LPS Induces MCP-1 And ICAM-1 Expression In HUVECs Via NADPH Oxidase And TLR2 Pathways

OBJECTIVEAtherosclerosis (As) with immune responses during initiation and progression is increasingly recognized as a kind of chronic inflammatory diseases. One potentially important source of vascular inflammation in As is bacterial endotoxin. Toll-like receptors (TLRs) associated with inflammatory responses in As, play an important role in innate immune signaling. Oxidative stress is reactive oxygen species (ROS) accumulation, which induces inflammatory gene expression and promotes the development of As. ROS, derived from nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, is the mainly source of vascular cells. As a second messenger, ROS, play an important role in many signaling pathways. In this studies, the aim was to investigate the effects of NADPH oxidase—TLR2 signaling on the expression of monocyte chemotactic protein-1 (MCP-1) and intercellular adhesion molecule-1 (ICAM-1) in human umbilical vein endothelial cells (HUVECs) incubated with LPS.METHODSHUVECs were incubated for 8 hours (h) with 0 ng/ml, 12.5 ng/ml, 25 ng/ml, 50 ng/ml, 100 ng/ml LPS, respectively. TLR2 mRNA and protein levels were determined by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting respectively. The optimal LPS concentration was adopted. HUVECs were incubated with 25 ng/ml LPS for 0 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, respectively. TLR2 mRNA and protein levels were determined by RT-PCR and Western blotting respectively. The optimally incubating time was adopted. HUVECs were incubated with 25 ng/ml LPS for 0 min, 20 min, 40 min, 1 h, 2 h, 4 h, respectively. The activities of NADPH oxidase were determined by NBT reduction experiment. The mRNA levels of NADPH oxidase submits: p22phox, p67phox and Rac-1 were determined by RT-PCR. HUVECs were pretreated for 0.5 h with 10 mmol/L acetylcysteine (NAC), 100 umol/L apocynin (APO) or 30 umol/L diphenylene iodonium chloride (DPI), and then were incubated for another 2 h with 25 ng/ml LPS. TLR2 mRNA and protein levels were determined by RT-PCR and Western blotting respectively. HUVECs were pretreated for 0.5 h with 10 mmol/L NAC, 100 umol/L APO and 30 umol/L DPI, respectively, and then were incubated for 8 h with 25 ng/ml LPS. MCP-1 and ICAM-1 mRNA levels were determined by RT-PCR. HUVECs were pretreated for 0.5 h with 10 ug/ml TLR2 blocking antibody or control antibody, and then were incubated for 8 h with 25 ng/ml LPS. MCP-1, ICAM-1 mRNA or protein levels were determined by RT-PCR or Western blotting.RESULTSThe expression of TLR2 mRNA and protein was up-regulated in HUVECs incubated with LPS, and the optimal LPS concentration was 25 ng/ml, the optimally incubating time was 2 h. The activity of NADPH oxidase was increased firstly, and then decreased, the levels of NADPH oxidase submits: p22phox, p67phox and Rac-1 mRNA were up-regulated, which consisted with the activity of NADPH oxidase. The expression of TLR2 mRNA and protein was down-regulated in HUVECs incubated with NAC, APO or DPI. MCP-1 and ICAM-1 mRNA expression was down-regulated in HUVECs incubated with NAC, APO or DPI. MCP-1, ICAM-1 mRNA or protein expression was down-regulated in HUVECs incubated with TLR2 blocking antibody.CONCLUSIONSLPS induces the expression of TLR2 in HUVECs. NADPH Oxidase regulates the expression of TLR2 in HUVECs. LPS up-regulates the expression of MCP-1 and ICAM-1. NADPH Oxidase and TLR2 pathways regulate the expression of MCP-1 and ICAM-1 in HUVECs.