补充生物活性肽QRPR对巨噬细胞RAW264.7抵御脂多糖刺激的作用

目的研究补充生物活性肽QRPR(Gln-Arg-Pro-AH)对巨噬细胞RAW264.7抵御脂多糖(lipopolysaccharides,LPS)刺激的作用。方法用不同浓度的LPS刺激RAW264.7细胞不同时间,ELISA法测定细胞培养上清中细胞因子白介素-6(interleukin-6,IL-6)及肿瘤坏死因子-α(tumor necrosis factor-alpha,TNF-α)的浓度,确定LPS刺激RAW264.7细胞的最适浓度和时间,建立RAW264.7细胞抵御LPS刺激模型。采用ELISA法测定QRPR肽对RAW264.7细胞抵御LPS刺激时IL-6和TNF-α表达量的影响。MTS法检测QRPR肽对RAW264.7细胞的毒性作用。结果100 ng/m L LPS诱导16 h为刺激RAW264.7细胞验证QRPR肽调节作用的最佳浓度和时间。QRPR肽可以抑制RAW264.7细胞受LPS刺激后IL-6和TNF-α的产生,QRPR肽浓度为250μmol/L时,其降低LPS刺激产生IL-6和TNF-α的能力最强。QRPR肽本身对RAW264.7细胞的生长既无促进作用,也无毒性和抑制作用。结论补充活性肽QRPR对巨噬细胞RAW264.7抵御LPS刺激具有积极的调节作用。     

激活素A对巨噬细胞系RAW264.7细胞吞饮活性及TLR4表达的双向调节作用

目的探讨激活素A(ActivinA)对巨噬细胞系RAW264.7细胞活性的调节作用及其可能的机制。方法取对数生长期的小鼠巨噬细胞系RAW264.7细胞,加入1μg/ml脂多糖(LPS),继续培养8h,采用ELISA法检测细胞分泌ActivinA水平;分别加入ActivinA、LPS和ActivinA+LPS,中性红染料法检测细胞吞饮活性;流式细胞术分析细胞表面分子MHCⅠ、MHCⅡ及Toll样受体4(TLR4)的表达水平。结果LPS呈时间依赖性刺激RAW264.7细胞分泌ActivinA;ActivinA可明显促进静息RAW264.7细胞的吞饮活性,而对MHCⅠ、MHCⅡ及TLR4的表达水平无明显影响;ActivinA和LPS共同作用,ActivinA明显抑制了LPS活化的RAW264.7细胞的吞饮活性,并下调TLR4的表达。结论ActivinA可能以自分泌/旁分泌形式参与巨噬细胞活性调节,其抑制LPS作用与TLR4表达有关。     

81种药用植物提取物影响小鼠巨噬细胞RAW264.7活化的研究

目的通过对81种药用植物提取物影响巨噬细胞活化的研究,获得相关活性数据,为后续研究开发和利用提供试验数据和物质基础。方法将提取物样品作用于RAW264.7细胞一定时间后,用ELISA法测定细胞分泌的TNF-α,以判断样品对细胞是否有活化作用;将LPS和81种提取物样品作用于RAW264.7细胞一定时间后,用ELISA法测定细胞分泌的TNF-α,以判断样品是否对LPS诱导的细胞活化有抑制作用。结果对81种药用植物提取物的研究结果表明,有34种植物提取物样品能够使RAW264.7细胞TNF-α表达上调,使其增加TNF-α的分泌;有2种植物提取物样品能够使LPS诱导的RAW264.7细胞TNF-α表达下调,使其减少TNF-α的分泌。结论通过对81种药用植物提取物的筛选研究表明,既有对巨噬细胞有活化作用的样品,也有能够抑制LPS诱导的巨噬细胞活化的样品,为进一步的研究,提供了相关活性数据和物质基础。     

脂多糖诱导小鼠巨噬细胞系RAW264.7细胞的活化凋亡作用

目的探讨脂多糖(LPS)诱导小鼠巨噬细胞系RAW264.7细胞活化凋亡的作用。方法体外培养小鼠巨噬细胞系RAW264.7细胞,分别用0.5、1.0、2.5μg/mlLPS刺激RAW264.7细胞24h,一氧化氮(NO)试剂盒检测细胞培养上清中NO水平;用1.0μg/mlLPS分别刺激细胞3d和6d,台盼蓝拒染法检测细胞增殖情况;用1.0μg/mlLPS刺激细胞6d,流式细胞术分析细胞周期及细胞凋亡情况。结果经LPS刺激后24h,RAW264.7细胞培养上清中NO含量明显增加,且具有剂量依赖性;LPS刺激3d和6d后,细胞的增殖均受到抑制,且呈时间依赖性;LPS刺激6d时,细胞周期被阻滞在S期,并出现明显的凋亡。结论LPS具有诱导小鼠巨噬细胞系RAW264.7细胞活化凋亡的作用。     

破骨细胞抑制因子生物活性定量测定方法的建立

目的以破骨细胞为模型,建立抑制破骨细胞功能药物的生物活性定量测定方法。方法将小鼠RAW264.7和SP2/0细胞在含地塞米松和1,25(OH)2VitD3的破骨细胞诱导分化液中混合培养,通过TRAP染色鉴定破骨细胞的成熟。加入不同浓度的rhOPG-Fc,检测其对RAW264.7细胞增殖、分化和成熟破骨细胞活性的抑制作用,计算药物的IC50,并定量计算活性单位。结果rhOPG-Fc对RAW264.7细胞增殖抑制的IC50为0.02mg/L,其生物活性为50U/mg;对RAW264.7细胞分化抑制的IC50为0.02mg/L,其生物活性为50U/mg;对成熟破骨细胞活性抑制的IC50为0.18mg/L,其生物活性为5.5U/mg。结论以破骨细胞抑制因子为候选药物所建立的细胞学模型,可方便快速地定量测定破骨细胞抑制类药物的生物活性,且能协助判断药物的作用机理。     

hCAP-18/LL-37基因转染对RAW264.7细胞活化功能的影响

目的探讨抗菌肽hCAP-18/LL-37基因转染对巨噬细胞RAW264.7活化功能的影响。方法将重组质粒pcD-NA4/Myc-His-hCAP-18/LL-37瞬时转染RAW264.7细胞,MTT法检测细胞的增殖活性;中性红吞噬试验检测细胞的吞噬能力;RT-PCR法分析细胞活化相关分子CD80、CD86、TLR4及细胞因子IL-1β、TNF-αmRNA的转录。结果 hCAP-18/LL-37基因转染可促进经脂多糖(Lipopolysaccharide,LPS)处理的RAW264.7细胞的增殖活性与吞噬能力(P<0.05);可上调RAW264.7细胞CD80、CD86、TLR4、IL-1β、TNF-α基因mRNA的转录水平。结论 hCAP-18/LL-37基因转染可通过促进RAW264.7细胞增殖活性、吞噬能力及活化相关分子表达,调控巨噬细胞的活化功能。     

激活素A抑制脂多糖活化巨噬细胞的作用机制

目的探讨激活素A(Activin A)抑制脂多糖(Lipopolysaccharides,LPS)活化巨噬细胞的作用机制。方法取小鼠巨噬细胞系RAW264.7细胞,分别添加Activin A(5 ng/ml)、LPS(1μg/ml)和Activin A(5 ng/ml)+LPS(1μg/ml),同时设以含单纯2.5%胎牛血清的DMEM培养液培养的细胞作为对照孔,培养24 h后,采用还原酶法检测细胞分泌一氧化氮(Nitric oxide,NO)的水平,流式细胞术分析TLR2和TLR4蛋白的表达水平,RT-PCR分析细胞ActRⅡA和ActRⅡB基因mRNA的转录水平。结果 Activin A和LPS单独作用均促进RAW264.7细胞分泌NO,但二者联合使用时,Activin A可抑制LPS刺激RAW264.7细胞的NO分泌;Activin A能抑制LPS上调RAW264.7细胞TLR4蛋白的表达,但对TLR2蛋白的表达无影响;LPS可促进RAW264.7细胞ActRⅡA基因mRNA的转录水平,但对ActRⅡB基因mRNA的转录水平无影响。结论 Activin A通过调控TLR4途径抑制LPS的作用,LPS可能通过促进ActRⅡA的表达进一步增强Activin A的负反馈调节作用。     

PPE68/Ipr1真核共表达质粒的构建及鉴定

目的构建结核分枝杆菌PPE68基因和小鼠胞内病原体抗性基因1(Intracellular pathogen resistance 1,Ipr1)的真核共表达质粒,并在小鼠巨噬细胞RAW264.7中表达。方法将结核分枝杆菌PPE68基因和小鼠Ipr1基因分别亚克隆至含多启动子的共表达载体pBudCE4.1中,构建真核共表达质粒pBud68-Ipr1,转染RAW264.7细胞,通过RT-PCR及Western blot法检测PPE68和Ipr1基因的转录和表达。结果重组表达质粒pBud68-Ipr1经PCR、双酶切及测序证实,插入的PPE68和Ipr1基因片段序列正确;质粒转染RAW264.7细胞后,PPE68和Ipr1基因进行了转录和表达,基因编码产物相对分子质量分别为37 000和50 000。结论已成功构建了真核共表达质粒pBud68-Ipr1,并在RAW264.7细胞中获得表达,为PPE68/Ipr1重组BCG的构建及其免疫保护作用的进一步研究奠定了基础。     

依克多因与羟基积雪草甙协同抗炎作用的研究

采用LPS诱导RAW264.7细胞炎症模型证实了依克多因、羟基积雪草甙及依克多因与羟基积雪草甙组合配比在设定的质量浓度条件下,对LPS诱导的RAW264.7细胞产生NO均具有良好的抑制作用,即在细胞水平具有抗炎活性。同时依克多因与羟基积雪草甙组合在设定的不同质量浓度及配比下对NO的抑制率大多高于单一组分,并且依克多因与羟基积雪草甙组合在设定的不同质量浓度及配比下的SI值1,以上说明依克多因与羟基积雪草甙组合在抗炎功效方面具有协同增效作用,关于其协同增效机理,需要进一步实验研究确定。     

激活素A对小鼠巨噬细胞RAW264.7吞噬活性的促进作用

目的探讨激活素A对巨噬细胞吞噬活性的促进作用及其机制。方法分别通过中性红试验、鸡红细胞法和流式细胞术,检测激活素A对巨噬细胞系RAW264.7细胞的吞饮活性、吞噬活性以及对MHCⅠ、Ⅱ类分子和CD68表达的影响。结果激活素A可明显促进RAW264.7细胞的吞饮及吞噬活性,对MHCI、II类分子表达没有影响,但可明显上调巨噬细胞表面分子CD68的表达。结论激活素A可促进巨噬细胞系RAW264.7细胞吞噬活性,这与其促进巨噬细胞成熟有关。     

β-Cypermethrin Alleviated the Inhibitory Effect of Medium from RAW 264.7 Cells on 3T3-L1 Cell Maturation into Adipocytes

Studies have elucidated that pyrethroids induce adipogenesis. It is also known that macrophages can affect the homeostasis of adipose tissue. However, whether and how the β-cypermethrin (β-CYP)-mediated inhibition of the macrophages affects adipogenesis remain unknown. To explore the effects of β-CYP on adipogenesis through modulating the function of macrophages, 3T3-L1 cells, a preadipocyte cell line, were exposed to culture medium from either RAW 264.7 cells, a macrophage cell line (RM), or β-CYP-treated RAW 264.7 cells (CRM). CRM decreased the inhibitory effects of RM treatment on cell proliferation and adipogenesis, as lipid accumulation, the CEBPA content, and Fasn and Acaca expression in 3T3-L1 cells were higher following CRM treatment than following RM treatment through the higher levels of the demethylated CEBPA promoter in 3T3-L1 cells. However, the medium from β-CYP- and N-acetyl-L-cysteine-cotreated RAW 264.7 cells (CNRM) partially restored the inhibitory effects of RAW 264.7 cells on 3T3-L1 cells that had been reduced by CRM, indicating that β-CYP might reduce the cytotoxicity and inhibitory effects of RAW 264.7 cells on the adipogenesis of 3T3-L1 cells through elevating ROS levels in RAW 264.7 cells. Moreover, exposure to β-CYP downregulated the TNF-α secretion in RAW 264.7 cells. In conclusion, these data demonstrated that β-CYP affected the function of RAW 264.7 cells, alleviating their inhibitory effects on adipogenesis and CEBPA demethylation in 3T3-L1 cells. β-CYP might achieve these effects through downregulating the secretion of TNF-α via elevating ROS levels in RAW 264.7 cells. Our experiments provide a new perspective on the obesogenic effect of pyrethroids.     

In Vitro Studies Regarding the Safety of Chitosan and Hyaluronic Acid-Based Nanohydrogels Containing Contrast Agents for Magnetic Resonance Imaging

The aim of this study was to investigate the biocompatibility of contrast agents, such as gadolinium 1, 4, 7, 10 tetraazacyclo-dodecane tetraacetic acid (GdDOTA) and gadolinium dioctyl terephthalate (GdDOTP), encapsulated in a polymeric matrix containing chitosan and hyaluronic acid using RAW264.7 murine macrophages and human blood samples. The cell viability and cytotoxicity were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays, while cell cycle analysis was determined in RAW264.7 cells using flow cytometry. The mitochondrial membrane potential (MMP), hemolytic index, complement activation, and thrombogenic potential of gadolinium (Gd) containing nanohydrogels were measured by fluorometric and spectrophotometric methods. Taken together, our results demonstrate the good bio- and hemocompatibility of chitosan-based nanohydrogels with the RAW264.7 cell line and human blood cells, suggesting that these could be used as injectable formulations for the magnetic resonance imaging diagnostic of lymph nodes.     

Dietary Methionine Deficiency Enhances Genetic Instability in Murine Immune Cells

Both cell and animal studies have shown that complete or partial deficiency of methionine inhibits tumor growth. Consequently, the potential implementation of this nutritional intervention has recently been of great interest for the treatment of cancer patients. Unfortunately, diet alteration can also affect healthy immune cells such as monocytes/macrophages and their precursor cells in bone marrow. As around half of cancer patients are treated with radiotherapy, the potential deleterious effect of dietary methionine deficiency on immune cells prior to and/or following irradiation needs to be evaluated. Therefore, we examined whether modulation of methionine content alters genetic stability in the murine RAW 264.7 monocyte/macrophage cell line in vitro by chromosomal analysis after 1-month culture in a methionine-deficient or supplemented medium. We also analyzed chromosomal aberrations in the bone marrow cells of CBA/J mice fed with methionine-deficient or supplemented diet for 2 months. While all RAW 264.7 cells revealed a complex translocation involving three chromosomes, three different clones based on the banding pattern of chromosome 9 were identified. Methionine deficiency altered the ratio of the three clones and increased chromosomal aberrations and DNA damage in RAW 264.7. Methionine deficiency also increased radiation-induced chromosomal aberration and DNA damage in RAW 264.7 cells. Furthermore, mice maintained on a methionine-deficient diet showed more chromosomal aberrations in bone marrow cells than those given methionine-adequate or supplemented diets. These findings suggest that caution is warranted for clinical implementation of methionine-deficient diet concurrent with conventional cancer therapy.     

Escherichia coli Maltose-Binding Protein Induces M1 Polarity of RAW264.7 Macrophage Cells via a TLR2- and TLR4-Dependent Manner

Maltose-binding protein (MBP) is a critical player of the maltose/maltodextrin transport system in Escherichia coli. Our previous studies have revealed that MBP nonspecifically induces T helper type 1 (Th1) cell activation and activates peritoneal macrophages obtained from mouse. In the present study, we reported a direct stimulatory effect of MBP on RAW264.7 cells, a murine macrophage cell line. When stimulated with MBP, the production of nitric oxide (NO), IL-1β, IL-6 and IL-12p70, and the expressions of CD80, MHC class II and inducible nitric oxide synthase (iNOS) were all increased in RAW264.7 cells, indicating the activation and polarization of RAW264.7 cells into M1 macrophages induced by MBP. Further study showed that MBP stimulation upregulated the expression of TLR2 and TLR4 on RAW264.7 cells, which was accompanied by subsequent phosphorylation of IκB-α and p38 MAPK. Pretreatment with anti-TLR2 or anti-TLR4 antibodies largely inhibited the phosphorylation of IκB-α and p38 MAPK, and greatly reduced MBP-induced NO and IL-12p70 production, suggesting that the MBP-induced macrophage activation and polarization were mediated by TLR2 and TLR4 signaling pathways. The observed results were independent of lipopolysaccharide contamination. Our study provides a new insight into a mechanism by which MBP enhances immune responses and warrants the potential application of MBP as an immune adjuvant in immune therapies.     

Bazedoxifene,a Selective Estrogen Receptor Modulator,Promotes Functional Recovery in a Spinal Cord Injury Rat Model

In research on various central nervous system injuries, bazedoxifene acetate (BZA) has shown two main effects: neuroprotection by suppressing the inflammatory response and remyelination by enhancing oligodendrocyte precursor cell differentiation and oligodendrocyte proliferation. We examined the effects of BZA in a rat spinal cord injury (SCI) model. Anti-inflammatory and anti-apoptotic effects were investigated in RAW 264.7 cells, and blood-spinal cord barrier (BSCB) permeability and angiogenesis were evaluated in a human brain endothelial cell line (hCMEC/D3). In vivo experiments were carried out on female Sprague Dawley rats subjected to moderate static compression SCI. The rats were intraperitoneally injected with either vehicle or BZA (1mg/kg pre-SCI and 3 mg/kg for 7 days post-SCI) daily. BZA decreased the lipopolysaccharide-induced production of proinflammatory cytokines and nitric oxide in RAW 264.7 cells and preserved BSCB disruption in hCMEC/D3 cells. In the rats, BZA reduced caspase-3 activity at 1 day post-injury (dpi) and suppressed phosphorylation of MAPK (p38 and ERK) at dpi 2, hence reducing the expression of IL-6, a proinflammatory cytokine. BZA also led to remyelination at dpi 20. BZA contributed to improvements in locomotor recovery after compressive SCI. This evidence suggests that BZA may have therapeutic potential to promote neuroprotection, remyelination, and functional outcomes following SCI.     

Endoplasmic Reticulum Stress Cooperates in Zearalenone-Induced Cell Death of RAW 264.7 Macrophages

Zearalenone (ZEA) is a fungal mycotoxin that causes cell apoptosis and necrosis. However, little is known about the molecular mechanisms of ZEA toxicity. The objective of this study was to explore the effects of ZEA on the proliferation and apoptosis of RAW 264.7 macrophages and to uncover the signaling pathway underlying the cytotoxicity of ZEA in RAW 264.7 macrophages. This study demonstrates that the endoplasmic reticulum (ER) stress pathway cooperated in ZEA-induced cell death of the RAW 264.7 macrophages. Our results show that ZEA treatment reduced the viability of RAW 264.7 macrophages in a dose- and time-dependent manner as shown by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay (MTT) and flow cytometry assay. Western blots analysis revealed that ZEA increased the expression of glucose-regulated protein 78 (GRP78) and CCAAT/enhancer binding protein homologous protein (CHOP), two ER stress-related marker genes. Furthermore, treating the cells with the ER stress inhibitors 4-phenylbutyrate (4-PBA) or knocking down CHOP, using lentivirus encoded short hairpin interfering RNAs (shRNAs), significantly diminished the ZEA-induced increases in GRP78 and CHOP, and cell death. In summary, our results suggest that ZEA induces the apoptosis and necrosis of RAW 264.7 macrophages in a dose- and time-dependent manner via the ER stress pathway in which the activation of CHOP plays a critical role.