Lipopolysaccharide-induced Inducible Nitric Oxide Synthase Influence On Smooth Muscle Cell In Atherosclerosis

AS(Atherosclerosis)is a chronic inflammatory and degenerative disease.Smooth muscle cell play an important role in the pathology of vascular occlusive diseases.Under the healthy condition,inflammation is a normal steady state.But under the long-term inflammation condition,inflammation will convert protection mechanism into harmful response,and maybe lead to further damage.i NOS(Inducible nitric oxide synthase)is the key enzyme to produce high levels NO(nitric oxide)in pathological conditions.Too much NO reacts with O2-to form ONOO-,and it is harmful for tissues and participates in AS.Purpose: The purpose of this study is to investigate how the i NOS effect on the VSMC(vascular smooth muscle cell)in the formation process of AS.Methods: In this study,the experiment had been done into two fields: human coronary arteries tissues(degree of stenosis less than 50%)and HVSMC(human vascular smooth muscle cell).We divided coronary arteries tissues into two groups: group of normal coronary arteries and groupof atherosclerotic coronary arteries(degree of stenosis less than 50%).Two groups both extracted vascular without bifurcation,then did western blot,pcr,immunohistochemistry,HE staining,Weigert staining and Griess kit assay.We divided HVSMC into two groups: group of normal HVSMC and group of HVSMC within LPS(lipopolysaccharide),then did western blot,pcr,immunofluorescence analysis,CCK8,cell scratch-wound assay,migration and invasion assay and Griess kit assay.In addition,we down the i NOS gene,then cells divided into four groups: LPS+i NOS downregulation group,LPS+ i NOS downregulation negative control group,i NOS downregulation group,i NOS downregulation negative control group,then we did migration assay in this four groups.We also down the p65 gene,divided cells into four groups: LPS+p65 downregulation group,LPS+ p65 downregulation negative control group,p65 downregulation group,p65 downregulation negative control group,then did western blot to detect the i NOS expression.Results: Our results showed i NOS and NO expression were higher in the AS tissues by western blot,pcr and Griess kit.Immunohistochemistry results showed that i NOS expressed higher in foam cell cytoplasm root in smooth muscle cell.Weigert staining results showed that elastic fibers migrated from middle membrane to inner membrane.i NOS and NO expression were higher in group LPS than in group normal by western blot,pcr and Griess kit.Immunofluorescence analysis results showed that i NOS expressed higher in group LPS and expressed in cytoplasm.CCK8,cell scratch-wound assay,migration assay results showed that cells of group LPS had higher ability to prolife and migrate.Conclusion: These findings suggest that the i NOS expresses higher in AS,and various inflammatory factors such as LPS can induce SMC to express high i NOS to promote proliferation and migration of HVSMC and make HVSMC migrate from middle membrane to inner membrane,but no invasion ability,to participate in AS development via NF-κB signaling pathway.