| Atherosclerosis (AS)is vascular disease characterized by lipid deposition and chronic inflammation within the arterial wall. When vascular endothelium is injured, a wide range of inflammatory factors produced by vascular smooth muscle cells(VSMCs), endothelial cells and macrophages stimulate the synthesis and secretion of adhesion-related proteins from endothelial cells, such as vascular cell adhesion molecule-1(VCAM-1), intercellular adhesion molecule-1(ICAM-1), osteopontin(OPN) and tenascin-c, which promote the adherence of platelets and monocytes to vascular endothelial cells, and increase the release of inflammatory mediators from endothelial cells. As a result, vascular inflammatory reaction is further aggravated by these inflammatory factors.Nuclear factor-κB(NF-κB)acts as a central mediator of the immune response and controls the expression of various genes involved in inflammation and proliferation. NF-κB activation can regulate the expression of many genes, including inflammatory mediators, adhesion molecules, proliferation and apoptosis factors involved in inflammation-mediated immune response, cell proliferation and apoptosis pathophysiological process. NF-κB activation is considered as one of starting mechanisms of vessel endothelial cell injury. Therefore, blocking the signaling pathway of inflammatory response by inhibiting NF-κB activation plays an important role in the prevention and treatment of cardiovascular disease.Inula Britannica is a traditional Chinese medicinal herb that has been used to treat vomiting, bronchitis and inflammation. 1,6-O,O-diacetylbritannilactone ( ABLO2 ) is a new active monomer isolated from Inula Britannica-F.To understand whether ABLO2 can inhibit vascular inflammation, the present study examined the effects of ABLO2 on vascular endothelial cell inflammation.MethodsThe NF-κB/DNA binding activity in nuclear extracts of ECV304 cells was determined by electrophoretic mobility shift assay; NF-κB p65 expression and nuclear translocation and level of IKK, p-IKK, IκBα, p-IκBαand adhesion molecule were analyzed by Western blotting; cell adhesion and cell transmembrane migration activity was detected by endothelial cell adhesion and monocyte migration experiment.Results1 ABLO2 inhibits nuclear translocation and activation of NF-κBWestern blot showed that the level of nuclear NF-κB p65 increased significantly after treated with LPS/IFN-γfor 30 min in ECV304 cells and peaked at 1 hour. After the cells were incubated in the medium containing 20μmol/L ABLO2, the increased level of nuclear p65 in ECV304 cells induced by LPS/IFN-γdeclined to some extent. These findings suggested that ABLO2 inhibits the nuclear translocation of NF-κB induced by LPS/ IFN-γ.2 ABLO2 inhibits the DNA-binding activity of NF-κBAs shown by EMSA, the DNA binding activity of NF-κB increased remarkably by stimulation of LPS/IFN-γ, and DNA binding of NF-κB induced by LPS/IFN-γwas significantly inhibited by pre-incubation with ABLO2 for 2 hours in ECV304 cells. This result indicated that ABLO2 inhibits the DNA binding activity of NF-κB.3 ABLO2 inhibits IκBαphosphorylation and degradation.Western blot showed that LPS/IFN-γtriggered IκBαphosphorylation, which was maximal at 10 min, with remarkable reduction of IκBαlevel. However, pretreatment of the cells with 20μmol/L ABLO2 before LPS/IFN-γstimulation obviously attenuated the phosphorylation and degradation of IκBαinduced by LPS/IFN-γ. These results suggested that ABLO2 can inhibit the phosphorylation and degradation of IκBαin ECV304 cells.4 ABLO2 inhibits the activation of IKKWestern blot showed that IKK phosphorylation induced by LPS/IFN-γdecreased by 73% in the cells pretreated with ABLO2. The effect of ABLO2 was the strongest at 30 min. The results indicated that ABLO2 blocks the phosphorylation of IKK induced by LPS/IFN-γin ECV304 cells .5 ABLO2 inhibits the expression of NF-κB-dependent adhesion molecules The expression of VCAM-1, OPN, MMP-9 and Tenascin-c significantly increased in response to inflammatory stimulation, which promotes the adhesion of circulating leukocytes to vascular endothelium. The level of MMP-9, OPN and VCAM-1 protein increased by 3.14-fold, 5.2-fold, 2.3-fold in ECV304 cells treated by 10μg/ml LPS/IFN-γfor 6 hours, respectively, and Tenascin-c expression increased by 4.5-fold at 6 hours, compared with control group. In ECV304 cells pre-incubated with ABLO2 for 2 hours, the expression of VCAM-1,OPN,MMP-9 and Tenascin-c stimulated by LPS/IFN-γdecreased by 61.3 %, 77.5 %, 62.6 %, 78.4 %, respectively. These results indicated that ABLO2 can inhibit the expression of NF-κB-dependent adhesion molecules.6 ABLO2 inhibits the adhesion between monocyte and endothelial cellsThe results of cell adhesion assay demonstrated that the adhesion between monocyte and endothelial cells stimulated by LPS/IFN-γincreased by 4.7 folds, compared with control group. The adhesion rate decreased by 52% in the cells pretreated with ABLO2.7 ABLO2 suppresses the transmembrane migration of RAW264.7 cellsCell transmembrane migration analysis showed that the number of transmembrane RAW264.7 cells stimulated by LPS/IFN-γwas significantly higher than that of control group. RAW264.7 cells were pre-treated by ABLO2 for 2 hours following stimulation with LPS/IFN-γfor 6 hours, the transmembrane cells reduced by 15.4 %.Conclusions1 ABLO2 suppresses NF-κB activation and the nuclear translocation of p65 via inhibition of IKK and IκBαphosphorylation and degradation in the classic pathway of NF-κB activation.2 ABLO2 inhibits the expression of NF-κB-dependent adhesion molecules .3 ABLO2 downregulates LPS/IFN-γ-induced adhesion between monocyte and endothelial cells and transmembrane migration of RAW264.7 cells.
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