The Mechanism And Influence On IL-6Screting Inducing By Lipopolysaccharide In Human THP-1Mononuclear Cells

Coronary atherosclerotic heart disease refers to bureaucratic tone, narrowed or blocked blood vessels because of coronary atherosclerosis, or (and) coronary functional changes (spasm) leading to myocardial ischemia or hypoxia necrosis,leading to heart disease, referred to as coronary heart disease. Since the21st century,coronary heart disease is a serious hazard to human life and health of the common, with the increase in the proportion of the elderly population, population aging, and high salt, high-fat diet, and the obese population increased tension, the coronary heart disease patients increasingly "young", the coronary heart disease morbidity and mortality significantly increased. The pathogenesis of coronary heart disease has been the focus of attention and hot spots around the world, but its mechanism is still unclear, which is the main difficulty for coronary heart disease prevention and treatment progress. Atherosclerosis (AS) is the main pathological basis of coronary heart disease. Studies have shown that atherosclerosis is caused by a variety of factors acting on different aspects of these factors are called risk factors, the major risk factors such as old age, lipids increased, smoking, high blood pressure, elevated blood sugar, high and half cystine hyperlipidemia, and hyperuricemia. These risk factors promoted AS occurrence and development in certain. There are many incidence doctrines of atherosclerosis (AS) mechanism in recent years and in the past,for example the doctrine of lipid infiltration, endothelial damage response doctrine, thrombosis doctrine, the monoclonal doctrine of smooth muscle cells, homocysteine theory, arginine doctrine, endothelial dysfunction theory, theory of oxidative stress,but these all so not enough to completely explain the AS disease incidence foundation.However, in recent years, a large number of studies have confirmed that the inflammatory response has been recognized as the formation mechanism of atherosclerosis:Atherosclerosis is a chronic inflammatory disease,, inflammation AS also has the basic pathological characteristics:degeneration, exudative and proliferative,the same with other inflammatory diseases. With further research, it is found that AS is not only local vascular inflammatory lesions but also systemic immune abnormalities caused by a variety of immune cell disorders. The development and progression of atherosclerotic lesions, even from the beginning of the fatty streaks to fibrous plaque and unstable plaque rupture and thrombosis, a variety of inflammatory cells and participate in a variety of inflammatory mediators coronary local or systemic inflammatory response plays an important role in the occurrence of AS in the development process and its complications caused. The study confirmed that the major factors as plaque instability may be associated with the activation of the inflammatory response.Mononuclear macrophage are important immune cells, which are the most common inflammatory cells in the formation of atherosclerosis,and are the important part of the AS plaque and also are the important pathophysiological basic of vulnerable plaque. Under normal circumstances, the vascular endothelial cells can prevent leukocyte aggregates and can facilitate the dissolution of fibrin. When endothelial cells stimulate by hyperlipidemia, hypertension, diabetes, smoking, obesity, and inflammation, leading to the initial vascular inflammation injury and produce inflammatory cytokines,which can attract mononuclear cells to reach the injury partial and then into the artery membrane under the gap, and then the vascular intima swallow phagocytic lipoprotein change into foam cells, and then the plaque preliminary molding. Mononuclear macrophage exogenous antigens presented to T lymphocytes, activate the acquired immune system, aggravate vascular inflammatory lesions. While macrophages also secrete tissue factor and metal proteinases, dissolve the extracellular matrix in the intima of the artery, resulting in plaque fibrous cap digestion and plaque rupture.As plaque in the monocyte-macrophages secrete a variety of inflammatory cytokines, such as IL-6, monocyte chemoattractant protein1(MCP-1) and TNF-a, due to monocyte accumulation exacerbating, macrophages cell proliferation,and the migration and proliferation of smooth muscle cells, prompting As plaque continuous progress. More evidence that the inflammatory response in patients with acute coronary syndrome (ACS) is not limited to the lesion site and even across the vascular bed spread, and associated with IL-6and increased levels of CRP and other inflammatory markers and the systemic circulation leukocyte activation.There are some studies have shown that atherosclerotic plaque monocytes stimulated secretion of IL-6is a central inflammatory response adjustment factor, plays a key role in vascular injury of coronary heart disease and acute myocardial ischemia.Lipopolysaccharide, also referred as bacterial endotoxin, and constitutes the main component of the cell wall of Gram-negative bacteria. Lipopolysaccharide can be slowly released from viable body and massive release into the surrounding environment when the bacterial cell is broken. LPS is an amphiphilic molecules, including a hydrophobic phospholipid portion (phospholipid A) constituting the outermost part of the bacterial outer membrane, the hydrophilic portion of the polysaccharide moiety, projecting to the outside of the microbial cells.lipopolysaccharide molecules on the lipid fraction and the polysaccharide moiety are involved in the biological activity of the lipopolysaccharides. Complete bacterial lipopolysaccharide can bind specifically to the cell surface in human monocytes and macrophages LPS-induced inflammation is caused by its interaction with the host cell, the sensitivity of the organism against the lipopolysaccharide from Soluble and cell surface binding protein mediated and adjustable. LPS-mediated inflammatory process found that its main lure to cause inflammatory cytokines such as interleukin a single chemoattractant protein, expression of oxygen free radicals, resulting in promotion of the imbalance between inflammation and fight infection system, resulting in inflammation. Interleukin6, also known as proinflammatory cytokines, which affect inflammation, tissue injury and host defense humoral and cellular immune function. IL-6is a multifunctional cytokine that is produced in vascular endothelial cells, activated T lymphocytes, peripheral blood mononuclear cells, macrophages and smooth muscle cells. At present IL-6is considered participate in the formation of coronary artery atherosclerosis (AS) through a variety of channels and progress, such as IL-6promote macrophage cell surface LDL receptor synthesis and uptake of LDL, promote atheroma formation; IL-6can stimulate macrophages which secretion of monocyte chemoattractant protein and that promote monocyte accumulation into the vascular endothelial involved in plaque formation; IL-6stimulate cell proliferation of vascular smooth muscle by the way of Autocrine; IL-6can induce liver cells to produce plasminogen activator inhibitor (PAI), as well as the liver to produce acute phase proteins, to promote coronary thrombosis. IL-6, can stimulate the proliferation of smooth muscle cells in the plaque resulting plaque rupture. IL-6is important predictors of AS and other cardiovascular diseases, and IL-6levels correlated with the prognosis of the disease and cardiovascular events happen again closely related Rotablator plaques extracted from AS patients found IL-6expression was significantly increased, suggesting that IL-6is closely related to unstable plaque, shows that elevated plasma IL-6values have predictive value for the occurrence of ACS patients.Cilostazol is a novel anti-platelet drugs, phosphodiesterase inhibitor, reversible, selective inhibition of phosphodiesterase activity and blocking cAMP degradation, while inhibition of adenosine uptake, and can inhibit platelet aggregation and the blood vessels to dilate. Cilostazol has the function of preventing atherosclerosis, anti arterial thrombosis and vascular stenosis, and can be widely used for the treatment of diabetic complications, regulate blood lipids and dilation of the arteries, to play the role of plaque stabilization antiplatelet pull aggregation in the secondary prevention of ischemic stroke,and also improve endothelial function and reduce the risk of bleeding (cardiology).Recent studies have found that cilostazol application reduces neointimal hyperplasia and remodeling after stenting (PCI), resistance to late restenosis role, but the development of azole anti-As cilostazol and may mechanism of action currently unclear the Lee study found, cilostazol can reduce low-density lipoprotein (LDL) receptor-deficient mice atherosclerotic area and reduces the synthesis of TNF-a, while reducing the accumulation of plaque local macrophages, MCP-1decreased. In vitro tests showed that cilostazol inhibit MCP-1and MMP-9mRNA expression in THP-1monocytes, of these mechanisms may reduce inflammation. Another study also showed that, Cilostazol inhibits LPS-induced macrophage IL-1β,IL-6and TNF-a synthesis, and this effect is caused by inhibiting the activity of NF-KB to play the anti-atherosclerotic effect, play an important role in the treatment of coronary heart disease.The monocyte macrophages is the most common inflammatory cells in the formation process of atherosclerosis, is also the important pathophysiological basis in vulnerable plaque formation, not only play a key role in the initiation and progression of atherosclerosis, also plays an important role in plaque rupture and thrombosis. There are some studies have shown that atherosclerotic plaque monocytes stimulated secretion of IL-6is a central inflammatory response adjustment factor, plays a key role in vascular injury of coronary heart disease and acute myocardial ischemia.Cilostazol is a new type of anti-platelet drugs, not only has anti-platelet aggregation, but also can promote arterial dilation, inhibition of smooth muscle cell proliferation and intimal thickening. Cilostazol plays an important role in the prevention and treatment of coronary heart disease, but the mechanism of cilostazol anti-atherosclerosis is not clear. Cilostazol whether influence on LPS-induced human THP-1monocyte cells to secrete IL-6, and what is the mechanism of this action.This subject divided into two parts, the human THP-1monocytes cultured in vitro as the object of study, and were divided into a control group, LPS group, cilostazol azole low solubility group, cilostazol in solubility group,cilostazol high concentrations.The cilostazol groups add with different solubility (10,20,40μmol/L) intervention12h and then adding LPS stimulation24h.(1)Detect the cell culture supernatant IL-6levels by ELISA, and to observe the changes of IL-6levels, explore the affection of cilostazol on IL-6secrete by LPS-induced human THP-1monocytes. (2) Detect the expression of each group the cytoplasm the IκBα and nucleus NF-κB P65by Western blot,to observe the changes of expression of IκBα and nucleus NF-κB P65explore the mechanism of cilostazol on IL-6secreted by LPS-induced human THP-1monocyte cells. The arm of the research is offering the rational use of drugs designed for clinical coronary heart disease treatment. The results are summarized as follows:1Cilostazol can inhibit the interleukin-6synthesis by LPS-induced human THP-1monocyte1.1Different concentrations of cilostazol intervention on human THP-1monocyteThe Human THP-1monocytes as experimental subjects, were divided into five groups, the control group, LPS group, cilostazol azole low concentration group (LPS100μg/L+cilostazol10μmol/L), cilostazol concentration group (LPS100μg/L+cilostazol-20μmol/L), scirocco his the azole high concentration group (LPS100μg/L+cilostazo-40μmol/L), the experimental group, adding different concentrations of cilostazol azole intervention12h, and then give a final concentration of100μg/L LPS for24h.1.2CCK-8detects the impact of drugs on cell proliferation activityCilostazol pre intervention for12hours, the then combined effects of LPS was added after24hours, compared with the control group, cilostazol10μmol/L,20μmol/L,40μmol/L the plus LPS100μg/L combined effect on the cell survival rate without significant effect (P>0.05), while the combined effects of cilostazol80μmol/LL plus LPS1OOμg/L cells have a significant effect (P<0.05).1.3Enzyme-linked immunosorbent assay (ELISA), to detect the level of IL-6Different concentrations of cilostazol can inhibit LPS100μg/L induced human THP-1single-nucleated cells synthesis of interleukin-6, with the dose increased, and this inhibition is becoming increasingly obvious, when cilostazol dose of40μmol/L, suppression role with LPS group compared to the differences were statistically significant (P<0.05).2The influence on LPS-induced human THP-1single nuclear cytoplasmic protein IκBα and nuclear protein NF-κB P65level by cilostazol 2.1Western blot detection of cytoplasmic protein IκBα and nuclear protein NF-κB P65expressionControl group of human monocyte plasma protein IκBα highest level, the nucleoprotein NF-κB P65lowest levels, LPS group human monocytes sarcoplasmic proteins IκBα lowest nucleoprotein NF-κB P65highest level (P<0.05). Added Helo-pre-intervention12h, the TPH-1monocyte cytoplasmic protein IκBα levels decline and Helo-inhibit LPS-induced NF-κB P65nucleoprotein level of increase, with increasing concentration, this inhibition the stronger the effect, the difference was significant (P<0.05).Through the two part of the experiment, we can draw the following conclusions:①Cilostazol was concentration-dependently inhibited the LPS-induced THP-1monocyte IL-6synthesis.②Cilostazol inhibited LPS-induced THP-1monocyte IL-6synthesis, may refer to the reduction degradation of IκBα cell activation process and P65moving from the cytoplasm to the nucleus translocation thereby inhibiting NF-κB activation.