involved in vein graft stenosis progression. As a crucial transcription factor in inflammatory responses, nuclear factor kappa B (NF-κB) controls transcription of many genes. Blocking the key transcription factor NF-κB, can inhibit cell proliferation and inflammatory pathway activation, and effectively prevent vein graft restenosis development.Objective:In vitro experimental study, VSMC was transfected by NF-κB p65 siRNA plasmid liposome complexes (small interfering RNA targeting nuclear transcription factor kappa B gene p65 subunit). We observe its effect on VSMC proliferation activity and the inflammatory cytokines expressions in cell experimental study. We construct rat model of autologous external jugular vein to the abdominal aorta, and animal experiments research on the p65 siRNA transfection function, and we determine the expressions of NF-κB p65 protein, inflammatory factors such as TNF-a and MCP-1, studying the siRNA prevention effect on neointimal hyperplasia and some inflammatory factors expressions, and observing its inhibition on inflammatory response and vein grafts restenosis.Methods:This experiment research using gene silencing technique known as RNA interference (RNAi) to inhibit the NF-κB activation, block the inflammatory reaction and reduce many inflammatory cytokines expressions in its downstream, so we can inhibit the VSMC proliferation, and prevent intimal hyperplasia or vein graft restenosis. According to NF-κB (RelA, p65) gene sequence from genetic data, we first design and encode the NF-κB specific short hairpin RNA (shRNA) with 21nt sequence (5’-AAGCACAGATACCAC TAAGAC-3’) that located in 211-231bp of p65 subunit, and clone it into the linearized plasmid vector pGenesil-1.2 with an mU6 promoter. A SacI restriction site in the plasmid pGenesil-1.2 between the HindⅢ and mU6 promoter, the multiple cloning site of plasmid pGenesil-1.2 was EGFP (enhanced green fluorescence protein)-SacI-CMV(cytomegalovirus) promoter-MLUI-HindⅢ-SacI-shRNA-mU6 promoter. Enzyme analysis and DNA sequencing confirmed that the inserted sequences have been successfully cloned in the vector. After sequence identified, purify and amplify, NF-κB siRNA plasmid can be for experiments. Briefly, male Wistar rats from experimental animal center (200g～300g), anesthetized with 10% chloral hydrate solution (3ml/kg) by intraperitoneal injection, then wistar rats thoracic aorta smooth muscle tissue was obtained by tissue-piece inoculation. The aortic medial tissue was cut into tissue pieces about the size 1.5*1.5mm, VSMC were purified by differential attachment and conventional trypsinization subculturing, and cells were cultured in Dulbecco modified Eagle medium (DMEM/F12) supplemented with 20% fetal bovine serum at 37℃ in 5% CO2. The digestive cell suspension after 15 min was transfer to the next bottle, and relatively pure smooth muscle cells can be obtained with repeated culture. Cells were randomly divided into group A (the non-treated normal cells), group B (blank plasmid liposome complex treatment) and group C (NF-κB p65 siRNA plasmid liposome complexes treatment). VSMC were cultured and used for morphological observation, immunohistochemical identification, then for transfection experiments. We observe the transfection effect on VSMC with MTT method to test proliferation activity, FCM (flow cytometry) to detect cell apoptosis and WesternBlotting to measure NF-κB p65 protein.Then animal experimental study, animal were randomly divided into four groups:group A: normal vein (n=10), group B:simple vein graft (n=10), group C:vein graft with negative transfection (n=20), group D:vein graft with NF=κB siRNA (n=20). The rat model of groups B, C and D need to an autologous external jugular vein to abdominal aorta reverse interposition. Vein graft was transfected by siRNA with soaking for 20min, pressure perfusion 30mmHg for 5min and adventitia smeared with 1ml siRNA plasmid liposome complexes. At the 7 days assessment, vein graft tissues were examined inflammatory factors MCP-1 and TNF-α by polymerase chain reaction (PCR) and Westernbloting analysis for NF-κB p65 protein. At corresponding time we measure neointimal thickness and assay proliferating cell nuclear antigen (PCNA) by immunohistochemical detection. The endogenous house keeping gene β-actin was used for statistical analysis as internal reference. Results:Smooth muscle cells were characterized by peak and valley growth pattern and showed a-actin positive expression with immunohistochemical staining. WesternBloting, Flow Cytometry and MTT testing results showed that compared with group A and B, the group C cell proliferation activity was obviously inhibited, and the proportion of apoptotic cells increased obviously, the NF-κB p65 protein expression was significantly decreased (P<0.05), there is no significant difference between group A and B. In animal experiment, PCR and WesternBloting results indicated that compared with group B and C, in the group D, NF-κB p65 protein level was significantly inhibitted, MCP-1 mRNA and TNF-a mRNA expression were decreased significantly (P<0.05), the difference was statistically significant. Vein graft in Group B and C appears obvious intimal hyperplasia thickening and has a large number of PCNA positive cells, but in the group D, the neointimal hyperplasia is reduced and PCNA positive cells was decreased significantly(P<0.05), and vascular tissue pathological and immunohistochemical results is visible.Conclusion:Cells and vein tissue were Transfected with NF-κB p65 siRNA may block the trigger of inflammatory cascades, and seems to inhibit intimal hyperplasia and proliferation development induced by endothelial cell injury. The cytological results suggest NF-κB p65 siRNA can effectively inhibit the VSMC proliferating activity, increase cell apoptosis and inflammatory factor expression in vitro. In animal experiment model the NF-κB gene RNA interference can significantly inhibit NF-κB activation, reduce the inflammatory factor NF-κB, MCP-1 and TNF-a expressions, effectively inhibited vein graft thickening through dampening inflammation cascades and neointimal hyperplasia, RNAi technology can provide a new way for atherosclerotic restenosis, that may useful approach to prevent early stenosis and late occlusion.