姜黄素对大鼠血管平滑肌细胞单核细胞趋化蛋白-1表达、增殖和凋亡影响及其机制的研究
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摘要
研究背景
冠心病(Coronary Artery Disease, CAD)严重危害人民健康,是目前是世界及我国首要致死原因。根据流行病学调查冠心病发病率逐年递增。冠心病的病理基础为动脉粥样硬化(Atherosclerosis, AS),也是导致冠心病病理生理变化的重要原因,随着AS的进展,最终有可能导致管腔狭窄、斑块破裂而引起急性心脏事件危害生命。血管平滑肌细胞(Vscular smooth muscle cells, VSMCs)是构成血管壁的重要组成成分,平滑肌细胞增生、迁移贯穿动脉粥样硬化全过程,因此探讨调节VSMCs生理病理功能,对防治AS具有重要意义。
氧化型低密度脂蛋白(Oxidized low-density lipoprotein, Ox-LDL)促进AS形成的作用己被大量的研究所证实。目前认为AS形成初期步骤是低密度脂蛋白(Low-density lipoprotein cholesterol, LDL)进入内皮损伤部位的血管壁,氧化、诱导多种趋化因子,并激活单核细胞分泌不同的生长因子和细胞因子,刺激平滑肌细胞迁移、增殖。单核细胞趋化蛋白-1(monocyte chemoattractant protein-1, MCP-1)是具有趋化及活化单核/巨噬细胞作用的重要趋化细胞因子之一,可由内皮细胞、巨噬细胞、血管平滑肌细胞产生,血液中的单核细胞在MCP-1作用下发生迁移并聚集在血管内膜下发挥重要的生物学效应如吞噬脂质,形成泡沫细胞。另外,AS的过程中血管平滑肌的增殖也在其形成过程中起到了重要作用,动脉中的血管平滑肌细胞由收缩型转变为合成型,平滑肌细胞从内膜基底部移行入内膜层,而且大量增生并产生胶原纤维和蛋白多糖,最终导致内膜增厚,使病变演变为纤维斑块。在AS病变初期,血管壁细胞数量增多、内膜增厚、管腔变窄,除了与VSMCs过度增殖有关,还与凋亡率下降导致细胞累积、数量增多密切相关。由此可见,MCP-1在AS的发生发展中起到扮演重要角色,提示抑制MCP-1的表达分泌和细胞过度增殖可以作为一个AS治疗的重要靶点。姜黄素(Curcumin)是从姜黄、郁金、莪术等植物中提取的一种中药单体,属于生物多酚化合物,研究显示姜黄素具有抗炎、抗氧化、促细胞凋亡和抑制细胞增殖等药理作用,这些作用理论上可有效拮抗AS的发生与发展,但是其具体作用机制不甚清楚。因此,本研究以VSMCs作为模型,运用现代分子生物学技术,观察姜黄素对Ox-LDL诱导MCP-1表达分泌和增殖,及促凋亡影响,初步探讨其可能的机制。
研究目的
以大鼠主动脉分离后原代培养的VSMCs为研究对象,运用现代分子生物学技术,如蛋白免疫印迹(Western blot)、逆转录-聚合酶链反应(Reverse Transcription-Polymerase Chain Reaction, RT-PCR)、四甲基偶氮唑盐微量酶反应比色法(MTT)、流式细胞术检测等,观察姜黄素对其分泌MCP-1和增殖、促凋亡的影响,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
第一章姜黄素对Ox-LDL诱导血管平滑肌细胞MCP-1分泌的影响及相关信号通路的研究
取SD雄性大鼠,处死后分离胸主动脉,去除外膜和内膜,按常规组织贴块方法原代培养并传代,实验取用3-8代细胞,免疫组化法通过显示α平滑肌肌动蛋白(a-smooth muscle actin, a-SM-actin)阳性表达鉴定为血管平滑肌细胞。
细胞毒性试验：实验分组：1)正常对照组,2)姜黄素(5μM)组,3)姜黄素(1OμM)组,4)姜黄素(20μM)组,5)姜黄素(40μM)组,6)姜黄素(80μM)组；放入孵箱中干预24h,采用四甲基偶氮唑盐微量酶反应比色法(MTT)检测存活率,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；
Ox-LDL对大鼠血管平滑肌细胞MVP-1分泌的影响：实验(1)分组：1)正常对照组,2)Ox-LDL (10μg/ml)组,3)Ox-LDL (50μg/ml)组,4) Ox-LDL (100μg/ml)组,干预24h；实验(2)分组：100μg/ml Ox-LDL (0、6、12、24h)作用不同时间作用组。ELISA检测细胞上清中MCP-1的浓度。
姜黄塞控制Ox-LDL诱导血管平滑肌细胞MCP-1表达的影响：实验分组：1)正常对照组,2) Ox-LDL (100μg/ml)组,3) Ox-LDL (100μg/ml)+姜黄素(5μM)组,4) Ox-LDL (100μg/ml)+姜黄素(10μM)组,5) Ox-LDL (100μg/ml)+姜黄素(30μM)组,通过ELISA和RT-PCR检测细胞上清MCP-1浓度和细胞中MCP-1mRNA表达情况。
不促分裂原活化蛋白激酶MAPK (Mitogen activated protein kinase, MAPK)信号通(JNK, ERK1/2, p38MAPK)抑制剂和核转录因子-κB通路抑制剂(BAY11-7082)对Ox-LDL诱导大鼠血管平滑肌细胞MCP-1分泌的影响响：实验分组：1)正常对照组,2) Ox-LDL (100μg/ml)组,3) Ox-LDL (100μg/ml)+SP600125(JNK抑制剂)组,4)Ox-LDL (100μg/ml)+PD98059(ERK1/2抑制剂)组,5) Ox-LDL (100μg/ml)+SB203580(p38抑制剂)组,6) Ox-LDL (100μg/ml)+BAY11-7082(NF-κB抑制剂)组,通过ELISA对细胞上清MCP-1进行检测,通过此实验确定引起MCP-1分泌可能被介导的信号通路,为下一个实验提供参考。
p38抑制剂(SB203580)和NF-κB抑制剂(BAY11-7082)对Ox-LDL诱导大鼠血管平滑肌细胞磷酸化p38和核内NF-κBp65表达的影响：实验(1)分组：1)正常对照组,2)Ox-LDL (100μg/ml)组,3) Ox-LDL (100μg/ml)+SB203580组；通过Western blot检测磷酸化p38表达水平；实验(2)分组：1)正常对照组,2)Ox-LDL (100μg/ml)组,3)Ox-LDL (100μg/ml)+BAY11-7082组；Western blot检测核内NF-κBp65表达,结合上一实验进一步证实介导的信号通路。
姜黄素对Ox-LDL诱导大鼠血管平滑肌细胞磷酸化p38和核内NF-κBp65表达的影响：实验(1)分组：1)正常对照组,2)Ox-LDL (100μg/ml)组,3)Ox-LDL (100μg/ml)+姜黄素(5μM)组,4) Ox-LDL (100μg/ml)+姜黄素(10μM)组,5) Ox-LDL (100μg/ml)+姜黄素(30μM),通过Western blot检测磷酸化p38MAPK表达情况；实验(2)分组：1)正常对照组,2) Ox-LDL (100μg/ml)组,3)Ox-LDL (100μg/ml)+姜黄素(5μM)组,4)Ox-LDL (100μ/ml)+姜黄素(1OμM)组,5) Ox-LDL (100μg/ml)+姜黄素(30μM),通过Western blot检测核内NF-κB p65的表达情况。
第二章姜黄素对Ox-LDL诱导大鼠血管平滑肌细胞增殖的影响及相关机制初步探讨
Ox-LDL对大鼠血管平滑肌细胞(VSMCs)增殖的影响：实验分组：1)正常对照组,2)Ox-LDL (50μg/ml)组,3) Ox-LDL (100μg/ml)组,4) Ox-LDL (200μg/ml)组,5)Ox-LDL (300μg/ml)组,采用四甲基偶氮唑盐微量酶反应比色法(MTT)检测,在酶标仪上测得OD值代表细胞增殖程度；
姜黄素对Ox-LDL (100μg/ml)诱导的VSMCs增殖的影响：实验分组：1)正常对照组,2)Ox-LDL (100μg/ml)组,3) Ox-LDL (100μg/ml)+姜黄素(20μM)组,4)Ox-LDL (100μg/ml)+姜黄素(40μM)组,5)Ox-LDL (100μg/ml)+姜黄素(80μM)组,采用MTT检测,在酶标仪上测得OD值代表细胞增殖程度；
血红素氧合酶-1Heme oxygenase-1, HO-1)控制剂(ZnPPIX)对姜黄素抑制Ox-LDL诱导VSMCs增殖的影响：实验分组：1)正常对照组,2) Ox-LDL(100μg/m1)组,3) Ox-LDL (100μg/ml)+姜黄素(80μM)组,4) Ox-LDL (100μg/ml)+姜黄素(80μM)+ZnPPIX组,采用MTT检测,在酶标仪上测得OD值代表细胞增殖程度；
姜黄素对大鼠血管平滑肌细胞HO-1mRNA和蛋白表达的影响：实验分组：1)正常对照组,2)姜黄素(20μM)组,3)姜黄素(40μM)组,4)姜黄素(80μM)组,通过RT-PCR和Western blot检测细胞HO-1mRNA表达和蛋白表达的情况；
HO-1抑制剂(ZnPPIX对姜黄素抑制Ox-LDL诱导VSMCs增殖细胞核抗原(proliferating cell nuclear antigen, PCNA)表达的影响：实验分组：1)正常对照组,2) Ox-LDL (100μg/ml)组,3) Ox-LDL (100μg/ml)+姜黄素(80μM)组,4)Ox-LDL (100μg/ml)+姜黄素(80μM)+ZnPPIX组,通过Western blot检测细胞中PCNA表达水平；
第三章姜黄素诱导血管平滑肌细胞凋亡的影响的初步探讨
姜黄素对大鼠血管平滑肌细胞凋亡影响：实验(1)分组：1)正常对照组,2)姜黄素(50μM)组,3)姜黄素(100μM)组；通过HOECHST33342细胞染色荧光显微镜观察姜黄素对大鼠血管平滑肌细胞凋亡形态；实验(2)分组：)正常对照组,2)姜黄素(50μM)组,3)姜黄素(100μM)组,4)姜黄素(120μM)；通过Annexin V-FITC和PI双染流式细胞仪检测细胞凋亡
姜黄素对大鼠血管平滑肌细胞caspase-8p10的影响：实验分组：1)正常对照组,2)姜黄素(50μM)组,3)姜黄素(100μM)组,4)姜黄素(120μM)组；通过Western blot检测细胞中caspase-8p10表达水平；
研究结果
一、姜黄素对Ox-LDL诱导血管平滑肌细胞MCP-1分泌的影响及相关信号通路的研究
1)姜黄素在浓度范围(0-80μM)范围内细胞存活率无显著变化：姜黄素加入VSMCs后,与正常对照组比较,5μM组(P=0.634)、10μM (P=0.309)、20μM (P=0.243)、40μM (P=0.202)、80μM (P=0.163)组细胞活性差异无统计学意义。
2) Ox-LDL可诱导血管平滑肌细胞分泌MCP-1,在本实验浓度范围呈浓度依赖性：不同浓度(10、50、100μg/ml) Ox-LDL作用于血管平滑肌细胞后,细胞上清中分泌的MCP-1不同程度的增加(P<0.05),并呈浓度依赖性,此实验中在100μg/ml达到最高,故此以后实验均采用100μg/ml浓度进行细胞干预。
3) Ox-LDL可诱导血管平滑肌细胞分泌MCP-1,在本实验时间范围呈时间依赖性:将100μg/ml浓度的Ox-LDL作用于血管平滑肌细胞不同时间点(0、6、12、24h)后,检测得细胞上清中的MCP-1不同程度的增加(P<0.05),呈现时间依赖性。
4)姜黄素可抑制Ox-LDL诱导血管平滑血细胞MCP-1蛋白表达：将不同浓度的姜黄素预处理,再给予Ox-LDL刺激24h (100μg/ml),与对照组相比,其余各组细胞上清MCP-1明显偏高(P=0.000,P=0.000,P=0.000,P=0.003)差异具有显著性。与Ox-LDL组单独作用组比较,Ox-LDL+姜黄素(10μM)和Ox-LDL+姜黄素(30μM)组细胞上清中MCP-1明显减低(P=0.000和P=0.000),差异具有显著性。而Ox-LDL+姜黄素(5μM)组和Ox-LDL组单独作用组比较无显著差异(P=0.565)。
姜黄素可抑制Ox-LDL诱导血管平滑肌细胞MCP-1mRNA表达：RT-PCR检测结果显示：与对照组相比,其余各组(Ox-LDL/姜黄素干预组)MCP-1mRNA明显偏高(P=0.000,P=0.000,P=0.001,P=0.024),差异具有统计学意义。与Ox-LDL组单独作用组比较,Ox-LDL+姜黄素(10μM)和Ox-LDL+姜黄素(30μM)组细胞MCP-1mRNA明显减低(P=0.000和P=0.000),差异具有显著性。而Ox-LDL+姜黄素(5μM)组和Ox-LDL组单独作用组比较无显著差异(P=0.054)。
5) p38MAPK和NF-κB介导Ox-LDL透导VSMCs表达分泌MCP-1：将不同浓度的姜黄素预处理,MAPK信号通路(JNK, ERK1/2, p38MAPK)抑制剂(SP600125, PD98059, SB203580)或NF-κB抑制剂(BAY11-7082)处理细胞1h,再给予Ox-LDL刺激30min (100μg/ml)。与Control组相比,Ox-LDL单独作用组细胞上清中的MCP-1明显升高,差异具有显著性(P=0.000)。与Ox-LDL组相比,Ox-LDL+SB组和Ox-LDL+BAY组细胞上清中的MCP-1明显降低(P=0.000和P=0.002),差异具有统计学意义；但是,Ox-LDL+SP组和Ox-LDL+PD组与Ox-LDL组无统计学差异(P=0.536和P=0.438),通过此实验确定引起MCP-1分泌可能被介导的信号通路,为下一个实验提供参考。
6) Ox-LDL促进p38MAPK磷酸化和核内NF-κB p65表达上调：根据上个实验结果,给予p38MAPK和NF-κB的抑制剂(SB203580和BAY11-7082)预处理细胞1h,再给与Ox-LDL(100μg/ml)组刺激30min。与对照组比较,Ox-LDL组磷酸化p38及核NF-κB p65明显表达增加(P=0.000和P=0.000),差异具有显著性；与Ox-LDL组比较,Ox-LDL+SB组和Ox-LDL+BAY组中的细胞磷酸化p38和核NF-KBp65的水平明显降低(P=0.006和P=0.006),差异具有显著性。
7)姜黄素抑制Ox-LDL诱导血管平滑肌细胞p38MAPK磷酸化及核内NF-κBp65表达：给予不同浓度姜黄素(5、10、30μM)预处理后,再给予Ox-LDL(100μg/m1)刺激30min。与对照组比较,Ox-LDL单独作用组磷酸化p38表达水平明显升高(P=0.000),差异具有显著性；与Ox-LDL单独作用组比较,姜黄素(10、30μM)+Ox-LDL组可明显下调磷酸化p38(P=0.000和P=0.000),差异具有显著性。Ox-LDL单独作用组与姜黄素(5μM)+Ox-LDL组比较磷酸化p38表达水平无统计学差异(P=0.108)。同样地,与对照组比较,Ox-LDL单独作用组核内NF-KBp65表达水平明显升高(P=0.000),差异具有显著性；与Ox-LDL单独作用组比较,姜黄素(10、30μM)+Ox-LDL组可明显下调核内NF-κBp65(P=0.001和P=0.000),差异具有显著性。Ox-LDL单独作用组与姜黄素(5μM)+Ox-LDL组比较,核内NF-κB p65表达水平无统计学差异(P=0.796)
二、姜黄素对Ox-LDL诱导大鼠血管平滑肌细胞增殖影响及相关机制初步探讨
1) Ox-LDL可诱导血管平滑肌细胞增殖：将不同浓度的(50、100、200、300μg/ml) Ox-LDL刺激VSMCs24小时后,与对照组相比,50、100、200μg/ml组OD值明显升高(P=0.015,P=0.009,P=0.035),差异显著,具有统计学意义。
2)姜黄素抑制Ox-LDL诱导大鼠血管平滑肌增殖：前面的实验结果已经证实Ox-LDL诱导增殖的效应明显,因此在本实验中我们着重观察姜黄素是否存在对Ox-LDL诱导增殖的抑制效应；给予姜黄素处理细胞,再给予Ox-LDL(100μg/m1)刺激。与对照组比较,Ox-LDL组OD值明显升高,差异具有显著性(P=0.002)。予以姜黄素预处理,Ox-LDL+姜黄素(20μM)与Ox-LDL组比较无统计学差异(P==0.717)。与Ox-LDL组比较,Ox-LDL+姜黄素(40μM)和Ox-LDL+姜黄素(80μM)组OD值明显降低,差异具有统计学意义(P=0.047和P=0.009)。
3)HO-1可能介导姜黄素抑制Ox-LDL所诱导的VSMCs增殖效应：为了观察HO-1是否介导了姜黄素抑制Ox-LDL诱导血管平滑肌细胞增殖的效应,我们用HO-1抑制剂ZnPPIX、姜黄素单独或共同预处理细胞,再给予Ox-LDL刺激。与对照组比较,Ox-LDL组OD值明显升高,差异具有显著性(P=0.004)与Ox-LDL组比较,Ox-LDL+姜黄素(80μM)组OD值明显降低,差异具有统计学意义(P=0.016)。与Ox-LDL+姜黄素(80μM)组比较,Ox-LDL+姜黄素+ZnPPIX组OD值明显升高,差异具有统计学意义(P=0.036)
4)姜黄素可上调大鼠血管平滑肌细胞HO-1mRNA和蛋白表达：为进一步证实HO-1的介导,用不同浓度姜黄素刺激VSMCs。 RT-PCR结果显示：0μM姜黄素组仍可见微量HO-1mRNA表达(0.14+0.03),与0μM组相比,20μM、40μM和80μM组HO-1mRNA水平明显升高,且差异具有显著性(P=0.006,P=0.000和P=0.006)。与20μM组相比,40μM组HO-1mRNA水平明显升高,且差异具有显著性(P=0.003)。与40μM组相比,80μM组HO-1mRNA水平明显升高,且差异具有统计学意义(P=0.025)。同样地,Western blot结果显示与0μM组相比,20μM和80μM组HO-1蛋白表达水平明显升高,且差异具有显著性(P=0.014,P=0.000和P=0.000)。与20μM组相比,40μM组HO-1蛋白表达水平明显升高,且差异具有显著性(P=0.006)。与40μM组相比,80μM组HO-1表达水平明显升高,且差异具有统计学意义(P=0.000)。以上提示：姜黄素可上调大鼠血管平滑肌细胞HO-1表达,结合上一个实验,认为姜黄素上调HO-1表达介导抑制平滑肌细胞增殖的效应。
5)姜黄素抑制Ox-LDL诱导的PCNA表达可被HO-1抑制剂ZnPPIX下调：为了观察姜黄素诱导的HO-1的上调表达是否影响PCNA的表达,我们用HO-1抑制剂ZnPPIX姜黄素单独或共同预处理细胞,再给予Ox-LDL刺激,Western blot检测PCNA水平。结果显示：经Ox-LDL (100μg/ml)处理细胞后,Ox-LDL组PCNA的表达显著上调,差异具有显著性(P=0.000)。与Ox-LDL组比较,Ox-LDL+姜黄素组PCNA的表达显著下调,差异具有显著性(P=0.000)。与Ox-LDL+姜黄素组比较,Ox-LDL+姜黄素+ZnPPIX组PCNA的表达增加,差异具有统计学意义(P=0.006)。
三、姜黄素诱导血管平滑肌细胞凋亡的影响的初步探讨
1)姜黄素可诱导大鼠血管平滑肌细胞凋亡：大鼠平滑肌细胞经姜黄素干预之后经Hoechst33342染色,在荧光显微镜下观察：正常对照组VSMCs核呈现蓝色；给予50μM姜黄素处理24h后,可见少量细胞核会致密浓染,呈白色发亮状态；当给予100gM姜黄素处理以后,大量细胞核呈致密浓染,略呈白色。
2)流式细胞仪检测进一步证实凋亡情况：与0μM组比较,50μM、100μM、120gM姜黄素组细胞早期凋亡数明显增加,差异具有显著性(P=0.024,P=0.000,P=0.000)。与50μM比较,100μM姜黄素组细胞早期凋亡数显著增加,差异有统计学意义(P=0.000)。与100μM比较,120gM姜黄素组细胞早期凋亡数显著增加,差异也有统计学意义(P=0.016)。
3)姜黄素上调caspase-8p10蛋自表达水平：不同浓度的姜黄素(50、100、120μM)对大鼠平滑肌细胞干预以后,与0μM组相比,50μM、100μM和120μM姜黄素干预组caspase-8p10蛋白表达水平明显上调(P=0.043,P=0.001,P=0.000),差异显著。与50μM组相比,100μM姜黄素干预组caspase-8p10蛋白表达水平明显上调(P=0.044),差异显著。与100gM组相比,120μM组caspase-8P10蛋白表达水平也明显上调(P=0.011),差异有统计学意义。
结论
1)姜黄素在浓度范围(0-80μM)范围内无细胞毒性。
2) Ox-LDL诱导大鼠血管平滑肌细胞中p38MAPK的磷酸化及NF-κB入核促进MCP-1分泌表达。
3)姜黄素通过抑制p38MAPK和NF-κB信号通路,下调Ox-LDL诱导的大鼠血管平滑肌细胞表达分泌MCP-1。
4)姜黄素通过上调HO-1的表达,从而下调PCNA的蛋白表达,抑制Ox-LDL诱导大鼠血管平滑肌细胞增殖。
5)姜黄素可诱导大鼠血管平滑肌细胞凋亡,Caspase-8激活有可能是姜黄素诱导大鼠血管平滑肌细胞凋亡的机制之一。
Background
Coronary 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.
Objectives
Primary 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.
Methods
Part 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.
Results
Part 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).
Conclusions
1) 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.
冠心病(Coronary Artery Disease, CAD)严重危害人民健康,是目前是世界及我国首要致死原因。根据流行病学调查冠心病发病率逐年递增。冠心病的病理基础为动脉粥样硬化(Atherosclerosis, AS),也是导致冠心病病理生理变化的重要原因,随着AS的进展,最终有可能导致管腔狭窄、斑块破裂而引起急性心脏事件危害生命。血管平滑肌细胞(Vscular smooth muscle cells, VSMCs)是构成血管壁的重要组成成分,平滑肌细胞增生、迁移贯穿动脉粥样硬化全过程,因此探讨调节VSMCs生理病理功能,对防治AS具有重要意义。
氧化型低密度脂蛋白(Oxidized low-density lipoprotein, Ox-LDL)促进AS形成的作用己被大量的研究所证实。目前认为AS形成初期步骤是低密度脂蛋白(Low-density lipoprotein cholesterol, LDL)进入内皮损伤部位的血管壁,氧化、诱导多种趋化因子,并激活单核细胞分泌不同的生长因子和细胞因子,刺激平滑肌细胞迁移、增殖。单核细胞趋化蛋白-1(monocyte chemoattractant protein-1, MCP-1)是具有趋化及活化单核/巨噬细胞作用的重要趋化细胞因子之一,可由内皮细胞、巨噬细胞、血管平滑肌细胞产生,血液中的单核细胞在MCP-1作用下发生迁移并聚集在血管内膜下发挥重要的生物学效应如吞噬脂质,形成泡沫细胞。另外,AS的过程中血管平滑肌的增殖也在其形成过程中起到了重要作用,动脉中的血管平滑肌细胞由收缩型转变为合成型,平滑肌细胞从内膜基底部移行入内膜层,而且大量增生并产生胶原纤维和蛋白多糖,最终导致内膜增厚,使病变演变为纤维斑块。在AS病变初期,血管壁细胞数量增多、内膜增厚、管腔变窄,除了与VSMCs过度增殖有关,还与凋亡率下降导致细胞累积、数量增多密切相关。由此可见,MCP-1在AS的发生发展中起到扮演重要角色,提示抑制MCP-1的表达分泌和细胞过度增殖可以作为一个AS治疗的重要靶点。姜黄素(Curcumin)是从姜黄、郁金、莪术等植物中提取的一种中药单体,属于生物多酚化合物,研究显示姜黄素具有抗炎、抗氧化、促细胞凋亡和抑制细胞增殖等药理作用,这些作用理论上可有效拮抗AS的发生与发展,但是其具体作用机制不甚清楚。因此,本研究以VSMCs作为模型,运用现代分子生物学技术,观察姜黄素对Ox-LDL诱导MCP-1表达分泌和增殖,及促凋亡影响,初步探讨其可能的机制。
研究目的
以大鼠主动脉分离后原代培养的VSMCs为研究对象,运用现代分子生物学技术,如蛋白免疫印迹(Western blot)、逆转录-聚合酶链反应(Reverse Transcription-Polymerase Chain Reaction, RT-PCR)、四甲基偶氮唑盐微量酶反应比色法(MTT)、流式细胞术检测等,观察姜黄素对其分泌MCP-1和增殖、促凋亡的影响,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
第一章姜黄素对Ox-LDL诱导血管平滑肌细胞MCP-1分泌的影响及相关信号通路的研究
取SD雄性大鼠,处死后分离胸主动脉,去除外膜和内膜,按常规组织贴块方法原代培养并传代,实验取用3-8代细胞,免疫组化法通过显示α平滑肌肌动蛋白(a-smooth muscle actin, a-SM-actin)阳性表达鉴定为血管平滑肌细胞。
细胞毒性试验：实验分组：1)正常对照组,2)姜黄素(5μM)组,3)姜黄素(1OμM)组,4)姜黄素(20μM)组,5)姜黄素(40μM)组,6)姜黄素(80μM)组；放入孵箱中干预24h,采用四甲基偶氮唑盐微量酶反应比色法(MTT)检测存活率,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；
Ox-LDL对大鼠血管平滑肌细胞MVP-1分泌的影响：实验(1)分组：1)正常对照组,2)Ox-LDL (10μg/ml)组,3)Ox-LDL (50μg/ml)组,4) Ox-LDL (100μg/ml)组,干预24h；实验(2)分组：100μg/ml Ox-LDL (0、6、12、24h)作用不同时间作用组。ELISA检测细胞上清中MCP-1的浓度。
姜黄塞控制Ox-LDL诱导血管平滑肌细胞MCP-1表达的影响：实验分组：1)正常对照组,2) Ox-LDL (100μg/ml)组,3) Ox-LDL (100μg/ml)+姜黄素(5μM)组,4) Ox-LDL (100μg/ml)+姜黄素(10μM)组,5) Ox-LDL (100μg/ml)+姜黄素(30μM)组,通过ELISA和RT-PCR检测细胞上清MCP-1浓度和细胞中MCP-1mRNA表达情况。
不促分裂原活化蛋白激酶MAPK (Mitogen activated protein kinase, MAPK)信号通(JNK, ERK1/2, p38MAPK)抑制剂和核转录因子-κB通路抑制剂(BAY11-7082)对Ox-LDL诱导大鼠血管平滑肌细胞MCP-1分泌的影响响：实验分组：1)正常对照组,2) Ox-LDL (100μg/ml)组,3) Ox-LDL (100μg/ml)+SP600125(JNK抑制剂)组,4)Ox-LDL (100μg/ml)+PD98059(ERK1/2抑制剂)组,5) Ox-LDL (100μg/ml)+SB203580(p38抑制剂)组,6) Ox-LDL (100μg/ml)+BAY11-7082(NF-κB抑制剂)组,通过ELISA对细胞上清MCP-1进行检测,通过此实验确定引起MCP-1分泌可能被介导的信号通路,为下一个实验提供参考。
p38抑制剂(SB203580)和NF-κB抑制剂(BAY11-7082)对Ox-LDL诱导大鼠血管平滑肌细胞磷酸化p38和核内NF-κBp65表达的影响：实验(1)分组：1)正常对照组,2)Ox-LDL (100μg/ml)组,3) Ox-LDL (100μg/ml)+SB203580组；通过Western blot检测磷酸化p38表达水平；实验(2)分组：1)正常对照组,2)Ox-LDL (100μg/ml)组,3)Ox-LDL (100μg/ml)+BAY11-7082组；Western blot检测核内NF-κBp65表达,结合上一实验进一步证实介导的信号通路。
姜黄素对Ox-LDL诱导大鼠血管平滑肌细胞磷酸化p38和核内NF-κBp65表达的影响：实验(1)分组：1)正常对照组,2)Ox-LDL (100μg/ml)组,3)Ox-LDL (100μg/ml)+姜黄素(5μM)组,4) Ox-LDL (100μg/ml)+姜黄素(10μM)组,5) Ox-LDL (100μg/ml)+姜黄素(30μM),通过Western blot检测磷酸化p38MAPK表达情况；实验(2)分组：1)正常对照组,2) Ox-LDL (100μg/ml)组,3)Ox-LDL (100μg/ml)+姜黄素(5μM)组,4)Ox-LDL (100μ/ml)+姜黄素(1OμM)组,5) Ox-LDL (100μg/ml)+姜黄素(30μM),通过Western blot检测核内NF-κB p65的表达情况。
第二章姜黄素对Ox-LDL诱导大鼠血管平滑肌细胞增殖的影响及相关机制初步探讨
Ox-LDL对大鼠血管平滑肌细胞(VSMCs)增殖的影响：实验分组：1)正常对照组,2)Ox-LDL (50μg/ml)组,3) Ox-LDL (100μg/ml)组,4) Ox-LDL (200μg/ml)组,5)Ox-LDL (300μg/ml)组,采用四甲基偶氮唑盐微量酶反应比色法(MTT)检测,在酶标仪上测得OD值代表细胞增殖程度；
姜黄素对Ox-LDL (100μg/ml)诱导的VSMCs增殖的影响：实验分组：1)正常对照组,2)Ox-LDL (100μg/ml)组,3) Ox-LDL (100μg/ml)+姜黄素(20μM)组,4)Ox-LDL (100μg/ml)+姜黄素(40μM)组,5)Ox-LDL (100μg/ml)+姜黄素(80μM)组,采用MTT检测,在酶标仪上测得OD值代表细胞增殖程度；
血红素氧合酶-1Heme oxygenase-1, HO-1)控制剂(ZnPPIX)对姜黄素抑制Ox-LDL诱导VSMCs增殖的影响：实验分组：1)正常对照组,2) Ox-LDL(100μg/m1)组,3) Ox-LDL (100μg/ml)+姜黄素(80μM)组,4) Ox-LDL (100μg/ml)+姜黄素(80μM)+ZnPPIX组,采用MTT检测,在酶标仪上测得OD值代表细胞增殖程度；
姜黄素对大鼠血管平滑肌细胞HO-1mRNA和蛋白表达的影响：实验分组：1)正常对照组,2)姜黄素(20μM)组,3)姜黄素(40μM)组,4)姜黄素(80μM)组,通过RT-PCR和Western blot检测细胞HO-1mRNA表达和蛋白表达的情况；
HO-1抑制剂(ZnPPIX对姜黄素抑制Ox-LDL诱导VSMCs增殖细胞核抗原(proliferating cell nuclear antigen, PCNA)表达的影响：实验分组：1)正常对照组,2) Ox-LDL (100μg/ml)组,3) Ox-LDL (100μg/ml)+姜黄素(80μM)组,4)Ox-LDL (100μg/ml)+姜黄素(80μM)+ZnPPIX组,通过Western blot检测细胞中PCNA表达水平；
第三章姜黄素诱导血管平滑肌细胞凋亡的影响的初步探讨
姜黄素对大鼠血管平滑肌细胞凋亡影响：实验(1)分组：1)正常对照组,2)姜黄素(50μM)组,3)姜黄素(100μM)组；通过HOECHST33342细胞染色荧光显微镜观察姜黄素对大鼠血管平滑肌细胞凋亡形态；实验(2)分组：)正常对照组,2)姜黄素(50μM)组,3)姜黄素(100μM)组,4)姜黄素(120μM)；通过Annexin V-FITC和PI双染流式细胞仪检测细胞凋亡
姜黄素对大鼠血管平滑肌细胞caspase-8p10的影响：实验分组：1)正常对照组,2)姜黄素(50μM)组,3)姜黄素(100μM)组,4)姜黄素(120μM)组；通过Western blot检测细胞中caspase-8p10表达水平；
研究结果
一、姜黄素对Ox-LDL诱导血管平滑肌细胞MCP-1分泌的影响及相关信号通路的研究
1)姜黄素在浓度范围(0-80μM)范围内细胞存活率无显著变化：姜黄素加入VSMCs后,与正常对照组比较,5μM组(P=0.634)、10μM (P=0.309)、20μM (P=0.243)、40μM (P=0.202)、80μM (P=0.163)组细胞活性差异无统计学意义。
2) Ox-LDL可诱导血管平滑肌细胞分泌MCP-1,在本实验浓度范围呈浓度依赖性：不同浓度(10、50、100μg/ml) Ox-LDL作用于血管平滑肌细胞后,细胞上清中分泌的MCP-1不同程度的增加(P<0.05),并呈浓度依赖性,此实验中在100μg/ml达到最高,故此以后实验均采用100μg/ml浓度进行细胞干预。
3) Ox-LDL可诱导血管平滑肌细胞分泌MCP-1,在本实验时间范围呈时间依赖性:将100μg/ml浓度的Ox-LDL作用于血管平滑肌细胞不同时间点(0、6、12、24h)后,检测得细胞上清中的MCP-1不同程度的增加(P<0.05),呈现时间依赖性。
4)姜黄素可抑制Ox-LDL诱导血管平滑血细胞MCP-1蛋白表达：将不同浓度的姜黄素预处理,再给予Ox-LDL刺激24h (100μg/ml),与对照组相比,其余各组细胞上清MCP-1明显偏高(P=0.000,P=0.000,P=0.000,P=0.003)差异具有显著性。与Ox-LDL组单独作用组比较,Ox-LDL+姜黄素(10μM)和Ox-LDL+姜黄素(30μM)组细胞上清中MCP-1明显减低(P=0.000和P=0.000),差异具有显著性。而Ox-LDL+姜黄素(5μM)组和Ox-LDL组单独作用组比较无显著差异(P=0.565)。
姜黄素可抑制Ox-LDL诱导血管平滑肌细胞MCP-1mRNA表达：RT-PCR检测结果显示：与对照组相比,其余各组(Ox-LDL/姜黄素干预组)MCP-1mRNA明显偏高(P=0.000,P=0.000,P=0.001,P=0.024),差异具有统计学意义。与Ox-LDL组单独作用组比较,Ox-LDL+姜黄素(10μM)和Ox-LDL+姜黄素(30μM)组细胞MCP-1mRNA明显减低(P=0.000和P=0.000),差异具有显著性。而Ox-LDL+姜黄素(5μM)组和Ox-LDL组单独作用组比较无显著差异(P=0.054)。
5) p38MAPK和NF-κB介导Ox-LDL透导VSMCs表达分泌MCP-1：将不同浓度的姜黄素预处理,MAPK信号通路(JNK, ERK1/2, p38MAPK)抑制剂(SP600125, PD98059, SB203580)或NF-κB抑制剂(BAY11-7082)处理细胞1h,再给予Ox-LDL刺激30min (100μg/ml)。与Control组相比,Ox-LDL单独作用组细胞上清中的MCP-1明显升高,差异具有显著性(P=0.000)。与Ox-LDL组相比,Ox-LDL+SB组和Ox-LDL+BAY组细胞上清中的MCP-1明显降低(P=0.000和P=0.002),差异具有统计学意义；但是,Ox-LDL+SP组和Ox-LDL+PD组与Ox-LDL组无统计学差异(P=0.536和P=0.438),通过此实验确定引起MCP-1分泌可能被介导的信号通路,为下一个实验提供参考。
6) Ox-LDL促进p38MAPK磷酸化和核内NF-κB p65表达上调：根据上个实验结果,给予p38MAPK和NF-κB的抑制剂(SB203580和BAY11-7082)预处理细胞1h,再给与Ox-LDL(100μg/ml)组刺激30min。与对照组比较,Ox-LDL组磷酸化p38及核NF-κB p65明显表达增加(P=0.000和P=0.000),差异具有显著性；与Ox-LDL组比较,Ox-LDL+SB组和Ox-LDL+BAY组中的细胞磷酸化p38和核NF-KBp65的水平明显降低(P=0.006和P=0.006),差异具有显著性。
7)姜黄素抑制Ox-LDL诱导血管平滑肌细胞p38MAPK磷酸化及核内NF-κBp65表达：给予不同浓度姜黄素(5、10、30μM)预处理后,再给予Ox-LDL(100μg/m1)刺激30min。与对照组比较,Ox-LDL单独作用组磷酸化p38表达水平明显升高(P=0.000),差异具有显著性；与Ox-LDL单独作用组比较,姜黄素(10、30μM)+Ox-LDL组可明显下调磷酸化p38(P=0.000和P=0.000),差异具有显著性。Ox-LDL单独作用组与姜黄素(5μM)+Ox-LDL组比较磷酸化p38表达水平无统计学差异(P=0.108)。同样地,与对照组比较,Ox-LDL单独作用组核内NF-KBp65表达水平明显升高(P=0.000),差异具有显著性；与Ox-LDL单独作用组比较,姜黄素(10、30μM)+Ox-LDL组可明显下调核内NF-κBp65(P=0.001和P=0.000),差异具有显著性。Ox-LDL单独作用组与姜黄素(5μM)+Ox-LDL组比较,核内NF-κB p65表达水平无统计学差异(P=0.796)
二、姜黄素对Ox-LDL诱导大鼠血管平滑肌细胞增殖影响及相关机制初步探讨
1) Ox-LDL可诱导血管平滑肌细胞增殖：将不同浓度的(50、100、200、300μg/ml) Ox-LDL刺激VSMCs24小时后,与对照组相比,50、100、200μg/ml组OD值明显升高(P=0.015,P=0.009,P=0.035),差异显著,具有统计学意义。
2)姜黄素抑制Ox-LDL诱导大鼠血管平滑肌增殖：前面的实验结果已经证实Ox-LDL诱导增殖的效应明显,因此在本实验中我们着重观察姜黄素是否存在对Ox-LDL诱导增殖的抑制效应；给予姜黄素处理细胞,再给予Ox-LDL(100μg/m1)刺激。与对照组比较,Ox-LDL组OD值明显升高,差异具有显著性(P=0.002)。予以姜黄素预处理,Ox-LDL+姜黄素(20μM)与Ox-LDL组比较无统计学差异(P==0.717)。与Ox-LDL组比较,Ox-LDL+姜黄素(40μM)和Ox-LDL+姜黄素(80μM)组OD值明显降低,差异具有统计学意义(P=0.047和P=0.009)。
3)HO-1可能介导姜黄素抑制Ox-LDL所诱导的VSMCs增殖效应：为了观察HO-1是否介导了姜黄素抑制Ox-LDL诱导血管平滑肌细胞增殖的效应,我们用HO-1抑制剂ZnPPIX、姜黄素单独或共同预处理细胞,再给予Ox-LDL刺激。与对照组比较,Ox-LDL组OD值明显升高,差异具有显著性(P=0.004)与Ox-LDL组比较,Ox-LDL+姜黄素(80μM)组OD值明显降低,差异具有统计学意义(P=0.016)。与Ox-LDL+姜黄素(80μM)组比较,Ox-LDL+姜黄素+ZnPPIX组OD值明显升高,差异具有统计学意义(P=0.036)
4)姜黄素可上调大鼠血管平滑肌细胞HO-1mRNA和蛋白表达：为进一步证实HO-1的介导,用不同浓度姜黄素刺激VSMCs。 RT-PCR结果显示：0μM姜黄素组仍可见微量HO-1mRNA表达(0.14+0.03),与0μM组相比,20μM、40μM和80μM组HO-1mRNA水平明显升高,且差异具有显著性(P=0.006,P=0.000和P=0.006)。与20μM组相比,40μM组HO-1mRNA水平明显升高,且差异具有显著性(P=0.003)。与40μM组相比,80μM组HO-1mRNA水平明显升高,且差异具有统计学意义(P=0.025)。同样地,Western blot结果显示与0μM组相比,20μM和80μM组HO-1蛋白表达水平明显升高,且差异具有显著性(P=0.014,P=0.000和P=0.000)。与20μM组相比,40μM组HO-1蛋白表达水平明显升高,且差异具有显著性(P=0.006)。与40μM组相比,80μM组HO-1表达水平明显升高,且差异具有统计学意义(P=0.000)。以上提示：姜黄素可上调大鼠血管平滑肌细胞HO-1表达,结合上一个实验,认为姜黄素上调HO-1表达介导抑制平滑肌细胞增殖的效应。
5)姜黄素抑制Ox-LDL诱导的PCNA表达可被HO-1抑制剂ZnPPIX下调：为了观察姜黄素诱导的HO-1的上调表达是否影响PCNA的表达,我们用HO-1抑制剂ZnPPIX姜黄素单独或共同预处理细胞,再给予Ox-LDL刺激,Western blot检测PCNA水平。结果显示：经Ox-LDL (100μg/ml)处理细胞后,Ox-LDL组PCNA的表达显著上调,差异具有显著性(P=0.000)。与Ox-LDL组比较,Ox-LDL+姜黄素组PCNA的表达显著下调,差异具有显著性(P=0.000)。与Ox-LDL+姜黄素组比较,Ox-LDL+姜黄素+ZnPPIX组PCNA的表达增加,差异具有统计学意义(P=0.006)。
三、姜黄素诱导血管平滑肌细胞凋亡的影响的初步探讨
1)姜黄素可诱导大鼠血管平滑肌细胞凋亡：大鼠平滑肌细胞经姜黄素干预之后经Hoechst33342染色,在荧光显微镜下观察：正常对照组VSMCs核呈现蓝色；给予50μM姜黄素处理24h后,可见少量细胞核会致密浓染,呈白色发亮状态；当给予100gM姜黄素处理以后,大量细胞核呈致密浓染,略呈白色。
2)流式细胞仪检测进一步证实凋亡情况：与0μM组比较,50μM、100μM、120gM姜黄素组细胞早期凋亡数明显增加,差异具有显著性(P=0.024,P=0.000,P=0.000)。与50μM比较,100μM姜黄素组细胞早期凋亡数显著增加,差异有统计学意义(P=0.000)。与100μM比较,120gM姜黄素组细胞早期凋亡数显著增加,差异也有统计学意义(P=0.016)。
3)姜黄素上调caspase-8p10蛋自表达水平：不同浓度的姜黄素(50、100、120μM)对大鼠平滑肌细胞干预以后,与0μM组相比,50μM、100μM和120μM姜黄素干预组caspase-8p10蛋白表达水平明显上调(P=0.043,P=0.001,P=0.000),差异显著。与50μM组相比,100μM姜黄素干预组caspase-8p10蛋白表达水平明显上调(P=0.044),差异显著。与100gM组相比,120μM组caspase-8P10蛋白表达水平也明显上调(P=0.011),差异有统计学意义。
结论
1)姜黄素在浓度范围(0-80μM)范围内无细胞毒性。
2) Ox-LDL诱导大鼠血管平滑肌细胞中p38MAPK的磷酸化及NF-κB入核促进MCP-1分泌表达。
3)姜黄素通过抑制p38MAPK和NF-κB信号通路,下调Ox-LDL诱导的大鼠血管平滑肌细胞表达分泌MCP-1。
4)姜黄素通过上调HO-1的表达,从而下调PCNA的蛋白表达,抑制Ox-LDL诱导大鼠血管平滑肌细胞增殖。
5)姜黄素可诱导大鼠血管平滑肌细胞凋亡,Caspase-8激活有可能是姜黄素诱导大鼠血管平滑肌细胞凋亡的机制之一。
Background
Coronary 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.
Objectives
Primary 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.
Methods
Part 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.
Results
Part 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).
Conclusions
1) 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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