姜黄素对巨噬细胞中单核细胞趋化蛋白-1表达及逆胆固醇转运的影响及其机制研究
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摘要
研究背景
冠状动脉粥样硬化性心脏病(coronary heart disease,CAD)已成为临床最常见的心脏疾病。动脉粥样硬化(atherosclerosis, AS)则是冠心病最基本的的病理生理学基础,但目前AS的发病机理仍未完全明确。目前公认的脂质沉积、氧化修饰和炎症反应均是动脉粥样硬化斑块形成的基本病理特征。因此血脂代谢异常是其最重要的危险因素之一,经过氧化修饰后的低密度脂蛋白(Oxdized low-density lipoprotein, Ox-LDL)则极易损伤内皮功能、促进胆固醇的内皮下沉积和激活巨噬细胞迁移与吞噬,最终形成泡沫细胞并激活进一步的炎症反应,加速AS的演变。因此Ox-LDL和巨噬细胞在AS病变早期,无论在炎症激活和脂质代谢方面均发挥有重要作用。
单核细胞趋化蛋白1(monocyte chemoattractant protein-1, MCP-1)属于趋化因子(chemotactic cytokines, C-C)亚族的主要成员之一。斑块结构内的主要组成细胞,包括单核／巨噬细胞、内皮细胞、成纤维细胞和平滑肌细胞等在多种炎症刺激下均可被诱导分泌MCP-1,进一步诱导单核／巨噬细胞迁移至血管内皮下吞噬脂质,形成泡沫细胞,从而加速AS的进展。因此,MCP-1在AS的发生发展中扮演着重要角色,提示MCP-1可能为动脉粥样硬化的重要治疗靶点之一。
逆胆固醇转运(reverse cholesterol transport, RCT),即是指机体将胆固醇从周围组织(包括动脉粥样硬化斑块)转运到肝脏进行再循环或以胆酸的形式排泄的这一整个过程,它包括了主要在肝脏进行的胆固醇清除和在外周组织进行的胆固醇外流(cholesterol efflux)两个主要环节。参与外周组织胆固醇外流的转运蛋白如ATP结合盒转运蛋白A1(ATP-binding cassette transport protein A1, ABCA1)ABCA1、ATP结合盒转运蛋白Gl (ATP-binding cassette transport protein G1,ABCG1)和清道夫受体-B族Ⅰ型(Scavenger receptor class B type I, SR-BI)等,均存在于巨噬细胞膜表面,参与了巨噬细胞中将脂质从细胞内移至细胞外并与高密度脂蛋白(High-density lipoprotein, HDL)结合的过程,从而对胆固醇外流进行调节。因此,如何改善胆固醇转运蛋白所介导的逆胆固醇转运,同样是目前防治AS的一项研究热点。
姜黄素是一种从生姜中提取的活性药物单体,已经有诸多研究证实姜黄素表现出抗炎、抗氧化、抑制肿瘤和调节能量代谢等多种生物活性,广泛涉及到细胞增殖、炎症、凋亡等多种信号通路调节机制。另外有研究发现姜黄素可以抑制炎症介质分泌、升高动物体内HDL-C水平以及促进脂质代谢等抗AS和心血管保护作用,但其中许多具体机制仍有待研究。
研究目的
分别以小鼠源性的Raw264.7巨噬细胞和人源性的THP-1细胞作为研究对象,运用分子生物学技术,如酶联免疫吸附实验(ELISA)、蛋白免疫印迹实验(Western blot)、放射性同位素标记等方法,观察姜黄素对Ox-LDL刺激下巨噬细胞分泌MCP-1和逆胆固醇转运的影响,并探讨其可能的相关机制,进一步揭示姜黄素在心血管药理学方面的分子机制,为姜黄素在心血管疾病治疗上的应用提供理论依据。
研究方法
第一部分姜黄素对Ox-LDL诱导巨噬细胞MCP-1分泌的影响及相关信号通路的研究
1、药物毒性试验
实验分组：1)正常对照组,2)姜黄素5μM组,3)姜黄素10μM组,4)姜黄素20μM组,5)姜黄素40μM组,6)姜黄素80μM组；按分组给予刺激后,于细胞培养箱中干预24h,采用四甲基偶氮唑盐微量酶反应比色法(MTT)检测细胞生存活性,目的是排除在实验范围内引起细胞因子分泌水平减少是由于药物引起细胞数量减少所致的可能性,同时寻找合适的药物刺激浓度范围。
2、Ox-LDL对Raw264.7巨噬细胞及THP-1巨噬细胞MCP-1分泌的影响
1、浓度效应：实验分组：1)正常对照组,2) Ox-LDL (10μg/ml)组,3)Ox-LDL (20μg/ml)组,4) Ox-LDL (40μg/ml)组,5) Ox-LDL (80μg/ml)组,干预24h；
2、时间效应：实验分组：1)0h对照组,2)6h刺激组,3)12h刺激组,4)24h刺激组,均以80μg/ml Ox-LDL干预：
通过ELISA方法检测细胞培养基中MCP-1的浓度。
3、姜黄素对Ox-LDL诱导Raw264.7巨噬细胞及THP-1巨噬细胞MCP-1分泌的影响
实验分组：1)正常对照组,2) Ox-LDL (80μg/ml)组,3) Ox-LDL (80μg/ml)+姜黄素(5μM)组,4) Ox-LDL (80μg/ml)+姜黄素(10μM)组,5)Ox-LDL (80μg/ml)＋姜黄素(20μM)组,6) Ox-LDL (80μg/ml)+姜黄素(40μM)组。
通过ELISA方法检测细胞培养基中MCP-1浓度。
4、不同促分裂原活化蛋白激酶MAPK (Mitogen activated protein kinase, MAPK)信号通路(p38MAPK, JNK,ERK1/2)抑制剂及核转录因子-κB(NF-κB)通路抑制剂对Ox-LDL诱导Raw264.7巨噬细胞MCP-1分泌的影响
实验分组：1)正常对照组,2) Ox-LDL (80μg/ml)组,2) Ox-LDL (80μg/ml)+SB203580(p38抑制剂)组,3) Ox-LDL (80μg/ml)+SP600125(JNK抑制剂)组,4)Ox-LDL (80μg/ml)+PD98059(ERK1/2抑制剂)组,5) Ox-LDL (80μg/ml)+BAY11-7082(NF-κB抑制剂)组。
通过ELISA方法对Raw264.7细胞培养基MCP-1进行检测,通过此实验确定OxLDL诱导细胞分泌MCP-1可能被介导的信号通路,为下一步实验提供参考。
5、JNK抑制剂(SP600125)对Ox-LDL诱导Raw264.7巨噬细胞JNK磷酸化的影响
实验分组：1)正常对照组,2) Ox-LDL (80μg/ml)组,3) Ox-LDL (80μg/ml)+SP600126组。
通过Western blot检测JNK通路磷酸化水平。
6.NF-κB抑制剂(BAY11-7082)对Ox-LDL诱导Raw264.7巨噬细胞核内NF-κBp65表达的影响
实验分组：1)正常对照组,2) Ox-LDL (80μg/ml)组,3) Ox-LDL (80μg/ml)+BAY11-7082组。
提取核蛋白,通过Western blot检测核内NF-κB p65蛋白水平。
7、姜黄素对Ox-LDL诱导Raw264.7巨噬细胞JNK磷酸化和核内NF-κB p65的影响
实验分组：1)正常对照组,2) Ox-LDL (80μg/ml)组,3) Ox-LDL (80μg/ml)+姜黄素(5μM)组,4) Ox-LDL (80μg/ml)+姜黄素(10μM)组,5) Ox-LDL (80μg/ml)＋姜黄素,6) Ox-LDL (80μg/ml)+姜黄素(40μM)。
分别提取细胞蛋白及核蛋白,通过Western blot检测JNK通路磷酸化和核内NF-κB p65蛋白水平。
第二部分姜黄素对Ox-LDL诱导下巨噬细胞逆胆固醇转运的影响及相关机制探讨
1、姜黄素对Ox-LDL诱导下Raw264.7细胞及THP-1巨噬细胞中胆固醇流出率的影响
实验分组：1)对照组,2)姜黄素(5μM)组,3)姜黄素(10μM)组,4)姜黄素(20μM)组,5)姜黄素(40μM)组；
通过放射性同位素标记法来检测各组样品的胆固醇流出率。
2、姜黄素对Ox-LDL诱导下Raw264.7巨噬细胞胆固醇转运相关蛋白表达的影响
实验分组:1)对照组,2)姜黄素(5μM)组,3)姜黄素(10μM)组,4)姜黄素(20μM)组,5)姜黄素(40μM)组；
提取蛋白,通过Western blot检测脂质转运相关蛋白表达情况。
3. MAPK通路抑制剂及NF-κB抑制剂对Ox-LDL诱导下Raw264.7巨噬细胞胆固醇流出率的影响
实验分组:1)对照组,2)SB203580(p38抑制剂)组,3)SP600125(JNK抑制剂)组,4)PD98059(ERK1/2抑制剂)组,5) BAY11-7082(NF-κB抑制剂)组；
通过放射性同位素标记法来检测细胞样品的胆固醇流出率。
4、JNK抑制剂对Ox-LDL诱导下Raw264.7巨噬细胞胆固醇转运相关蛋白表达的影响
实验分组：1)对照组,2)SP600125(10μM)组,3) SP600125(20μM)组；
按分组行不同刺激后,提取细胞蛋白,通过Western blot检测脂质转运相关蛋白表达情况。
研究结果
第一部分姜黄素对Ox-LDL诱导巨噬细胞MCP-1分泌的影响及相关信号通路的研究
1、姜黄素在0-80μM浓度范围内对巨噬细胞生存活性的影响
用不同浓度姜黄素(0,5,10,20,40,80μM)刺激Raw264.7细胞及THP-1巨噬细胞后,各组之间行整体比较,组间生存活性呈显著性差异(F=56.651,P<0.001)(F=17.911,P<0.001)。进一步做组间两两比较发现,80μM刺激浓度下细胞数目急剧下降,对细胞产生明显毒性作用；而0-40μM浓度范围的姜黄素分别对Raw264.7细胞及THP-1巨噬细胞的生存活性均无明显影响。因此此实验提供了姜黄素合适的实验浓度范围参考(0-40μM),排除了在后续实验中引起的细胞因子分泌水平减少是由于药物毒性作用引起细胞数量减少或活性降低所致的可能干扰因素。
2、Ox-LDL可诱导巨噬细胞分泌MCP-1,在本实验的浓度范围内分别呈浓度依赖性和时间依赖性
不同浓度(0,10,20,40,80μg/ml) Ox-LDL作用于Raw264.7细胞及THP-1巨噬细胞后,细胞培养基中MCP-1的分泌有不同程度的增加(F=58.69,P<0.001)(F=86.98,P<0.001),并呈浓度依赖性。与对照组比较,此实验中Ox-LDL刺激效应在80μg/ml浓度时达到最高,故此以后实验均采用80μg/ml浓度进行细胞干预。
将80μg/ml浓度的Ox-LDL作用于Raw264.7细胞及THP-1巨噬细胞不同时间点(0、6、12、24h)后,检测得细胞培养基中的MCP-1均有不同程度的增加(F=99.80, P<0.001)(F=68.62, P<0.001),并呈现时间依赖性。此实验中MCP-1分泌在24h达到最高,故此以后实验中用Ox-LDL干预细胞时间均设为24h。
3、姜黄素可抑制Ox-LDL诱导巨噬细胞MCP-1分泌
先加入不同浓度(0,5,10,20,40μM)的姜黄素预处理细胞1h,再给予80μg/ml浓度的Ox-LDL刺激24h后,检测培养基中MCP-1浓度。两种细胞系所得结果相一致,结果显示不同组间具有显著性差异(F=75.96,P<0.001)(F=71.39,P<0.001)。与阴性对照组相比,其余各组细胞培养基中MCP-1均明显偏高。而与Ox-LDL单独刺激组比较,Ox-LDL+姜黄素(10μM)、Ox-LDL+姜黄素(20μM)组和Ox-LDL+姜黄素(40μM)组细胞培养基中MCP-1水平明显减低,具有显著性差异,并且总体呈剂量依赖性。而两组细胞系中Ox-LDL+姜黄素(5gM)组和Ox-LDL单独刺激组比较,无显著性差异(P=0.090)(P=0.769)
4、JNK通路和NF-κB通路介导了Ox-LDL诱导Raw264.7巨噬细胞分泌MCP-1
将不同的MAPK信号通路(p38MAPK、JNK、ERK1/2)抑制剂(SB203580、SP600125、PD98059)以及NF-κB通路抑制剂(BAY11-7082)分别预处理细胞1h,再给予Ox-LDL (80μg/ml)刺激24h。与对照组相比,Ox-LDL单独刺激组细胞培养基中的MCP-1明显升高(802.44±83.86pg/ml),具有显著性差异(P<0.001)。与Ox-LDL单独刺激组相比,Ox-LDL+SP组及Ox-LDL+BAY组细胞培养基中的MCP-1分别明显降低(576.20±41.82pg/ml)(P<0.001)(658.34±63.28pg/ml)(P<0.001),差异具有统计学意义；但是,Ox-LDL+SB组和Ox-LDL+PD组与Ox-LDL组无统计学差异(P=0.301和P=0.404),通过此实验发现了MCP-1分泌可能被JNK信号通路及NF-κB通路所介导,为下一步实验提供参考。
5、Ox-LDL促进Raw264.7巨噬细胞JNK磷酸化而JNK抑制剂(SP600125)可有效的抑制该通路激活
根据上一个实验结果,我们给予JNK抑制剂(SP600125)预处理细胞1h,再给与Ox-LDL(80μg/ml)组刺激30main。与对照组比较,Ox-LDL刺激组JNK磷酸化水平明显增加(P=0.003)；而与Ox-LDL组比较,Ox-LDL+SP组中的细胞磷酸化JNK的水平则出现明显降低(P=0.028),组间比较均具有显著性差异。
6、Ox-LDL增加Raw264.7巨噬细胞核内NF-κB p65蛋白水平而NF-κB抑制剂(BAYll-7082)可有效的抑制该通路激活
与上一步相似,我们给予NF-κB通路抑制剂(BAY11-7082)预处理细胞1h,再给与Ox-LDL (80μg/ml)组刺激30min。与对照组比较,Ox-LDL刺激组核内NF-κB p65水平明显增加(P=0.002)；而与Ox-LDL组比较,Ox-LDL+BAY组中的细胞核内的NF-κB p65水平则出现明显降低(P=0.032),组间比较均具有显著性差异。
7、姜黄素可抑制Ox-LDL诱导Raw264.7巨噬细胞JNK磷酸化及核内NF-κBp65表达
给予不同浓度姜黄素(0-40μM)预处理后,再给予Ox-LDL (80μg/ml)刺激30main。与对照组比较,Ox-LDL刺激组磷酸化JNK表达水平明显升高(P<0.01),差异具有显著性；与Ox-LDL刺激组比较,姜黄素(10、20、40μM)+Ox-LDL组均可明显下调JNK磷酸化水平(P=0.004、P<0.001、P<0.001),具有显著性差异。
与抑制JNK激活效应相类似的,与对照组比较,Ox-LDL刺激组核内NF-κBp65表达水平明显上升,而姜黄素(20、40μM)+Ox-LDL组均可明显下调核内NF-κB p65水平(P<0.001、P<0.001),具有显著性差异。两组实验总体效应均呈剂量依赖性。而两组实验中Ox-LDL刺激组与姜黄素(5、10mM)+Ox-LDL组比较均未见统计学差异(P=0.491,P=0.060)。综上实验结果提示:姜黄素可以通过抑制JNK通路及NF-κB通路的激活,降低Ox-LDL诱导的巨噬细胞MCP-1分泌。
第二部分姜黄素对巨噬细胞逆胆固醇转运的影响及其相关机制研究
1、姜黄素可增强Ox-LDL诱导下巨噬细胞的胆固醇流出率
将不同浓度(0,5,10,20,40μM)的姜黄素刺激Raw264.7细胞后,与对照组胆固醇流出率(5.60±0.61%)相比,姜黄素10、20和40μM刺激组胆固醇流出率明显上升(分别为6.95±0.67%,9.15±0.68%和10.68±0.79%)(P=0.016、P<0.001和P<0.001),且在实验浓度范围内成剂量依赖性。而对照组与5μM刺激组(5.90±0.76%)之间比较则无显著性差异。
与上述结果相类似,在不同浓度姜黄素刺激THP-1巨噬细胞后,与对照组流出率(7.39±1.25%)相比,姜黄素10、20和40μM刺激组胆固醇流出率也呈逐渐上升趋势(分别为11.77±1.84%,14.43±2.11%和15.67±1.71%)(P=0.002、P<0.001和P<0.001),结果均有显著性差异。而对照组与5μM刺激组(7.89±1.49%)之间比较则无显著性差异。
2、姜黄素可上调Ox-LDL诱导下Raw264.7巨噬细胞的胆固醇代谢相关蛋白(ABCAl、SR-BI、LXRα)的表达
上一步的实验结果已经证实姜黄素对逆胆固醇转运功能的影响,因此在本实验中我们着重观察姜黄素对胆固醇转运蛋白表达的调节机制：给予不同浓度(0,5,10,20,40gM)姜黄素处理细胞后,ABCA1、SR-BI、和LXRa三个蛋白表达指标的组间差异均十分显著(F=27.604,P<0.001；F=48.577,P<0.001；F=33.054,P<0.001),而ABCG1组间差异无统计学意义(F=0.693,P=0.614)与对照组比较,姜黄素(10μM、20μM与40μM)明显提高了ABCA1、SR-BI及LXRa三个蛋白的表达水平,且在实验浓度范围内呈剂量依赖效应,差异均具有统计学意义。以上实验结果提示:姜黄素可上调胆固醇代谢蛋白的表达来增强逆胆固醇转运功能。
3、JNK抑制剂可增强Ox-LDL诱导下Raw264.7巨噬细胞的胆固醇流出率
为了进一步观察上一部分实验中讨论的MAPK及NF-κB信号通路是否参与了姜黄素增强细胞中逆胆固醇转运的效应,我们分别加入不同的通路抑制剂处理细胞。与对照组比较,JNK抑制剂(SP600125)组胆固醇流出率明显升高(5.35±0.59%vs.7.88±0.67%),具有显著性差异(P<0.001)。而与对照组比较,p38抑制剂(SB203580)组、ERK抑制剂(PD98059)组和NF-κB抑制剂(BAY11-7082)组胆固醇流出率未见明显变化,差异不具有统计学意义(P=0.819,P=0.612,P=0.900)。本部分实验结果提示JNK通路可能介导了胆固醇转运的调节。
4、JNK抑制剂可上调Ox-LDL诱导下Raw264.7巨噬细胞的ABCA1、SR-BI蛋白表达,但对LXRa蛋白表达无影响
为进一步证实JNK通路介导了胆固醇逆转运变化,用不同浓度JNK抑制剂刺激Raw264.7巨噬细胞。Western Blot结果显示：JNK抑制剂可以显著上调ABCA1和SR-BI的表达,具有显著性差异(F=18.034,P=0.003；以及F=16.469,P=0.004)；但对细胞核蛋白LXRa的调节则未见明显影响,无显著性差异(F=0.352,P=0.717)。前面的实验结果已经证实,姜黄素具有JNK抑制剂样作用,可抑制JNK通路的磷酸化激活,结合本部分3、4结果,提示姜黄素可通过抑制JNK磷酸化途径调节逆胆固醇转运蛋白(ABCA1与SR-BI)的表达和胆固醇流出率,并且可能与姜黄素上调LXRa表达的通路调节效应相互独立。
结论
1)姜黄素在(0-40μM)浓度范围内无细胞毒性；
2)Ox-LDL诱导巨噬细胞MCP-1分泌,并呈浓度依赖效应和时间依赖效应；
3)JNK通路和NF-κB通路介导了Ox-LDL诱导下的巨噬细胞MCP-1分泌；
4)姜黄素分别通过抑制JNK通路磷酸化和抑制NF-κB通路激活,减少Ox-LDL诱导的巨噬细胞分泌MCP-1；
5)姜黄素可以上调巨噬细胞细胞内LXRa-ABCA1/SR-BI这一经典通路的表达,进而增强细胞内的胆固醇流出率；
6)姜黄素可以抑制Ox-LDL诱导下的JNK磷酸化途径,增强Ox-LDL诱导的巨噬细胞中胆固醇膜转运蛋白(ABCA1/SR-BI)的表达和胆固醇流出率,且该效应独立于姜黄素对LXRa-ABCA1/SR-BI通路的激活效应。
Background
Coronary 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.
Objectives
Raw264.7cells 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.
Methods
Part Ⅰ. Effect of curcumin on ox-LDL-induced MCP-1expression and the related molecular mechanisms in macrophages
1. 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 macrophages
Concentration 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 macrophages
Cells 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 Raw264.7cells
Cells 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 Raw264.7cells
Cells 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 macrophages
Cells 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 Raw264.7cells
Cells 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 Raw264.7cells
Cells 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 Raw264.7cells
Cells were divided into3groups:1) control group,2)10μM SP600125group,3)20μM SP600125group.
The expression of lipid transfer protein was detected by Western blot.
Results
Part I. Effect of curcumin on ox-LDL-induced MCP-1expression and the related molecular mechanisms in macrophages
1. Curcumin-induced cell toxicity was negligible at concentrations of0-40μM in macrophages
There 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 manner
Raw264.7cells 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 Raw264.7cells
Cells 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 cells
Based 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 Raw264.7cells
Raw264.7cells 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 Raw264.7cells
Cells 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 protein
Raw264.7cells 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.
Conclusions
1) 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.
冠状动脉粥样硬化性心脏病(coronary heart disease,CAD)已成为临床最常见的心脏疾病。动脉粥样硬化(atherosclerosis, AS)则是冠心病最基本的的病理生理学基础,但目前AS的发病机理仍未完全明确。目前公认的脂质沉积、氧化修饰和炎症反应均是动脉粥样硬化斑块形成的基本病理特征。因此血脂代谢异常是其最重要的危险因素之一,经过氧化修饰后的低密度脂蛋白(Oxdized low-density lipoprotein, Ox-LDL)则极易损伤内皮功能、促进胆固醇的内皮下沉积和激活巨噬细胞迁移与吞噬,最终形成泡沫细胞并激活进一步的炎症反应,加速AS的演变。因此Ox-LDL和巨噬细胞在AS病变早期,无论在炎症激活和脂质代谢方面均发挥有重要作用。
单核细胞趋化蛋白1(monocyte chemoattractant protein-1, MCP-1)属于趋化因子(chemotactic cytokines, C-C)亚族的主要成员之一。斑块结构内的主要组成细胞,包括单核／巨噬细胞、内皮细胞、成纤维细胞和平滑肌细胞等在多种炎症刺激下均可被诱导分泌MCP-1,进一步诱导单核／巨噬细胞迁移至血管内皮下吞噬脂质,形成泡沫细胞,从而加速AS的进展。因此,MCP-1在AS的发生发展中扮演着重要角色,提示MCP-1可能为动脉粥样硬化的重要治疗靶点之一。
逆胆固醇转运(reverse cholesterol transport, RCT),即是指机体将胆固醇从周围组织(包括动脉粥样硬化斑块)转运到肝脏进行再循环或以胆酸的形式排泄的这一整个过程,它包括了主要在肝脏进行的胆固醇清除和在外周组织进行的胆固醇外流(cholesterol efflux)两个主要环节。参与外周组织胆固醇外流的转运蛋白如ATP结合盒转运蛋白A1(ATP-binding cassette transport protein A1, ABCA1)ABCA1、ATP结合盒转运蛋白Gl (ATP-binding cassette transport protein G1,ABCG1)和清道夫受体-B族Ⅰ型(Scavenger receptor class B type I, SR-BI)等,均存在于巨噬细胞膜表面,参与了巨噬细胞中将脂质从细胞内移至细胞外并与高密度脂蛋白(High-density lipoprotein, HDL)结合的过程,从而对胆固醇外流进行调节。因此,如何改善胆固醇转运蛋白所介导的逆胆固醇转运,同样是目前防治AS的一项研究热点。
姜黄素是一种从生姜中提取的活性药物单体,已经有诸多研究证实姜黄素表现出抗炎、抗氧化、抑制肿瘤和调节能量代谢等多种生物活性,广泛涉及到细胞增殖、炎症、凋亡等多种信号通路调节机制。另外有研究发现姜黄素可以抑制炎症介质分泌、升高动物体内HDL-C水平以及促进脂质代谢等抗AS和心血管保护作用,但其中许多具体机制仍有待研究。
研究目的
分别以小鼠源性的Raw264.7巨噬细胞和人源性的THP-1细胞作为研究对象,运用分子生物学技术,如酶联免疫吸附实验(ELISA)、蛋白免疫印迹实验(Western blot)、放射性同位素标记等方法,观察姜黄素对Ox-LDL刺激下巨噬细胞分泌MCP-1和逆胆固醇转运的影响,并探讨其可能的相关机制,进一步揭示姜黄素在心血管药理学方面的分子机制,为姜黄素在心血管疾病治疗上的应用提供理论依据。
研究方法
第一部分姜黄素对Ox-LDL诱导巨噬细胞MCP-1分泌的影响及相关信号通路的研究
1、药物毒性试验
实验分组：1)正常对照组,2)姜黄素5μM组,3)姜黄素10μM组,4)姜黄素20μM组,5)姜黄素40μM组,6)姜黄素80μM组；按分组给予刺激后,于细胞培养箱中干预24h,采用四甲基偶氮唑盐微量酶反应比色法(MTT)检测细胞生存活性,目的是排除在实验范围内引起细胞因子分泌水平减少是由于药物引起细胞数量减少所致的可能性,同时寻找合适的药物刺激浓度范围。
2、Ox-LDL对Raw264.7巨噬细胞及THP-1巨噬细胞MCP-1分泌的影响
1、浓度效应：实验分组：1)正常对照组,2) Ox-LDL (10μg/ml)组,3)Ox-LDL (20μg/ml)组,4) Ox-LDL (40μg/ml)组,5) Ox-LDL (80μg/ml)组,干预24h；
2、时间效应：实验分组：1)0h对照组,2)6h刺激组,3)12h刺激组,4)24h刺激组,均以80μg/ml Ox-LDL干预：
通过ELISA方法检测细胞培养基中MCP-1的浓度。
3、姜黄素对Ox-LDL诱导Raw264.7巨噬细胞及THP-1巨噬细胞MCP-1分泌的影响
实验分组：1)正常对照组,2) Ox-LDL (80μg/ml)组,3) Ox-LDL (80μg/ml)+姜黄素(5μM)组,4) Ox-LDL (80μg/ml)+姜黄素(10μM)组,5)Ox-LDL (80μg/ml)＋姜黄素(20μM)组,6) Ox-LDL (80μg/ml)+姜黄素(40μM)组。
通过ELISA方法检测细胞培养基中MCP-1浓度。
4、不同促分裂原活化蛋白激酶MAPK (Mitogen activated protein kinase, MAPK)信号通路(p38MAPK, JNK,ERK1/2)抑制剂及核转录因子-κB(NF-κB)通路抑制剂对Ox-LDL诱导Raw264.7巨噬细胞MCP-1分泌的影响
实验分组：1)正常对照组,2) Ox-LDL (80μg/ml)组,2) Ox-LDL (80μg/ml)+SB203580(p38抑制剂)组,3) Ox-LDL (80μg/ml)+SP600125(JNK抑制剂)组,4)Ox-LDL (80μg/ml)+PD98059(ERK1/2抑制剂)组,5) Ox-LDL (80μg/ml)+BAY11-7082(NF-κB抑制剂)组。
通过ELISA方法对Raw264.7细胞培养基MCP-1进行检测,通过此实验确定OxLDL诱导细胞分泌MCP-1可能被介导的信号通路,为下一步实验提供参考。
5、JNK抑制剂(SP600125)对Ox-LDL诱导Raw264.7巨噬细胞JNK磷酸化的影响
实验分组：1)正常对照组,2) Ox-LDL (80μg/ml)组,3) Ox-LDL (80μg/ml)+SP600126组。
通过Western blot检测JNK通路磷酸化水平。
6.NF-κB抑制剂(BAY11-7082)对Ox-LDL诱导Raw264.7巨噬细胞核内NF-κBp65表达的影响
实验分组：1)正常对照组,2) Ox-LDL (80μg/ml)组,3) Ox-LDL (80μg/ml)+BAY11-7082组。
提取核蛋白,通过Western blot检测核内NF-κB p65蛋白水平。
7、姜黄素对Ox-LDL诱导Raw264.7巨噬细胞JNK磷酸化和核内NF-κB p65的影响
实验分组：1)正常对照组,2) Ox-LDL (80μg/ml)组,3) Ox-LDL (80μg/ml)+姜黄素(5μM)组,4) Ox-LDL (80μg/ml)+姜黄素(10μM)组,5) Ox-LDL (80μg/ml)＋姜黄素,6) Ox-LDL (80μg/ml)+姜黄素(40μM)。
分别提取细胞蛋白及核蛋白,通过Western blot检测JNK通路磷酸化和核内NF-κB p65蛋白水平。
第二部分姜黄素对Ox-LDL诱导下巨噬细胞逆胆固醇转运的影响及相关机制探讨
1、姜黄素对Ox-LDL诱导下Raw264.7细胞及THP-1巨噬细胞中胆固醇流出率的影响
实验分组：1)对照组,2)姜黄素(5μM)组,3)姜黄素(10μM)组,4)姜黄素(20μM)组,5)姜黄素(40μM)组；
通过放射性同位素标记法来检测各组样品的胆固醇流出率。
2、姜黄素对Ox-LDL诱导下Raw264.7巨噬细胞胆固醇转运相关蛋白表达的影响
实验分组:1)对照组,2)姜黄素(5μM)组,3)姜黄素(10μM)组,4)姜黄素(20μM)组,5)姜黄素(40μM)组；
提取蛋白,通过Western blot检测脂质转运相关蛋白表达情况。
3. MAPK通路抑制剂及NF-κB抑制剂对Ox-LDL诱导下Raw264.7巨噬细胞胆固醇流出率的影响
实验分组:1)对照组,2)SB203580(p38抑制剂)组,3)SP600125(JNK抑制剂)组,4)PD98059(ERK1/2抑制剂)组,5) BAY11-7082(NF-κB抑制剂)组；
通过放射性同位素标记法来检测细胞样品的胆固醇流出率。
4、JNK抑制剂对Ox-LDL诱导下Raw264.7巨噬细胞胆固醇转运相关蛋白表达的影响
实验分组：1)对照组,2)SP600125(10μM)组,3) SP600125(20μM)组；
按分组行不同刺激后,提取细胞蛋白,通过Western blot检测脂质转运相关蛋白表达情况。
研究结果
第一部分姜黄素对Ox-LDL诱导巨噬细胞MCP-1分泌的影响及相关信号通路的研究
1、姜黄素在0-80μM浓度范围内对巨噬细胞生存活性的影响
用不同浓度姜黄素(0,5,10,20,40,80μM)刺激Raw264.7细胞及THP-1巨噬细胞后,各组之间行整体比较,组间生存活性呈显著性差异(F=56.651,P<0.001)(F=17.911,P<0.001)。进一步做组间两两比较发现,80μM刺激浓度下细胞数目急剧下降,对细胞产生明显毒性作用；而0-40μM浓度范围的姜黄素分别对Raw264.7细胞及THP-1巨噬细胞的生存活性均无明显影响。因此此实验提供了姜黄素合适的实验浓度范围参考(0-40μM),排除了在后续实验中引起的细胞因子分泌水平减少是由于药物毒性作用引起细胞数量减少或活性降低所致的可能干扰因素。
2、Ox-LDL可诱导巨噬细胞分泌MCP-1,在本实验的浓度范围内分别呈浓度依赖性和时间依赖性
不同浓度(0,10,20,40,80μg/ml) Ox-LDL作用于Raw264.7细胞及THP-1巨噬细胞后,细胞培养基中MCP-1的分泌有不同程度的增加(F=58.69,P<0.001)(F=86.98,P<0.001),并呈浓度依赖性。与对照组比较,此实验中Ox-LDL刺激效应在80μg/ml浓度时达到最高,故此以后实验均采用80μg/ml浓度进行细胞干预。
将80μg/ml浓度的Ox-LDL作用于Raw264.7细胞及THP-1巨噬细胞不同时间点(0、6、12、24h)后,检测得细胞培养基中的MCP-1均有不同程度的增加(F=99.80, P<0.001)(F=68.62, P<0.001),并呈现时间依赖性。此实验中MCP-1分泌在24h达到最高,故此以后实验中用Ox-LDL干预细胞时间均设为24h。
3、姜黄素可抑制Ox-LDL诱导巨噬细胞MCP-1分泌
先加入不同浓度(0,5,10,20,40μM)的姜黄素预处理细胞1h,再给予80μg/ml浓度的Ox-LDL刺激24h后,检测培养基中MCP-1浓度。两种细胞系所得结果相一致,结果显示不同组间具有显著性差异(F=75.96,P<0.001)(F=71.39,P<0.001)。与阴性对照组相比,其余各组细胞培养基中MCP-1均明显偏高。而与Ox-LDL单独刺激组比较,Ox-LDL+姜黄素(10μM)、Ox-LDL+姜黄素(20μM)组和Ox-LDL+姜黄素(40μM)组细胞培养基中MCP-1水平明显减低,具有显著性差异,并且总体呈剂量依赖性。而两组细胞系中Ox-LDL+姜黄素(5gM)组和Ox-LDL单独刺激组比较,无显著性差异(P=0.090)(P=0.769)
4、JNK通路和NF-κB通路介导了Ox-LDL诱导Raw264.7巨噬细胞分泌MCP-1
将不同的MAPK信号通路(p38MAPK、JNK、ERK1/2)抑制剂(SB203580、SP600125、PD98059)以及NF-κB通路抑制剂(BAY11-7082)分别预处理细胞1h,再给予Ox-LDL (80μg/ml)刺激24h。与对照组相比,Ox-LDL单独刺激组细胞培养基中的MCP-1明显升高(802.44±83.86pg/ml),具有显著性差异(P<0.001)。与Ox-LDL单独刺激组相比,Ox-LDL+SP组及Ox-LDL+BAY组细胞培养基中的MCP-1分别明显降低(576.20±41.82pg/ml)(P<0.001)(658.34±63.28pg/ml)(P<0.001),差异具有统计学意义；但是,Ox-LDL+SB组和Ox-LDL+PD组与Ox-LDL组无统计学差异(P=0.301和P=0.404),通过此实验发现了MCP-1分泌可能被JNK信号通路及NF-κB通路所介导,为下一步实验提供参考。
5、Ox-LDL促进Raw264.7巨噬细胞JNK磷酸化而JNK抑制剂(SP600125)可有效的抑制该通路激活
根据上一个实验结果,我们给予JNK抑制剂(SP600125)预处理细胞1h,再给与Ox-LDL(80μg/ml)组刺激30main。与对照组比较,Ox-LDL刺激组JNK磷酸化水平明显增加(P=0.003)；而与Ox-LDL组比较,Ox-LDL+SP组中的细胞磷酸化JNK的水平则出现明显降低(P=0.028),组间比较均具有显著性差异。
6、Ox-LDL增加Raw264.7巨噬细胞核内NF-κB p65蛋白水平而NF-κB抑制剂(BAYll-7082)可有效的抑制该通路激活
与上一步相似,我们给予NF-κB通路抑制剂(BAY11-7082)预处理细胞1h,再给与Ox-LDL (80μg/ml)组刺激30min。与对照组比较,Ox-LDL刺激组核内NF-κB p65水平明显增加(P=0.002)；而与Ox-LDL组比较,Ox-LDL+BAY组中的细胞核内的NF-κB p65水平则出现明显降低(P=0.032),组间比较均具有显著性差异。
7、姜黄素可抑制Ox-LDL诱导Raw264.7巨噬细胞JNK磷酸化及核内NF-κBp65表达
给予不同浓度姜黄素(0-40μM)预处理后,再给予Ox-LDL (80μg/ml)刺激30main。与对照组比较,Ox-LDL刺激组磷酸化JNK表达水平明显升高(P<0.01),差异具有显著性；与Ox-LDL刺激组比较,姜黄素(10、20、40μM)+Ox-LDL组均可明显下调JNK磷酸化水平(P=0.004、P<0.001、P<0.001),具有显著性差异。
与抑制JNK激活效应相类似的,与对照组比较,Ox-LDL刺激组核内NF-κBp65表达水平明显上升,而姜黄素(20、40μM)+Ox-LDL组均可明显下调核内NF-κB p65水平(P<0.001、P<0.001),具有显著性差异。两组实验总体效应均呈剂量依赖性。而两组实验中Ox-LDL刺激组与姜黄素(5、10mM)+Ox-LDL组比较均未见统计学差异(P=0.491,P=0.060)。综上实验结果提示:姜黄素可以通过抑制JNK通路及NF-κB通路的激活,降低Ox-LDL诱导的巨噬细胞MCP-1分泌。
第二部分姜黄素对巨噬细胞逆胆固醇转运的影响及其相关机制研究
1、姜黄素可增强Ox-LDL诱导下巨噬细胞的胆固醇流出率
将不同浓度(0,5,10,20,40μM)的姜黄素刺激Raw264.7细胞后,与对照组胆固醇流出率(5.60±0.61%)相比,姜黄素10、20和40μM刺激组胆固醇流出率明显上升(分别为6.95±0.67%,9.15±0.68%和10.68±0.79%)(P=0.016、P<0.001和P<0.001),且在实验浓度范围内成剂量依赖性。而对照组与5μM刺激组(5.90±0.76%)之间比较则无显著性差异。
与上述结果相类似,在不同浓度姜黄素刺激THP-1巨噬细胞后,与对照组流出率(7.39±1.25%)相比,姜黄素10、20和40μM刺激组胆固醇流出率也呈逐渐上升趋势(分别为11.77±1.84%,14.43±2.11%和15.67±1.71%)(P=0.002、P<0.001和P<0.001),结果均有显著性差异。而对照组与5μM刺激组(7.89±1.49%)之间比较则无显著性差异。
2、姜黄素可上调Ox-LDL诱导下Raw264.7巨噬细胞的胆固醇代谢相关蛋白(ABCAl、SR-BI、LXRα)的表达
上一步的实验结果已经证实姜黄素对逆胆固醇转运功能的影响,因此在本实验中我们着重观察姜黄素对胆固醇转运蛋白表达的调节机制：给予不同浓度(0,5,10,20,40gM)姜黄素处理细胞后,ABCA1、SR-BI、和LXRa三个蛋白表达指标的组间差异均十分显著(F=27.604,P<0.001；F=48.577,P<0.001；F=33.054,P<0.001),而ABCG1组间差异无统计学意义(F=0.693,P=0.614)与对照组比较,姜黄素(10μM、20μM与40μM)明显提高了ABCA1、SR-BI及LXRa三个蛋白的表达水平,且在实验浓度范围内呈剂量依赖效应,差异均具有统计学意义。以上实验结果提示:姜黄素可上调胆固醇代谢蛋白的表达来增强逆胆固醇转运功能。
3、JNK抑制剂可增强Ox-LDL诱导下Raw264.7巨噬细胞的胆固醇流出率
为了进一步观察上一部分实验中讨论的MAPK及NF-κB信号通路是否参与了姜黄素增强细胞中逆胆固醇转运的效应,我们分别加入不同的通路抑制剂处理细胞。与对照组比较,JNK抑制剂(SP600125)组胆固醇流出率明显升高(5.35±0.59%vs.7.88±0.67%),具有显著性差异(P<0.001)。而与对照组比较,p38抑制剂(SB203580)组、ERK抑制剂(PD98059)组和NF-κB抑制剂(BAY11-7082)组胆固醇流出率未见明显变化,差异不具有统计学意义(P=0.819,P=0.612,P=0.900)。本部分实验结果提示JNK通路可能介导了胆固醇转运的调节。
4、JNK抑制剂可上调Ox-LDL诱导下Raw264.7巨噬细胞的ABCA1、SR-BI蛋白表达,但对LXRa蛋白表达无影响
为进一步证实JNK通路介导了胆固醇逆转运变化,用不同浓度JNK抑制剂刺激Raw264.7巨噬细胞。Western Blot结果显示：JNK抑制剂可以显著上调ABCA1和SR-BI的表达,具有显著性差异(F=18.034,P=0.003；以及F=16.469,P=0.004)；但对细胞核蛋白LXRa的调节则未见明显影响,无显著性差异(F=0.352,P=0.717)。前面的实验结果已经证实,姜黄素具有JNK抑制剂样作用,可抑制JNK通路的磷酸化激活,结合本部分3、4结果,提示姜黄素可通过抑制JNK磷酸化途径调节逆胆固醇转运蛋白(ABCA1与SR-BI)的表达和胆固醇流出率,并且可能与姜黄素上调LXRa表达的通路调节效应相互独立。
结论
1)姜黄素在(0-40μM)浓度范围内无细胞毒性；
2)Ox-LDL诱导巨噬细胞MCP-1分泌,并呈浓度依赖效应和时间依赖效应；
3)JNK通路和NF-κB通路介导了Ox-LDL诱导下的巨噬细胞MCP-1分泌；
4)姜黄素分别通过抑制JNK通路磷酸化和抑制NF-κB通路激活,减少Ox-LDL诱导的巨噬细胞分泌MCP-1；
5)姜黄素可以上调巨噬细胞细胞内LXRa-ABCA1/SR-BI这一经典通路的表达,进而增强细胞内的胆固醇流出率；
6)姜黄素可以抑制Ox-LDL诱导下的JNK磷酸化途径,增强Ox-LDL诱导的巨噬细胞中胆固醇膜转运蛋白(ABCA1/SR-BI)的表达和胆固醇流出率,且该效应独立于姜黄素对LXRa-ABCA1/SR-BI通路的激活效应。
Background
Coronary 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.
Objectives
Raw264.7cells 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.
Methods
Part Ⅰ. Effect of curcumin on ox-LDL-induced MCP-1expression and the related molecular mechanisms in macrophages
1. 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 macrophages
Concentration 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 macrophages
Cells 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 Raw264.7cells
Cells 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 Raw264.7cells
Cells 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 macrophages
Cells 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 Raw264.7cells
Cells 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 Raw264.7cells
Cells 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 Raw264.7cells
Cells were divided into3groups:1) control group,2)10μM SP600125group,3)20μM SP600125group.
The expression of lipid transfer protein was detected by Western blot.
Results
Part I. Effect of curcumin on ox-LDL-induced MCP-1expression and the related molecular mechanisms in macrophages
1. Curcumin-induced cell toxicity was negligible at concentrations of0-40μM in macrophages
There 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 manner
Raw264.7cells 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 Raw264.7cells
Cells 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 cells
Based 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 Raw264.7cells
Raw264.7cells 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 Raw264.7cells
Cells 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 protein
Raw264.7cells 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.
Conclusions
1) 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.
引文
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