Exploration of Fibronectin Role in Coronary Heart Disease with Blood Stasis Syndrome Based on NF-κB Inflammatory Pathway
Objective To search for the key protein of inhibiting blood stasis syndrome of coronary heart disease by proteomics,and to verify the effect of fibronectin 1(Fn)on blood stasis syndrome of coronary heart disease based on nuclear factoer kappa-B(NF-κB)inflammatory signal pathway and endothelial cell injury model.Methods the serum of patients with coronary heart disease of blood stasis syndrome and healthy subjects were analyzed by ITRAQ marker proteomics,and the differential proteins were enriched by GO and KEGG functions.Fn,a key protein,was screened from the enrichment pathway closely related to coronary heart disease.A model of endothelial cell activation was established using 60 μg/mL of oxidized Low-density lipoprotein(oxLDL),three concentrations of plasma Fn(5μg/cm2,10μg/cm2,20 μg/cm2)or knockdown of endothelial cell-derived Fn were added to the endothelial cell activation model.The nuclear translocation of NF-κB and the expression of NF-κB protein were detected by immunofluorescence and western blotting in Blank group,oxLDL Group,control group,FnEC-KD Group,Fn5 Group,Fn10 Group and Fn20 group,the levels of ICAM-1,VCAM-1,Prostacyclin-I-2 and endothelin in the culture supernatant were detected by ELISA.Results The results of serum proteomics showed that there were 121 different proteins in the healthy group and the coronary heart disease with blood stasis syndrome group,the 51 down-regulated proteins in the blood stasis syndrome groups were concentrated in the complement-coagulation cascade,the regulation of actin cytoskeleton,and the ECM-receptor interaction pathway,the 70 up-regulated proteins in the blood stasis syndrome groups were concentrated in the complement and coagulation cascade,the regulation of actin cytoskeleton,platelet activation and phagocytic pathway.Fn is closely related to coronary heart disease and down-regulated in patients with blood stasis syndrome.There was no significant difference in the total protein expression of NF-κB among the three groups(P>0.05).Compared with the control group,the nuclear translocation of NF-κB p65,the expression of ICAM-1,VCAM-1,endothelin in oxLDL group were significantly increased and the secretion of PGI2 was significantly decreased(P<0.05).In Fn5,Fn10 and Fn20 groups,the nuclear translocation of NF-κB and the secretion of VCAM-1,ICAM-1,and endothelin were decreased(P<0.05),and the secretion of PGI2 was increased significantly(P<0.05).In FnEC-KD Group,the nuclear translocation of NF-κB and the expression of VCAM-1,ICAM-1,endothelin were decreased,and the expression of PGI2 was increased significantly(P<0.05).Conclusion Plasma fibronectin inhibits endothelial cell activation and dysfunction,and endothelial cell-derived Fn promotes endothelial cell activation and dysfunction.