The stimulation of ox-LDL and LPS both increased the expression of ICAM-1, IL-6 and IL-8, but did not change TLR2 protein expression in both normal and high glucose conditions

The stimulation of ox-LDL and LPS both increased the expression of ICAM-1, IL-6 and IL-8, but did not change TLR2 protein expression in both normal and high glucose conditions. without neutralizing anti-TLR2 antibody. After that, HUVECs were treated with ox-LDL (20, or 40 g/ml) or LPS (200 ng/ml) under normal and high glucose conditions. The expressions of ICAM-1 and TLR2 protein were analyzed by immunoblotting, and IL-6 and IL-8 were measured by ELISA. Results: Compared with those in normal glucose condition, IL-6 and IL-8 expression were increased in high glucose condition. The activation of ox-LDL and LPS both increased the expression of ICAM-1, IL-6 and IL-8, but did not switch TLR2 protein expression in both normal and high glucose conditions. Additionally, the expression of ICAM-1, IL-6 and IL-8 was not changed when TLR2 was knocked out under these two conditions. Conclusion: The inflammatory responses induced by Ox-LDL were not changed with or without TLR2 under both normal and high glucose conditions in HUVECs. Our study indicates TLR2 is not involved in the ox-LDL mediated endothelial injury under high glucose conditions, which is an important step of atherosclerosis formation in diabetes. strong class=”kwd-title” Keywords: TLR2, inflammation, HUVECs, ox-LDL Introduction Diabetes millitus is an important risk factor for the AZD-4635 (HTL1071) development of atherosclerosis. Endothelial AZD-4635 (HTL1071) dysfunction induced by oxidized low density cholesterol (ox-LDL) is regarded as an initial step in the pathogenesis of atherosclerosis plaque formation. It is known that ox-LDL functions via binding to a number of scavenger receptors, such as SR-A1, SR-A2 and lectin-like oxidized low-density lipoprotein receptor (LOX-1). LOX-1 facilitates the uptake of ox-LDL, induces endothelial dysfunction and mediates numerous ox-LDL-induced proatherogenic effects, resulting in ox-LDL accumulation in the vessel wall [1]. LOX-1 is the main ox-LDL receptor of endothelial cells. Ox-LDL also regulates some other receptors, especially inflammatory receptors such as Toll-like receptors (TLRs) in nuclear cells. In diabetes millitus, the effect of ox-LDL around the inflammatory receptors is still interesting. TLRs, pathogen pattern acknowledgement receptors, are characterized by the expression and release of cytokines and chemokines which is usually implicated in the AZD-4635 (HTL1071) development and progression of atherosclerosis. Scavenger receptors and TLRs cooperate in response to danger signals to adjust the host immune response [2]. TLR2 has a central role in innate immunity and inflammation [3]. Ox-LDL induced TLR2 and TLR4 expression at mRNA level and caused a significant activation of NF-B in monocytes [4,5]. CTSS TLRs are involved in the LPS/PGN-mediated inflammatory responses in endothelial cells [6], and it could be also involved in the inflammation induced by ox-LDL. The advanced glycation end-product of low-density-lipoprotein activates the TLR4 pathway implications for diabetic atherosclerosis [7]. TLRs activation and ligands are found to be increasing in recently diagnosed type 2 diabetic subjects [8]. We also found that TLR2/4 activation enhances endothelial inflammation in type 1 diabetes [6]. So we want to know the effect of ox-LDL on TLR2 pathway in endothelial cells, especially in diabetic condition. TLR2 expression is usually enhanced by LPS in HUVECs under high glucose condition [9]. It has not been decided whether TLR2 is usually enhanced in the inflammation induced by ox-LDL in human umbilical vein endothelial cells (HUVECs). We wanted to study the role of TLR2 plays in the inflammatory response induced by ox-LDL in HUVECs under high glucose condition. To test this, we treated HUVECs with ox-LDL under high glucose conditions in vitro. The high glucose condition is usually modeling as diabetic condition in vivo [9]. Under high glucose condition, the purposes of this study are to determine: 1) the effect of ox-LDL around the inflammatory responses in HUVECs, 2) whether TLR2 levels are increased by different concentration of ox-LDL, and 3) whether the switch of TLR2 level could alter the inflammation in HUVECs. Materials and methods Materials HUVECs were obtained from American type culture collection (ATCC). HUVECs were cultured in endothelial cell medium (25 ml of fetal bovine serum at 5%, 5 ml of endothelial cell growth product (EBM-2, contains 2% FBS) (Lonza, Boulder, CO, USA) at 1% and 5 ml of penicillin/streptomycin answer at 1% was added into 500 ml) were from Scien Cell Research Laboratories (San Diego, California, USA); Ox-LDL (oxidized using Cu2SO4 (oxidant) in PBS) were obtained from Yiyuan Biotech (Guangzhou, Guangdong, China); TLR2 Antibody was obtained from Santa Cruz Biotechnology (Dallas, Texas, USA); neutralizing anti-TLR2 antibody (T2.5) was purchased from Invivogen (San Diego, California, USA) and ICAM1 was from Abcam (Cambridge, MA, USA); GAPDH antibody and HRP-conjugated goat anti-rabbit IgG was purchased from PTG (Pro Teintech Group, USA); IL-6 and IL-8 ELISA.