Inhibition Of NF-κB Pathway Activation To Inhibit Intimal Hyperplasia Of Autologous Vein Grafting After Bypass Graft

Partâ… Establish Abdominal External Jugular Vein Transplatation Model With Cuff Technique in RatsObjectiveTo establish an rat mode of autologous vein grafts restenosis with cuff technique, the pathological process of which is similar with cononary artery bypass grafting.MethodsTwenty male Wistar rats(weight,350 to 400 g)were obtained and housed in conformity with international guidelines for the care and use of laboratory animals. After the rat was anesthetized,a segment of the left external jugular vein was excised and a segment of the abdominal aorta was denuded and disconnected.Then the autologous external jugular vein graft was interposed into the aortic nick according to cuff technique.Subsequently,rats were euthanized at 2 and 4 weeks respectively after grafting and vein grafts were processed for morphometric analysis.Meanwile,the right external jugular vein was obtained as own control.ResultsAbdominal external jugular vein transplantation was performed in twenty rats, seventeen survived while three died.The reasons of death were the lumbar artery damage,the delayed bleeding of abdominal aorta stump,and the intestinal obstruction respectively.The achievement ratio of operation is 85%.The mean operative time was 60min.The cold ischemia time and wam isehemia time of the donor was 10-20min and 5-10min,respectively.Histopathology showed that the intima of grafts thickened gradually during 4 weeks after surgery accompanyed with inflammatory cell infiltration and smooth muscle cell proliferation.ConclusionsThe cuff technique is feasible to establish rat model of abdominal external jugular vein transplantation.This technique would be ideal because it is simple and easier to peform with higher achievement ratio.It is also able to reflect the pathological process of intimal hyperplasia of autologous vein grafting objectively and could be an ideal model for further preventing restenosis after cononary artery bypass grafting. Partâ…¡Proteasome Inhibitor Bortezomib Inhibit Intimal Hyperplasia of Autologous Vein Grafting in Rat ModelObjectiveA autologous vein transplantation model was used to detect whether inflammation played an important role in intimal hyperplasia induced by bypass graft. On this basis,to explore the feasibility and efficiency of proteasome inhibitor bortezomib in the inhibition of neointima formation in transplant-induced vasculopathy for its anti-inflammatory effect.MethodsEighty-eight male Wistar rats(weight,350 to 400 g)were obtained and housed in conformity with international guidelines for the care and use of laboratory animals. After the rat was anesthetized,a segment of the left external jugular vein was excised and interposed into the abdominal aorta nick according to cuff technique.All post-surgical rats were divided into two groups randomly and treated with bortezomib or placebo.After 24 and 72 hours,rats were humanly killed and vein grafts were processed for real-time RT-PCR(24 and 72 hours)to test CINC-2β,MCP-1,IL-1, IL-6 and TNF-α,for ELISA(24 hours)to test C1NC-2β,MCP-1,IL-1,IL-6 and TNF-α,and for neutrophil chemotaxis assay(24 hours).Subsequently,rats were euthanized at 2 and 4 weeks after grafting and samples were processed for morphometric analysis.Meanwile,the right external jugular vein was obtained as own control.ResultsMorphometric analysis showed that the intima of grafts thickened gradually during 4 weeks after surgery.Bortezomib resulted in significant inhibition of intimal hyperplasia in each time point,compared with untreated controls(P＜0.05).The expression of mRNA for selective chemokine(CINC-2βand MCP-1)and cytokines (IL-1,6 and TNF-α)increased markedly in injured vessels during the first day after surgery and declined gradually in the following three days(except MCP-1).ELISA showed chemokine(C1NC-2βand MCP-1)and cytokines(IL-1,6 and TNF-α)also had significant rise accordingly in 24 hours after surgry(P＜0.05).Compared with control,Bortezomib significantly attenuated gene expression and protein level in most of the inflammatory mediators and simultaneously inhibited neutrophil chemotactic activity of the vessel homogenates(P＜0.05).ConclusionsIncreasing evidence indicated that inflammation played an important role in intimal hyperplasia induced by autologous vein grafting.Bortezomib could inhibit neointima formation significantly by attenuating the inflammatory response in transplant-induced vasculopathy and should be used as a novel vaso-protective remedium in the clinical field. Partâ…¢Construction of Plasmid Vector Expressing Rat NF-KB Small Interfering RNA and In Vitro Function IdentificationObjectiveTo construct one RNA interference(RNAi)expression vector targeting rat NF-κB(Rela,P65)and identify its inhibitory effect to corresponding gene expression in SVAREC cells transfected with the recombinant plasmid,preparing for the next in vivo study.MethodsDesign small interfering RNA(siRNA)sequence targeting rat NF-κB p65,and synthesize two complementary oligodeoxyribonucleotides encoding NF-κB p65 short hairpin RNA(shRNA).Two complementary oligodeoxyribonucleotides were annealed and ligated into linearized pgenesil-1.2 to construct the recombinant plasmid pGenesil-1.2-p65siRNA.The ligation mixtures were transformed into competent E.coli DH5a.The recombinant plasmids were extracted from small-scale bacterial cultures by Alkaline Lysis and then identified by enzyme analysis and DNA sequence analysis.QIAGEN plasmid maxikit was employed tbr large-scale preparation of recombinant plasmids.Lipofectamine2000 was used for the transfection of SVAREC cells by pGenesil-1.2-p65siRNA.After the transfection,LPS(lμg/ml)was used to stimulate SVAREC cells for activating the NF-κB signal pathway,total protein was isolated from cells and NF-κB p65 protein was analyzed by Western blotting.Then,flow cytometry and MTT assay were performed to observe the relationship between cell proliferation and NF-κB pathway activation,and the suppression effect due to the pGenesil-1.2-p65siRNA transfection.Results.It was confirmed by enzyme analysis and DNA sequencing that the insert sequence was successfully cloned into the vector.Western blotting showed that protein level of NF-κB p65 in transfected SVAREC cells reduced significantly as stimulated by LPS(P＜0.05).Flow cytometry with MTT assay suggested that the activation of NF-κB signal pathway stimulated by LPS would prompt cell cycle converting from G₀/G₁ phase to S phase,thereby promoting the cell proliferation. Meanwhile,Rreal transfection could significantly inhibit the increased cell proportion in S phase and cell proliferation index induced by LPS stimulation in SVAREC cells respectively(P＜0.05).ConclusionThe plasmids vector pGenesil-1.2-p65siRNA which express rat NF-κB p65 siRNA were successfully designed and constructed.It was demonstrated in initiatory study that transfected pGenesil-1.2-p65siRNA in vitro rat endothelial cells could effectively suppress the NF-κB p65 expression,and significantly inhibit cells proliferation induced by NF-κB signal pathway activation,which prepare for further in vivo study.