| Aim: Endothelial injury and inflammation play an important role in atherosclerosis. Modified lipoproteins and local or distant infections may be two major triggers. A number of studies have proved that oxidatively modified low-density lipoprotein (ox-LDL) is involved in endothelial dysfunction via a lectin-like receptor for ox-LDL (LOX-1). Recent studies show that Toll-like receptor 4 (TLR4), an immune and inflammatory response receptor, is associated with the initiation and progression of atherosclerotic disease. TLR4 induces activation of transcription factor NF-κB and regulates transcription of a variety of cellular genes. TLR4 and LOX-1 have been detected in endothelial cells of atherosclerotic plaques, ox-LDL upregulated TLR4 mRNA expression in cultured human monocyte-derived macrophages, whether TLR4 can modulate endothelial LOX-1 expression and involve in LOX-1-mediated endothelial inflammation and injury remains to be analyzed. Here we stimulated human umbilical vein endothelial cells (HUVECs) with TLR4 special agonist (purified LPS) in vitro, to observe the regulation of TLR4/NF-κB signal activation on LOX-1expression and role for them in human monocyte adhesion to endothelium, to analyze the interaction between ox-LDL and infections at receptor level, to investigate the role of TLR4/NF-κB signal activation in atherosclerosis,and to assess the intervention of Atorvastatin and Ginkgo biloba extract (GBE50) on TLR4/NF-κB sinals and their possible anti-atherosclerotic mechanism. Method: 1. HUVECs were incubated by LPS(10～1000ng/ml) for 24 hours. TLR4,LOX-1,ICAM-1,E-selectin mRNA expression were measured by RT-PCR. TLR4 and LOX-1 protein expression were observed by Western blot analysis. The expression percentage of TLR4 and LOX-1 positive cells was detected by flow cytometry. The adhesive percentage between monocytes and HUVECs were determined by counting, and the role of LOX-1,ICAM-1,E-selectin in monocytes adhesion to endothelium was examined by adding anti-LOX-1,anti-ICAM-1,anti-E-selectin (10μg/ml) at 30 min before adhesion experiment. 2. HUVECs were pretreated by NF-κB inhibitor CAPE (20μg/ml) for 30 min, then was incubated by LPS for 24h. Translocation of NF-κB p65 was detected by immunohistochemistry;NF-κB activation was measured by nuclear protein Western blot analysis. The effects of CAPE on LPS-mediated TLR4,LOX-1,ICAM-1,E-selectin expression and monocytes adhesion to endothelium were investigated by the same methods above. 3. HUVECs were pretreated by Atorvastatin (10μmol/L) and GBE50 (80μg/ml) for 30 min, then was incubated by LPS for 24h. The effects of Atorvastatin and GBE50 on LPS-mediated TLR4,LOX-1,ICAM-1,E-selectin expression,NF-κB activation and monocytes adhesion to endothelium were investigated by the same methods above. Results: 1. LPS(10-1000ng/ml) upregulated TLR4,LOX-1,ICAM-1,E-selectin mRNA and TLR4 ,LOX-1 protein expression , in a dose-dependent fashion. LPS (1000ng/ml) increased the percentage ofTLR4 and LOX-1 positive cell in HUVECs. LPS (1000ng/ml) increased the percentage of monocytes adhesion to endothelium, pretreatment of HUVECs with anti-LOX-1,ICAM-1 or E-selectin all partly abolished the increase of monocytes adhesion to endothelium. 2. LPS (1000ng/ml) led to NF-κB activation. Pretreatment of cell with CAPE inhibited LPS-mediated NF-κB activation, attenuated upregualtion of TLR4,LOX-1,ICAM-1,E-selectin mRNA and TLR4,LOX-1 protein expression, and abolished the increase of monocyte adhesion to endothelium. 3. Atorvastatin and GBE50 both blocked NF-κB activation, suppressed LPS-induced upregulation of TLR4,LOX-1,ICAM-1,E-selectin expression and the increase of monocyte adhesion to endothelium. Conclusion: 1. TLR4/NF-κB signals activation by LPS modulates self-receptor TLR4 in a positive feedback fashion, the upregulation of TLR4 may increase the injury in HUVECs. 2. TLR4/NF-κB signals activation by LPS upregualtes LOX-1 in HUVECs, suggesting one of the mechanisms by which TLR4 is involved in atherogenesis maybe th...
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