炎症应激通过激活mTOR通路诱导THP-1巨噬细胞泡沫化的实验研究

目的研究脂多糖(LPS)诱导的炎症应激是否通过激活m TOR通路,增加SCAP/SREBP2表达,干扰SCAP/SREBP2负反馈调控,增加THP-1巨噬细胞对非修饰低密度脂蛋白胆固醇(LDL)摄取,导致泡沫细胞形成。方法诱导分化成功的THP-1源性巨噬细胞在无血清培养基中培养4 h后,分为对照组(5 mg·L~(-1)LDL)、炎症刺激组(5 mg·L~(-1)LDL+200μg·L~(-1)LPS)、炎症+雷帕霉素组(5 mg·L~(-1)LDL+200μg·L~(-1)LPS+10μg·L~(-1)Rapamycin)。以上各组细胞培养24 h后收获。油红O染色法检测各组细胞内脂质沉积情况,Real-time PCR法检测LDLr、SREBP2、SCAP、S6K1和m TOR mRNA水平,Western blot法检测LDLr、S6K1、mTOR蛋白表达,激光共聚焦法检测SCAP在内质网与高尔基体间的转位情况。结果油红O染色发现,炎症应激增加THP-1巨噬细胞内脂质沉积,雷帕霉素抑制炎症应激诱导的脂质聚积。Real-time PCR检测显示,炎症应激上调THP-1巨噬细胞LDLr、SREBP2、SCAP、S6K1和mTOR mRNA水平(P<0.05),雷帕霉素减少炎症应激诱导的LDLr、SREBP2、SCAP、S6K1和mTOR mRNA水平升高(P<0.05)。Western blot检测发现,炎症应激上调LDLr、S6K1和m TOR蛋白表达(P<0.05),雷帕霉素减少炎症应激诱导的LDLr、S6K1和mTOR蛋白表达水平升高(P<0.05)。激光共聚焦检测发现,炎症应激增加SCAP/SREBP2从内质网转位到高尔基体,雷帕霉素则抑制炎症应激诱导的SCAP/SREBP2复合物从内质网转位到高尔基体。结论炎症应激通过激活mTOR通路,上调SCAP/SREBP2表达,致SCAP/SREBP2复合体转位至高尔基体异常增加,LDLr表达上调,致使胆固醇聚积于细胞内,泡沫细胞形成。雷帕霉素能够逆转炎症应激诱导mTOR通路激活,减少细胞内胆固醇沉积,提示炎症应激状态下,mTOR可能是泡沫细胞形成的关键通路。     

替罗非班对体外培养THP-1源性巨噬细胞CD40及其配体表达的影响

目的探讨替罗非班对氧化型低密度脂蛋白(ox-LDL)刺激下THP-1源性巨噬细胞CD40和CD40配体(CD40L)表达的影响,探讨该类药物的抗炎及稳定斑块的作用机制。方法向体外培养的THP-1细胞中加入ox-LDL(80mg/L)共同孵育24h,然后与替罗非班共同孵育不同时间(分别为12、24、48h),应用流式细胞技术检测CD40和CD40L在细胞上的表达。采用反转录-聚合酶链反应(RT-PCR)检测CD40和CD40L mRNA表达。结果替罗非班既可下调细胞表面CD40和CD40L的表达,又能使CD40和CD40LmRNA表达下降,其阻断作用呈时间依赖性(P<0.05)。结论替罗非班可以拮抗巨噬细胞CD40和CD40L表达,并呈时间依赖性,这种作用可能是其减轻动脉粥样斑块炎症及稳定斑块的机制之一。     

甲壳低聚糖对THP-1源性巨噬细胞脂质转运相关基因表达的影响

目的观察甲壳低聚糖(COS)对离体培养的THP-1源性巨噬细胞脂质转运相关基因表达的影响,以初步探讨COS降低血脂的可能分子机制。方法离体培养人THP-1单核细胞,诱导分化成巨噬细胞后,用100μg/mL的COS处理THP-1源性巨噬细胞6,12,24,48 h,运用半定量RT-PCR方法检测其LDL受体、SR-BⅠ、ABCA1和CD36mRNA表达水平的变化。结果COS处理THP-1源性巨噬细胞24和48 h后,LDL受体mRNA的表达与对照组比较分别下调了15.6%(P<0.05)和30.7%(P<0.01);处理6,12,24,48 h后,SR-BⅠmRNA的表达与对照组比较分别上调了14.7%,23.0%,14.6%(P<0.05)和33.9%(P<0.01);处理6和12 h后,ABCA1 mRNA的表达与对照组比较分别上调了12.6%和14.5%(P<0.05);处理6和12 h后,CD36 mRNA的表达与对照组比较分别下调了25.5%和10.2%(P<0.05)。结论通过细胞离体培养实验表明,COS具有的降低血脂作用可能与其能上调巨噬细胞中AB-CA1和SR-BⅠmRNA表达水平以及降低LDL受体和CD36 mRNA水平有关。     

苯扎贝特对THP1巨噬细胞PPARγ及ABCA1表达的影响

目的 探讨苯扎贝特对THP1巨噬细胞PPARγ及ABCA1表达的影响.方法 THP1细胞经PMA诱导24h后,添加不同浓度苯扎贝特(0、1、10、50μmol/L)继续作用24h,RT-PCR法测定PPARγ、ABCA1 mRNA表达.结果 PMA刺激THP1细胞表达PPARγ及ABCA1;苯扎贝特剂量依赖性上调THP-1巨噬细胞PPARγ及ABCA1的表达(P＜0.05),50μmol/L浓度时二者表达最高,且PPARγ与ABCA1的mRNA表达呈正相关(r=0.628,P=0.002).结论 苯扎贝特上调THP1细胞中PPARγ和ABCA1的表达.     

瘦素对THP-1细胞泡沫化过程中ACAT-1表达的影响

目的：研究单核或巨噬细胞转分化和泡沫细胞形成过程中瘦素对人酰基辅酶A1胆固醇酰基转移酶(ACAT-1)表达的影响。方法：在体外模拟动脉粥样硬化(AS)形成过程中，运用放射性同位素标记底物法、逆转录聚合酶链反应、蛋白质免疫印迹法检测ACAT-1 mRNA和蛋白表达水平。结果：单核或巨噬细胞转分化过程中ACAT-1酶活性、蛋白及mRNA水平均升高。巨噬细胞转变为泡沫细胞过程中ACAT-1表达水平无明显变化。高瘦素血症可明显增强单核或巨噬细胞转分化和泡沫细胞形成过程中ACAT-1表达。结论：高瘦素血症可能通过增强ACAT-1表达导致动脉粥样硬化提前发生。     

甲壳低聚糖-硒对THP-1源性巨噬细胞抗氧化酶相关基因表达的影响

目的初步探讨甲壳低聚糖-硒(COS-Se)抗氧化的可能分子机制。方法用100μg/mL的COS-Se处理THP-1源性巨噬细胞6,12,24,48 h,运用半定量RT-PCR方法检测其超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)mRNA表达水平的变化。结果COS-Se处理THP-1源性巨噬细胞24和48 h后,SOD mRNA的表达与对照组比较分别上调了14.4%,16.9%(P<0.05);处理6,12,24,48 h后,GSH-Px mRNA的表达与对照组比较分别上调了12.7%,10.9%,13.1%(P<0.05)和27.3%(P<0.01)。结论COS-Se具有抗氧化功能可能与其能上调巨噬细胞中SOD、GSH-Px mRNA表达水平有关。     

PMA诱导单核细胞分化过程中基质金属蛋白酶9的表达及其活性的变化

目的应用单核细胞向巨噬细胞分化的体外模型，研究单核细胞向巨噬细胞细胞分化过程中基质金属蛋白酶-9表达及其活性的变化．方法用乙酸肉豆蔻佛波酯（PMA）孵育THP1细胞不同时间，采用倒置显微镜观察细胞的形态学变化，RTPCR观察MMP-9mFINA表达的变化，Western blot观察MMP～9蛋白表达的变化，明胶酶谱（gelatin—zymogram）分析法测定MMP-9活性的变化。结果THP-1细胞经PMA孵育后，细胞由分散、悬浮转变为黏附、贴壁，且MMP-9的表达明显上调，活性增加．结论单核细胞向巨噬细胞分化的过程中，MMP-9的表达量及其活性明显增加。     

普罗布考在抗ox-LDL诱导THP-1巨噬细胞凋亡中对CD36、Caveolin-1表达的影响

目的研究普罗布考(Probucol)在抗ox-LDL诱导THP-1巨噬细胞凋亡中对CD36、Caveolin-1表达的影响。方法用流式细胞仪检测细胞凋亡;用RT-PCR、免疫荧光分别检测CD36、Caveolin-1mRNA水平和蛋白表达。结果普罗布考抗ox-LDL诱导THP-1巨噬细胞凋亡;RT-PCR检测发现普罗布考组与ox-LDL组CD36、Caveolin-1mRNA表达无明显差异;免疫荧光检测结果显示普罗布考下调Caveolin-1蛋白表达,但对CD36蛋白的表达没有影响。结论普罗布考在抗ox-LDL诱导THP-1巨噬细胞凋亡中下调Caveolin-1蛋白的表达。     

血晶素诱导HO-1表达增加及对急性肺损伤小鼠MMP-2、MMP-9 mRNA的影响

目的 通过复制急性肺损伤模型,探讨血晶素诱导血红素加氧酶-1（heme oxygenase-1, HO-1）的表达对于小鼠急性肺损伤的影响.方法 利用逆转录-聚合酶链反应和蛋白质印迹方法检测急性肺损伤小鼠肺组织基质金属蛋白酶-2 （matrix metalloproteinas-2, MMP-2）、MMP-9 mRNA和HO-1蛋白的表达.结果 血晶素加脂多糖组和对照组比较,HO-1蛋白表达明显增高 （P〈0.05）;脂多糖组与对照组比较,MMP-2 mRNA和MMP-9 mRNA表达差异有统计学意义（P〈0.05）,血晶素加脂多糖组与对照组比较,差异无统计学意义（P〉0.05）.结论 HO-1降低了急性肺损伤小鼠肺组织MMP-2、MMP-9 mRNA的表达,对于小鼠的急性肺损伤有保护作用.     

MMP-1在NIV毒素诱导软骨细胞损伤中的表达规律

目的观察NIV毒素对软骨细胞基质金属蛋白酶-1(MMP-1)基因表达的调节作用,在分子水平探讨软骨细胞损伤的机制。方法在体外单层培养的人胚软骨细胞中加入不同浓度(0.025,0.1,0.5 mg.L-1)的NIV毒素,连续作用5 d后收集软骨细胞和细胞培养液,用反转录—聚合酶反应(RT-PCR)检测软骨细胞MMP-1 mRNA表达;用酶联免疫吸附方法(ELISA)测定细胞培养液MMP-1的水平。结果 0.1～0.5 mg.L-1NIV毒素能引起MMP-1 mRNA表达增加(P0.05),并与毒素浓度呈剂量—效应关系;在0.025～0.5 mg.L-1NIV毒素作用下,细胞培养液中MMP-1的含量随毒素浓度升高逐渐增高(P0.05)。结论 NIV毒素可以促进软骨细胞合成MMP-1,这可能在软骨细胞损伤中发挥重要作用。     

姜黄素诱导急性单核细胞白血病细胞凋亡的实验研究

目的观察姜黄素对人白血病细胞株K562的致凋亡作用,及其对凋亡相关基因NF-κB与Caspase-3表达的影响。方法将姜黄素加入白血病细胞株THP-1中,用流式细胞仪检测其凋亡率,免疫组化方法检测NF-κB与Caspase-3的表达,比色法检测Caspase-3活性。结果流式细胞仪检测15μmol/L姜黄素作用24h后,凋亡率为(26.504±0.617)%,与对照组(1.232±0.120)%相比明显增高,差异有显著性(P<0.01),姜黄素诱导THP-1细胞凋亡具有量-效关系;免疫组化法检测发现姜黄素可降低NF-κB的表达,增加Caspase-3蛋白的表达,且表达量与姜黄素的作用呈剂量相关。结论姜黄素能诱导人白血病细胞株THP-1凋亡,其机制可能与姜黄素抑制NF-κB信号转导通路,下调NF-κB表达继而上调Caspase-3表达有关。     

Combined effects of low levels of palmitate on toxicity of ZnO nanoparticles to THP-1 macrophages

We have recently proposed that the interaction between food components and nanoparticles (NPs) should be considered when evaluating the toxicity of NPs. In the present study, we used THP-1 differentiated macrophages as a model for immune cells and investigated the combined toxicity of low levels of palmitate (PA; 10 or 50 μM) and ZnO NPs. The results showed that PA especially at 50 μM changed the size, Zeta potential and UV–vis spectra of ZnO NPs, indicating a possible coating effect. Up to 32 μg/mL ZnO NPs did not significantly affect mitochondrial activity, intracellular reactive oxygen species (ROS) or release of interleukin 6 (IL-6), but significantly impaired lysosomal function as assessed by neutral red uptake assay and acridine orange staining. The presence of 50 μM PA, but not 10 μM PA, further promoted the toxic effects of ZnO NPs to lysosomes but did not significantly affect other endpoints. In addition, ZnO NPs dose-dependently increased intracellular Zn ions in THP-1 macrophages, which was not significantly affected by PA. Taken together, the results of the present study showed a combined toxicity of low levels of PA and ZnO NPs especially to lysosomes in THP-1 macrophages.     

RalA exerts an inhibitory effect on IL-1β/IL-18 secretion by blocking NLRP3 inflammasome activation in levornidazole-treated human THP-1 macrophages

The NLRP3 inflammasome is an important mediator of inflammatory responses and its regulation is an active area of research. RalA is a Ras-like GTPase, which play pivotal roles in the biology of cells. So far, there have been very few studies on RalA regulating inflammatory responses. Bioinformatics analysis predicted that RalA might participate in the regulatory network of NLRP3 inflammasome, which has been confirmed in THP-1 macrophages. After virtual screening of compounds, it was found that levonidazole selected from our virtual small molecule compound library has the potential to bind to RalA. Of note, the interaction of RalA/levornidazole was verified by Surface Plasmon Resonance-Biacore T200, LC/MS analysis and Western blotting analysis. Molecular dynamics simulations revealed that the conformational changes of RalA might be regulated by levornidazole. Additionally, IL-1β/IL-18 secretion from ATP + LPS stimulated THP-1-derived macrophages was RalA-dependently suppressed by levornidazole, suggesting that RalA might have an inhibitory effect on NLRP3 inflammasome activation. The results of co-immunoprecipitation and RalA depletion experiments showed that levornidazole could induce RalA to block the assembly of NLRP3/ASC/pro-caspase-1 complex, thereby reducing the levels of cleaved-caspase-1 and IL-1β/IL-18 secretion. Our study has suggested an anti-inflammatory function of RalA and identified its targeting chemical compound. Overall, this study clarifies a novel pharmacological mechanism by which RalA/levornidazole inhibits NLRP3 inflammasome activation and IL-1β/IL-18 secretion.     

GD1a对基质金属蛋白酶-9在不同细胞类型中表达的调控作用

目的在不同的细胞类型中,检测神经节苷脂GD1a是否抑制基质金属蛋白酶-9(MMP-9)的表达。方法采用逆转录PCR法和酶谱法检测MMP-9及MMP-2的表达。结果在鼠黑色素瘤B16细胞中,神经节苷脂GD1a促进MMP-9的表达,而在小鼠肺癌Lewis细胞、人单核细胞THP-1及人子宫颈癌HeLa细胞中,神经节苷脂GD1a降低MMP-9的表达。在上述细胞中,神经节苷脂GD1a不影响基质金属蛋白酶MMP-2的表达;肿瘤坏死因子TNF-α在这几种细胞中均能促进MMP-9的表达,但对MMP-2无影响;在B16细胞中,GD1a通过胞窖膜介导信号传递,并且主要通过ERK促进MMP-9的表达,而在Lewis细胞中,GD1a不通过胞窖膜介导信号传递,但部分通过p38、ERK和JNK抑制MMP-9表达。结论在不同的细胞类型中,神经节苷脂GD1a能够激活不同的信号转导途径。     

The Effects of Shiga Toxin 1, 2 and Their Subunits on Cytokine and Chemokine Expression by Human Macrophage-Like THP-1 Cells

Infection by Shiga toxin (Stx)-producing enterohemorrhagic Escherichia coli (EHEC) results in severe diarrhea, hemorrhagic colitis, and, occasionally, hemolytic-uremic syndrome (HUS). HUS is associated with an increase in pro-inflammatory cytokines and chemokines, many of which are produced by macrophages in the kidneys, indicating that localized host innate immunity likely plays a role in renal pathogenesis. EHEC serotypes may express one or two classes of serologically defined but structurally and functionally-related Shiga toxins called Stx1 and Stx2. Of these, Stx2 appears to be linked to higher rates of HUS than Stx1. To investigate a possible reason for this, we exposed human macrophage-like THP-1 cells to Stx1 or Stx2 and then used the Luminex multiplex system to assess cytokine/chemokine concentrations in culture supernatant solutions. This analysis revealed that, relative to Stx1, Stx2 significantly caused increased expression of GRO, G-CSF, IL-1β, IL-8 and TNFα in macrophage-like THP-1 cells. This was determined to not be due to a difference in cytotoxicity since both Stx1 and Stx2 displayed similar cytotoxic activities on macrophage-like THP-1 cells. These observations indicate that, in vitro, Stx2 can provoke a greater pro-inflammatory response than Stx1 in macrophages and provides a possible partial explanation for higher rates of HUS in patients infected with EHEC strains expressing Stx2. To begin to determine a mechanism for Shiga toxin-mediated cytokine production, we exposed macrophage-like THP-1 cells to Stx1 or Stx2 A and B subunits. Luminex analysis of cytokines in cell culture supernatant solutions demonstrated that neither subunit alone induced a cytokine response in THP-1 cells.