| Chapter One The Effect of LOX-1/NF-κB Signals on the Expression of Toll-like Receptor 4 in Cultured Human Umbilical Vein Endothelial Cells Induced by Oxidized Low-density Lipoprotein and Modulated by FluvastatinBackgrounds and objectivesAtherosclerosis is considered as an immune-inflammatory disease. Toll-like receptor 4, a pattern recognition receptor, mediates innate immunity and inflammatory responses and plays important roles in the processs of atherosclerosis. Oxidized low-density lipoprotein(ox-LDL) to be an independent risk factor that gave rise to the atherosclerosis. Lectin-like oxidized low density lipoprotein receptor-1 (LOX-1), a special receptor of ox-LDL,can bind,intake,degrade and mediate its biological function. The overexpression of TLR4 and LOX-1 have been detected in endothelial cells of atherosclerotic plaques. It was established that the upregulation of TLR4 in macrophages is induced by ox-LDL, which suggest that TLR4 exert important roles in proinflammation responces mediated by lipid. The upregulation of LOX-linduced by LPS through the TLR4/NF-κB signals, can improve the precess of atherosclerosis. These data revealed that the close relationships between TLR4 and LOX-1 exist in the atherosclerotic immune-inflammatory responses. As a nucleus transcription factor, NF-κB can induced the expression of TLR4 and regulates its signal transduction. So it was speculated that the signaling pathway of LOX-1/NF-κB—TLR4 may be exist. Thus, this study investigated the interaction between TLR4 and LOX-1, and the regulatory mechanicsms of this signaling pathway by Fluvastatin. It has provided a new basis for the prevention and control of atherosclerosis.MethodsHUVECs were incubated with ox-LDL for 24 hours with or without pretreated by LOX-1 neutralizing antibody, NF-κB blockers pyrrolidinedithiocarbamate (PDTC) or Fluvastatin. TLR4 and LOX-1 mRNA expression were evaluated by RT-PCR.ResultsThe levels of mRNA of TLR4 and LOX-1 increased after cells were incubated with different concertrations of ox-LDL, when compared with the control group. Within the dosage range of 25 to 50 mg/L, the abovementioned indexes significantly changed in a concentration-dependent manner(P＜0.01). HUVECs were pretreated with LOX-1 neutralizing antibody or PDTC decreased the expression of LOX-1 and TLR4 mRNA induced by ox-LDL. Pretreatment of HUVECs with Fluvastatin in different concentrations for 24 hours decreased the expression of mRNA of TLR4 and LOX-1 induce by ox-LDL in a dose-dependent manner. Conclusions1. LOX-1 is not only a special and high affinity receptor for HUVECs to uptake ox-LDL, but also a signal molecule Transferring biological signals to upregulate itself.2. LOX-1/NF-κB signals activated by ox-LDL upregulates TLR4 in HUVECs and aggravates endothelial cell injuries, suggesting one of the mechanisms by which TLR4 is invovled in atherogenisis. Meanwhile, there are close relationships between lipoprotein and inflammation/immunity in pathogenesis of atherosclerosis from receptor level.3. NF-κB play a key role in ox-LDL effects described above. Therefore, LOX-1/NF-κB signal may become the new target which prevents and cures atherosclerosis.4. Fluvastatin inhibit the expression of TLR4 induced by ox- LDL, which may be one of the mechanisms of anti-inflammation or anti-immunity in addition to lipid-lowering capacity. Chapter Two Effects of LOX-1/NF-κB Signals on Expression of Inflammatory Cytokines Induced By Ox-LDL in Endothelial cells and Modulated by FluvastatinBackgrounds and objectivesAtherosclerosis is now understood to be a chronic inflammatory disease. Inflammatory mechanisms are involved in causing endothelial injury and dysfunction, the formation of foam cells, the initiation and rupturing of atherosclerotic plaque, thus they play vital roles in the process of atherosclerosis. A number of studies have proved that oxidized low-density lipoprotein(ox-LDL) to be an important factor that gave rise to the atherosclerosis. Lectin-like oxidized low density lipoprotein receptor-1(LOX-1) is a major special receptor of ox-LDL in endothelial cells.Its inducible expression is involved in ox-LDL-mediated multiple roles in atherogenesis. The present study is to determine whether LOX-1 mediates the inflammatory cytokines IL-6 and TNF-a expression from endothelial cells stimulated by ox-LDL and the modulation of Fluvastatin. This will provide the new theoretical basis for prevention and treatment of atherosclerosis.MethodsHUVECs were incubated with ox-LDL or intervened by LOX-1 neutralizing antibody, NF-κB blockers pyrrolidinedithiocarbamate (PDTC) or Fluvastatin. The supematant IL-6 and TNF-a levels were measured by ELISA.ResultsThe levels of IL-6 and TNF-a from culture supematants increased after cells were incubated with different concertrations of ox-LDL, when compared with the control group. Within the dosage range of 25 to 50 mg/L, these indexes significantly changed in a concentration-dependent manner(P＜0.01). HUVECs were pretreated with LOX-1 neutralizing antibody or PDTC decreased the expression of IL-6 and TNF-a induced by ox-LDL. Pretreatment of HUVECs with Fluvastatin in different concentrations for 24 hours decreased the expression of IL-6 and TNF-a induced by ox-LDL in a dose-dependent manner.Conclusions1. ox-LDL upregulates the expression of IL-6 and TNF-a from the supematant of HUVECs. Within the dosage range of 25 to 50 mg/L, their expression significantly changed in a concentration-dependent manner.2. LOX-1/NF-κB signals involved in the inflammation through upregulating the expression of endothelial inflammatory cytokines TNF-a and IL-6, suggested that LOX-1/NF-κB signals play important role in the process of athersclerosis.3. Fluvastatin decreases the endothelial inflammatory injury through inhibiting the expression of IL-6 and TNF-a induced by ox-LDL in a concentration-dependent manner. It suggest that fluvastatin may play the anti-inflammatory actions in addition to lipid-lowering capacity.
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