Effects And Mechanism Of Leflunomide On The Expression Of ICAM-1 In Human Umbilical Vein Endothelial Cells Induced By HMGB-1

Objective: Rheumatoid arthritis (RA) is a chronic systemic inflammatory disease. Along with the wide application of immunosuppressant, disability rate has dropped significantly. But as the chronic complications, cardiovascular disease have become one of the main death causes of RA patients. Moreover early onset of atherosclerosis possess higher proportion. Now, most of scholars think that atherosclerosis is an inflammatory disease. Inflammatory reaction network formed by a variety of cytokines and monocyte/macrophage, endothelial cells penetrate in the entire process of atherosclerosis occurrence and development. Therefore, the research effect of cytokines in atherosclerosis of RA patients plays an important role in disease prevention.High mobility group box protein-1 (HMGB-1) is an important proinflammatory factor in the pathogenesis of RA. HMGB-1 can also activate monocyte/macrophage system to accelerate atherosclerosis occurrence. There were many evidences indicate that HMGB-1 was related to atherosclerosis. But the mechanism of HMGB-1 was still not very clear. Toll like receptor 4 (TLR4) is a kind of signaling transmembrane receptors on the cells surface, it is one of receptors of HMGB-1. TLR4 can result in atherosclerosis by various channels. So, Whether the combination of HMGB-1 and TLR4 can romote atherosclerosis events by activating the endothelial functions?Clinical studies showed that, early application of disease-modifying anti-rheumatic drugs could reduce the incidence of cardioascular disease in RA patients. Leflunomide (LEF) belongs to one of disease-modifying anti-rheumatic drugs, which has the effect of anti-inflammatory and immunodepression. It can reduce the rate of atherosclerosis by improving on lipid metabolism. But there were no reports about the function of LEF on endothelial cell. This experiment was adopted method of cell culture in vitro, observed the influence of HMGB-1 on human umbilical vein endothelial cells (HUVEC) and to explore whether leflunomide can protect endothelial cells. Moreover, we also provided theoretical basis on early onset atherosclerosis prevention of RA patients by researching its possible mechanism.Methods : HUVEC were maintained at 37℃in a humidified atmosphere of 5% CO2 in Dulbecco's modified eagle's medium (DMEM) containing penicillin/streptomycin (100 KU/L and 100 mg/L, respectively) and 10% fetal bovine serum (FBS). MTT assay was carried out to study the inhibitory effects of LEF with different concentrations on the growth of HUVEC. The cells were divided into five groups based on the results of MTT: control group, HMGB-1 (10ng/ml) group, 10ng/ml HMGB-1 and different concentration LEF(10, 20 and 40μg/ml) respectively. Immunocytochemistry and Western blotting were used to detect the proteins expression of ICAM-1.The mRNA of ICAM-1 and TLR4 were detected by RT-PCR.Results :1The inhibitory effects of LEF with different concentrations on the proliferation of HUVECMTT data indicated LEF could inhibit the growth of HUVEC stimulated by HMGB-1 in a dose-dependent manner. The inhibitory effects of LEF2.5μg/ml and 5μg/ml were lower. However, 82% HUVEC can be inhibited by LEF of 160μg/ml after 24h.2 The expression of ICAM-1 protein of HUVECThe results of immunocytochemistry staining show that the expression of ICAM-1 protein was located in cytoplasmic primarily. There were low level expression in normal control group. The expression of ICAM-1 protein increased after HMGB-1 stimulation for 24h . Compared with HMGB-1 group, ICAM-1 protein expressions in LEF groups were decreased in a dose-dependent manner.Western blotting results indicated that HMGB-1 could up-regulated the expression of ICAM-1 protein compared with control group(P<0.01). The levels of ICAM-1 were decreased in the HUVEC exposed to LEF in a dose-dependent manner too.3 The expression of ICAM-1 mRNA of HUVECRT-PCR indicated that compared with control group, HMGB-1 could up-regulated the expression of ICAM-1 mRNA(P<0.01). LEF could down-regulated mRNA expression of ICAM-1 obviously, and the lowest expression occurred in 40μg/ml LEF group (P<0.01).4 The expression of TLR4 mRNA of HUVECCompared with control group, TLR4 mRNA expression in the cells stimulated by HMGB-1 obviously increased. Compared with HMGB-1 group, LEF could decrease the expression of TLR4 mRNA in endothelial cells in a dose-dependent manner (P<0.01).5 Correlation analysisThere were positive correlation significantly between the mRNA expressions of ICAM-1 and TLR4(r=0.977,P=0.004).Conclusions:1 HMGB-1 could damage the function of human umbilical vein endothelial cells by increasing the expression of ICAM-1. However, LEF could protect endothelial cells.2 HMGB-1 increased the expression of ICAM-1 and the role of LEF through TLR4.