| BackgroundCoronary artery disease is a kind of common disease in clinical endangering the human health seriously. Indeed, atherosclerosis (Atherosclerosis, AS) is the pathological basis and the key cause of pathophysiological changes in coronary heart disease, which eventually may lead to plaque rupture and trigger acute ischemic heart disease events, but the explicit mechanisms were not clear. Lipid deposition, oxidation and inflammation were recognized as the basic pathological characteristics of the formation of atherosclerotic plaque. Therefore the abnormal metabolism of lipid is one of the most important risk factors. Oxidative modificated low density lipoproteinthe would injury endothelial function, promote cholesterol deposition under endothelium and macrophage migration, which leading to the formation of foam cells and activation of inflammatory response and accelerating the progress of AS. So Ox-LDL and macrophages in the AS early lesions, both play an important role in terms of inflammatory activation and lipid metabolism.Monocyte chemotactic protein1belongs to the chemokine (chemotactic cytokines, C-C) family, which is produced from endothelial cell, macrophage and smooth muscle cell. In the atherosclerotic plaque, monocytes stimulated with MCP-1migrate and accumulate in the intima and play essential biological effects such as phagocytosing lipid and becoming foamy cells. MCP-1plays an important role in the development of AS, suggesting that MCP-1may be one of important therapeutic target for atherosclerosis.Reverse cholesterol transport (RCT) means transporting cholesterol from peripheral tissues (including atherosclerotic plaque) into the liver for recycling or being excreted in bile, which includes the cholesterol removal in liver and the cholesterol efflux in peripheral tissue. Cholesterol efflux transport proteins in peripheral tissue such as ABCA1, ABCG1and SR-BI, are expressed in macrophage membrane surface, and participate the process of lipid transfer in macrophages. Therefore, how to improve the reverse cholesterol transport cholesterol transporter mediated current control, is also a research hotspot in AS.Curcumin is an active drug extracted from ginger monomer, and its molecular formula is C21H20O6, corresponding to a molecular weight of368.37. Cuecumin is slightly soluble in water, while soluble in organic solvents such as ethanol. There have been many studies confirmed that curcumin exhibit anti-inflammatory, antioxidant, inhibiting tumor and regulate energy metabolism and other biological activity, widely involved in cell proliferation, inflammation, apoptosis signal pathway regulating mechanism. Other studies have found that curcumin inhibit the secretion of inflammatory mediators, elevate HDL-C levels in animal models and promote lipid metabolism, but many of the specific mechanisms remain to be investigated.ObjectivesRaw264.7cells and THP-1-derived macrophages were chosen as the models to identify the effect of curcumin on MCP-1production and lipid metabolism, and we sequentially elucidate the related molecular mechanisms by preforming modern molecular biology, such as ELISA, Western blot, and radioactive isotope labeling technique. These results may provide theoretical basis for the application of curcumin in the treatment of cardiovascular disease.MethodsPart Ⅰ. Effect of curcumin on ox-LDL-induced MCP-1expression and the related molecular mechanisms in macrophages1. Assessment of cell toxicity of curcumin:Cells were divided into6groups:1)control group,2)5μM curcumin group,3)10μM curcumin group,4)20μM curcumin group,5)40μM curcumin group,6)80μM curcumin group; After cells were treated for24h, MTT was performed to detect the cell viability (OD value), which exclude the possibility that reductions of the levels of inflammatory cytokine from the cells were due to direct toxicity of curcumin to the cells.2. The effect of Ox-LDL-induced MCP-1production in macrophagesConcentration effect:Cells were divided into5groups:1) control group,2) Ox-LDL(10μg/ml) group,3) Ox-LDL(20μg/ml) group,4) Ox-LDL(40μg/ml) group,5) Ox-LDL(80μg/ml) group, and cells were treated for24h;Concentration effect:Cells were divided into various indicated time groups(0,6,12,24h). Cells were treated by Ox-LDL(80μg/ml).ELISA was performed to determined the MCP-1concentrations in cell supernatants.3. The inhibitory effect of curcumin on Ox-LDL-induced MCP-1expression in macrophagesCells were divided into6groups:1) control group,2) Ox-LDL(80μg/ml) group,3) Ox-LDL(80μg/ml)+curcumin(5μM)group,4) Ox-LDL(80μg/ml)+curcumin(10μM) group,5) Ox-LDL(80μg/ml)+curcumin(20μM) group,6) Ox-LDL(80μg/ml)+curcumin(40μM) group. ELISA was performed to examine MCP-1concentrations in cell supernatants. 4. Effect of different MAPK inhibitor (p38MAPK, JNK, ERK1/2) and NF-κB inhibitor on Ox-LDL-induced MCP-1production in Raw264.7cellsCells were divided into6groups:1) control group,2) Ox-LDL(80μg/ml) group,3) Ox-LDL(80μg/ml)+SB203580(p38inhibitor) group,4) Ox-LDL(80μg/ml)+SP600125(JNK inhibitor) group,5) Ox-LDL(80μg/ml)+PD98059(ERKl/2inhibitor) group,6) Ox-LDL(80μg/ml)+BAY11-7082(NF-κB) group.ELISA was performed to examine MCP-1production in cell supernatants. These data show the possible pathways that involved in the Ox-LDL-induced MCP-1production, which would provide reference for the next experiments.5. Effect of JNK inhibitor (SP600125) on Ox-LDL-induced the phosphorylation of JNK in Raw264.7cellsCells were divided into3groups:1) control group,2) Ox-LDL(80μg/ml) group,3) Ox-LDL(80μg/ml)+SP600125group.Western blot was performed to determine the phosphorylation of JNK pathway, which further confirms the involved pathways.6. Effect of NF-κB inhibitor (BAY11-7082) on Ox-LDL-induced the NF-κB p65in nuleus in Raw264.7cells:Cells were divided into3groups:1) control group,2) Ox-LDL(80μg/ml) group,3) Ox-LDL(80μg/ml)+BAY11-7082group.Western blot was performed to determine the NF-κB p65level in nuleus, which further confirms the involved pathways.7. Effect of curcumin on Ox-LDL-induced the phosphorylation of JNK and NF-κB p65in nuleus in Raw264.7cells:Cells were divided into6groups:1) control group,2) Ox-LDL(80μg/ml) group,3) Ox-LDL+curcumin (5μM) group,4) Ox-LDL+curcumin(10μM) group,5) Ox-LDL+curcumin(20μM) group,5) Ox-LDL+curcumin(40μM) group.Western blot was performed to determine the phosphorylation of JNK expression and the NF-κB p65level in nuleus. Part II. Effect of curcumin on Ox-LDL-induced cholesterol efflux and the related molecular mechanisms in macrophages.1. Effect of curcumin on Ox-LDL induced cholesterol efflux rate in macrophagesCells were divided into5groups:1) control group,2) curcumin (5μM) group,3) curcumin(10μM) group,4) curcumin(20μM) group,5) curcumin(40μM) group.The radioactive isotope labeling method was performed to detect the cholesterol efflux rate of Raw264.7cells and THP-1-derived macrophages.2. Effect of curcumin on Ox-LDL induced expression of cholesterol transport related proteins in Raw264.7cellsCells were divided into5groups:1) control group,2) curcumin (5μM) group,3) curcumin(10μM) group,4) curcumin(20μM) group,5) curcumin(40μM) group.The expression of lipid transfer protein was detected by Western blot.3. Effects of different MAFK pathway inhibitor or NF-κB pathway inhibitor on cells cholesterol efflux rate in Raw264.7cellsCells were divided into5groups:1) control group,2) SB203580(p38inhibitor) group,3) SP600125(JNK inhibitor) group,4) PD98059(ERK1/2inhibitor) group,5) BAY11-7082(NF-κB inhibitor) group;The radioactive isotope labeling method was performed to detect the cholesterol efflux rate of Raw264.7cells..4. Effect of JNK inhibitor on Ox-LDL induced expression of cholesterol transport related proteins in Raw264.7cellsCells were divided into3groups:1) control group,2)10μM SP600125group,3)20μM SP600125group.The expression of lipid transfer protein was detected by Western blot. ResultsPart I. Effect of curcumin on ox-LDL-induced MCP-1expression and the related molecular mechanisms in macrophages1. Curcumin-induced cell toxicity was negligible at concentrations of0-40μM in macrophagesThere was statistically significant difference between control group and80μM curcumin group, but no statistically significant difference was noted between control group and various concentrations curcumin group (0-40μM). Thus there was no significant effect on the survival activity of0-40μM concentration of curcumin in macrophages.2. MCP-1production induced by Ox-LDL in macrophages was markedly increased in a concentrations-dependent manner and in a time-dependent mannerRaw264.7cells were stimulated with various concentrations Ox-LDL (0-80μg/ml) for24h. MCP-1production was markly elavated in a concentrations-dependent manner (F=58.91, P<0.001and F=86.98, P<0.001). Microscale of MCP-1in control group still existed in cell supernatant (93.22±13.32pg/ml). Compared with control group, MCP-1production was markly elavated in10μg/ml (P<0.001),20μg/ml (P<0.001),40μg/ml (P<0.001) and80μg/ml (P<0.001) group, respectively. Compared with control group, MCP-1production in80μg/ml Ox-LDL group increased to996.32±122.87pg/ml.And the MCP-1of THP-1-derived macrophages induced by ox-LDL were as similar as Raw264.7cells.Raw264.7cells were stimulated with80μg/ml Ox-LDL for indicated times (0,6,12,24h). Compared with control group, MCP-1production was markly elavated in6h,12h and24h group, respectively (F=99.80, P<0.001and F=68.62, P<0.001). Compared with control group, MCP-1production in24h group increased obviously (894.30±85.89pg/ml).And the results of THP-1-derived macrophages were as similar as Raw264.7cells. 3. Curcumin inhibit Ox-LDL-induced MCP-1production in Raw264.7cells and THP-1-derived macrophages:Cells were pretreated with curcumin and then exposed to80μg/ml ox-LDL. MCP-1production was significantly higher in other groups than in control group (F-75.69, P<0.001). Compared with Ox-LDL alone group, Ox-LDL+curcumin (10μM), Ox-LDL+curcumin (20μM) and Ox-LDL+curcumin (40μM) showed a significantly lower level of MCP-1(P<0.001, P<0.001, P<0.001). However, there were no significant differences between Ox-LDL+curcumin (5μM) group and Ox-LDL alone group (P=0.09). And the results of THP-1-derived macrophages were as similar as Raw264.7cells (F=71.39, P<0.001).4. The JNK pathway and NF-κB pathway were involved in MCP-1production by ox-LDL in Raw264.7cellsCells were pretreated with curcumin and different MAPK inhibitor (p38MAPK, JNK, ERK1/2), then exposed to ox-LDL (80μg/ml). Compared with control group, MCP-1production was markly elavated in Ox-LDL alone group (802.44±83.86pg/ml)(P<0.01). MCP-1production in Ox-LDL+SP group and Ox-LDL+BAY group were significantly lower than in Ox-LDL alone group (576.20±41.82pg/ml)(P<0.01)()(P<0.01). However, There were no significant differences between Ox-LDL+SB group and Ox-LDL+PD group (P=0.404and P=0.301). Based on results above, we ensured the possible pathway to provide a reference for the next experiment.5. The Ox-LDL activated the phosphorylation of JNK pathway in Raw264.7cells:Based on results above, cells were pretreated with JNK inhibitor(SP600125) for1h, then exposed to ox-LDL for30min. Compared with control group, phosphorylation of JNK was markly elavated in Ox-LDL alone group (P=0.003). The effect in Ox-LDL+SP group is significantly lower than in Ox-LDL alone group (P=0.028).6. The Ox-LDL upregulated the level of NF-κB p65in nucleus in Raw264.7 cellsBased on results above, cells were pretreated with NF-κB inhibitor (BAY11-7082) for lh, then exposed to ox-LDL for30min. Compared with control group, NF-κB p65in nucleus was markly elavated in Ox-LDL alone group (P=0.002). The effect in Ox-LDL+BAY group is significantly lower than in Ox-LDL alone group (P=0.032).7. Curcumin inhibits Ox-LDL-induced the phosphorylation of JNK pathway and the level of NF-κB p65in nucleus in Raw264.7cells:Cells were pretreated with curcumin for1h and then exposed to40μg/ml Ox-LDL for30min. Compared with control group, phosphorylation of JNK and NF-κB p65in nucleus were markly elavated in Ox-LDL alone group(P<0.001). The phosphorylation of JNK in Ox-LDL+curcumin (10μM), Ox-LDL+curcumin (20μM) and Ox-LDL+curcumin (40μM) group significantly lower than in Ox-LDL alone group(P=0.004, P<0.001and P<0.001). However, there were no significant differences between Ox-LDL alone group and Ox-LDL+curcumin (5μM) group (P=0.625).And the results of curcumin on NF-κB p65level in nucleus were as similar as JNK pathway. The NF-κB p65level in nucleus in Ox-LDL+curcumin (20μM) and Ox-LDL+curcumin (40μM) group significantly lower than in Ox-LDL alone group(P<0.001and P<0.001). However, there were no significant differences between Ox-LDL group, Ox-LDL+curcumin (5μM) group (P=0.491) and Ox-LDL+curcumin (10μM) group (P=0.060). These results revealed that curcumin reduce the MCP-1production in macrophages by inhibiting Ox-LDL-induced the phosphorylation of JNK pathway and the level of NF-κB p65in nucleus.Part Ⅱ. Effect of curcumin on Ox-LDL-induced cholesterol efflux and the related molecular mechanisms in macrophages.1. Curcumin enhanced the Ox-LDL induced cholesterol efflux rate in macrophages Raw264.7cells were stimulated by different concentrations (0-40uM) curcumin. Compared with the control group (5.60±0.61%), cholesterol efflux rate was significantly increased in curcumin10,20and40μM stimulation group (cholesterol efflux rate were6.95±0.67%,9.15±0.68%and10.68±0.79%, respectively)(P=0.016, P<0.001and P<0.001), and the effect was in a dose-dependent manner in the experimental concentration range. But there was no different noted between control group and5uM stimulation group (5.90±0.76%).The results of THP-1-derived macrophages were as similar as Raw264.7cells. Compared with the control group (7.39±1.25%), cholesterol efflux rate was significantly increased in curcumin10,20and40μM stimulation group (cholesterol efflux rate were11.77±0.666%,14.43±0.684%and15.67±0.786%, respectively)(P=0.002, P<0.001and P<0.001), and the effect was in a dose-dependent manner in the experimental concentration range. But there was no different noted between control group and5uM stimulation group (7.89±1.49%).2. Curcumin up-regulated cholesterol metabolism related proteins expression (ABCA1, SR-BI, LXR a) induced by Ox-LDL in Raw264.7cellsRaw264.7cells were stimulated by different concentrations of curcumin. Compared with the control group, curcumin (10μM,20uM and40uM) significantly increased the expression level of ABCA1, SR-BI and LXRa proteins (F=27.604, P<0.001; F=48.577, P<0.001; F=33.054, P<0.001), and effect was in a dose-dependent manner. But there is no significant difference on ABCG1protein expression between each group(F=0.693, P=0.614). These results revealed that curcumin up-regulated expression of cholesterol transport proteins to enhance reverse cholesterol transport function.3. Inhibitors of JNK enhanced Ox-LDL induced cholesterol efflux rate in Raw264.7cellsCells were treated by different MAPK inhibitors () and NF-kB inhibitor (BAY11-7082). Compared with the control group, JNK inhibitor (SP600125) group of cholesterol efflux rate increased significantly (5.348±0.589%vs.7.878± 0.668%)(P<0.001).But there was no obvious change of cholesterol efflux rate in other groups. The results showed JNK pathway may be involved in change of cholesterol transport.4. Inhibitors of JNK raise the expression of ABCA1, SR-BI protein induced by Ox-LDL in Raw264.7cells, but do not affect the expression of LXRa proteinRaw264.7cells were stimulated by different concentrations of JNK inhibitor. JNK inhibitor significantly unregulated the expression of ABCA1and SR-BI proteins (F=18.034, P=0.003and F=16.469, P-0.004). But the regulation of nuclear protein LXR alpha was not obviously influenced (F=0.352, P=0.717). Combining with the results of part1, curcumin could inhibit the phosphorylation of JNK pathway then enhance the expression of transportant proteins and cholesterol efflux function, which is independent of the effect that up-regulate the expression of the nuclear protein LXRa.Conclusions1) Curcumin-induced cell toxicity was negligible at concentrations of0-40μM in macrophages.2) Ox-LDL induced MCP-1secretion in macrophages, and the effect was in concentration dependent manner and time dependent manner.3) The JNK pathway and NF-kB pathway mediated the regulation of MCP-1secretion induced by Ox-LDL in macrophages.4) Curcumin inhibited JNK phosphorylation and NF-kB activation, then reduced Ox-LDL induced MCP-1secretion in macrophages.5) Curcumin up-regulated the expression of LXRa, then enhanced reverse cholesterol transport channel protein ABCA1, SR-BI protein expression. Thus the intracellular cholesterol efflux rate was enhanced by this classic pathway. 6) Curcumin enhanced the expression of lipid transport membrane proteins (ABCAl and SR-BI) and cholesterol efflux rate by inhibiting the phosphorylation of JNK pathway induced by Ox-LDL in macrophages. And this effect would be independent of the activation effect of LXR alpha-ABCA1/SR-BI by curcumin. |
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