Molecular Mechanism Of NFKB1 Gene Mutant Mediated Vascular Endothelial Cell Injury In The Susceptibility Of Coronary Artery Disease

Objective:(1)To explore the association between NFKB1 gene promoter-94 ATTG ins/del mutant and coronary artery disease and its susceptibility and severity from the perspective of population genetic epidemiology.(2)To investigate the correlation between NFKB1 gene and coronary artery disease(CAD)related factors’ level of eNOS,IL-6,IL-8,MDA and SOD.(3)To clarify the molecular mechanisms of NFKB1 gene mutant in the pathogenesis of CAD.Methods:(1)Genotyping the polymorphism of NFKB1 gene in 1184 CAD patients and 1112 healthy controls by polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP)and using Logistic regression to analyze its correlation with CAD.Using Gensini score and coronary artery lesion count to evaluate NFKB1 gene and pathological changes of coronary artery stenosis lesion.(2)To measurement the plasma levels of endothelial function factor eNOS,inflammatory cytokines IL-6,IL-8 and oxidative stress injury factor MDA and SOD in CAD patients and healthy controls by ELISA.(3)Using vWF antibody and CD31 antibody to detect the purity of isolated and cultured primary human umbilical vein endothelial cells(HUVECs)by immunofluorescence and flow cytometry;To test oxidative stress injury between different genotypes cells by CCK-8;To detect the expressions of p50 and other moleculars by Western blot,real-time PCR and immunofluorescence;To detect nuclear transcription factor p50 by electrophoretic mobility shift assay(EMSA).Results:(1)(1)The distribution frequency of II,ID and DD genotypes were significantly different between CAD patients ans control subjects,the frequency of DD genotype was significantly higher in CAD than that in healthy controls(18.2% vs.14.1%,P=0.016).(2)The frequency of I and D alleles were significantly different between CAD patients ans control subjects,the frequency of D allele was significantly higher in CAD than that in healthy controls(41.9% vs.37.9%,P=0.006).(3)Logistic regression analysis showed that NFKB1 gene was association with the incidence of CAD,NFKB1 gene DD genotype is a risk factor for CAD(OR=1.37,95% CI 1.087 1.726,P=0.008).(4)The Gensini score was significantly higher in CAD patients who with NFKB1 gene DD genotype than who with II or ID genotype(DD: 35.61 ± 8.78 point,ID: 26.86 ± 7.58 point,II: 25.95 ± 7.2,P < 0.05).The severity of coronary artery stenosis was worse in CAD patients who with NFKB1 gene DD genotype than who with II or ID genotype(DD: 2.42 ± 0.32,ID:2.14 ± 0.35,II: 2.03 ± 0.35,P<0.05).(2)(1)The activity of plasma eNOS in CAD patients with DD genotype was significantly lower than who with ID or II genotype(DD: 15.85 ± 3.15 ng/ml,ID: 20.55 ± 3.74 ng/ml,II: 19.57 ± 3.32 ng/ml,P<0.05).(2)The plasma level of IL-6 in CAD patients who with DD genotype was significantly higher than who with ID or II genotype(DD: 4.23 ± 1.05 pg/ml,ID: 3.62 ± 1.05 pg/ml,II: 3.59 ± 1.08 pg/ml,P<0.05).(3)The activity of plasma SOD in CAD patients who with DD genotype was significantly lower than who with ID or II genotype(DD: 379.56 ± 6.02 pg/ml,ID: 397.90 ± 4.40 pg/ml,II: 388.14 ± 5.23 pg/ml,P=0.027).(3)(1)The purity of the isolated primary HUVECs was 93.3% in our experiment.(2)The expression of p50 protein was lower in mutant cells than that in wild cells.(3)The transcription activity of p50 protein was significantly lower in mutant cells than that in wild cells.(4)The apoptosis rate and the expression of Bax protein were significantly higher in mutant cells than that in wild cells under the exposure of oxidative stress.(5)The IL-6 gene mRNA expression was significantly higher in mutant cells than that in wild cells under the exposure of oxidative stress.(6)The activity and expression of eNOS were significantly decreased in mutant cells than that in wild cells under the exposure of oxidative stress.Conclusion:(1)NFKB1 gene was association with the incidence of CAD and lesion of coronary artery stenosis and NFKB1 gene DD genotype was the risk factor for CAD.(2)NFKB1 gene mutations can increase the body’s endothelial cell dysfunction,inflammatory response and oxidative stress injury.(3)NFKB1 gene mutations can reduce the expression of p50 protein and its transcription function.(4)NFKB1 gene mutation can cause NF-κB signal pathway extremely activated under the oxidative stress condition and cause higher apoptosis rate in mutant cells.(5)NFKB1 gene mutation can significantly increase IL-6 gene mRNA level and elevate the level of inflammatory response.(6)NFKB1 gene mutations can significantly inhibit eNOS gene mRNA expression and reduce the eNOS activity result in endothelial cell dysfunction.Therefore,NFKB1 gene promoter region-94 ATTG ins/del mutation increased the susceptibility of CAD may due to the mutation of NFKB1 gene decreases the expression and nuclear transcription function of p50 and increase endothelial cell dysfunction,inflammatory response and oxidative stress injury.