| BackgroundCoronary artery disease is a kind of common disease endangering the human health seriously and the primary cause of mortality in China and around the world. Taking China as an example, the incidence of coronary heart disease increased year by year. In addition, atherosclerosis (Atherosclerosis, AS) is the 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. Therefore, the aim to explore effective drug for regulating the physiological function in VSMCs has became a significance job for the prevention and treatment of AS.A large amount of new studies manifested that oxidized low density lipoprotein(Ox-LDL) is an incitant factor in the progress of AS. The initial stages of AS is the low density lipoprotein permeate in vascular wall where endothelial cell injuried and induce multiple chemokines secretion. After that, the various cytokines and growth factors produced from activated of mononuclear cells stimulate smooth muscle cell migration, proliferation.Monocyte chemoattractant protein-1is an important chemotacticcytokine that produced from endothelial cell, macrophage, smooth muscle cell,which have an ability to promotes chemotaxis and activation of monocytes/macrophages. In the blood, monocytes stimulated with MCP-1migrate and accumulate in the intima and play essential biological effects such as phagocytosing lipid and becoming foamy cells. Collectively, MCP-1plays an important role in the pathogenesis of progress of atherosclerosis, which suggest that suppression of MCP-1expression and excessive proliferation in VSMCs may have great therapeutic potential for atherosclerosis. Curcumin extracted from turmeric, radix, rhizoma is a monomer of polyphenol compounds in chinese medicine. Several lines of evidence have showed that the therapeutic effects of curcumin, such as its anti-inflammatory, anti-oxidant, pro-apoptotic and proliferation inhibition, have been demonstrated. However, the underlying molecular mechanisms of such protective effect that may be benefit for anti-atherosclerosis are not fully understood. Therefore, these experiments were conducted to examined the effect of curcumin on MCP-1expression and proliferation/apoptosis in VSMCs and elucidated the related molecular mechanisms.ObjectivesPrimary cultured VSMCs separated from rat aorta was chosen as a model to identify the effect of curcumin on MCP-1production, proliferation and pro-apoptotic in VSMCs, and sequentially elucidate the related molecular mechanisms by preforming modern molecular biology, such as Western blot, RT-PCR, MTT, flow cytometer methods and so on. These results may be 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 rat vascular smooth muscle cells.Rat aortic smooth muscle cells were obtained from the thoracic aorta of male Sprague-Dawley rats. Isolated VSMCs were cultured in DMEM at37℃in a humidified atmosphere. The purity and identity of VSMCs were verified using a monoclonal antibody against smooth muscle a-actin. Cells from passages3-8were used in all experiments.Assessment of cell toxicity of curcumin:VSMCs were divided into six groups: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 (%), 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.The effect of Ox-LDL-induced MCP-1production in VSMCs:Experiment1, VSMCs were divided into four groups:1)control group,2)Ox-LDL(10μg/ml) group,3)Ox-LDL(50μg/ml) group,4)Ox-LDL(100μg/ml) group, cells were treated for24h; Experiment2, VSMCs were divided into various indicated time groups(0,6,12,24h). ELISA was performed to determined the MCP-1concentrations in cell supernatants.The inhibitory effect of curcumin on Ox-LDL-induced MCP-1expression: VSMCs were divided into five groups:1)control group,2)Ox-LDL(100μg/ml) group,3)Ox-LDL(100μg/ml)+curcumin(5μM)group,4)Ox-LDL(100μg/ml)+curcumin(10μM) group,5)Ox-LDL(100μg/ml)+curcumin(30μM) group, RT-PCR was performed to determine MCP-1mRNA, ELISA was performed to examine MCP-1concentrations in cell supernatants.Effect of different MAPK inhibitor (JNK, ERK1/2, p38MAPK) and nuclear factor-KB inhibitor(BAY11-7082) on Ox-LDL-induced MCP-1production: VSMCs were divided into six groups:1)control group,2)Ox-LDL(100μg/ml) group,3)Ox-LDL(100μg/ml)+SP600125(JNK inhibitor) group,4)Ox-LDL(100μg/ml)+PD98059(ERK1/2inhibitor) group,5)Ox-LDL(100μg/ml)+SB203580(p38inhibitor) group,6)Ox-LDL(100μg/ml)+BAY11-7082(NF-KB inhibitor) 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.Effect of p38inhibitor(SB203580) and NF-κB inhibitor(BAY11-7082) on Ox-LDL-induced the phosphorvlation of p38MAPK and nuclear NF-κBp65 expression:Experiment1, VSMCs were divided into three groups:1)control group,2)Ox-LDL(100μg/ml) group,3)Ox-LDL(100μg/ml)+SB203580group; Experiment2,1)control group,2)Ox-LDL(100μg/ml) group,3)Ox-LDL(100μg/ml)+BAY11-7082group; Western blot was performed to determine the phosphorylation of p38MAPK and nuclear NF-KBp65expression, which further confirm the involved pathways.Effect of curcumin on Ox-LDL-induced the phosphorvlation of p38MAPK and nuclear NF-κBp65expression:Experiment1, VSMCs were divided into five groups:1)control group,2)Ox-LDL(100μg/ml) group,3)Ox-LDL+curcumin (5μM) group,4)Ox-LDL+curcumin(10μM) group,5)Ox-LDL+curcumin(30μM) group; Western blot was performed to determine the phosphorylation of p38MAPK expression; Experiment2, VSMCs were divided into five groups:1)control group,2)Ox-LDL(100μg/ml)group,3)Ox-LDL+curcumin(5μM)group,4)Ox-LDL+curcumin(10μM)group,5)Ox-LDL+curcumin(30μM)group; Western blot was performed to determine the nuclear NF-KBp65expression.Part II. Effect of curcumin on Ox-LDL-induced proliferation and the related molecular mechanisms in rat vascular smooth muscle cells.The effect of Ox-LDL-induced proliferation in VSMCs:VSMCs were divided into five groups:1)control group,2)Ox-LDL(50μg/ml) group,3)Ox-LDL(100μg/ml) group,4)Ox-LDL(200μg/ml) group,5)Ox-LDL(300μg/ml) group. MTT was performed to examine the OD value on behalf of cell proliferation.Effect of curcumin on Ox-LDL-induced proliferation in VSMCs:VSMCs were divided into five groups:1)control group,2)Ox-LDL(100μg/ml) group,3) Ox-LDL+curcumin(20μM) group,4)Ox-LDL+curcumin(40μM) group,5)Ox-LDL+curcumin(80μM) group; MTT was performed to examine the OD value on behalf of cell proliferation.Effect of HO-1inhibitor (ZnPPⅨ) on Ox-LDL-induced proliferation in VSMCs:VSMCs were divided into four groups:1)control group,2)Ox-LDL (100μg/ml) group,3)Ox-LDL+curcumin(80μM) group,4)Ox-LDL+curcumin (80μM)+ZnPPⅨ group; MTT was performed to determined the OD value on behalf of cell proliferation. Effect of curcumin on HO-1mRNA and protein levels inVSMCs:VSMCs were divided into four groups:1) control group,2)20μM curcumin group,3)40μM curcumin group,4)80μM curcumin group, RT-PCR was performed to determine HO-1mRNA, Western blot was used to examine protein levels of HO-1.Effect of HO-1inhibitor(ZnPPⅨ) on curcumin inhibit Ox-LDL-induced proliferating cell nuclear antigen(PCNA) expression in VSMCs:VSMCs were divided into four groups:1)control group,2)Ox-LDL(100μg/ml) group,3)Ox-LDL+curcumin(80μM) group,4)Ox-LDL+curcumin(80μM)+ZnPPⅨ group; Western blot was performed to determine the PCNA expression.Part Ⅲ. Curcumin induced the apoptosis and the related molecular mechanisms in rat vascular smooth muscle cells.Effect of curcumin induced the apoptosis in rat vascular smooth muscle cells:Experiment1, VSMCs were divided into three groups:1) control group,2)50μM curcumin group,3)100μM curcumin group, Hoechst33342dye was performed to examine apoptotic morphology. Experiment2, VSMCs were divided into four groups:1) control group,2)50μM curcumin group,3)100μM curcumin group,4)120μM curcumin group; The apoptosis and death of VSMCs were determined by flow cytometry with Annexin V-FITC/PI double staining.Effect of curcumin on caspase-8p10expression in rat vascular smooth muscle cells:VSMCs were divided into four groups:1)control group,2)50μM curcumin group,3)100μM curcumin group,4)120μM curcumin group; Western blot was performed to determine the caspase-8p10expression.ResultsPart Ⅰ. Effect of curcumin on ox-LDL-induced MCP-1expression and the related molecular mechanisms in rat vascular smooth muscle cells.1) Curcumin-induced cell toxicity was negligible at concentrations of0-80μM in VSMCs:There was no statistically significant difference in control group and various concentrations curcumin group, respectively. The various concentrations curcumin group including5μM(P=0.634),10μM(P=0.309),20μM(P=0.243),40μM(P=0.202) and80μM(P=0.163).2) MCP-1production induced by Ox-LDL in VSMCs was markedly increased in a concentrations-dependent manner:VSMCs were stimulated with various concentrations Ox-LDL(10、50、100μg/ml) for24h. After that, micro amounts MCP-1in the control group still existed in cell supernatant(93.22±13.32pg/ml). Compared with control group, MCP-1production was markly elavated in10|μg/ml(P=0.000),50μg/ml(P=0.000) and100μg/ml (P=0.000) group, respectively. Compared with lOμg/mlOx-LDL group, MCP-1production in50μg/mlOx-LDL group increased to792.45±72.78pg/ml(P=0.008).100μg/mlOx-LDL group was substantially than that of the MCP-1production in50μg/mlOx-LDL group(P=0.031).3) MCP-1production induced by Ox-LDL in VSMCs was markedly increased in a time-dependent manner: VSMCs were stimulated with Ox-LDL for indicated times(0,6,12,24h). After that, micro amounts MCP-1in Oh group still existed in cell supernatant(88.89±8.83pg/ml). Compared with control group, MCP-1production was markly elavated in6h(P=0.001),12h(P=0.000) and24h(P=0.000) group, respectively. Compared with6h group, MCP-1production in12h group increased obviously (P=0.000).24h group was substantially than that of the MCP-1production in12h group(P=0.02).4) Curcumin inhibit Ox-LDL-induced MCP-1production and MCP-1mRNA expression in VSMCs:VSMCs were pretreated with curcumin and then exposed to ox-LDL. MCP-1production was significantly higher in other groups than in control group(P=0.000, P=0.000, P=0.000, P=0.003). Compared with Ox-LDL alone group, Ox-LDL+curcumin(10μM) group and Ox-LDL+curcumin(30μM) showed a significantly lower level of MCP-1(P=0.000, P=0.000). However, there were no significant differences between Ox-LDL+curcumin(5μM) group and Ox-LDL alone group(P=0.565).RT-PCR show these data:Compared with control group, MCP-lmRNA is significantly increased in other groups(P=0.000, P=0.000, P=0.00, P=0.024). Compared with Ox-LDL alone group, Ox-LDL+curcumin(10μM) group and Ox-LDL+curcumin(30μM) showed a significantly lower level of MCP-1mRNA (P=0.000,P=0.000). However, there were no significant differences between Ox-LDL+curcumin(5μM) group and Ox-LDL alone group(P=0.054).5) The p38MAPK and NF-κB is involved in MCP-1production by ox-LDL in VSMCs:VSMCs were pretreated with curcumin and different MAPK inhibitor (JNK, ERK1/2, p38MAPK), nuclear factor-KB inhibitor(BAYl1-7082), then exposed to ox-LDL. Compared with control group, MCP-1production was markly elavated in Ox-LDL alone group(P=0.000). MCP-1production in Ox-LDL+SB group and Ox-LDL+BAY group significantly lower than in Ox-LDL alone group(P=0.000and P=0.002). However, There were no significant differences between Ox-LDL+SP group and Ox-LDL+PD group(P=0.536and P=0.438). Based on results above, we ensured the possible pathway to provide a reference for the next experiment.6) The Ox-LDL upregulates the phosphated p38MAPK and nuclear NF-κBp65expression in VSMCs:Based on results above, VSMCs were pretreated with p38MAPK and NF-κB inhibitor(SB203580and BAY11-7082) for1h, respectively, then exposed to ox-LDL for30min. Compared with control group, phosphorylation of p38MAPK and nuclear NF-κBp65expression was markly elavated in Ox-LDL alone group(P=0.000and P=0.000). These effect in Ox-LDL+SB group and Ox-LDL+BAY group significantly lower than in Ox-LDL alone group(P=0.006and P=0.006).7) Curcumin inhibits Ox-LDL-induced the phosphated p38MAPK and nuclear NF-κBp65expression in VSMCs:VSMCs were pretreated with curcumin and then exposed to ox-LDL.Compared with control group, phosphorylation of p38MAPK was markly elavated in Ox-LDL alone group(P=0.000). The phosphorylation of p38MAPK in Ox-LDL+curcumin (10μM) group and Ox-LDL+curcumin(30μM) group significantly lower than in Ox-LDL alone group(P=0.000and P=0.000). However, there were no significant differences between Ox-LDL alone group and Ox-LDL+curcumin(5μM) group(P=0.108). Similarly, the nuclear NF-KBp65in Ox-LDL alone group is significantly increased compared to control group(P=0.000). The nuclear NF-KBp65in Ox-LDL+curcumin(10μM) group and Ox-LDL+curcumin (30μM) group significantly lower than in Ox-LDL alone group(P=0.001and P=0.000). However, there were no significant differences between Ox-LDL alone group and Ox-LDL+curcumin(5μM) group(P=0.796).Part Ⅱ. Effect of curcumin on Ox-LDL-induced proliferation and the related molecular mechanisms in rat vascular smooth muscle cells.1) Ox-LDL induced proliferation in rat vascular smooth muscle:VSMCs were pretreated with various concentrations Ox-LDL. Compared with control group, OD value is significantly increased in50,100,200μg/ml group(P=0.015, P=0.009, P=0.035). However, OD value in300μg/ml group significantly lower than in control group(P=0.027).2) Curcumin inhibited ox-LDL-induced proliferation in VSMCs:The above experiment show that Ox-LDL induce proliferation in VSMCs. In this experiment, we focused on the inhibitory effect of curcumin on ox-LDL-induced proliferation in VSMCs. VSMCs were pretreated with curcumin and then exposed to ox-LDL. Compared with control group, OD value was markly elavated in Ox-LDL alone group(P=0.002). OD value in Ox-LDL+curcumin(40μM) group and Ox-LDL+curcumin(80μM) group significantly lower than in Ox-LDL alone group(P=0.047and P=0.009). However, there were no significant differences between Ox-LDL alone group and Ox-LDL+curcumin(20μM) group(P=0.717).3) HO-1may mediates the inhibitory effect of curcumin on Ox-LDL induce proliferation in VSMCs:In order to identify whether HO-1mediates the inhibitory effect of curcumin on Ox-LDL induce proliferation in VSMCs, we pretreated VSMCs with ZnPPⅨ and curcumin alone or jointly, then exposed to ox-LDL. Compared with control group, OD value was markly elavated in Ox-LDL alone group(P=0.004). OD value in Ox-LDL+curcumin(80μM) group significantly lower than in Ox-LDL alone group(P=0.016). OD value in Ox-LDL+curcumin(80μM) group significantly lower than in Ox-LDL+curcumin+ZnPPⅨ group(P=0.036).4) The curcumin upregulates HO-1mRNA and protein expression in VSMCs:In order to further identify the effect that HO-1-mediated the inhibitory effect of curcumin on Ox-LDL induce proliferation in VSMCs, we pretreated VSMCs with curcumin. RT-PCR results show: Micro amounts HO-1mRNA in the control group still existed in cell(0.14±0.03). Compared with OμM group, HO-1mRNA and was markly elavated in20μM、40μM and80μM curcumin group(P=0.006, P=0.000and P=0.000). Compared with20μM group, HO-1mRNA expression was significantly lower than in40μM group(P=0.003). Compared with40μM group, HO-1mRNA expression was markly elavated in80μM(P=0.025). Similarly, Western blot results show:Compared with0μM group, protein levels of HO-1was markly elavated in20μM、40μM and80μM curcumin group(P=0.014, P=0.000and P=0.000). Compared with20μM group, protein levels of HO-1was significantly lower than in40μM group(P=0.006). Compared with40μM group, protein levels of HO-1was markly elavated in80μM(P=0.000). These data show that curcumin significantly upregulates HO-1mRNA and protein expression in a concentration-dependent manner. Combined with the experiment above, these data show that HO-1upregulated by curcumin mediated the inhibition of Ox-LDL-induced smooth muscle cell proliferation.5) The inhibitory effect of curcumin on Ox-LDL-induced PCNA expression was attenuated by HO-1inhibitor(ZnPPⅨ):In order to identify whether curcumin-induced HO-1expression have an effect on PCNA expression, we pretreated VSMCs with ZnPPⅨ and curcumin alone or jointly, then exposed to ox-LDL. Compared with control group, PCNA expression is markly elavated in Ox-LDL alone group(P=0.000). Compared with Ox-LDL alone group, PCNA expression in Ox-LDL+curcumin group was significantly lower than in Ox-LDL alone group(P=0.000). PCNA expression was inhibited in Ox-LDL+curcumin group compare to Ox-LDL+curcumin+ZnPPⅨ(P=0.006).Part III. Curcumin induced the apoptosis and the related molecular mechanisms in rat vascular smooth muscle cells.1) Curcumin induced the apoptosis in VSMCs:VSMCs were pretreated with curcumin and stained with Hoechst33342. Under a fluorescence microscope, nucleus of VSMCs in control group appear blue; In50μM group, white shiny state, condensation and densely stained appeared in a small amount of the nucleus. In100μM group, a large number nucleus of cells appeared white shiny state, condensation and densely stained.2) Flow cvtometrv further confirmed that apoptosis appeared in VSMCs:The early apoptosis rates in50μM,100μM,120μM group were significantly higher than the OμM group(P=0.024, P=0.000,P=0.000). Compared with50μM group, the early apoptosis rates was significantly lower than in100μM group(P=0.000). Compared with100μM group, the early apoptosis rates was markly elavated in120μM group (P=0.016).3) Curcumin upregulates Caspase-8p10expression in VSMCs:Caspase-8p10expression in50μM,100μM,120μM group were substantially higher than that of the OμM group(P=0.043,P=0.001, P=0.000). Compared with50μM group, the Caspase-8p10expression was markly elavated in100μM(.P=0.044).Compared with100μM group, the Caspase-8p10expression was significantly lower than in120μM group(P=0.011).Conclusions1) Curcumin-induced cell toxicity was negligible at concentrations of0-80μM in VSMCs.2)The p38MAPK and NF-κB is involved Ox-LDL-induced MCP-1production in VSMCs.3) Curcumin inhibits Ox-LDL-induced MCP-1expression by suppressing the p38MAPK and NF-κB pathways in VSMCs.4) Curcumin inhibits Ox-LDL-induced VSMCs proliferation via HO-1-mediated down-regulation of PCNA expression.5) Caspase-8activation may be one of the possible pathways to induce apoptosis by curcumin in VSMCs. |
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