| The hallmark of atherosclerosis are intimal fibrofatty plaques,the atherosclerotic lesions,which develop principally in large and medium-sized muscular and elastic arteries and are characterized by SMC proliferaton,extracellular matrix formation and deposition,and lipid accumulation.NF-κB was discovered as a B-cell nuclear factor that binds to a site on the immunoglobulinκenhancer. Later, it was shown by other researchers that NF-κB is expressed and regulated in other cell types, and has binding sites in the promoters of genes other than immunoglobulinκenhancer. NF-κB can modulate the expression of many cytokines, cell adhesion molecules,inflammation mediators and enzymes involved in inflammatory and immune response,cellular proliferation and survival in addition to promoting angiogenesis and metastasis.Andrographolide is an active component isolated from the leaves of Andrographis paniculata, a Chinese herbal medicine used for the treatment laryngitis,diarrhea, and rheumatoid arthritis.However,little is known about the pharmacological mechanism of its anti-inflammatory actions. In the present study, we provide evidence that Andro forms a covalent adduct with reduced cysteine (62)of p50, thus blocking the binding of NF-κB oligonucleotide to nuclear proteins. In this present, we found that NF-κB activity was potently inhibited by Andro through luciferase assay. So, we want to know whether Andro can decrease Atherosclerosis by inhibiting NF-κB pathway. In the present study we want to investigate whether Andro can regulate NF-κB transcription activity in stimulated vascular endothelial cells and activated monocytes/macrophages. We further hope to assesse the functional significance of Andro in atherosclerosis and its mechanisms.The present study includes two parts: (1) In vitro experiments :Human adherent monocytes, murine macrophages, HUVECs and murine PVECs were obtained and cultured to be used in vitro experiments. Transfection and luciferase assay showed that Andro decreases the interaction of NF-κB with the binding site of E-selectin promoter and TF promoter. (2) In vivo experiments: ApoE deficient mice feed on western-type diet were used to assesse the the functional significance of Andro in modulation of atherosclerosis. H&E staining and Oil Red O staining were utilized to appreciate the effect of Andro treatment in the atherosclerotic plaques. Electrophoretic mobility shift assay (EMSA) was utilized to demonstrated the direct inhibition of Andro on NF-κB binding activity to the promoter in Atherosclerotic plaques. Futhermore, Quantitative PCR and immunohistochemical staining and immunohistoch- emical gray analysis were adopted to detect wether Andro-treated exhibited blunted TF, E-selectin,and VCAM-1 expression. Fibrin ,a hallmark of activity of TF,plays important role in thrombosis in Atherosclerosis.Body weight was measured every two weeks during all through experiment.Blood samples were collected from two groups fasted overnight,and plasma lipid levels were measured using an automated analyzer.Based on the above analysis, our study identifed that: (1)Andro inhibits luciferase activity in a dose-dependent manner , demonstrating its inhitory activity for NF-κB activation in stimulated 293 cells;(2)Andro ablates E-selectin in stimulated endothelial cells;(3)Andro decreases TF in stimulated endothelial cells and monocytes /macrophages;(4)Andro antagonizes the NF-κB site in human TF promoter;(5)Andro suppresses atherosclerotic plaques in apoE deficient mice;(6)Andro decreases TF expression and fibrin deposition;(7) Andro reduces the expression of NF-κB targeting genes in both mRNA and protein level in apoE deficient mice;(8) Andro has no effect on body weight and plasma lipid.In conclusion, the present study demonstrates that:(1) Andro regulates NF-κB binding activity in stimulated human and murine vascular endothelial cells and macrophages in vitro and in in vivo models of atherosclerosis.(2)Andro derivatives can be designed as small molecular antagonists of NF-κB activation and therapeutic prevention and treatment of atherosclerosis.
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