Nur77影响apoE~(-/-)小鼠动脉粥样硬化进展及相关机制研究
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摘要
背景
流行病学研究表明冠状动脉粥样硬化是西方发达国家中发病率和死亡率的首要原因。高胆固醇血症是动脉粥样硬化发病的重要原因和风险因素。临床试验的证据表明,通过饮食和／或药理学方法可以降低血浆胆固醇,减少心血管疾病的发病率和死亡率。实验研究发现,很多核受体超家族成员可以作为为减少高胆固醇血症和动脉粥样硬化的治疗靶点。这些核激素受体超家族的成员包括过氧化物酶体增殖物激活受体和肝X受体等。这些配体激活的转录因子通过调控特定的基因盒的表达来调节各种重要的代谢过程。
除了配体激活的转录因子,核受体家族还包括许多孤儿受体,其配体和生理功能尚不清楚。其中核受体NR4A就是重要的核孤儿受体超家族成员,包括Nur77(NR4A1),Nurr1基因(NR4A2)和Nor-1(NR4A3).NR4A与其他核受体家族成员相比,是一种“即早基因”,在各种环境刺激下能短暂和迅速被诱导反应。Nur77是NR4A家族中的成员,与该家族中其他核受体一样含有N-端的激活功能区域AF-1、带两个锌指结构的DNA结合域DBD以及C-终端配体结合域LBD。早期功能研究已经指出Nur77在调节分化,增殖和凋亡过程中起着关键作用。最近的研究表明Nur77在葡萄糖代谢和脂质代谢,脂肪形成,炎症反应以及血管重构中发挥重要的作用。最初的实验表明,Nur77能够促进肌肉中的脂肪水解。随后研究表明,Nur77可以调节小鼠血浆脂蛋白水平和肝脏脂质代谢。在最近的实验研究中发现,在高脂喂养Nur77缺陷小鼠中转录因子固醇调节元件结合蛋白1C(SREBP1C)的表达明显增加并出现明显的肝脏脂质蓄积。因此,本研究通过增强和抑制Nur77的表达,观察Nur77对apoE-/-小鼠主动脉粥样硬化病变的影响并探讨其相关的机制。
目的
1、Nur77是否可以影响细胞胆固醇吸收,细胞内胆固醇成分以及细胞内胆固醇流出。2、Nur77是否可以影响apoE-/-小鼠血脂水平和循环中炎症因子水平。3、Nur77是否可以影响apoE-/-小鼠肝脏脂质代谢。4、Nur77是否可以影响肠对脂质吸收相关基因表达。5、Nur77是否可以影响apoE-/-小鼠动脉粥样硬化病变。
方法
1、Nur77对THP-1巨噬细胞胆固醇吸收,对THP-1巨噬细胞源性泡沫细胞脂质成分和胆固醇流出影响的研究。
1.1THP-1细胞生长于含有10%新生小牛血清的RPMI-1640培养液中,37℃,5%C02培养箱中静置培养。培养液巾加100U/ml的青霉素和100μg/mL的链霉素。取对数生长期细胞进行实验,在每次实验前用100nM佛波酯(phorbol12-myristate13-acetate, PMA)(?)呼育THP-1细胞72h,使其诱导分化成巨噬细胞,更换培养基后加50mg/ml ox-LDL孵育48h时期转化成泡沫细胞。
1.2PCR-XL-TOPO载体,pIRES2-EGFP载体,platinum HIFI Taq Polymerase高保真扩增酶,Accuprime Pfx DNA Polymerase高保真扩增酶购自于Invitrogen(上海)公司。pCDNA3.1(+)载体,DH5α感受态细胞,XhoI、EcoRI限制性内切酶,T4DNA Ligase(?)勾自于TaKaRa(大连)公司。通过重叠PCR将PCR-XL-TOPO和PIRES2-EGFP2个片段拼接为EcoRI-NR4A1-IRES-EGFP-XhoI的全长目的片段,并将其连接到pcDNA3.1构建重组质粒pcDNA3.1-Nur77-IRES-EGFP.经电泳和测序验证后,通过脂质体2000将重组质粒转染到培养的细胞中,采用RT-PCR和Western blot检测转染效率。
1.3人类Nur77特异性siRNA(Nur77-siRNA)和对照siRNA合成自广州锐博生物公司。采用脂质体2000转染细胞(2×106/well)。转染后48小时采用实时定量PCR和Western blot检测干扰效果。Western blot分析结果表明,与对照组siRNA相比较,Nur77-siRNA抑制THP-1巨噬细胞源性泡沫细胞内Nur77蛋白表达效率为83%。
1.4根据Invitrogen公司RNA提取试剂盒操作说明提取培养细胞中提取总RNA。将1lμg,总RNA用逆转录试剂盒(TaKaRa)反转录成20μμlcDNA。实时定量PCR在应用生物系统ABI7500FAST上进行。溶解曲线分析表明PCR反应产物为单独的双链DNA。用ΔΔCt值法,以GAPDH表达为内参定量其它基因的表达。
1.5流式细胞术检测细胞胆固醇吸收。THP-1细胞经PMA诱导分化成巨噬细胞后分别经Nur77激动剂Cytosporone B (Csn-B10μg/ml),重组质粒过表达Nur77(Ad-Nur77)以及Nur77特异性siRNAs(si-Nur77)处理,然后加入荧光标记Dil-oxLDL培养细胞24小时。收集贴壁细胞,用磷酸盐缓冲液(PBS)洗三次后,采用Becton Dickinson公司流式细胞仪分析细胞荧光强度。
1.6高效液相色谱检测细胞内胆固醇成分。THP-1细胞经PMA诱导分化成巨噬细胞再经ox-LDL诱导转化成泡沫细胞后,分别经Nur77激动剂Cytosporone B(Csn-B10μg/m1)),重组质粒过表达Nur77(Ad-Nur77)以及Nur77特异性siRNAs(si-Nur77)处理。待细胞处理结束后,弃培养基,PBS洗3遍,加入细胞裂解液200μl,反复冻融3次裂解细胞,BCA法定量蛋白后,7.2%三氯乙酸沉淀蛋白,800×g离心10min,取上清进行胆固醇检测,以豆甾醇为内标。取100μl上清液,加入8.9mol/L氢氧化钾溶液200μl,水解胆固醇酯后为细胞内总胆固醇检测样品。各样品分别与内标液混匀,用正己烷和无水乙醇抽提后,1.5mol/L的三氧化铬进行氧化衍生并真空干燥,100μl乙晴-异丙醇(80：20)溶解样品,上样于高效液相色谱仪。采用Waters Corp公司2790高效液相色谱仪进行检测。
1.7采用液体闪烁技术检测细胞内胆固醇流出。THP-1细胞经PMA诱导分化成巨噬细胞再经ox-LDL诱导转化成泡沫细胞后,分别经Nur77激动剂Cytosporone B(Csn-B10μg/ml),重组质粒过表达Nur77(Ad-Nur77)以及Nur77特异性siRNAs(si-Nur77)处理。待细胞处理结束后,弃培养基,PBS洗3遍,用0.2μCi/ml[3H]胆固醇在含有10%小牛血清RPMI-1640培养液共同孵育72小时。用PBS液洗涤细胞,置含脂蛋白的无血清RPMI-1640培养液中培养24小时。PBS液洗涤细胞,闪烁液裂解细胞后,用闪烁计数法检测培养液和细胞中的[3H]胆固醇。胆固醇流出率用培养液中CPM除以总CPM(培养液CPM+细胞CPM),再乘以100%来表示。
2、Nur77对apoE-/-小鼠血浆脂质水平和血浆炎症因子表达水平影响的研究。
2.1血清IL-1β,IL-6和TNF-a浓度使用R&D公司商业ELISA试剂盒检测。血清CRP浓度使用Cusabio Biotech Co公司商业ELISA试剂盒检测。
2.2血清载脂蛋白A1(apoAl)和载脂蛋白B100(apoB100)浓度采用Cusabio公司商业ELISA试剂盒进行测定。采用全自动生化分析仪检测血清总胆固醇(T-Cho),总三酰甘油(TG),低密度脂蛋白胆固醇(LDL-C),高密度脂蛋白胆固醇(HDL-C)和极低密度脂蛋白胆固醇(VLDL-C)水平。
3、Nur77对apoE-/-小鼠肝脏脂质蓄积影响的研究。
3.1肝脏组织切片油红O染色。取肝组织样本在-30℃条件下滴加包埋剂进行包埋,用衡冷切片机进行切片。肝脏冰冻切片在60%异丙醇条件下固定10分钟,再采用含0.3%油红O染色液的60%异丙醇中孵育30分钟,随后采用60%的异丙醇洗净。采用苏木素复染后进行组织形态学定量分析。
3.2肝脏组织切片免疫组化。取肝组织样本在-30℃条件下滴加包埋剂进行包埋,用衡冷切片机进行切片。冰冻切片经多聚赖氨酸浸泡风干并以丙酮固定。采用Abcam公司兔抗Nur77一抗进行孵育,PBS洗后再采用酶标二抗孵育并显色。显色后采用奥林巴斯显微镜进行拍照分析。
4、Nur77对Caco-2细胞和apoE-/-小鼠肠脂质吸收相关基因表达影响的研究。
4.1Caco-2细胞生长于含有10%新生小牛血清的DMEM培养液中,37℃、5%CO2培养箱巾静置培养。培养液巾加100U/ml的青霉素和100gg/mL的链霉素。取对数生长期细胞进行实验。
4.2根据Invitrogen公司RNA提取试剂盒操作说明提取培养细胞和组织中总RNA。将1μg总RNA用逆转录试剂盒(TaKaRa)反转录成20μlcDNA。实时定量PCR在应用生物系统ABI7500FAST上进行。溶解曲线分析表明PCR反应产物为单独的双链DNA。用ΔΔCt值法,以GAPDH表达为内参定量其它基因的表达。
5、Nur77对apoE-/-小鼠动脉粥样硬化病变影响的研究。
5.1从腹主动脉分叉至主动脉弓将主动脉剪下,干净剔除主动脉血管外的脂肪组织后,将主动脉树纵向剪开,主动脉树以用油红O染色观察整个主动脉的病变。
5.2自主动脉根部将心脏与主动脉分离,随即将心脏置于4%的多聚甲醛溶液中、常温下固定3h；冰冻切片时,将心脏自心尖剪去约1/3(沿与心脏纵轴垂直方向),以O.C.T compound包埋剂包埋。心尖朝下、将心脏垂直固定与冰冻切片机,自主动脉根部(或心底)向心尖方向进行低温冰冻连续10μm切片。每组随机取5个标本,每一个心脏标本,将连续12张10μm冰冻切片进行油红O染色,以IMAGEPROPLUS软件计算As病变面积。
结果
1、Nur77对THP-1巨噬细胞胆固醇吸收,对THP-1巨噬细胞来源泡沫细胞脂质成分和胆固醇流出影响的研究。
我们首先检测THP-1细胞经PMA诱导分化成巨噬细胞后,分别经Nur77激动齐(?)Cytosporone B(Csn-B10μg/ml),重组质粒过表达Nur77(Ad-Nur77)以及Nur77特异性siRNAs(si-Nur77)处理后,细胞对胆固醇吸收的影响。THP-1巨噬细胞经Csn-B和Ad-Nur77处理后,巨噬细胞对荧光标记Dil-oxLDL吸收减少(P<0.001),而经si-Nur77处理后,巨噬细胞对荧光标记Dil-oxLDL吸收增加(P<0.001)。接着我们采用高效液相色谱法和液体闪烁技术检测Nur77对THP-1巨噬细胞源性泡沫细胞内胆固醇成分和细胞胆固醇流出的影响。经Csn-B和Ad-Nur77处理后细胞内胆固醇成分减少(P<0.05),而细胞内胆固醇流出增加(P<0.05)。相反地,THP-1巨噬细胞源性泡沫细胞经si-Nur77处理后,细胞内胆固醇成分增加(P<0.05),而细胞胆固醇流出减少(P<0.05)。
随后,我们采用实时定量PCR检测THP-1巨噬细胞源性泡沫细胞分别经Csn-B、Ad-Nur77和si-Nur77处理后胆固醇代谢相关基因的表达。结果表明Nur77上调ATP结合盒体Al(ABCA1),清道夫受体B类1型(SR-B1)的,C型尼曼-匹克蛋白(NPC1),小凹蛋白1(CAV-1)和中性胆固醇酯水解酶(nCEH)基因表达水平(P<0.05)。相反地,Nur77能下调的基因包括低密度脂蛋白受体(LDLR),胆固醇酯转移蛋白(CETP),清道夫受体CD36的SRA1(P<0.05)。此外,Nur77对ATP结合盒G1(ABCG1)的mRNA的表达影响没有统计学意义(P>0.05)。
接着我们观察THP-1巨噬细胞源性泡沫细胞分别经Csn-B、Ad-Nur77和Si-Nur77处理后炎症基因表达情况。我们发现Nur77上调炎症基因包括转化生长因子(TGF-p)和CD40(P<0.05),而Nur77下调基因包括白细胞介素-1p(IL-1p),白细胞介素-6(IL-6),白细胞介素12(IL-12),肿瘤坏死因子-α(TNF-a),C-反应蛋白(CRP),细胞间粘附分子1(ICAM-1)和核因子Kb(NF-KB)(P<0.05)。此外,Csn-B对血管细胞粘附分子(VCAM-1)的mRNA表达影响没有统计学意义(P>0.05),却能上调白细胞介素18(IL-18)mRNA的表达(P<0.05)。Ad-Nur77能抑制VCAM-1的mRNA表达而si-Nur77能促进VCAM-1的mRNA表达(P<0.05)。Ad-Nur77和si-Nur77对IL-18的mRNA表达影响没有统计学意义(P>0.05)。此外,THP-1巨噬细胞源性泡沫细胞分别经Csn-B、Ad-Nur77和si-Nur77处理后没有检测到Y-干扰素(IFN-γ)和白细胞介素10(IL-10)的mRNA表达。
2、Nur77对apoE-/-小鼠血浆脂质水平和血浆炎症因子表达水平影响的研究。
接着我们检测Nur77激动剂(Csn-B),Nur77慢病毒过表达载体(Ad-Nur77)和Nur77特异性siRNA慢病毒载体(si-Nur77)处理apoE-/-小鼠对后血浆脂质水平和血浆细胞因子水平的变化。我们发现apoE-/-小鼠经Csn-B和Ad-Nur77处理后,血浆高密度脂蛋白胆固醇(HDL-C)减少(P<0.05)。而且,apoE-/-小鼠经Csn-B和Ad-Nur77处理后血浆低密度脂蛋白胆固醇(LDL-C)增加(P<0.05)。相反地,apoE-/-小鼠经si-Nur77处理后,血浆中HDL-C明显增加(P<0.05),而LDL-C水平却减少(P<0.05)。然而,与对照组相比较,apoE-/-小鼠分别经Csn-B、 Ad-Nur77和Si-Nur77处理后,血浆中载脂蛋白Al(apoA1),载脂蛋白B(apoB),极低密度脂蛋白胆固醇(VLDL-C),总甘油三酯(TG)和总胆固醇(T-Cho)水平之间的差异没有统计学意义(P>0.05)。
接着我们采用ELISA检测apoE-/-小鼠体内Nur77表达的变化是否会引起血浆炎性细胞因子的相应变化。我们发现,apoE-/-小鼠分别经Csn-B和Ad-Nur77处理后血浆CRP浓度减少(P<0.05)。相反地,apoE-/-小鼠经si-Nur77处理后血浆CRP浓度增加(P<0.05)。然而,在各对照组和处理组之间,血浆炎症因子IL-1β,IL-6和TNF-a水平之间的差异没有统计学意义(P>0.05)。
3、Nur77对apoE-/-小鼠肝脏脂质蓄积影响的研究。
我们首先采用Western blot和免疫组化检测分别经Csn-B、Ad-Nur77和si-RNA处理后apoE-/-小鼠肝脏Nur77蛋白表达情况。我们发现基础组Nur77蛋白质水平表达较低,而对照组Nur77蛋白表达增加。此外,与对照组相比较,Csn-B和Ad-Nur77处理组Nur77蛋白表达增加而si-RNA处理组Nur77表达减少。接下来,我们采用油红O染色和酶法分析Nur77对apoE-/-小鼠肝脏脂质含量的影响。与对照组相比较,Csn-B和Ad-Nur77处理组肝脏三酰甘油水平减少(P<0.05),而si-Nur77处理组肝脏三酰甘油水平增加(P<0.05)。然而,apoE-/-小鼠肝脏胆固醇水平在各组之间的差异没有统计学意义(P>0.05)。
我们采用实时定量PCR检测肝脏组织中脂质代谢相关基因的表达以分析Nur77减少肝脏脂质蓄积的机制。与对照组相比较,Csn-B和Ad-Nur77处理组肝脏组织中LDLR,ATP结合盒G5(ABCG5),CRP,固醇调节元件结合蛋白-1C(SREBP1C)和固醇调节元件结合蛋白-2(SREBP2)的：mRNA表达较少(P<0.05),而SR-B1和肝脂酶(HL)的mRNA表达增加(P<0.05)。此外,si-RNA处理组与对照组相比较,肝脏组织中LDLR,ABCG5,CRP,SREBP1c和SREBP2的mRNA表达增加(P<0.05),而SR-B1和HL的mRNA表达减少(P<0.05)。虽然Csn-B处理组肝脏组织中HMG-CoA还原酶(HMGCR)的基因表达减少(P<0.05),而Ad-Nur77处理组HMGCR的nRNA表达增加(P<0.05),并且si-RNA处理组HMGCR的mRNA表达减少(P<0.05)。此外,Nur77对apoE-/-小鼠肝脏组织apoA1和卵磷脂胆固醇转移酶(LCAT)的mRNA表达影响没有统计学意义(P>0.05)。
4、Nur77对Caco-2细胞和apoE-/-小鼠肠脂质吸收相关基因表达影响的研究。
为了研究Nur77对肠脂质吸收的影响,我们通过培养Caco-2细胞,分别经Nur77激动剂(Csn-B10μg/ml),重组质粒过表达Nur77(Ad-Nur77)以及Nur77特异性siRNAs(si-Nur77)处理后,检测肠脂质转运相关基因表达水平。我们发现,与对照组相比较,Csn-B和Ad-Nur77处理后Caco-2细胞内微粒体甘油三酯转运蛋白(MTP),尼曼-匹克C1型类似蛋白1(NPC1L1)和ABCG5基因表达水平减少(P<0.05)。相反,经si-Nur77处理后Caco-2细胞内MTP,NPC1L1和ABCG5基因表达水平增加(P<0.05)。同样,我们也检测了apoE-/-小鼠分别经Csn-B、 Ad-Nur77和si-RNA处理后肠组织巾MTP,NPC1L1和ABCG5表达情况。我们发现MTP,NPC1L1和ABCG5基因表达水平变化与Csn-B、Ad-Nur77和si-RNA处理Caco-2细胞后基因表达水平变化一致。
5、Nur77对apoE-/-小鼠动脉粥样硬化病变影响的研究。
我们通过观察apoE-/-小鼠主动脉窦和整个主动脉树的动脉粥样硬化病变面积大小评估Nur77对动脉粥样硬化病变的影响。我们发现apoE-/-小鼠经Csn-B和Ad-Nur77处理后,主动脉窦和整个主动脉树动脉粥样硬化病变面积都减少(P<0.05),相反地,apoE-/-小鼠经si-Nur77处理后,主动脉窦和整个主动脉树动脉粥样硬化病变面积都增加(P<0.05)。
接下来,我们采用实时定量PCR检测主动脉组织炎症分子,粘附分子以及与胆固醇代谢相关基因表达的情况。与对照组相比较,apoE-/-小鼠分别经Csn-B和Ad-Nur77处理后,主动脉组织中SRA1,CD36,IL-1β,IL-6,CRP,ICAM-1,巨噬细胞炎症蛋白-1α(MIP-1α)和单核细胞趋化蛋白-1(MCP-1)基因表达减少(P<0.05),但ABCA1, SR-B1, NPC1和CD40分子基因表达增加(P<0.05)。相反地,与对照组相比较,apoE-/-小鼠经si-Nur77处理后,主动脉组织中SRA1,CD36,IL-1β,IL-6,CRP,ICAM-1,MIP-1α和MCP-1基因表达增加(P<0.05),而ABCA1,SRB1,NPC1和CD40基因表达减少(P<0.05)。另外,TNF-α基因表达水平在Ad-Nur77处理apoE-/-小鼠主动脉组织中减少(P<0.05),而在si-Nur77处理apoE-/-小鼠主动脉组织中增加(P<0.05)。但是Csn-B处理apoE-/-小鼠主动脉组织中TNF-α基因表达变化没有统计学意义(P>0.05)。
结论
1、Nur77能减少THP-1巨噬细胞胆固醇吸收,减少THP-1巨噬细胞源性泡沫细胞内脂质成分并增强THP-1巨噬细胞源性泡沫细胞内胆固醇流出。2、Nur77能减少apoE-/-小鼠血浆HDL胆固醇水平,增加LDL胆固醇水平并减少血浆CRP水平。3、Nur77能减少apoE-/-小鼠肝脏三酰甘油水平但不影响肝脏胆固醇水平。4、Nur77能减少肠脂质吸收相关基因的表达。5、Nur77能减少apoE-/-小鼠主动脉动脉粥样硬化病变面积。
Background
Coronary atherosclerosis represents the leading cause of morbidity and mortality of men and women throughout the western world. Hypercholesterolemia is a well-established risk factor for the incidence of atherosclerosis and its pathological complications. Evidence from clinical trials indicates that reducing plasma cholesterol by dietary and/or pharmacological means leads to reductions in the incidence of death from cardiovascular disease. Appealing therapeutic targets for reducing hypercholesterolemia and atherosclerosis include members of the nuclear hormone receptor superfamily, such as the peroxisome proliferator-activated receptor and the liver X receptor subfamilies, among others. These ligand-activated transcription factors regulate various metabolic processes by controlling the expression of specific gene cassettes.
In addition to these well-characterized ligand-activated transcription factors, the nuclear receptor (NR) superfamily comprises many orphan receptors, whose ligands and physiological functions remain unknown. Among this group of orphan receptors is the NR4A subfamily, including Nur77(NR4A1), Nurrl (NR4A2), and Nor-1(NR4A3). In contrast to other members of the superfamily, NR4A nuclear receptors are'immediate early genes', and are transiently and rapidly induced by a pleiotropy of environmental cues. Nur77is a member of the NR4A subfamily and consists, like other nuclear receptors, of an N-terminal activating function-1(AF-1) domain, a central two zinc-finger DNA-binding domain (DBD), and a C-terminal ligand binding domain (LBD). Early functional studies have pointed to a critical role of Nur77in regulating differentiation, proliferation, and apoptosis. More recent research has characterized Nur77as key transcriptional regulators of glucose and lipid homeostasis, adipogenesis, inflammation, and vascular remodeling. Initial experiments have demonstrated that Nur77promotes lipolysis in muscle. Subsequently, studies reveal that Nur77modulates plasma lipoprotein profiles and hepatic lipid metabolism in mice. Consistent with these data, the recent report have noted that the hepatic steatosis and increased transcription factors sterol regulatory element-binding binding protein lc (SREBPlc) expression can be observed in Nur77-deficient mice fed a high-fat diet. The present study evaluated the effect of Nur77on cholesterol metabolism in THP-1macrophage-derived foam cells, and on plasma lipoprotein profiles, circulating cytokine levels, hepatic lipid deposition and the development of aortic atherosclerosis in apoE-/-mice.
Aims
1. To study the effect of Nur77on lipid loading, lipid content and cholesterol efflux.2. To study the effect of Nur77on plasma lipid parameters and circulating cytokinelevels in apoE-/-Mice.3. To study the effect of Nur77on hepatic lipid deposition in apoE-/-mice.4. To study the effect of Nur77on gene expression involved in intestinal lipid absorption.5. To study the effect of Nur77on plaque formation in ApoE-/-Mice.
Methods
1. Effect of Nur77on lipid loading, lipid content and cholesterol efflux by in THP-1macrophage-derived foam cells by treatment with Nur77agonist Cytosporone B (Csn-B,10μg/ml), recombinant plasmid over-expressing Nur77(Ad-Nur77) and siRNAs against Nur77(si-Nur77).
1.1Human monocytic THP-1cells were maintained in RPMI1640medium containing10%fetal calf serum (FCS) in the presence of streptomycin (100μg/mL), penicillin (100U/ml) and differentiated for72h with100nM phorbol12-myristate13-acetate (PMA). Macrophages were transformed into foam cells by incubation in the presence or absence of50mg/ml ox-LDL in serum-free RPMI1640medium containing0.3%bovine serum albumin (BSA) for48h. Cells were seeded in6-or12-well plates or60-mm dishes and grown to80-90%confluence before use.
1.2The PCR-XL-TOPO vector containing Nur77, vector PIRES2-EGFP, Platinum HIFI Taq polymerase and Accuprime Pfx DNA polymerase were purchased from Invitrogen Biotechnology (Shanghai, China). The pCDNA3.1(+) vector, competent DH5a cells, Xhol and EcoRI restriction enzymes and T4DNA ligase were purchased from TaKaRa Biotechnology Co. Ltd.(Dalian, China). The fragment of EcoRI-Nur77-IRES-EGFP-Xhol was achieved from the PCR-XL-TOPO vector and PIRES2-EGFP vector by overlap extension PCR and was linked to pcDNA3.1to create the recombinant plasmid pcDNA3.1-Nur77-IRES-EGFP. The inserted gene was identified by electrophoresis and sequencing. The recombinant plasmid was then transfected into the cultured cells by Lipofectamine2000(Invitrogen), and the over-expression effects of Nur77was confirmed by RT-PCR and western blotting.
1.3Short-interfering RNA (siRNA) specific for human Nur77and nonsilencing control siRNA were synthesized by Guangzhou RiboBio, China. Cells (2×106/well) were transfected using Lipofectamine2000. Forty-eight h post-transfection, real-time RT-PCR and Western blotting were performed. Based on the Western blot analysis, the Nur77siRNA suppressed the expression of Nur77proteins by83%as compared to the control siRNA in THP-1macrophage-derived foam cells.
1.4Total RNA from cultured cells was extracted using TRIzol reagent (Invitrogen) in accordance with the manufacturer's instructions. Real-time quantitative PCR, using SYBR Green detection chemistry, was performed on the ABI7500Fast Real Time PCR system (Applied Biosystems, Foster City, CA, USA). Melt curve analyses of all real-time PCR products were performed and shown to produce a single DNA duplex. All samples were measured in triplicate and the mean value was considered for comparative analysis. Quantitative measurements were determined using the AACt method and GAPDH expression was used as the internal control.
1.5PMA-differentiated THP-1cells were treated with Nur77agonist Cytosporone B (Csn-B,10μg/ml), recombinant plasmids over-expressing Nur77(Ad-Nur77) and siRNAs against Nur77(si-Nur77) as indicated, then fluorescent-tagged Dil-oxLDL was added, and the cells were incubated for24h. Adherent cells were harvested, washed three times with phosphate buffer saline (PBS). Analysis was performed on a fluorescent activated cell sorting (FACS) calibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) with Cell Quest Pro software (BD Biosciences, San Jose, CA, USA).
1.6The sterol analyses were performed using a HPLC system (model2790, controlled with Empower Pro software; Waters Corp., Milford, MA, USA). Absorbance at216nm was monitored. Data were analyzed with TotalChrom software from PerkinElmer (Waltham, MA, USA).
1.7Cells were cultured and treated with Nur77agonist Cytosporone B (Csn-B,10μg/ml), recombinant plasmids over-expressing Nur77(Ad-Nur77) and siRNAs against Nur77(si-Nur77), as indicated above. Then, they were labeled with0.2μCi/ml [3H]cholesterol. After72h, cells were washed with PBS and incubated overnight in RPMI1640medium containing0.1%(w/v) BSA to allow equilibration of [3H]cholesterol in all cellular pools. Equilibrated [3H]cholesterol-labeled cells were washed with PBS and incubated in2ml of efflux medium containing RPMI1640medium and0.1%BSA with25μg/ml human plasma apoA-I. A150μl sample of efflux medium was obtained at the times designated and passed through a0.45-μm filter to remove any floating cells. Monolayers were washed twice with PBS, and cellular lipids were extracted with isopropanol. Medium and cell-associated [3H]cholesterol was then measured by liquid scintillation counting. Percent efflux was calculated by the following equation:[total media counts/(total cellular counts+total media counts)]×100%.
2. Effect of Nur77on plasma lipid parameters and circulating cytokine levels in apoE-/-mice by treatment with Nur77agonist, lentivirus encoding mouse Nur77(Ad-Nur77) and lentivirus encoding mouse Nur77(si-Nur77).
2.1The serum concentrations of IL-1β, IL-6and TNF-a were measured in duplicate using a commercial ELISA kit (R&D Systems, Minneapolis, MN, USA). Serum CRP amount was measured in duplicate using a commercial ELISA kit (Diagnostic System Laboratories, Webster, TX, USA).
2.2The serum apolipoprotein A1(apoA1) and apoB100concentrations were measured in duplicate using a commercial ELISA kit (Cusabio Biotech Co., Ltd., China). The T-Cho, TG, LDL-C, HDL-C and VLDL-C concentrations were determined enzymatically using an automated analyzer.
3. Effect of Nur77on hepatic lipid deposition in apoE-/-mice by treatment with Nur77agonist, lentivirus encoding mouse Nur77(Ad-Nur77) and lentivirus encoding mouse Nur77(si-Nur77).
3.1Hepatic lipid deposition was assessed in samples embedded in OCT compound by Oil Red O staining. Briefly, liver cryosections were fixed for10min in60%isopropanol and stained with0.3%Oil Red O in60%isopropanol for30min and subsequently washed with60%isopropanol. Sections were counterstained with Gill's hematoxylin, washed with acetic acid solution (4%), and mounted with aqueous solution. Once stained, sections were quantified by histomorphometry.
3.2For the procedure, each frozen liver tissue was sectioned at5μM thicknesses and fixed to microscope slides. Sections of frozen tissue specimens were mounted on polyl-lysine (Sigma, St. Louis, MO)-coated slides, air dried, and fixed with acetone. Immunohistochemical staining was performed for Nur77using Rabbit polyclonal to Nur77antibody at a dilution of1:100(Abcam, Cambridge, MA, USA). Images were acquired and quantitated on an Olympus BX50microscope using Optimis software (Version6.2) and digitized using a color video camera (three-charge coupled device; JVC, Wayne, NJ).
4. Effect of Nur77on gene expression involved in intestinal lipid absorption in Caco-2cells by treatment with Nur77agonist Cytosporone B (Csn-B,10μg/ml), recombinant plasmid over-expressing Nur77(Ad-Nur77) and siRNAs against Nur77(si-Nur77) and in apoE-/-mice by treatment with Nur77agonist, lentivirus encoding mouse Nur77(Ad-Nur77) and lentivirus encoding mouse Nur77(si-Nur77).
4.1Caco-2cells were grown in Dulbecco's modified Eagle's medium (DMEM) containing10%FCS with streptomycin (100μg/mL) and penicillin (100U/ml). All cells were incubated at37℃,5%CO2. Cells were seeded in6-or12-well plates or60-mm dishes and grown to80-90%confluence before use.Caco2cells by treatment with Nur77agonist Cytosporone B (Csn-B,10μg/ml), recombinant plasmid over-expressing Nur77(Ad-Nur77) and siRNAs against Nur77(si-Nur77).
4.2Total RNA from cultured cells was extracted using TRIzol reagent (Invitrogen) in accordance with the manufacturer's instructions. Real-time quantitative PCR, using SYBR Green detection chemistry, was performed on the ABI7500Fast Real Time PCR system (Applied Biosystems, Foster City, CA, USA). Melt curve analyses of all real-time PCR products were performed and shown to produce a single DNA duplex. All samples were measured in triplicate and the mean value was considered for comparative analysis. Quantitative measurements were determined using the AACt method and GAPDH expression was used as the internal control.
5. Effect of Nur77on plaque formation in ApoE-/-Mice by treatment with Nur77agonist, lentivirus encoding mouse Nur77(Ad-Nur77) and lentivirus encoding mouse Nur77(si-Nur77).
5.1For en face analysis, aortas from different groups were opened longitudinally from the heart to the iliac arteries, and lesions were stained with Oil Red O. En face aortic lesion areas were digitized by a Nikon S6digital camera, analyzed using Image-Pro Plus image analysis software (Media Cybernetics, Bethesda, MD, USA), and expressed as the percentage of the total aortic surface area covered by lesions.
5.2The upper portion of the heart and proximal aorta were obtained, embedded in Optimal Cutting Temperature (OCT) compound (Fisher, Tustin, CA), and stored at -70℃. Serial10-μm thick cryosections of aorta, beginning at the aortic root, were collected over a distance of400μm. Sections were stained with Oil Red O. The Oil Red O-positive areas in digitized color images of stained aortic root sections (three equally spaced sections per mouse; n=5per group) were quantified using Image-Pro Plus image analysis software (Media Cybernetics), and the data are expressed as percent of total section area.
Results
1. Nur77contributes to lipid loading, lipid content and cholesterol efflux
We first investigated the role of Nur77during lipid loading in THP-1macrophages, the effects of Nur77on lipid content and cholesterol efflux in THP-1macrophage-derived foam cells by treatment with Nur77agonist Cytosporone B (Csn-B,10μg/ml), recombinant plasmid over-expressing Nur77(Ad-Nur77) and siRNAs against Nur77(si-Nur77). DiI-labeled ox-LDL uptake was obviously decreased by Csn-B and Ad-Nur77(P<0.001), while obviously increased by si-Nur77(P<0.001). Next, we examined the effects of Nur77on cholesterol content and cholesterol efflux in THP-1macrophage-derived foam cells by HPLC and liquid scintillation counting assays, respectively. Cellular cholesterol content was decreased while cholesterol efflux was increased when cells were treated with Csn-B and Ad-Nur77(P<0.05). On the contrary, cellular cholesterol content was increased while cholesterol efflux was decreased when cells were treated with si-Nur77(P<0.05).
Subsequently, we performed mRNA expression analysis of a panel of genes involved in lipid uptake, lipid transport and cholesterol efflux in THP-1macrophage-derived foam cells by treatment with Csn-B, recombinant plasmid over-expressing Nur77and siRNAs against Nur77. Nur77up-regulated genes included ATP-binding cassette A1(ABCA1), scavenger receptor class B type1(SR-B1), Niemann-Pick C1protein (NPC1), Caveolin-1(CAV-1), neutral cholesterol ester hydrolase (nCEH)(P<0.05). Down-regulated genes through the expression of Nur77included low density lipoprotein receptor (LDLR), cholesteryl ester transfer protein (CETP), scavenger receptor CD36and SRA1(P<0.05). In addition, Nur77 had no effect on ATP-binding cassette G1(ABCG1) mRNA expression (P>0.05).
Next, we explored the effect of Nur77on inflammatory gene expression in THP-1macrophage-derived foam cells by treatment with Csn-B, Ad-Nur77and si-Nur77. Nur77up-regulated genes included transforming growth factor (TGF-(3) and CD40(P<0.05), whereas down-regulated genes through the expression of Nur77included interleukin-1β(IL-1β), interleukin-6(IL-6), interleukin-12(IL-12), tumor necrosis factor-a (TNF-a), C-reactive protein (CRP), intercellular adhesion molecule-1(ICAM-1) and nuclear factor κB (NF-κB)(P<0.05). In addition, Csn-B had no effect on vascular cell adhesion molecule (VCAM-1) mRNA expression (P>0.05), but up-regulated interleukin-18(IL-18) mRNA expression (P<0.05). However, VCAM-1mRNA expression was inhibited by Ad-Nur77(P<0.05), while enhanced by Si-Nur77(P<0.05). Treatment with both Ad-Nur77and Si-Nur77had no effect on IL-18mRNA expression (P>0.05). Furthermore, the gene expressions of interferon-y (INF-y) and interleukin-10(IL-10) were not detectable in THP-1macrophage-derived foam cells through treatment with Csn-B, Ad-Nur77and si-Nur77.
2. Nur77regulates plasma lipid parameters and circulating cytokine levels in apoE-/-Mice
We examined the terminal plasma lipid levels from experimental mice.the plasma high density lipoprotein cholesterol (HDL-C) showed a moderate reduction in the Csn-B group and Ad-Nur77group as compared to their control groups, respectively (P<0.05). Concomitantly, plasma low density lipoprotein cholesterol (LDL-C) increased in the Csn-B group and Ad-Nur77group as compared to their control groups, respectively (P<0.05). In contrast, treatment with si-Nur77led to an increase in plasma HDL-cholesterol (P<0.05) and a reduction in plasma LDL-cholesterol as compared to the si-Mock group (P<0.05). However, no significant alternation occurred in apoAl, apolipoprotein B (apoB), very low density lipoprotein cholesterol (VLDL-cholesterol), total triglyceride and cholesterol (P>0.05).
To investigate whether change in Nur77expression could result in corresponding changes in plasma inflammatory cytokines, we conducted a series of ELISAs. Consistent with the data of inflammatory gene expression in THP-1macrophage-derived foam cells, treatment with both Csn-B and Ad-Nur77resulted in down-regulation of CRP concentrations in plasma, respectively (P<0.05). In agreement with these data, si-Nur77-mediated knockdown of Nur77results in an up-regulation of CRP concentrations in plasma (P<0.05). However, no significant alteration occurred in levels of IL-1β, IL-6and TNF-a between groups at the end of the experiments (P>0.05).
3. Nur77affects hepatic lipid deposition in apoE-/-mice
The protein expression of Nur77in mouse liver was investigated by Western blot and immunohistochemistry analyses. We observed that minor expression levels were detectable in the baseline group, but expression of Nur77was markedly increased in the control group. In addition, the Csn-B group and Ad-Nur77group had significantly higher expression of Nur77than their control groups while the si-Nur77group had a lower expression of Nur77as compared to the si-Mock group. Next, the effects of Nur77on lipid content in the liver of apoE-/-mice were analyzed by Oil Red O staining and measured enzymatically. Hepatic triglyceride levels in the liver was reduced in mice treated with Csn-B and Ad-Nur77, respectively, compared with mice of the control group (P<0.05). In contrast, the hepatic triglyceride levels in the liver was increased in mice treated with si-Nur77in comparison to mice treated with si-Mock (P<0.05). However, hepatic cholesterol level had no change in response to regulation of expression of Nur77in the liver (P>0.05).
To investigate the mechanisms of Nur77reduction of hepatic lipid deposition, hepatic lipid metabolism gene expression levels in livers of apoE-/-mice were analyzed by real time PCR. As shown, Csn-B and Ad-Nur77treatment progressively reduced gene levels of LDLR, ATP-binding cassette G5(ABCG5), CRP, SREBP1C, SREBP2(P<0.05), and increased gene levels of SR-B1and hepatic lipase (HL)(P<0.05). In addition, treatment with si-Nur77could up-regulated gene expression of LDLR, ABCG5, CRP, SREBP1C and SREBP2(P<0.05). While SR-B1and HL gene expressions were down-regulated by treatment with si-Nur77(P<0.05). Although gene expression of HMG-CoA reductase (HMGCR) was repressed by treatment with Csn-B (P<0.05), the gene expression of HMGCR was increased by treatment with Ad-Nur77(P<0.05) while decreased by treatment with si-Nur77(P<0.05). In addition, Nur77had no effect on apoA1and LCAT expression (P>0.05).
4. Nur77inhibits gene expression involved in intestinal lipid absorption
To address the possibility that expression levels of intestinal lipid transporters are affected by Nur77, we incubated Caco-2cells with Csn-B, recombinant plasmid Ad-Nur77and siRNAs against Nur77(si-Nur77). Csn-B and Ad-Nur77treatment progressively reduced gene levels of microsomal triglyceride transfer protein (MTP), Niemann-Pick Cl-like1(NPC1L1) and ABCG5(P<0.05). On the contrary, si-Nur77treatment markedly induced gene expression of MTP, NPC1L1and ABCG5(P<0.05). To further investigate the Nur77mechanisms affecting intestinal cholesterol absorption in apoE-/-mice, gene expression in intestinal tissue of apoE-/-mice were analyzed by real time PCR. We also found that over-expression of Nur77reduced MTP, NPC1L1and ABCG5gene expression (P<0.05), while silenced Nur77increased these gene levels (P<0.05).
5. Nur77reduces plaque formation in ApoE-/-Mice
To investigate the impact of Nur77on atherogenesis in apoE-/-mice, atherosclerotic lesions were evaluated by aortic valve section and en face analyses. Mice receiving Csn-B and Ad-Nur77showed a decrease in the average lesion area compared with their controls by both en face and aortic valve section analyses (P<0.05). On the contrary, si-Nur77-treated mice showed an increase in average lesion area compared with si-Mock-treated mice by both en face and aortic valve section analyses (P>0.05).
Next, the gene expression changes of the inflammatory molecules, adhesion molecules and molecules related to cholesterol metabolism were investigated in aortic tissues. In both Csn-B-treated and Ad-Nur77-treated apoE-/-mice, gene expression of SRA1, CD36, IL-1β, IL-6, CRP, ICAM-1, macrophage inflammatory protein-1α (MIP-1α) and monocyte chemo-attractant protein-1(MCP-1) were markedly repressed (P<0.05), but gene expression of ABCA1, SR-B1, NPC1and CD40were up-regulated at week12(P<0.05). On the contrary, we found that gene expression of SRA1, CD36, IL-1β, IL-6, CRP, ICAM-1, MIP-1α and MCP-1were up-regulated (P<0.05), while gene expression of ABCA1, SRB1, NPC1and CD40were down-regulated (P<0.05) in the aorta of si-Nur77-treated ApoE-/-mice. In addition, gene expression of TNF-a was reduced in Ad-Nur77-treated ApoE-/-mice (P<0.05), while increased in si-Nur77-treated ApoE-/-mice (P<0.05). However, Csn-B-treated apoE-deficient mice had no change in TNF-a gene expression (P>0.05).
Conclusions
1. Nur77reduces lipid loading, lipid content and enhanced cholesterol efflux in THP-1macrophage-derived foam cells.2. Nur77decreases plasma HDL-cholesterol level, increases plasma LDL-cholesterol level and decreases CRP concentrations in apoE-/-Mice.3. Nur77down-regulates triglyceride levels but not cholesterol level in the liver.4. Nur77inhibits gene expression involved in intestinal lipid absorption.5. Nur77reduces plaque formation in ApoE-/-Mice.
流行病学研究表明冠状动脉粥样硬化是西方发达国家中发病率和死亡率的首要原因。高胆固醇血症是动脉粥样硬化发病的重要原因和风险因素。临床试验的证据表明,通过饮食和／或药理学方法可以降低血浆胆固醇,减少心血管疾病的发病率和死亡率。实验研究发现,很多核受体超家族成员可以作为为减少高胆固醇血症和动脉粥样硬化的治疗靶点。这些核激素受体超家族的成员包括过氧化物酶体增殖物激活受体和肝X受体等。这些配体激活的转录因子通过调控特定的基因盒的表达来调节各种重要的代谢过程。
除了配体激活的转录因子,核受体家族还包括许多孤儿受体,其配体和生理功能尚不清楚。其中核受体NR4A就是重要的核孤儿受体超家族成员,包括Nur77(NR4A1),Nurr1基因(NR4A2)和Nor-1(NR4A3).NR4A与其他核受体家族成员相比,是一种“即早基因”,在各种环境刺激下能短暂和迅速被诱导反应。Nur77是NR4A家族中的成员,与该家族中其他核受体一样含有N-端的激活功能区域AF-1、带两个锌指结构的DNA结合域DBD以及C-终端配体结合域LBD。早期功能研究已经指出Nur77在调节分化,增殖和凋亡过程中起着关键作用。最近的研究表明Nur77在葡萄糖代谢和脂质代谢,脂肪形成,炎症反应以及血管重构中发挥重要的作用。最初的实验表明,Nur77能够促进肌肉中的脂肪水解。随后研究表明,Nur77可以调节小鼠血浆脂蛋白水平和肝脏脂质代谢。在最近的实验研究中发现,在高脂喂养Nur77缺陷小鼠中转录因子固醇调节元件结合蛋白1C(SREBP1C)的表达明显增加并出现明显的肝脏脂质蓄积。因此,本研究通过增强和抑制Nur77的表达,观察Nur77对apoE-/-小鼠主动脉粥样硬化病变的影响并探讨其相关的机制。
目的
1、Nur77是否可以影响细胞胆固醇吸收,细胞内胆固醇成分以及细胞内胆固醇流出。2、Nur77是否可以影响apoE-/-小鼠血脂水平和循环中炎症因子水平。3、Nur77是否可以影响apoE-/-小鼠肝脏脂质代谢。4、Nur77是否可以影响肠对脂质吸收相关基因表达。5、Nur77是否可以影响apoE-/-小鼠动脉粥样硬化病变。
方法
1、Nur77对THP-1巨噬细胞胆固醇吸收,对THP-1巨噬细胞源性泡沫细胞脂质成分和胆固醇流出影响的研究。
1.1THP-1细胞生长于含有10%新生小牛血清的RPMI-1640培养液中,37℃,5%C02培养箱中静置培养。培养液巾加100U/ml的青霉素和100μg/mL的链霉素。取对数生长期细胞进行实验,在每次实验前用100nM佛波酯(phorbol12-myristate13-acetate, PMA)(?)呼育THP-1细胞72h,使其诱导分化成巨噬细胞,更换培养基后加50mg/ml ox-LDL孵育48h时期转化成泡沫细胞。
1.2PCR-XL-TOPO载体,pIRES2-EGFP载体,platinum HIFI Taq Polymerase高保真扩增酶,Accuprime Pfx DNA Polymerase高保真扩增酶购自于Invitrogen(上海)公司。pCDNA3.1(+)载体,DH5α感受态细胞,XhoI、EcoRI限制性内切酶,T4DNA Ligase(?)勾自于TaKaRa(大连)公司。通过重叠PCR将PCR-XL-TOPO和PIRES2-EGFP2个片段拼接为EcoRI-NR4A1-IRES-EGFP-XhoI的全长目的片段,并将其连接到pcDNA3.1构建重组质粒pcDNA3.1-Nur77-IRES-EGFP.经电泳和测序验证后,通过脂质体2000将重组质粒转染到培养的细胞中,采用RT-PCR和Western blot检测转染效率。
1.3人类Nur77特异性siRNA(Nur77-siRNA)和对照siRNA合成自广州锐博生物公司。采用脂质体2000转染细胞(2×106/well)。转染后48小时采用实时定量PCR和Western blot检测干扰效果。Western blot分析结果表明,与对照组siRNA相比较,Nur77-siRNA抑制THP-1巨噬细胞源性泡沫细胞内Nur77蛋白表达效率为83%。
1.4根据Invitrogen公司RNA提取试剂盒操作说明提取培养细胞中提取总RNA。将1lμg,总RNA用逆转录试剂盒(TaKaRa)反转录成20μμlcDNA。实时定量PCR在应用生物系统ABI7500FAST上进行。溶解曲线分析表明PCR反应产物为单独的双链DNA。用ΔΔCt值法,以GAPDH表达为内参定量其它基因的表达。
1.5流式细胞术检测细胞胆固醇吸收。THP-1细胞经PMA诱导分化成巨噬细胞后分别经Nur77激动剂Cytosporone B (Csn-B10μg/ml),重组质粒过表达Nur77(Ad-Nur77)以及Nur77特异性siRNAs(si-Nur77)处理,然后加入荧光标记Dil-oxLDL培养细胞24小时。收集贴壁细胞,用磷酸盐缓冲液(PBS)洗三次后,采用Becton Dickinson公司流式细胞仪分析细胞荧光强度。
1.6高效液相色谱检测细胞内胆固醇成分。THP-1细胞经PMA诱导分化成巨噬细胞再经ox-LDL诱导转化成泡沫细胞后,分别经Nur77激动剂Cytosporone B(Csn-B10μg/m1)),重组质粒过表达Nur77(Ad-Nur77)以及Nur77特异性siRNAs(si-Nur77)处理。待细胞处理结束后,弃培养基,PBS洗3遍,加入细胞裂解液200μl,反复冻融3次裂解细胞,BCA法定量蛋白后,7.2%三氯乙酸沉淀蛋白,800×g离心10min,取上清进行胆固醇检测,以豆甾醇为内标。取100μl上清液,加入8.9mol/L氢氧化钾溶液200μl,水解胆固醇酯后为细胞内总胆固醇检测样品。各样品分别与内标液混匀,用正己烷和无水乙醇抽提后,1.5mol/L的三氧化铬进行氧化衍生并真空干燥,100μl乙晴-异丙醇(80：20)溶解样品,上样于高效液相色谱仪。采用Waters Corp公司2790高效液相色谱仪进行检测。
1.7采用液体闪烁技术检测细胞内胆固醇流出。THP-1细胞经PMA诱导分化成巨噬细胞再经ox-LDL诱导转化成泡沫细胞后,分别经Nur77激动剂Cytosporone B(Csn-B10μg/ml),重组质粒过表达Nur77(Ad-Nur77)以及Nur77特异性siRNAs(si-Nur77)处理。待细胞处理结束后,弃培养基,PBS洗3遍,用0.2μCi/ml[3H]胆固醇在含有10%小牛血清RPMI-1640培养液共同孵育72小时。用PBS液洗涤细胞,置含脂蛋白的无血清RPMI-1640培养液中培养24小时。PBS液洗涤细胞,闪烁液裂解细胞后,用闪烁计数法检测培养液和细胞中的[3H]胆固醇。胆固醇流出率用培养液中CPM除以总CPM(培养液CPM+细胞CPM),再乘以100%来表示。
2、Nur77对apoE-/-小鼠血浆脂质水平和血浆炎症因子表达水平影响的研究。
2.1血清IL-1β,IL-6和TNF-a浓度使用R&D公司商业ELISA试剂盒检测。血清CRP浓度使用Cusabio Biotech Co公司商业ELISA试剂盒检测。
2.2血清载脂蛋白A1(apoAl)和载脂蛋白B100(apoB100)浓度采用Cusabio公司商业ELISA试剂盒进行测定。采用全自动生化分析仪检测血清总胆固醇(T-Cho),总三酰甘油(TG),低密度脂蛋白胆固醇(LDL-C),高密度脂蛋白胆固醇(HDL-C)和极低密度脂蛋白胆固醇(VLDL-C)水平。
3、Nur77对apoE-/-小鼠肝脏脂质蓄积影响的研究。
3.1肝脏组织切片油红O染色。取肝组织样本在-30℃条件下滴加包埋剂进行包埋,用衡冷切片机进行切片。肝脏冰冻切片在60%异丙醇条件下固定10分钟,再采用含0.3%油红O染色液的60%异丙醇中孵育30分钟,随后采用60%的异丙醇洗净。采用苏木素复染后进行组织形态学定量分析。
3.2肝脏组织切片免疫组化。取肝组织样本在-30℃条件下滴加包埋剂进行包埋,用衡冷切片机进行切片。冰冻切片经多聚赖氨酸浸泡风干并以丙酮固定。采用Abcam公司兔抗Nur77一抗进行孵育,PBS洗后再采用酶标二抗孵育并显色。显色后采用奥林巴斯显微镜进行拍照分析。
4、Nur77对Caco-2细胞和apoE-/-小鼠肠脂质吸收相关基因表达影响的研究。
4.1Caco-2细胞生长于含有10%新生小牛血清的DMEM培养液中,37℃、5%CO2培养箱巾静置培养。培养液巾加100U/ml的青霉素和100gg/mL的链霉素。取对数生长期细胞进行实验。
4.2根据Invitrogen公司RNA提取试剂盒操作说明提取培养细胞和组织中总RNA。将1μg总RNA用逆转录试剂盒(TaKaRa)反转录成20μlcDNA。实时定量PCR在应用生物系统ABI7500FAST上进行。溶解曲线分析表明PCR反应产物为单独的双链DNA。用ΔΔCt值法,以GAPDH表达为内参定量其它基因的表达。
5、Nur77对apoE-/-小鼠动脉粥样硬化病变影响的研究。
5.1从腹主动脉分叉至主动脉弓将主动脉剪下,干净剔除主动脉血管外的脂肪组织后,将主动脉树纵向剪开,主动脉树以用油红O染色观察整个主动脉的病变。
5.2自主动脉根部将心脏与主动脉分离,随即将心脏置于4%的多聚甲醛溶液中、常温下固定3h；冰冻切片时,将心脏自心尖剪去约1/3(沿与心脏纵轴垂直方向),以O.C.T compound包埋剂包埋。心尖朝下、将心脏垂直固定与冰冻切片机,自主动脉根部(或心底)向心尖方向进行低温冰冻连续10μm切片。每组随机取5个标本,每一个心脏标本,将连续12张10μm冰冻切片进行油红O染色,以IMAGEPROPLUS软件计算As病变面积。
结果
1、Nur77对THP-1巨噬细胞胆固醇吸收,对THP-1巨噬细胞来源泡沫细胞脂质成分和胆固醇流出影响的研究。
我们首先检测THP-1细胞经PMA诱导分化成巨噬细胞后,分别经Nur77激动齐(?)Cytosporone B(Csn-B10μg/ml),重组质粒过表达Nur77(Ad-Nur77)以及Nur77特异性siRNAs(si-Nur77)处理后,细胞对胆固醇吸收的影响。THP-1巨噬细胞经Csn-B和Ad-Nur77处理后,巨噬细胞对荧光标记Dil-oxLDL吸收减少(P<0.001),而经si-Nur77处理后,巨噬细胞对荧光标记Dil-oxLDL吸收增加(P<0.001)。接着我们采用高效液相色谱法和液体闪烁技术检测Nur77对THP-1巨噬细胞源性泡沫细胞内胆固醇成分和细胞胆固醇流出的影响。经Csn-B和Ad-Nur77处理后细胞内胆固醇成分减少(P<0.05),而细胞内胆固醇流出增加(P<0.05)。相反地,THP-1巨噬细胞源性泡沫细胞经si-Nur77处理后,细胞内胆固醇成分增加(P<0.05),而细胞胆固醇流出减少(P<0.05)。
随后,我们采用实时定量PCR检测THP-1巨噬细胞源性泡沫细胞分别经Csn-B、Ad-Nur77和si-Nur77处理后胆固醇代谢相关基因的表达。结果表明Nur77上调ATP结合盒体Al(ABCA1),清道夫受体B类1型(SR-B1)的,C型尼曼-匹克蛋白(NPC1),小凹蛋白1(CAV-1)和中性胆固醇酯水解酶(nCEH)基因表达水平(P<0.05)。相反地,Nur77能下调的基因包括低密度脂蛋白受体(LDLR),胆固醇酯转移蛋白(CETP),清道夫受体CD36的SRA1(P<0.05)。此外,Nur77对ATP结合盒G1(ABCG1)的mRNA的表达影响没有统计学意义(P>0.05)。
接着我们观察THP-1巨噬细胞源性泡沫细胞分别经Csn-B、Ad-Nur77和Si-Nur77处理后炎症基因表达情况。我们发现Nur77上调炎症基因包括转化生长因子(TGF-p)和CD40(P<0.05),而Nur77下调基因包括白细胞介素-1p(IL-1p),白细胞介素-6(IL-6),白细胞介素12(IL-12),肿瘤坏死因子-α(TNF-a),C-反应蛋白(CRP),细胞间粘附分子1(ICAM-1)和核因子Kb(NF-KB)(P<0.05)。此外,Csn-B对血管细胞粘附分子(VCAM-1)的mRNA表达影响没有统计学意义(P>0.05),却能上调白细胞介素18(IL-18)mRNA的表达(P<0.05)。Ad-Nur77能抑制VCAM-1的mRNA表达而si-Nur77能促进VCAM-1的mRNA表达(P<0.05)。Ad-Nur77和si-Nur77对IL-18的mRNA表达影响没有统计学意义(P>0.05)。此外,THP-1巨噬细胞源性泡沫细胞分别经Csn-B、Ad-Nur77和si-Nur77处理后没有检测到Y-干扰素(IFN-γ)和白细胞介素10(IL-10)的mRNA表达。
2、Nur77对apoE-/-小鼠血浆脂质水平和血浆炎症因子表达水平影响的研究。
接着我们检测Nur77激动剂(Csn-B),Nur77慢病毒过表达载体(Ad-Nur77)和Nur77特异性siRNA慢病毒载体(si-Nur77)处理apoE-/-小鼠对后血浆脂质水平和血浆细胞因子水平的变化。我们发现apoE-/-小鼠经Csn-B和Ad-Nur77处理后,血浆高密度脂蛋白胆固醇(HDL-C)减少(P<0.05)。而且,apoE-/-小鼠经Csn-B和Ad-Nur77处理后血浆低密度脂蛋白胆固醇(LDL-C)增加(P<0.05)。相反地,apoE-/-小鼠经si-Nur77处理后,血浆中HDL-C明显增加(P<0.05),而LDL-C水平却减少(P<0.05)。然而,与对照组相比较,apoE-/-小鼠分别经Csn-B、 Ad-Nur77和Si-Nur77处理后,血浆中载脂蛋白Al(apoA1),载脂蛋白B(apoB),极低密度脂蛋白胆固醇(VLDL-C),总甘油三酯(TG)和总胆固醇(T-Cho)水平之间的差异没有统计学意义(P>0.05)。
接着我们采用ELISA检测apoE-/-小鼠体内Nur77表达的变化是否会引起血浆炎性细胞因子的相应变化。我们发现,apoE-/-小鼠分别经Csn-B和Ad-Nur77处理后血浆CRP浓度减少(P<0.05)。相反地,apoE-/-小鼠经si-Nur77处理后血浆CRP浓度增加(P<0.05)。然而,在各对照组和处理组之间,血浆炎症因子IL-1β,IL-6和TNF-a水平之间的差异没有统计学意义(P>0.05)。
3、Nur77对apoE-/-小鼠肝脏脂质蓄积影响的研究。
我们首先采用Western blot和免疫组化检测分别经Csn-B、Ad-Nur77和si-RNA处理后apoE-/-小鼠肝脏Nur77蛋白表达情况。我们发现基础组Nur77蛋白质水平表达较低,而对照组Nur77蛋白表达增加。此外,与对照组相比较,Csn-B和Ad-Nur77处理组Nur77蛋白表达增加而si-RNA处理组Nur77表达减少。接下来,我们采用油红O染色和酶法分析Nur77对apoE-/-小鼠肝脏脂质含量的影响。与对照组相比较,Csn-B和Ad-Nur77处理组肝脏三酰甘油水平减少(P<0.05),而si-Nur77处理组肝脏三酰甘油水平增加(P<0.05)。然而,apoE-/-小鼠肝脏胆固醇水平在各组之间的差异没有统计学意义(P>0.05)。
我们采用实时定量PCR检测肝脏组织中脂质代谢相关基因的表达以分析Nur77减少肝脏脂质蓄积的机制。与对照组相比较,Csn-B和Ad-Nur77处理组肝脏组织中LDLR,ATP结合盒G5(ABCG5),CRP,固醇调节元件结合蛋白-1C(SREBP1C)和固醇调节元件结合蛋白-2(SREBP2)的：mRNA表达较少(P<0.05),而SR-B1和肝脂酶(HL)的mRNA表达增加(P<0.05)。此外,si-RNA处理组与对照组相比较,肝脏组织中LDLR,ABCG5,CRP,SREBP1c和SREBP2的mRNA表达增加(P<0.05),而SR-B1和HL的mRNA表达减少(P<0.05)。虽然Csn-B处理组肝脏组织中HMG-CoA还原酶(HMGCR)的基因表达减少(P<0.05),而Ad-Nur77处理组HMGCR的nRNA表达增加(P<0.05),并且si-RNA处理组HMGCR的mRNA表达减少(P<0.05)。此外,Nur77对apoE-/-小鼠肝脏组织apoA1和卵磷脂胆固醇转移酶(LCAT)的mRNA表达影响没有统计学意义(P>0.05)。
4、Nur77对Caco-2细胞和apoE-/-小鼠肠脂质吸收相关基因表达影响的研究。
为了研究Nur77对肠脂质吸收的影响,我们通过培养Caco-2细胞,分别经Nur77激动剂(Csn-B10μg/ml),重组质粒过表达Nur77(Ad-Nur77)以及Nur77特异性siRNAs(si-Nur77)处理后,检测肠脂质转运相关基因表达水平。我们发现,与对照组相比较,Csn-B和Ad-Nur77处理后Caco-2细胞内微粒体甘油三酯转运蛋白(MTP),尼曼-匹克C1型类似蛋白1(NPC1L1)和ABCG5基因表达水平减少(P<0.05)。相反,经si-Nur77处理后Caco-2细胞内MTP,NPC1L1和ABCG5基因表达水平增加(P<0.05)。同样,我们也检测了apoE-/-小鼠分别经Csn-B、 Ad-Nur77和si-RNA处理后肠组织巾MTP,NPC1L1和ABCG5表达情况。我们发现MTP,NPC1L1和ABCG5基因表达水平变化与Csn-B、Ad-Nur77和si-RNA处理Caco-2细胞后基因表达水平变化一致。
5、Nur77对apoE-/-小鼠动脉粥样硬化病变影响的研究。
我们通过观察apoE-/-小鼠主动脉窦和整个主动脉树的动脉粥样硬化病变面积大小评估Nur77对动脉粥样硬化病变的影响。我们发现apoE-/-小鼠经Csn-B和Ad-Nur77处理后,主动脉窦和整个主动脉树动脉粥样硬化病变面积都减少(P<0.05),相反地,apoE-/-小鼠经si-Nur77处理后,主动脉窦和整个主动脉树动脉粥样硬化病变面积都增加(P<0.05)。
接下来,我们采用实时定量PCR检测主动脉组织炎症分子,粘附分子以及与胆固醇代谢相关基因表达的情况。与对照组相比较,apoE-/-小鼠分别经Csn-B和Ad-Nur77处理后,主动脉组织中SRA1,CD36,IL-1β,IL-6,CRP,ICAM-1,巨噬细胞炎症蛋白-1α(MIP-1α)和单核细胞趋化蛋白-1(MCP-1)基因表达减少(P<0.05),但ABCA1, SR-B1, NPC1和CD40分子基因表达增加(P<0.05)。相反地,与对照组相比较,apoE-/-小鼠经si-Nur77处理后,主动脉组织中SRA1,CD36,IL-1β,IL-6,CRP,ICAM-1,MIP-1α和MCP-1基因表达增加(P<0.05),而ABCA1,SRB1,NPC1和CD40基因表达减少(P<0.05)。另外,TNF-α基因表达水平在Ad-Nur77处理apoE-/-小鼠主动脉组织中减少(P<0.05),而在si-Nur77处理apoE-/-小鼠主动脉组织中增加(P<0.05)。但是Csn-B处理apoE-/-小鼠主动脉组织中TNF-α基因表达变化没有统计学意义(P>0.05)。
结论
1、Nur77能减少THP-1巨噬细胞胆固醇吸收,减少THP-1巨噬细胞源性泡沫细胞内脂质成分并增强THP-1巨噬细胞源性泡沫细胞内胆固醇流出。2、Nur77能减少apoE-/-小鼠血浆HDL胆固醇水平,增加LDL胆固醇水平并减少血浆CRP水平。3、Nur77能减少apoE-/-小鼠肝脏三酰甘油水平但不影响肝脏胆固醇水平。4、Nur77能减少肠脂质吸收相关基因的表达。5、Nur77能减少apoE-/-小鼠主动脉动脉粥样硬化病变面积。
Background
Coronary atherosclerosis represents the leading cause of morbidity and mortality of men and women throughout the western world. Hypercholesterolemia is a well-established risk factor for the incidence of atherosclerosis and its pathological complications. Evidence from clinical trials indicates that reducing plasma cholesterol by dietary and/or pharmacological means leads to reductions in the incidence of death from cardiovascular disease. Appealing therapeutic targets for reducing hypercholesterolemia and atherosclerosis include members of the nuclear hormone receptor superfamily, such as the peroxisome proliferator-activated receptor and the liver X receptor subfamilies, among others. These ligand-activated transcription factors regulate various metabolic processes by controlling the expression of specific gene cassettes.
In addition to these well-characterized ligand-activated transcription factors, the nuclear receptor (NR) superfamily comprises many orphan receptors, whose ligands and physiological functions remain unknown. Among this group of orphan receptors is the NR4A subfamily, including Nur77(NR4A1), Nurrl (NR4A2), and Nor-1(NR4A3). In contrast to other members of the superfamily, NR4A nuclear receptors are'immediate early genes', and are transiently and rapidly induced by a pleiotropy of environmental cues. Nur77is a member of the NR4A subfamily and consists, like other nuclear receptors, of an N-terminal activating function-1(AF-1) domain, a central two zinc-finger DNA-binding domain (DBD), and a C-terminal ligand binding domain (LBD). Early functional studies have pointed to a critical role of Nur77in regulating differentiation, proliferation, and apoptosis. More recent research has characterized Nur77as key transcriptional regulators of glucose and lipid homeostasis, adipogenesis, inflammation, and vascular remodeling. Initial experiments have demonstrated that Nur77promotes lipolysis in muscle. Subsequently, studies reveal that Nur77modulates plasma lipoprotein profiles and hepatic lipid metabolism in mice. Consistent with these data, the recent report have noted that the hepatic steatosis and increased transcription factors sterol regulatory element-binding binding protein lc (SREBPlc) expression can be observed in Nur77-deficient mice fed a high-fat diet. The present study evaluated the effect of Nur77on cholesterol metabolism in THP-1macrophage-derived foam cells, and on plasma lipoprotein profiles, circulating cytokine levels, hepatic lipid deposition and the development of aortic atherosclerosis in apoE-/-mice.
Aims
1. To study the effect of Nur77on lipid loading, lipid content and cholesterol efflux.2. To study the effect of Nur77on plasma lipid parameters and circulating cytokinelevels in apoE-/-Mice.3. To study the effect of Nur77on hepatic lipid deposition in apoE-/-mice.4. To study the effect of Nur77on gene expression involved in intestinal lipid absorption.5. To study the effect of Nur77on plaque formation in ApoE-/-Mice.
Methods
1. Effect of Nur77on lipid loading, lipid content and cholesterol efflux by in THP-1macrophage-derived foam cells by treatment with Nur77agonist Cytosporone B (Csn-B,10μg/ml), recombinant plasmid over-expressing Nur77(Ad-Nur77) and siRNAs against Nur77(si-Nur77).
1.1Human monocytic THP-1cells were maintained in RPMI1640medium containing10%fetal calf serum (FCS) in the presence of streptomycin (100μg/mL), penicillin (100U/ml) and differentiated for72h with100nM phorbol12-myristate13-acetate (PMA). Macrophages were transformed into foam cells by incubation in the presence or absence of50mg/ml ox-LDL in serum-free RPMI1640medium containing0.3%bovine serum albumin (BSA) for48h. Cells were seeded in6-or12-well plates or60-mm dishes and grown to80-90%confluence before use.
1.2The PCR-XL-TOPO vector containing Nur77, vector PIRES2-EGFP, Platinum HIFI Taq polymerase and Accuprime Pfx DNA polymerase were purchased from Invitrogen Biotechnology (Shanghai, China). The pCDNA3.1(+) vector, competent DH5a cells, Xhol and EcoRI restriction enzymes and T4DNA ligase were purchased from TaKaRa Biotechnology Co. Ltd.(Dalian, China). The fragment of EcoRI-Nur77-IRES-EGFP-Xhol was achieved from the PCR-XL-TOPO vector and PIRES2-EGFP vector by overlap extension PCR and was linked to pcDNA3.1to create the recombinant plasmid pcDNA3.1-Nur77-IRES-EGFP. The inserted gene was identified by electrophoresis and sequencing. The recombinant plasmid was then transfected into the cultured cells by Lipofectamine2000(Invitrogen), and the over-expression effects of Nur77was confirmed by RT-PCR and western blotting.
1.3Short-interfering RNA (siRNA) specific for human Nur77and nonsilencing control siRNA were synthesized by Guangzhou RiboBio, China. Cells (2×106/well) were transfected using Lipofectamine2000. Forty-eight h post-transfection, real-time RT-PCR and Western blotting were performed. Based on the Western blot analysis, the Nur77siRNA suppressed the expression of Nur77proteins by83%as compared to the control siRNA in THP-1macrophage-derived foam cells.
1.4Total RNA from cultured cells was extracted using TRIzol reagent (Invitrogen) in accordance with the manufacturer's instructions. Real-time quantitative PCR, using SYBR Green detection chemistry, was performed on the ABI7500Fast Real Time PCR system (Applied Biosystems, Foster City, CA, USA). Melt curve analyses of all real-time PCR products were performed and shown to produce a single DNA duplex. All samples were measured in triplicate and the mean value was considered for comparative analysis. Quantitative measurements were determined using the AACt method and GAPDH expression was used as the internal control.
1.5PMA-differentiated THP-1cells were treated with Nur77agonist Cytosporone B (Csn-B,10μg/ml), recombinant plasmids over-expressing Nur77(Ad-Nur77) and siRNAs against Nur77(si-Nur77) as indicated, then fluorescent-tagged Dil-oxLDL was added, and the cells were incubated for24h. Adherent cells were harvested, washed three times with phosphate buffer saline (PBS). Analysis was performed on a fluorescent activated cell sorting (FACS) calibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) with Cell Quest Pro software (BD Biosciences, San Jose, CA, USA).
1.6The sterol analyses were performed using a HPLC system (model2790, controlled with Empower Pro software; Waters Corp., Milford, MA, USA). Absorbance at216nm was monitored. Data were analyzed with TotalChrom software from PerkinElmer (Waltham, MA, USA).
1.7Cells were cultured and treated with Nur77agonist Cytosporone B (Csn-B,10μg/ml), recombinant plasmids over-expressing Nur77(Ad-Nur77) and siRNAs against Nur77(si-Nur77), as indicated above. Then, they were labeled with0.2μCi/ml [3H]cholesterol. After72h, cells were washed with PBS and incubated overnight in RPMI1640medium containing0.1%(w/v) BSA to allow equilibration of [3H]cholesterol in all cellular pools. Equilibrated [3H]cholesterol-labeled cells were washed with PBS and incubated in2ml of efflux medium containing RPMI1640medium and0.1%BSA with25μg/ml human plasma apoA-I. A150μl sample of efflux medium was obtained at the times designated and passed through a0.45-μm filter to remove any floating cells. Monolayers were washed twice with PBS, and cellular lipids were extracted with isopropanol. Medium and cell-associated [3H]cholesterol was then measured by liquid scintillation counting. Percent efflux was calculated by the following equation:[total media counts/(total cellular counts+total media counts)]×100%.
2. Effect of Nur77on plasma lipid parameters and circulating cytokine levels in apoE-/-mice by treatment with Nur77agonist, lentivirus encoding mouse Nur77(Ad-Nur77) and lentivirus encoding mouse Nur77(si-Nur77).
2.1The serum concentrations of IL-1β, IL-6and TNF-a were measured in duplicate using a commercial ELISA kit (R&D Systems, Minneapolis, MN, USA). Serum CRP amount was measured in duplicate using a commercial ELISA kit (Diagnostic System Laboratories, Webster, TX, USA).
2.2The serum apolipoprotein A1(apoA1) and apoB100concentrations were measured in duplicate using a commercial ELISA kit (Cusabio Biotech Co., Ltd., China). The T-Cho, TG, LDL-C, HDL-C and VLDL-C concentrations were determined enzymatically using an automated analyzer.
3. Effect of Nur77on hepatic lipid deposition in apoE-/-mice by treatment with Nur77agonist, lentivirus encoding mouse Nur77(Ad-Nur77) and lentivirus encoding mouse Nur77(si-Nur77).
3.1Hepatic lipid deposition was assessed in samples embedded in OCT compound by Oil Red O staining. Briefly, liver cryosections were fixed for10min in60%isopropanol and stained with0.3%Oil Red O in60%isopropanol for30min and subsequently washed with60%isopropanol. Sections were counterstained with Gill's hematoxylin, washed with acetic acid solution (4%), and mounted with aqueous solution. Once stained, sections were quantified by histomorphometry.
3.2For the procedure, each frozen liver tissue was sectioned at5μM thicknesses and fixed to microscope slides. Sections of frozen tissue specimens were mounted on polyl-lysine (Sigma, St. Louis, MO)-coated slides, air dried, and fixed with acetone. Immunohistochemical staining was performed for Nur77using Rabbit polyclonal to Nur77antibody at a dilution of1:100(Abcam, Cambridge, MA, USA). Images were acquired and quantitated on an Olympus BX50microscope using Optimis software (Version6.2) and digitized using a color video camera (three-charge coupled device; JVC, Wayne, NJ).
4. Effect of Nur77on gene expression involved in intestinal lipid absorption in Caco-2cells by treatment with Nur77agonist Cytosporone B (Csn-B,10μg/ml), recombinant plasmid over-expressing Nur77(Ad-Nur77) and siRNAs against Nur77(si-Nur77) and in apoE-/-mice by treatment with Nur77agonist, lentivirus encoding mouse Nur77(Ad-Nur77) and lentivirus encoding mouse Nur77(si-Nur77).
4.1Caco-2cells were grown in Dulbecco's modified Eagle's medium (DMEM) containing10%FCS with streptomycin (100μg/mL) and penicillin (100U/ml). All cells were incubated at37℃,5%CO2. Cells were seeded in6-or12-well plates or60-mm dishes and grown to80-90%confluence before use.Caco2cells by treatment with Nur77agonist Cytosporone B (Csn-B,10μg/ml), recombinant plasmid over-expressing Nur77(Ad-Nur77) and siRNAs against Nur77(si-Nur77).
4.2Total RNA from cultured cells was extracted using TRIzol reagent (Invitrogen) in accordance with the manufacturer's instructions. Real-time quantitative PCR, using SYBR Green detection chemistry, was performed on the ABI7500Fast Real Time PCR system (Applied Biosystems, Foster City, CA, USA). Melt curve analyses of all real-time PCR products were performed and shown to produce a single DNA duplex. All samples were measured in triplicate and the mean value was considered for comparative analysis. Quantitative measurements were determined using the AACt method and GAPDH expression was used as the internal control.
5. Effect of Nur77on plaque formation in ApoE-/-Mice by treatment with Nur77agonist, lentivirus encoding mouse Nur77(Ad-Nur77) and lentivirus encoding mouse Nur77(si-Nur77).
5.1For en face analysis, aortas from different groups were opened longitudinally from the heart to the iliac arteries, and lesions were stained with Oil Red O. En face aortic lesion areas were digitized by a Nikon S6digital camera, analyzed using Image-Pro Plus image analysis software (Media Cybernetics, Bethesda, MD, USA), and expressed as the percentage of the total aortic surface area covered by lesions.
5.2The upper portion of the heart and proximal aorta were obtained, embedded in Optimal Cutting Temperature (OCT) compound (Fisher, Tustin, CA), and stored at -70℃. Serial10-μm thick cryosections of aorta, beginning at the aortic root, were collected over a distance of400μm. Sections were stained with Oil Red O. The Oil Red O-positive areas in digitized color images of stained aortic root sections (three equally spaced sections per mouse; n=5per group) were quantified using Image-Pro Plus image analysis software (Media Cybernetics), and the data are expressed as percent of total section area.
Results
1. Nur77contributes to lipid loading, lipid content and cholesterol efflux
We first investigated the role of Nur77during lipid loading in THP-1macrophages, the effects of Nur77on lipid content and cholesterol efflux in THP-1macrophage-derived foam cells by treatment with Nur77agonist Cytosporone B (Csn-B,10μg/ml), recombinant plasmid over-expressing Nur77(Ad-Nur77) and siRNAs against Nur77(si-Nur77). DiI-labeled ox-LDL uptake was obviously decreased by Csn-B and Ad-Nur77(P<0.001), while obviously increased by si-Nur77(P<0.001). Next, we examined the effects of Nur77on cholesterol content and cholesterol efflux in THP-1macrophage-derived foam cells by HPLC and liquid scintillation counting assays, respectively. Cellular cholesterol content was decreased while cholesterol efflux was increased when cells were treated with Csn-B and Ad-Nur77(P<0.05). On the contrary, cellular cholesterol content was increased while cholesterol efflux was decreased when cells were treated with si-Nur77(P<0.05).
Subsequently, we performed mRNA expression analysis of a panel of genes involved in lipid uptake, lipid transport and cholesterol efflux in THP-1macrophage-derived foam cells by treatment with Csn-B, recombinant plasmid over-expressing Nur77and siRNAs against Nur77. Nur77up-regulated genes included ATP-binding cassette A1(ABCA1), scavenger receptor class B type1(SR-B1), Niemann-Pick C1protein (NPC1), Caveolin-1(CAV-1), neutral cholesterol ester hydrolase (nCEH)(P<0.05). Down-regulated genes through the expression of Nur77included low density lipoprotein receptor (LDLR), cholesteryl ester transfer protein (CETP), scavenger receptor CD36and SRA1(P<0.05). In addition, Nur77 had no effect on ATP-binding cassette G1(ABCG1) mRNA expression (P>0.05).
Next, we explored the effect of Nur77on inflammatory gene expression in THP-1macrophage-derived foam cells by treatment with Csn-B, Ad-Nur77and si-Nur77. Nur77up-regulated genes included transforming growth factor (TGF-(3) and CD40(P<0.05), whereas down-regulated genes through the expression of Nur77included interleukin-1β(IL-1β), interleukin-6(IL-6), interleukin-12(IL-12), tumor necrosis factor-a (TNF-a), C-reactive protein (CRP), intercellular adhesion molecule-1(ICAM-1) and nuclear factor κB (NF-κB)(P<0.05). In addition, Csn-B had no effect on vascular cell adhesion molecule (VCAM-1) mRNA expression (P>0.05), but up-regulated interleukin-18(IL-18) mRNA expression (P<0.05). However, VCAM-1mRNA expression was inhibited by Ad-Nur77(P<0.05), while enhanced by Si-Nur77(P<0.05). Treatment with both Ad-Nur77and Si-Nur77had no effect on IL-18mRNA expression (P>0.05). Furthermore, the gene expressions of interferon-y (INF-y) and interleukin-10(IL-10) were not detectable in THP-1macrophage-derived foam cells through treatment with Csn-B, Ad-Nur77and si-Nur77.
2. Nur77regulates plasma lipid parameters and circulating cytokine levels in apoE-/-Mice
We examined the terminal plasma lipid levels from experimental mice.the plasma high density lipoprotein cholesterol (HDL-C) showed a moderate reduction in the Csn-B group and Ad-Nur77group as compared to their control groups, respectively (P<0.05). Concomitantly, plasma low density lipoprotein cholesterol (LDL-C) increased in the Csn-B group and Ad-Nur77group as compared to their control groups, respectively (P<0.05). In contrast, treatment with si-Nur77led to an increase in plasma HDL-cholesterol (P<0.05) and a reduction in plasma LDL-cholesterol as compared to the si-Mock group (P<0.05). However, no significant alternation occurred in apoAl, apolipoprotein B (apoB), very low density lipoprotein cholesterol (VLDL-cholesterol), total triglyceride and cholesterol (P>0.05).
To investigate whether change in Nur77expression could result in corresponding changes in plasma inflammatory cytokines, we conducted a series of ELISAs. Consistent with the data of inflammatory gene expression in THP-1macrophage-derived foam cells, treatment with both Csn-B and Ad-Nur77resulted in down-regulation of CRP concentrations in plasma, respectively (P<0.05). In agreement with these data, si-Nur77-mediated knockdown of Nur77results in an up-regulation of CRP concentrations in plasma (P<0.05). However, no significant alteration occurred in levels of IL-1β, IL-6and TNF-a between groups at the end of the experiments (P>0.05).
3. Nur77affects hepatic lipid deposition in apoE-/-mice
The protein expression of Nur77in mouse liver was investigated by Western blot and immunohistochemistry analyses. We observed that minor expression levels were detectable in the baseline group, but expression of Nur77was markedly increased in the control group. In addition, the Csn-B group and Ad-Nur77group had significantly higher expression of Nur77than their control groups while the si-Nur77group had a lower expression of Nur77as compared to the si-Mock group. Next, the effects of Nur77on lipid content in the liver of apoE-/-mice were analyzed by Oil Red O staining and measured enzymatically. Hepatic triglyceride levels in the liver was reduced in mice treated with Csn-B and Ad-Nur77, respectively, compared with mice of the control group (P<0.05). In contrast, the hepatic triglyceride levels in the liver was increased in mice treated with si-Nur77in comparison to mice treated with si-Mock (P<0.05). However, hepatic cholesterol level had no change in response to regulation of expression of Nur77in the liver (P>0.05).
To investigate the mechanisms of Nur77reduction of hepatic lipid deposition, hepatic lipid metabolism gene expression levels in livers of apoE-/-mice were analyzed by real time PCR. As shown, Csn-B and Ad-Nur77treatment progressively reduced gene levels of LDLR, ATP-binding cassette G5(ABCG5), CRP, SREBP1C, SREBP2(P<0.05), and increased gene levels of SR-B1and hepatic lipase (HL)(P<0.05). In addition, treatment with si-Nur77could up-regulated gene expression of LDLR, ABCG5, CRP, SREBP1C and SREBP2(P<0.05). While SR-B1and HL gene expressions were down-regulated by treatment with si-Nur77(P<0.05). Although gene expression of HMG-CoA reductase (HMGCR) was repressed by treatment with Csn-B (P<0.05), the gene expression of HMGCR was increased by treatment with Ad-Nur77(P<0.05) while decreased by treatment with si-Nur77(P<0.05). In addition, Nur77had no effect on apoA1and LCAT expression (P>0.05).
4. Nur77inhibits gene expression involved in intestinal lipid absorption
To address the possibility that expression levels of intestinal lipid transporters are affected by Nur77, we incubated Caco-2cells with Csn-B, recombinant plasmid Ad-Nur77and siRNAs against Nur77(si-Nur77). Csn-B and Ad-Nur77treatment progressively reduced gene levels of microsomal triglyceride transfer protein (MTP), Niemann-Pick Cl-like1(NPC1L1) and ABCG5(P<0.05). On the contrary, si-Nur77treatment markedly induced gene expression of MTP, NPC1L1and ABCG5(P<0.05). To further investigate the Nur77mechanisms affecting intestinal cholesterol absorption in apoE-/-mice, gene expression in intestinal tissue of apoE-/-mice were analyzed by real time PCR. We also found that over-expression of Nur77reduced MTP, NPC1L1and ABCG5gene expression (P<0.05), while silenced Nur77increased these gene levels (P<0.05).
5. Nur77reduces plaque formation in ApoE-/-Mice
To investigate the impact of Nur77on atherogenesis in apoE-/-mice, atherosclerotic lesions were evaluated by aortic valve section and en face analyses. Mice receiving Csn-B and Ad-Nur77showed a decrease in the average lesion area compared with their controls by both en face and aortic valve section analyses (P<0.05). On the contrary, si-Nur77-treated mice showed an increase in average lesion area compared with si-Mock-treated mice by both en face and aortic valve section analyses (P>0.05).
Next, the gene expression changes of the inflammatory molecules, adhesion molecules and molecules related to cholesterol metabolism were investigated in aortic tissues. In both Csn-B-treated and Ad-Nur77-treated apoE-/-mice, gene expression of SRA1, CD36, IL-1β, IL-6, CRP, ICAM-1, macrophage inflammatory protein-1α (MIP-1α) and monocyte chemo-attractant protein-1(MCP-1) were markedly repressed (P<0.05), but gene expression of ABCA1, SR-B1, NPC1and CD40were up-regulated at week12(P<0.05). On the contrary, we found that gene expression of SRA1, CD36, IL-1β, IL-6, CRP, ICAM-1, MIP-1α and MCP-1were up-regulated (P<0.05), while gene expression of ABCA1, SRB1, NPC1and CD40were down-regulated (P<0.05) in the aorta of si-Nur77-treated ApoE-/-mice. In addition, gene expression of TNF-a was reduced in Ad-Nur77-treated ApoE-/-mice (P<0.05), while increased in si-Nur77-treated ApoE-/-mice (P<0.05). However, Csn-B-treated apoE-deficient mice had no change in TNF-a gene expression (P>0.05).
Conclusions
1. Nur77reduces lipid loading, lipid content and enhanced cholesterol efflux in THP-1macrophage-derived foam cells.2. Nur77decreases plasma HDL-cholesterol level, increases plasma LDL-cholesterol level and decreases CRP concentrations in apoE-/-Mice.3. Nur77down-regulates triglyceride levels but not cholesterol level in the liver.4. Nur77inhibits gene expression involved in intestinal lipid absorption.5. Nur77reduces plaque formation in ApoE-/-Mice.
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[11]Dai XY, Ou X, Hao XR, et al. The Effect of T0901317 on ATP-Binding Cassette Transporter A1 and Niemann-Pick Type C1 in ApoE-/- Mice. J Cardiovasc Pharmacol.2008; 51(5):467-475.
[12]Ou X, Dai X, Long Z, et al. Liver X receptor agonist T0901317 reduces atherosclerotic lesions in apoE-/- mice by up-regulating NPC1 expression. Sci China C Life Sci.2008; 51(5):418-429.
[13]Bonta PI, Pols TW, de Vries CJ. NR4A nuclear receptors in atherosclerosis and vein-graft disease. Trends Cardiovasc Med.2007; 17(3):105-111.
[14]Pols TW, Ottenhoff R, Vos M, et al. Nur77 modulates hepatic lipid metabolism through suppression of SREBPlc activity. Biochem Biophys Res Commun. 2008; 366(4):910-916.
[15]Pei L, Castrillo A, Chen M, et al. Induction of NR4A orphan nuclear receptor expression in macrophages in response to inflammatory stimuli. J Biol Chem. 2005; 280(32):29256-29262.
[16]Perlmann T, Jansson L. A novel pathway for vitamin A signaling mediated by RXR heterodimerization with NGFI-B and NURR1. Genes Dev.1995; 9(7): 769-782
[17]Zetterstrom RH, Solomin L, Mitsiadis T, et al. Retinoid X receptor heterodimerization and developmental expression distinguish the orphan nuclear receptors NGFI-B, Nurrl, and Norl. Mol Endocrinol.1996; 10(12):1656-1666.
[18]Maxwell MA, Muscat GE. The NR4A subgroup:immediate early response genes with pleiotropic physiological roles. Nucl Recept Signal.2006; 4:e002.
[19]Pei L, Waki H, Vaitheesvaran B, et al. NR4A orphan nuclear receptors are transcriptional regulators of hepatic glucose metabolism. Nat Med.2006; 12(9):1048-1055.
[20]Chao LC, Wroblewski K, Zhang Z, et al. Insulin resistance and altered systemic glucose metabolism in mice lacking Nur77. Diabetes.2009; 58(12): 2788-2796.
[21]Bonta PI, van Tiel CM, Vos M, et al. Nuclear receptors Nur77, Nurr1, and NOR-1 expressed in atherosclerotic lesion macrophages reduce lipid loading and inflammatory responses. Arterioscler Thromb Vasc Biol.2006; 26(10): 2288-2294.
[22]Hummasti S, Tontonoz P. Adopting new orphans into the family of metabolic regulators. Mol Endocrinol.2008; 22(8):1743-1753.
[23]胡刘华,何奔,沈玲红等.孤儿核受体Nur77抑制小鼠巨噬细胞脂质沉积.中华心血管病杂志.2008,36(11)：1032-1036.
[24]Tiwari RL, Singh V, Barthwal MK. Macrophages:an elusive yet emerging therapeutic target of atherosclerosis. Med Res Rev.2008; 28(4):483-544.
[25]Chen M, Li W, Wang N, Zhu Y, Wang X.. ROS and NF-kappaB but not LXR mediate IL-lbeta signaling for the downregulation of ATP-binding cassette transporter A1. Am J Physiol Cell Physiol.2007; 292 (4):C1493-1501.
[26]Hao XR, Cao DL, Hu YW, et al. IFN-gamma down-regulates ABCA1 expression by inhibiting LXRalpha in a JAK/STAT signaling pathway-dependent manner. Atherosclerosis.2009; 203(2):417-428.
[27]Khovidhunkit W, Kim MS, Memon RA, et al. Effects of infection and inflammation on lipid and lipoprotein metabolism:mechanisms and consequences to the host. J Lipid Res.2004; 45(7):1169-1196.
[28]李桃园,章国良.代谢性核受体功能及转录活性调控机制的研究进[J].中国临床药理学杂志,2009,25(4)：346-349.
[29]Rigamonti E, Chinetti-Gbaguidi G, Staels B. Regulation of macrophage functions by PPAR-alpha, PPAR-gamma, and LXRs in mice and men. Arterioscler Thromb Vasc Biol.2008; 28(6):1050-1059.
[30]Ghosh S. Macrophage cholesterol homeostasis and metabolic diseases:critical role of cholesteryl ester mobilization. Expert Rev Cardiovasc Ther.2011; 9(3): 329-340.
[31]Babaev VR, Gleaves LA, Carter KJ, et al. Reduced atherosclerotic lesions in mice deficient for total or macrophage-specific expression of scavenger receptor-A. Arterioscler Thromb Vasc Biol.2000; 20(12):2593-2599.
[32]Febbraio M, Guy E, Silverstein RL. Stem cell transplantation reveals that absence of macrophage CD36 is protective against atherosclerosis. Arterioscler Thromb Vase Biol.2004; 24(12):2333-2338.
[33]Linton MF, Fazio S. Macrophages, inflammation, and atherosclerosis. Int J Obes Relat Metab Disord.2003; 27 Suppl 3:S35-40.
[34]Gargalovic P, Dory L. Caveolins and macrophage lipid metabolism. J Lipid Res. 2003; 44(1):11-21.
[35]Oram JF, Heinecke JW. ATP-binding cassette transporter Al:a cell cholesterol exporter that protects against cardiovascular disease. Physiol Rev.2005; 85(4): 1343-1372.
[36]Moore KJ, Freeman MW. Scavenger receptors in atherosclerosis:beyond lipid uptake. Arterioscler Thromb Vase Biol.2006; 26(8):1702-1711.
[37]Phang YL, Soga T, Kitahashi T, et al. Cloning and functional expression of novel cholesterol transporters ABCG1 and ABCG4 in gonadotropin-releasing hormone neurons of the tilapia. Neuroscience.2012; 203:39-49.
[38]Quazi F, Molday RS. Lipid transport by mammalian ABC proteins. Essays Biochem.2011; 50(1):265-290.
[39][39] Nation DA, Gonzales JA, Mendez AJ, et al. The effect of social environment on markers of vascular oxidative stress and inflammation in the Watanabe heritable hyperlipidemic rabbit. Psychosom Med.2008; 70(3): 269-275.
[40]Im SS, Osborne TF. Liver x receptors in atherosclerosis and inflammation. Circ Res.2011; 108(8):996-1001.
[41]Wu JT, Wu LL. Association of soluble markers with various stages and major events of atherosclerosis. Ann Clin Lab Sci.2005; 35(3):240-250.
[42]Tedgui A, Mallat Z. Cytokines in atherosclerosis:pathogenic and regulatory pathways. Physiol Rev.2006; 86(2):515-581.
[43]Stoll G, Bendszus M. Inflammation and atherosclerosis:novel insights into plaque formation and destabilization. Stroke.2006; 37(7):1923-1932.
[44]Choudhury RP, Fisher EA. Molecular imaging in atherosclerosis, thrombosis, and vascular inflammation. Arterioscler Thromb Vasc Biol.2009; 29(7):983-991.
[45]Kontush A, Chapman MJ. Functionally defective high-density lipoprotein:a new therapeutic target at the crossroads of dyslipidemia, inflammation, and atherosclerosis. Pharmacol Rev.2006; 58(3):342-374.
[46]骆庆峰,孙兰,杜冠华.胆固醇逆向转运过程中新靶点及其药物的研究进展[J].中国药理学通报,2006,22(8)：904-907.
[47]Hu YW, Ma X, Li XX, et al. Eicosapentaenoic acid reduces ABCA1 serine phosphorylation and impairs ABCA1-dependent cholesterol efflux through cyclic AMP/protein kinase A signaling pathway in THP-1 macrophage-derived foam cells. Atherosclerosis.2009; 204(2):e35-43.
[48]Mo ZC, Xiao J, Liu XH, et al. AOPPs inhibits cholesterol efflux by down-regulating ABCA1 expression in a JAK/STAT signaling pathway-dependent manner. J Atheroscler Thromb.2011; 18(9):796-807.
[49]Scanu AM, Edelstein C. HDL:bridging past and present with a look at the future. FASEB J.2008; 22(12):4044-4054.
[50]Watanabe M, Houten SM, Wang L, et al. Bile acids lower triglyceride levels via a pathway involving FXR, SHP, and SREBP-lc. J Clin Invest.2004; 113(10): 1408-1418.
[51]Yuan HD, Yuan HY, Chung SH, et al. attenuates hepatic lipid accumulation through activating AMP-activated protein kinase in human HepG2 cells. Biosci Biotechnol Biochem.2010; 74(2):322-328.
[52]Hui DY, Labonte ED, Howles PN. Development and physiological regulation of intestinal lipid absorption.Ⅲ.Intestinal transporters and cholesterol absorption. Am J Physiol Gastrointest Liver Physiol.2008; 294(4):G839-843.
[53]Iqbal J, Hussain MM. Intestinal lipid absorption. Am J Physiol Endocrinol Metab.2009; 296(6):E1183-1194.
[54]姜津,唐朝克.影响胆固醇在肠道吸收的相关蛋白[J].中南医学科学杂志,2011,39(5)：582-585.
[55]Betters JL, Yu L. NPC1L1 and cholesterol transport. FEBS Lett.2010; 584(13): 2740-2747.
[56]Davis HR Jr, Altmann SW. Niemann-Pick C1 Like 1 (NPC1L1) an intestinal sterol transporter. Biochim Biophys Acta.2009; 1791(7):679-683.
[57]Sabeva NS, Liu J, Graf GA. The ABCG5 ABCG8 sterol transporter and phytosterols:implications for cardiometabolic disease. Curr Opin Endocrinol Diabetes Obes.2009; 16(2):172-177.
[58]Kidambi S, Patel SB. Cholesterol and non-cholesterol sterol transporters: ABCG5, ABCG8 and NPC1L1:a review. Xenobiotica.2008; 38(7-8):1119-1139.
[59]Dashti N, Gandhi M, Liu X, et al. The N-terminal 1000 residues of apolipoprotein B associate with microsomal triglyceride transfer protein to create a lipid transfer pocket required for lipoprotein assembly. Biochemistry. 2002; 41(22):6978-6987.
[60]Hussain MM, Rava P, Walsh M, et al. Multiple functions of microsomal triglyceride transfer protein. Nutr Metab (Lond).2012;9 (1):14
[61]Doran AC, Meller N, McNamara CA. Role of smooth muscle cells in the initiation and early progression of atherosclerosis. Arterioscler Thromb Vasc Biol.2008; 28(5):812-819.
[62]Allahverdian S, Pannu PS, Francis GA. Contribution of monocyte-derived macrophages and smooth muscle cells to arterial foam cell formation Cardiovasc Res.2012 Feb 15. [Epub ahead of print]
[63]Arkenbout EK, de Waard V, van Bragt M, et al. Protective function of transcription factor TR3 orphan receptor in atherogenesis:decreased lesion formation in carotid artery ligation model in TR3 transgenic mice. Circulation. 2002; 106(12):1530-1535.
[64]Pires NM, Pols TW, de Vries MR, et al. Activation of nuclear receptor Nur77 by 6-mercaptopurine protects against neointima formation. Circulation.2007; 115(4):493-500.
[2]Poli G, Sottero B, Gargiulo S, et al. Cholesterol oxidation pro ducts in the vascular remodeling due to atherosclerosis. Mol Aspects Med.2009; 30: 180-189.
[3]Webb NR, Moore KJ. Macrophage-derived foam cells in atherosclerosis:lessons from murine models and implications for therapy. Curr Drug Targets.2007; 8: 1249-1263.
[4]Hu YW, Zheng L, Wang Q. Regulation of cholesterol homeostasis by liver X receptors. Clin Chim Acta.2010 Jan 6. [Epub ahead of print]
[5]毛奇琦,孙旭.核受体在胆汁酸和胆固醇代谢中的作用[J].国际消化病杂志,2008,28：243-245.
[6]廖端芳,唐朝克主编.胆固醇逆向转运基础与临床.科学出版社,北京,2009.
[7]Giguere V. Orphan nuclear receptors:from gene to function. Endocr Rev.1999; 20:689-725.
[8]Hu YW, Wang Q, Ma X, et al. TGF-beta1 Up-Regulates Expression of ABCA1, ABCG1 and SR-BI through Liver X Receptor alpha Signaling Pathway in THP-1 Macrophage-Derived Foam Cells. J Atheroscler Thromb.2010; 17(5): 493-502.
[9]Ma X, Hu YW, Mo ZC, et al. NO-1886 up-regulates Niemann-Pick C1 protein (NPC1) expression through liver X receptor alpha signaling pathway in THP-1 macrophage-derived foam cells. Cardiovasc Drugs Ther.2009; 23(3):199-206.
[10]Hao XR, Cao DL, Hu YW, et al. IFN-gamma down-regulates ABCA1 expression by inhibiting LXRalpha in a JAK/STAT signaling pathway-dependent manner. Atherosclerosis.2009; 203(2):417-28.
[11]Dai XY, Ou X, Hao XR, et al. The Effect of T0901317 on ATP-Binding Cassette Transporter A1 and Niemann-Pick Type C1 in ApoE-/- Mice. J Cardiovasc Pharmacol.2008; 51(5):467-475.
[12]Ou X, Dai X, Long Z, et al. Liver X receptor agonist T0901317 reduces atherosclerotic lesions in apoE-/- mice by up-regulating NPC1 expression. Sci China C Life Sci.2008; 51(5):418-429.
[13]Bonta PI, Pols TW, de Vries CJ. NR4A nuclear receptors in atherosclerosis and vein-graft disease. Trends Cardiovasc Med.2007; 17(3):105-111.
[14]Pols TW, Ottenhoff R, Vos M, et al. Nur77 modulates hepatic lipid metabolism through suppression of SREBPlc activity. Biochem Biophys Res Commun. 2008; 366(4):910-916.
[15]Pei L, Castrillo A, Chen M, et al. Induction of NR4A orphan nuclear receptor expression in macrophages in response to inflammatory stimuli. J Biol Chem. 2005; 280(32):29256-29262.
[16]Perlmann T, Jansson L. A novel pathway for vitamin A signaling mediated by RXR heterodimerization with NGFI-B and NURR1. Genes Dev.1995; 9(7): 769-782
[17]Zetterstrom RH, Solomin L, Mitsiadis T, et al. Retinoid X receptor heterodimerization and developmental expression distinguish the orphan nuclear receptors NGFI-B, Nurrl, and Norl. Mol Endocrinol.1996; 10(12):1656-1666.
[18]Maxwell MA, Muscat GE. The NR4A subgroup:immediate early response genes with pleiotropic physiological roles. Nucl Recept Signal.2006; 4:e002.
[19]Pei L, Waki H, Vaitheesvaran B, et al. NR4A orphan nuclear receptors are transcriptional regulators of hepatic glucose metabolism. Nat Med.2006; 12(9):1048-1055.
[20]Chao LC, Wroblewski K, Zhang Z, et al. Insulin resistance and altered systemic glucose metabolism in mice lacking Nur77. Diabetes.2009; 58(12): 2788-2796.
[21]Bonta PI, van Tiel CM, Vos M, et al. Nuclear receptors Nur77, Nurr1, and NOR-1 expressed in atherosclerotic lesion macrophages reduce lipid loading and inflammatory responses. Arterioscler Thromb Vasc Biol.2006; 26(10): 2288-2294.
[22]Hummasti S, Tontonoz P. Adopting new orphans into the family of metabolic regulators. Mol Endocrinol.2008; 22(8):1743-1753.
[23]胡刘华,何奔,沈玲红等.孤儿核受体Nur77抑制小鼠巨噬细胞脂质沉积.中华心血管病杂志.2008,36(11)：1032-1036.
[24]Tiwari RL, Singh V, Barthwal MK. Macrophages:an elusive yet emerging therapeutic target of atherosclerosis. Med Res Rev.2008; 28(4):483-544.
[25]Chen M, Li W, Wang N, Zhu Y, Wang X.. ROS and NF-kappaB but not LXR mediate IL-lbeta signaling for the downregulation of ATP-binding cassette transporter A1. Am J Physiol Cell Physiol.2007; 292 (4):C1493-1501.
[26]Hao XR, Cao DL, Hu YW, et al. IFN-gamma down-regulates ABCA1 expression by inhibiting LXRalpha in a JAK/STAT signaling pathway-dependent manner. Atherosclerosis.2009; 203(2):417-428.
[27]Khovidhunkit W, Kim MS, Memon RA, et al. Effects of infection and inflammation on lipid and lipoprotein metabolism:mechanisms and consequences to the host. J Lipid Res.2004; 45(7):1169-1196.
[28]李桃园,章国良.代谢性核受体功能及转录活性调控机制的研究进[J].中国临床药理学杂志,2009,25(4)：346-349.
[29]Rigamonti E, Chinetti-Gbaguidi G, Staels B. Regulation of macrophage functions by PPAR-alpha, PPAR-gamma, and LXRs in mice and men. Arterioscler Thromb Vasc Biol.2008; 28(6):1050-1059.
[30]Ghosh S. Macrophage cholesterol homeostasis and metabolic diseases:critical role of cholesteryl ester mobilization. Expert Rev Cardiovasc Ther.2011; 9(3): 329-340.
[31]Babaev VR, Gleaves LA, Carter KJ, et al. Reduced atherosclerotic lesions in mice deficient for total or macrophage-specific expression of scavenger receptor-A. Arterioscler Thromb Vasc Biol.2000; 20(12):2593-2599.
[32]Febbraio M, Guy E, Silverstein RL. Stem cell transplantation reveals that absence of macrophage CD36 is protective against atherosclerosis. Arterioscler Thromb Vase Biol.2004; 24(12):2333-2338.
[33]Linton MF, Fazio S. Macrophages, inflammation, and atherosclerosis. Int J Obes Relat Metab Disord.2003; 27 Suppl 3:S35-40.
[34]Gargalovic P, Dory L. Caveolins and macrophage lipid metabolism. J Lipid Res. 2003; 44(1):11-21.
[35]Oram JF, Heinecke JW. ATP-binding cassette transporter Al:a cell cholesterol exporter that protects against cardiovascular disease. Physiol Rev.2005; 85(4): 1343-1372.
[36]Moore KJ, Freeman MW. Scavenger receptors in atherosclerosis:beyond lipid uptake. Arterioscler Thromb Vase Biol.2006; 26(8):1702-1711.
[37]Phang YL, Soga T, Kitahashi T, et al. Cloning and functional expression of novel cholesterol transporters ABCG1 and ABCG4 in gonadotropin-releasing hormone neurons of the tilapia. Neuroscience.2012; 203:39-49.
[38]Quazi F, Molday RS. Lipid transport by mammalian ABC proteins. Essays Biochem.2011; 50(1):265-290.
[39][39] Nation DA, Gonzales JA, Mendez AJ, et al. The effect of social environment on markers of vascular oxidative stress and inflammation in the Watanabe heritable hyperlipidemic rabbit. Psychosom Med.2008; 70(3): 269-275.
[40]Im SS, Osborne TF. Liver x receptors in atherosclerosis and inflammation. Circ Res.2011; 108(8):996-1001.
[41]Wu JT, Wu LL. Association of soluble markers with various stages and major events of atherosclerosis. Ann Clin Lab Sci.2005; 35(3):240-250.
[42]Tedgui A, Mallat Z. Cytokines in atherosclerosis:pathogenic and regulatory pathways. Physiol Rev.2006; 86(2):515-581.
[43]Stoll G, Bendszus M. Inflammation and atherosclerosis:novel insights into plaque formation and destabilization. Stroke.2006; 37(7):1923-1932.
[44]Choudhury RP, Fisher EA. Molecular imaging in atherosclerosis, thrombosis, and vascular inflammation. Arterioscler Thromb Vasc Biol.2009; 29(7):983-991.
[45]Kontush A, Chapman MJ. Functionally defective high-density lipoprotein:a new therapeutic target at the crossroads of dyslipidemia, inflammation, and atherosclerosis. Pharmacol Rev.2006; 58(3):342-374.
[46]骆庆峰,孙兰,杜冠华.胆固醇逆向转运过程中新靶点及其药物的研究进展[J].中国药理学通报,2006,22(8)：904-907.
[47]Hu YW, Ma X, Li XX, et al. Eicosapentaenoic acid reduces ABCA1 serine phosphorylation and impairs ABCA1-dependent cholesterol efflux through cyclic AMP/protein kinase A signaling pathway in THP-1 macrophage-derived foam cells. Atherosclerosis.2009; 204(2):e35-43.
[48]Mo ZC, Xiao J, Liu XH, et al. AOPPs inhibits cholesterol efflux by down-regulating ABCA1 expression in a JAK/STAT signaling pathway-dependent manner. J Atheroscler Thromb.2011; 18(9):796-807.
[49]Scanu AM, Edelstein C. HDL:bridging past and present with a look at the future. FASEB J.2008; 22(12):4044-4054.
[50]Watanabe M, Houten SM, Wang L, et al. Bile acids lower triglyceride levels via a pathway involving FXR, SHP, and SREBP-lc. J Clin Invest.2004; 113(10): 1408-1418.
[51]Yuan HD, Yuan HY, Chung SH, et al. attenuates hepatic lipid accumulation through activating AMP-activated protein kinase in human HepG2 cells. Biosci Biotechnol Biochem.2010; 74(2):322-328.
[52]Hui DY, Labonte ED, Howles PN. Development and physiological regulation of intestinal lipid absorption.Ⅲ.Intestinal transporters and cholesterol absorption. Am J Physiol Gastrointest Liver Physiol.2008; 294(4):G839-843.
[53]Iqbal J, Hussain MM. Intestinal lipid absorption. Am J Physiol Endocrinol Metab.2009; 296(6):E1183-1194.
[54]姜津,唐朝克.影响胆固醇在肠道吸收的相关蛋白[J].中南医学科学杂志,2011,39(5)：582-585.
[55]Betters JL, Yu L. NPC1L1 and cholesterol transport. FEBS Lett.2010; 584(13): 2740-2747.
[56]Davis HR Jr, Altmann SW. Niemann-Pick C1 Like 1 (NPC1L1) an intestinal sterol transporter. Biochim Biophys Acta.2009; 1791(7):679-683.
[57]Sabeva NS, Liu J, Graf GA. The ABCG5 ABCG8 sterol transporter and phytosterols:implications for cardiometabolic disease. Curr Opin Endocrinol Diabetes Obes.2009; 16(2):172-177.
[58]Kidambi S, Patel SB. Cholesterol and non-cholesterol sterol transporters: ABCG5, ABCG8 and NPC1L1:a review. Xenobiotica.2008; 38(7-8):1119-1139.
[59]Dashti N, Gandhi M, Liu X, et al. The N-terminal 1000 residues of apolipoprotein B associate with microsomal triglyceride transfer protein to create a lipid transfer pocket required for lipoprotein assembly. Biochemistry. 2002; 41(22):6978-6987.
[60]Hussain MM, Rava P, Walsh M, et al. Multiple functions of microsomal triglyceride transfer protein. Nutr Metab (Lond).2012;9 (1):14
[61]Doran AC, Meller N, McNamara CA. Role of smooth muscle cells in the initiation and early progression of atherosclerosis. Arterioscler Thromb Vasc Biol.2008; 28(5):812-819.
[62]Allahverdian S, Pannu PS, Francis GA. Contribution of monocyte-derived macrophages and smooth muscle cells to arterial foam cell formation Cardiovasc Res.2012 Feb 15. [Epub ahead of print]
[63]Arkenbout EK, de Waard V, van Bragt M, et al. Protective function of transcription factor TR3 orphan receptor in atherogenesis:decreased lesion formation in carotid artery ligation model in TR3 transgenic mice. Circulation. 2002; 106(12):1530-1535.
[64]Pires NM, Pols TW, de Vries MR, et al. Activation of nuclear receptor Nur77 by 6-mercaptopurine protects against neointima formation. Circulation.2007; 115(4):493-500.
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