| Background and Objective: It has become evident that atherosclerosis is an inflammatory disease with immune responses during initiation and progression of this disease. In general,the immune system responds effectively to harmful agents by two closely related pathways: the innate and the adaptive immune recognition systems. The innate immune response is the first line of defense and is limited to recognize highly conserved pathogen motifs, entitled pathogen-associated molecular patterns (PAMPs) . Recently, Toll-like receptors (TLRs) have been identified as key recognition components of the innate immune system in mammals. In addition to their crucial role in innate immunity, TLRs ( especially TLR4) have recently been associated with initiation and progression of atherosclerosis. Some evidences showed that Toll-like receptor 4 was expressed by different cell types in the atherosclerotic vessel wall: endothelial cells , macrophages, adventitional fibroblasts and dendritic cells, it is prominently expressed and activated in lipid-rich macrophage- infiltrated human atherosclerotic plaques. Expression of TLR4 in athero- sclerotic plaques has led to the hypothesis that TLR4 might be involved in the development and progression of atherosclerotic disease.Dynamic instability of a coronary atherosclerotic plaque is now seen as the foundation for the development of the clinical syndromes which we recognize as unstable angina and myocardial infarction. A complex intravascular inflammatory response is an integral component of this dynamic instability. Compared to patients with chronic stable angina, patients with acute coronary syndromes have coronary plaques with much more extensive macrophage-rich areas. Activated macrophages within plaques produce a range of proinflammatory cytokines such as tumor necrosis factor-α, and interleukin (IL)-6, etc, which induce the aggravation of intravascular inflammatory responses and lead to onset of coronary events. It was demonstrated that TLR4 was involved in the instability of atherosclerotic plaque. Some studies indicated that genetic deficiency of TLR4 reduced aortic atherosclerosis in apoE-deficient mice. Furthermore, TLR4 deficiency was associated with alterations in plaque composition, including reduction in lipid and macrophage content, and markedly decreased expression of the pro-inflammatory factors. These changes revealed greater structural stability. Therefore, it has been suggested that TLR4 signaling might be important in plaque destabilization. The purpose of this study was to compare the expression of TLR4 on circulating CD14+ monocytes in patients with various clinical stages of atherosclerosis (stable angina and acute coronary syndrome) and to better understand the association of TLR4 with the progression and deterioration of coronary artery disease.HMG-CoA reductase inhibitors, statins, inhibit the biosynthesis of cholesterol and associated precursors, which are isoprenoid products of mevalonate. In addition, they reduce morbidity and mortality of patients with atherosclerotic diseases.However, benefits from statin therapy appear to beyond their cholesterol- lowering effect. Statin treatment results in inhibition of NF-κB activity and subsequent reduction of the proinflammatory cytokines such as TNF-α, IL-6, etc. Furthermore, statins inhibit lipopolysaccharide (LPS) mediated activation of human peripheral mononuclear cells and endothelial cells. The goal of our study was to investigate the impact of statins on expression of TLR4 in CD14+ monocytes in patients with ACS and to explore their mechanisms of antiinflammatory and plaque stability effect.Magnolol (Mag), an active constituent isolated from the Chinese herb Hou p'u (Magnolia officinalis), is known to possess therapeutic properties such as antiplatelet aggregation, vessel dilatation, anticancer and anti-inflammatory activities. Mag may have therapeutic potential for the treatment of chronic inflammatory diseases associated with atherosclerosis, asthma, and cancer. Mag possesses anti-inflammatory properties that might contribute to its atheroprotective effects. However, the detailed mechanisms of its anti-inflammatory effect remain to be defined. Our study was to investigate the effect of Mag on expression of TLR4 in monocytes in patients with ACS and to explore their mechanisms of atheroprotective effects.Methods:(1)121 cases (include controls:22, stable angina:17, acute coronary syndrome:82) were investigated. Blood samples were obtained from all cases and the the concentration of hsCRP,IL-12, TNF-αin serum, the expression of TLR4 on circulating monocytes were determined. TLR4 expression on CD14+ monocytes were quantified via flow-cytometry. IL-12 and TNF-αwere measured by ELISA.(2) 41patients with ACS were randomized to prescribe either atorvastatin 10mg/d (standard lipid-lowering therapy) or atorvastatin 40mg/d (intensive lipid-lowering therapy) for one month in addition to their routine anti- anginal treatment. Expression of TLR4 on monocyets and serum level of hsCRP,IL-12 and TNF-αwere investigated. Isolated and cultured peripheral blood mononuclear cells in patients with ACS were divided into five groups: A: controls (RPMI1640 medium), B:LPS(1mg/L), C: statin (atorvastating 10umol/L),D:LPS+atorvastatin,E:LPS+atorvastatin+ mevalonate(500umol/L). TLR4 expression in monocytes after incubation for 24 hours was measured via quantitative RT-PCR. The effects of atorvastatin on cytokine protein expression in monocytes were analyzed by ELISA. (TNF-αand IL-12 in the supernatants).(3) Isolated and cultured peripheral blood mononuclear cells from patients with ACS were divided into four roups: A: controls(RPMI1640 medium), B:LPS(1mg/L), C: magnolol(20uM), D: LPS+magnolol. TLR4 expression in monocytes after incubation for 24 hours was measured via quantitative RT-PCR. The effects of magnolol on cytokine protein expression in monocytes were analyzed by ELISA (TNF-αand IL-12 in the supernatants). Results:(1) TLR4 expression on CD14+ monocytes in patients with ACS was higher than patients with stable angina(p<0.05)and controls (p<0.05). The concentration of hsCRP, IL-12 and TNF-αin patients with ACS were also higher than that in patients with stable angina(p<0.05)and normal persons(p<0.05).(2) TLR4 expression on CD14+ monocytes was downregulated and the serum level of hsCRP, IL-12, TNF-αwere decreased after one month treatment with atorvastatin(p<0.05). The efficacy of intensive lipid- lowering therapy on them was more effective than that of standard lipid- lowering therapy(p<0.05). Atorvastatin incubation resulted in reduction of TLR4 expression compared with untreated monocytes(p<0.05). TNF-αand IL-12 in the supernatants decreased after incubation with atorvastatin (p<0.05), and these effects could be reversed in part by mevalonate.(3) Magnolol incubation resulted in reduction of TLR4 expression compared with untreated monocytes(p<0.05). TNF-αand IL-12 in the supernatants decreased after incubation with magnolol(p<0.05).Conclusions:(1)ACS is associated with enhanced expression of TLR4 in circulating monocytes and increased inflammatory responses.(2)The effect of intensive lipid-lowering on plaque stability may be associated with its antiinflammation effect through inhibition of TLR4 signaling pathway, which is in part by mevalonate path.(3)Magnolol has anti-inflammatory effects. The mechanisms of its anti-inflammatory effect may be mediated by inhibition of TLR4 signaling. |
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